G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian)
G00000888 (Mus musculus)

Databases (9)

Curated Gene
OTTHUMG00000032417 (Vega human gene)
ENSG00000197122 (Ensembl human gene)
6714 (Entrez Gene)
15 (G2Cdb plasticity & disease)
SRC (GeneCards)
190090 (OMIM)
Marker Symbol
HGNC:11283 (HGNC)
Protein Expression
4023 (human protein atlas)
Protein Sequence
P12931 (UniProt)

Synonyms (2)

  • ASV
  • c-src

Diseases (4)

Disease Nervous effect Mutations Found Literature Mutations Type Genetic association?
D00000100: Myeloid leukaemia N Y (2506951) Deletion (D) Y
D00000045: Endometrial carcinoma N Y (10804287) Single nucleotide polymorphism (SNP) Y
D00000021: Colon cancer N Y (9988270) Single nucleotide polymorphism (SNP) Y
D00000022: Colorectal cancer N Y (11161376) Single nucleotide polymorphism (SNP) N
D00000100: Myeloid leukaemia N Y (8241509) Deletion (D) Y


  • Lack of mutation at codon 531 of SRC in advanced colorectal cancers from Italian patients.

    Laghi L, Bianchi P, Orbetegli O, Gennari L, Roncalli M and Malesci A

    Department of Gastroenterology, Istituto Clinico Humanitas, Via Manzoni 56, Rozzano, Milan, 20089, Italy.

    A truncating mutation (C to T transition) at codon 531 of the human protooncogene c-src, possibly accounting for the activation of c-src tyrosine kinase, has been recently identified in a subset of advanced colorectal cancer from North-American patients. However, two subsequent studies have failed to confirm the occurrence of SRC 531 mutation in colorectal cancers from North-European and Asiatic patients, raising the hypothesis that the genetic activation of src in colon cancer might be restricted to patients belonging to specific ethnic groups. We investigated a large series of colorectal cancers from Italian patients (155 cases) with a high prevalence of liver metastasis (43%). Using a PCR-RFLP assay, the occurrence of a SRC 531 mutation was ruled out in all the investigated specimens of primary tumours and/or metastases. Our results demonstrate that SRC Gln531AMB plays no role in the development or in the progression of colorectal cancer among Italian patients.

    British journal of cancer 2001;84;2;196-8

  • Mutation of the SRC gene in endometrial carcinoma.

    Sugimura M, Kobayashi K, Sagae S, Nishioka Y, Ishioka S, Terasawa K, Tokino T and Kudo R

    Department of Obstetrics and Gynecology, Cancer Research Institute, Sapporo Medical University School of Medicine, Chuo-ku, Sapporo 060-0061, Japan.

    Recently, an activating mutation of the SRC gene has been implicated in about one-tenth of advanced colon cancers. The SRC 531 mutation results in truncation of SRC directly C-terminal to the regulatory Tyr 530 and appears to activate the Tyr 530. To investigate whether mutation of SRC plays an important role in the development and progression of gynecological tumors, we performed mutational analysis of the entire coding region of SRC in 70 ovarian carcinomas, 68 endometrial carcinomas and 3 endometrial stromal sarcomas by means of polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) followed by nucleotide sequencing and restriction fragment length polymorphism (RFLP) analysis. We found one truncated mutation at codon 531 (Gln to Stop) in an endometrial carcinoma. However, we found no mutation of this gene in ovarian carcinoma or endometrial stromal sarcoma. Our results suggest that mutation of SRC may be implicated in a small proportion of endometrial carcinomas.

    Japanese journal of cancer research : Gann 2000;91;4;395-8

  • Activating SRC mutation in a subset of advanced human colon cancers.

    Irby RB, Mao W, Coppola D, Kang J, Loubeau JM, Trudeau W, Karl R, Fujita DJ, Jove R and Yeatman TJ

    Department of Surgery, H. Lee Moffitt Cancer Center & Research Institute, University of South Florida College of Medicine, Tampa 33612, USA.

    The discovery of Rous sarcoma virus (RSV) led to the identification of cellular Src (c-Src), a non-receptor tyrosine kinase, which has since been implicated in the development of numerous human cancers. c-Src has been found to be highly activated in colon cancers, particularly in those metastatic to the liver. Studies of the mechanism of c-Src regulation have suggested that c-Src kinase activity is downregulated by phosphorylation of a critical carboxy-terminal tyrosine (Tyr 530 in human c-Src, equivalent to Tyr 527 in chicken Src) and have implied the existence of activating mutations in this C-terminal regulatory region. We report here the identification of a truncating mutation in SRC at codon 531 in 12% of cases of advanced human colon cancer tested and demonstrate that the mutation is activating, transforming, tumorigenic and promotes metastasis. These results provide, for the first time, genetic evidence that activating SRC mutations may have a role in the malignant progression of human colon cancer.

    Funded by: NCI NIH HHS: CA55652, CA65512

    Nature genetics 1999;21;2;187-90

  • Molecular genetics of myeloid leukemia: identification of the commonly deleted segment of chromosome 20.

    Roulston D, Espinosa R, Stoffel M, Bell GI and Le Beau MM

    Department of Medicine, University of Chicago Cancer Research Center, IL.

    A deletion of the long arm of chromosome 20 [del(20q)] is a recurring abnormality in malignant myeloid disorders. The occurrence of the del(20q) in a broad spectrum of myeloid disorders suggests that the loss of genetic material on 20q could provide a proliferative advantage to myeloid cells, possibly through the loss of a tumor-suppressor gene. We have examined a series of patients with the del(20q) using fluorescence in situ hybridization (FISH) with unique sequence probes that map along the length of 20q, and have delineated a segment that is deleted in 95% of all patients examined (18 of 19). In addition, we have shown that the deletions are interstitial rather than terminal. This region of deletion extends from 20q11.2 to q12, and is flanked by the RPN2 (proximal) and D20S17 loci (distal). The SRC and ADA genes are located within the commonly deleted segment. Our findings emphasize the importance of FISH and other molecular mapping techniques in defining such a region. The delineation of a commonly deleted segment in 20q11.2-q12 will facilitate the identification of candidate tumor-suppressor genes on 20q.

    Funded by: NCI NIH HHS: CA40046

    Blood 1993;82;11;3424-9

  • Localization of the SRC oncogene to chromosome band 20q11.2 and loss of this gene with deletion (20q) in two leukemic patients.

    Morris CM, Honeybone LM, Hollings PE and Fitzgerald PH

    Cytogenetic and Molecular Oncology Unit, Christchurch Hospital, New Zealand.

    In situ hybridization of the pHul-c-src probe to metaphase cells from three normal donors and two leukemic patients showed significant labeling in the proximal region of the long arm of chromosome 20q, with modal peaks of grains consistently at band 20q11.2. A secondary peak of grains was detected in the region 20q13.2-qter, the localization of SRC suggested by previous in situ studies. The exact localization of SRC is important for understanding the del(20q) chromosomal abnormality in myeloid neoplasias. Chromosome in situ hybridization and genomic studies showed loss of one allele of SRC in two patients with the deletion (20q). These results differ from previously published findings and suggest heterogeneity of the breakpoint at 20q11.2 in interstitial deletions of 20q, which characterize myeloid disorders.

    Blood 1989;74;5;1768-73

Literature (670)

Pubmed - human_disease

  • Lack of mutation at codon 531 of SRC in advanced colorectal cancers from Italian patients.

    Laghi L, Bianchi P, Orbetegli O, Gennari L, Roncalli M and Malesci A

    Department of Gastroenterology, Istituto Clinico Humanitas, Via Manzoni 56, Rozzano, Milan, 20089, Italy.

    A truncating mutation (C to T transition) at codon 531 of the human protooncogene c-src, possibly accounting for the activation of c-src tyrosine kinase, has been recently identified in a subset of advanced colorectal cancer from North-American patients. However, two subsequent studies have failed to confirm the occurrence of SRC 531 mutation in colorectal cancers from North-European and Asiatic patients, raising the hypothesis that the genetic activation of src in colon cancer might be restricted to patients belonging to specific ethnic groups. We investigated a large series of colorectal cancers from Italian patients (155 cases) with a high prevalence of liver metastasis (43%). Using a PCR-RFLP assay, the occurrence of a SRC 531 mutation was ruled out in all the investigated specimens of primary tumours and/or metastases. Our results demonstrate that SRC Gln531AMB plays no role in the development or in the progression of colorectal cancer among Italian patients.

    British journal of cancer 2001;84;2;196-8

  • Mutation of the SRC gene in endometrial carcinoma.

    Sugimura M, Kobayashi K, Sagae S, Nishioka Y, Ishioka S, Terasawa K, Tokino T and Kudo R

    Department of Obstetrics and Gynecology, Cancer Research Institute, Sapporo Medical University School of Medicine, Chuo-ku, Sapporo 060-0061, Japan.

    Recently, an activating mutation of the SRC gene has been implicated in about one-tenth of advanced colon cancers. The SRC 531 mutation results in truncation of SRC directly C-terminal to the regulatory Tyr 530 and appears to activate the Tyr 530. To investigate whether mutation of SRC plays an important role in the development and progression of gynecological tumors, we performed mutational analysis of the entire coding region of SRC in 70 ovarian carcinomas, 68 endometrial carcinomas and 3 endometrial stromal sarcomas by means of polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) followed by nucleotide sequencing and restriction fragment length polymorphism (RFLP) analysis. We found one truncated mutation at codon 531 (Gln to Stop) in an endometrial carcinoma. However, we found no mutation of this gene in ovarian carcinoma or endometrial stromal sarcoma. Our results suggest that mutation of SRC may be implicated in a small proportion of endometrial carcinomas.

    Japanese journal of cancer research : Gann 2000;91;4;395-8

  • Molecular genetics of myeloid leukemia: identification of the commonly deleted segment of chromosome 20.

    Roulston D, Espinosa R, Stoffel M, Bell GI and Le Beau MM

    Department of Medicine, University of Chicago Cancer Research Center, IL.

    A deletion of the long arm of chromosome 20 [del(20q)] is a recurring abnormality in malignant myeloid disorders. The occurrence of the del(20q) in a broad spectrum of myeloid disorders suggests that the loss of genetic material on 20q could provide a proliferative advantage to myeloid cells, possibly through the loss of a tumor-suppressor gene. We have examined a series of patients with the del(20q) using fluorescence in situ hybridization (FISH) with unique sequence probes that map along the length of 20q, and have delineated a segment that is deleted in 95% of all patients examined (18 of 19). In addition, we have shown that the deletions are interstitial rather than terminal. This region of deletion extends from 20q11.2 to q12, and is flanked by the RPN2 (proximal) and D20S17 loci (distal). The SRC and ADA genes are located within the commonly deleted segment. Our findings emphasize the importance of FISH and other molecular mapping techniques in defining such a region. The delineation of a commonly deleted segment in 20q11.2-q12 will facilitate the identification of candidate tumor-suppressor genes on 20q.

    Funded by: NCI NIH HHS: CA40046

    Blood 1993;82;11;3424-9

Pubmed - other

  • Dab1 tyrosine phosphorylation sites relay positional signals during mouse brain development.

    Howell BW, Herrick TM, Hildebrand JD, Zhang Y and Cooper JA

    Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA. HowellB@ninds.nih.gov

    Background: The extracellular protein Reln controls neuronal migrations in parts of the cortex, hippocampus and cerebellum. In vivo, absence of Reln correlates with up-regulation of the docking protein Dab1 and decreased Dab1 tyrosine phosphorylation. Loss of the Reln receptor proteins, apolipoprotein receptor 2 and very low density lipoprotein receptor, results in a Reln-like phenotype accompanied by increased Dab1 protein expression. Complete loss of Dab1, however, recapitulates the Reln phenotype.

    Results: To determine whether Dab1 tyrosine phosphorylation affects Dab1 protein expression and positioning of embryonic neurons, we have identified Dab1 tyrosine phosphorylation sites. We then generated mice in which the Dab1 protein had all the potential tyrosine phosphorylation sites mutated. This mutant protein is not tyrosine phosphorylated during brain development and is not upregulated to the extent observed in the Reln or the apoER2 and VLDLR receptor mutants. Animals expressing the non-phosphorylated Dab1 protein have a phenotype similar to the dab1-null mutant.

    Conclusions: Dab1 is downregulated by the Reln signal in neurons in the absence of tyrosine phosphorylation. Dab1 tyrosine phosphorylation sites and not downregulation of Dab1 protein are required for Reln signaling.

    Funded by: NCI NIH HHS: 5P30-CA15704-26, R37-CA41072

    Current biology : CB 

  • A non-catalytic function of the Src family tyrosine kinases controls prolactin-induced Jak2 signaling.

    García-Martínez JM, Calcabrini A, González L, Martín-Forero E, Agulló-Ortuño MT, Simon V, Watkin H, Anderson SM, Roche S and Martín-Pérez J

    Instituto de Investigaciones Biomédicas A. Sols (CSIC/UAM), Arturo Duperier 4, 28029 Madrid, Spain.

    The cytokine prolactin (PRL) plays important roles in the proliferation and differentiation of the mammary gland and it has been implicated in tumorigenesis. The prolactin receptor (PRLR) is devoid of catalytic activity and its mitogenic response is controlled by cytoplasmic tyrosine kinases of the Src (SFK) and Jak families. How PRLR uses these kinases for signaling is not well understood. Previous studies indicated that PRLR-induced Jak2 activation does not require SFK catalytic activity in favor of separate signaling operating on this cellular response. Here we show that, nevertheless, PRLR requires Src-SH2 and -SH3 domains for Jak2 signaling. In W53 lymphoid cells, conditional expression of two c-Src non-catalytic mutants, either SrcK295M/Y527F or SrcK, whose SH3 and SH2 domains are exposed, controls Jak2/Stat5 activation by recruiting Jak2, avoiding its activation by endogenous active SFK. In contrast, the kinase inactive SrcK295M mutant, with inaccessible SH3 and SH2 domains, does not. Furthermore, all three mutants attenuate PRLR-induced Akt and p70S6K activation. Accordingly, PRLR-induced Jak2/Stat5 signaling is inhibited in MCF7 breast cancer cells by Src depletion, expression of SrcK295M/Y527F or active Src harboring an inactive SH2 (SrcR175L) or SH3 domain (SrcW118A). Finally, Jak2/Stat5 pathway is also reduced in Src-/- mice mammary glands. We thus conclude that, in addition to Akt and p70S6K, SFK regulate PRLR-induced Jak2 signaling through a kinase-independent mechanism.

    Funded by: NCI NIH HHS: R01CA085736-P01HD38129

    Cellular signalling 2010;22;3;415-26

  • Initiation factor eIF2-independent mode of c-Src mRNA translation occurs via an internal ribosome entry site.

    Allam H and Ali N

    Department of Biological Sciences, University of Denver, Denver, Colorado 80208-0183, USA.

    Overexpression and activation of the c-Src protein have been linked to the development of a wide variety of cancers. The molecular mechanism(s) of c-Src overexpression in cancer cells is not clear. We report here an internal ribosome entry site (IRES) in the c-Src mRNA that is constituted by both 5'-noncoding and -coding regions. The inhibition of cap-dependent translation by m(7)GDP in the cell-free translation system or induction of endoplasmic reticulum stress in hepatoma-derived cells resulted in stimulation of the c-Src IRES activities. Sucrose density gradient analyses revealed formation of a stable binary complex between the c-Src IRES and purified HeLa 40 S ribosomal subunit in the absence of initiation factors. We further demonstrate eIF2-independent assembly of 80 S initiation complex on the c-Src IRES. These features of the c-Src IRES appear to be reminiscent of that of hepatitis C virus-like IRESs and translation initiation in prokaryotes. Transfection studies and genetic analysis revealed that the c-Src IRES permitted initiation at the authentic AUG351, which is also used for conventional translation initiation of the c-Src mRNA. Our studies unveiled a novel regulatory mechanism of c-Src synthesis mediated by an IRES element, which exhibits enhanced activity during cellular stress and is likely to cause c-Src overexpression during oncogenesis and metastasis.

    Funded by: NIAID NIH HHS: R03AI063046

    The Journal of biological chemistry 2010;285;8;5713-25

  • Differential phosphorylation of the docking protein Gab1 by c-Src and the hepatocyte growth factor receptor regulates different aspects of cell functions.

    Chan PC, Sudhakar JN, Lai CC and Chen HC

    Department of Life Science, National Chung Hsing University, Taichung, Taiwan.

    The docking protein Grb2-associated binder1 (Gab1) has a central role in the integration of the growth-factor signaling. In this study, we aimed to examine the significance of Src-mediated Gab1 phosphorylation in the hepatocyte growth factor (HGF) signaling. Using both mutagenesis and mass spectrometry approaches, Y242, Y259, Y317, Y373 and Y627 of Gab1 were identified to be phosphorylated by c-Src. It is interesting to note that the binding of the tyrosine phosphatase SHP2 to the Y627 antagonized the effect of c-Src on the phosphorylation of the other four tyrosine residues. Moreover, the tyrosine residues predominantly phosphorylated by c-Src were different from those predominantly phosphorylated by the HGF receptor. Gab1 overexpression potentiated both mitogenic and motogenic activities of HGF. However, a Gab1 mutant with substitutions of the Src phosphorylation sites (Y242, Y259, Y317 and Y373) failed to promote HGF-induced DNA synthesis, but retained its ability to facilitate HGF-induced chemotaxis. Taken together, our results not only suggest that the phosphorylation of Gab1 by c-Src is important for HGF-induced DNA synthesis, but also provide an example to illustrate how a docking protein (for example, Gab1) is differentially phosphorylated by c-Src and a receptor tyrosine kinase to emanate full spectrum of signals to the downstream.

    Oncogene 2010;29;5;698-710

  • BCAR3 regulates Src/p130 Cas association, Src kinase activity, and breast cancer adhesion signaling.

    Schuh NR, Guerrero MS, Schrecengost RS and Bouton AH

    Department of Microbiology, University of Virginia Health System, Charlottesville, Virginia 22908, USA.

    The nonreceptor protein-tyrosine kinase c-Src is frequently overexpressed and/or activated in a variety of cancers, including those of the breast. Several heterologous binding partners of c-Src have been shown to regulate its catalytic activity by relieving intramolecular autoinhibitory interactions. One such protein, p130(Cas) (Cas), is expressed at high levels in both breast cancer cell lines and breast tumors, providing a potential mechanism for c-Src activation in breast cancers. The Cas-binding protein BCAR3 (breast cancer antiestrogen resistance-3) is expressed at high levels in invasive breast cancer cell lines, and this molecule has previously been shown to coordinate with Cas to increase c-Src activity in COS-1 cells. In this study, we show for the first time using gain- and loss-of-function approaches that BCAR3 regulates c-Src activity in the endogenous setting of breast cancer cells. We further show that BCAR3 regulates the interaction between Cas and c-Src, both qualitatively as well as quantitatively. Finally, we present evidence that the coordinated activity of these proteins contributes to breast cancer cell adhesion signaling and spreading. Based on these data, we propose that the c-Src/Cas/BCAR3 signaling axis is a prominent regulator of c-Src activity, which in turn controls cell behaviors that lead to aggressive and invasive breast tumor phenotypes.

    Funded by: NCI NIH HHS: CA130168, F31 CA130168, R01 CA096846, T32 CA009109; NIDDK NIH HHS: P30 DK067629

    The Journal of biological chemistry 2010;285;4;2309-17

  • Src tyrosine kinase preactivation is associated with platelet hypersensitivity in essential thrombocythemia and polycythemia vera.

    Randi ML, Brunati AM, Scapin M, Frasson M, Deana R, Magrin E, Fabris F and Donella-Deana A

    Department of Medical and Surgical Sciences, University of Padova, Padova, Italy.

    Polycythemia vera (PV) and essential thrombocythemia (ET) are chronic myeloproliferative disorders characterized by an increased incidence of thrombo-hemorrhagic complications. The acquired somatic Janus kinase 2 (JAK2) V617F mutation is present in the majority of PV and ET patients. Because aberrant protein Tyr-phosphorylation has been associated with hematopoietic malignancies, the activity of the tyrosine kinases Src and JAK2 was analyzed in resting and thrombin-stimulated platelets from 13 PV and 42 ET patients. JAK2 was found inactive in healthy and pathological resting cells regardless of the V617F mutation. In addition, Src was inactive in all resting platelets, but in the pathological specimens it was present in a preactivated conformation as a consequence of anomalous dephosphorylation of its inhibitory phospho-Tyr527 residue, likely mediated by Src homology-2 domain-containing protein Tyr-phosphatase-2 (SHP-2), whose constitutive activity correlated with its recruitment to Src. Low thrombin concentration triggered a more rapid Src-signaling activation, higher [Ca(2+)](c) increase, and aggregation in pathological platelets compared with controls. Thrombin-induced Src activation preceded JAK2 activation, which occurred simultaneously in normal and pathological platelets. Our results indicate that a constitutive Src kinase preactivation is implicated in platelet hypersensitivity and likely involved, at least partially, in the functional abnormalities of PV and ET platelets.

    Blood 2010;115;3;667-76

  • Tyrosine phosphorylation of cofilin at Y68 by v-Src leads to its degradation through ubiquitin-proteasome pathway.

    Yoo Y, Ho HJ, Wang C and Guan JL

    Division of Molecular Medicine and Genetics, Departments of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA.

    Cofilin is a major regulator of actin dynamics involved in the regulation of cell spreading and migration through its actin depolymerizing and severing activities. v-Src is an activated Src tyrosine kinase and a potent oncogene known to phosphorylate a variety of cellular proteins in cell transformation process including altered cell adhesion, spreading and migration. Recently, it has been suggested that cofilin is a potential substrate of v-Src (Rush et al., 2005). Here, we show direct tyrosine phosphorylation of cofilin by v-Src and identify Y68 as the major phosphorylation site. Cofilin phosphorylation at Y68 did not change its activity per se, but induced increased ubiquitination of cofilin and its degradation through the proteosome pathway. Furthermore, the negative effect of cofilin on cellular F-actin contents was inhibited by coexpression of v-Src, whereas that of cofilin mutant Y68F (Y68 mutated to F) was not affected, suggesting that v-Src-mediated cofilin phosphorylation at Y68 is required for the degradation of cofilin in vivo. Lastly, inhibition of cell spreading by v-Src was rescued partially by coexpression of cofilin, and to a greater extent by the Y68F mutant, which is not subjected to v-Src-induced degradation through phosphorylation, suggesting that v-Src-mediated changes in cell spreading is, at least in part, through inhibiting the function of cofilin through phosphorylating it at Y68. Together, these results suggest a novel mechanism by which cofilin is regulated by v-Src through tyrosine phosphorylation at Y68 that triggers the degradation of cofilin through ubiquitination-proteosome pathway and consequently inhibits cofilin activity in reducing cellular F-actin contents and cell spreading.

    Funded by: NCI NIH HHS: R01 CA150926; NIGMS NIH HHS: GM48050, R01 GM048050, R01 GM048050-17

    Oncogene 2010;29;2;263-72

  • Involvement of Src tyrosine kinase in Escherichia coli invasion of human brain microvascular endothelial cells.

    Liu W, Zhao WD, Yan JC, Ren ZY, Fang WG, Zhu L, Shang DS and Chen YH

    Department of Developmental Biology, Key Laboratory of Cell Biology, Ministry of Public Health, Heping Distric, Shenyang, China.

    Invasion of brain microvascular endothelial cells is a prerequisite for successful crossing of the blood-brain barrier by Escherichia coli (E. coli), but the underlying mechanism remains unclear. Here we showed activation of Src tyrosine kinase in E. coli K1 invasion of human brain microvascular endothelial cells (HBMEC). E. coli invasion of HBMEC and the E. coli-induced rearrangement of actin filaments were blocked by Src inhibitors. Overexpression of dominant-negative Src in HBMEC significantly attenuated E. coli invasion and the concomitant actin filaments rearrangement. Furthermore, E. coli K1-triggered phosphatidylinositol 3-kinase (PI3K) activation in HBMEC was effectively blocked by Src inhibitors and dominant-negative Src. These results demonstrated the involvement of Src and its interaction with PI3K in E. coli K1 invasion of HBMEC.

    FEBS letters 2010;584;1;27-32

  • ADAM12 localizes with c-Src to actin-rich structures at the cell periphery and regulates Src kinase activity.

    Stautz D, Sanjay A, Hansen MT, Albrechtsen R, Wewer UM and Kveiborg M

    Department of Biomedical Sciences and Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark.

    ADAM12 is an active metalloprotease playing an important role in tumour progression. Human ADAM12 exists in two splice variants: a long transmembrane form, ADAM12-L, and a secreted form, ADAM12-S. The subcellular localization of ADAM12-L is tightly regulated and involves intracellular interaction partners and signalling proteins. We demonstrate here a c-Src-dependent redistribution of ADAM12-L from perinuclear areas to actin-rich Src-positive structures at the cell periphery, and identified two separate c-Src binding sites in the cytoplasmic tail of ADAM12-L that interact with the SH3 domain of c-Src with different binding affinities. The association between ADAM12-L and c-Src is transient, but greatly stabilized when the c-Src kinase activity is disrupted. In agreement with this observation, kinase-active forms of c-Src induce ADAM12-L tyrosine phosphorylation. Interestingly, ADAM12-L was also found to enhance Src kinase activity in response to external signals, such as integrin engagement. Thus, we suggest that activated c-Src binds, phosphorylates, and redistributes ADAM12-L to specific sites at the cell periphery, which may in turn promote signalling mechanisms regulating cellular processes with importance in cancer.

    Experimental cell research 2010;316;1;55-67

  • Alpha5beta1 integrin engagement increases large conductance, Ca2+-activated K+ channel current and Ca2+ sensitivity through c-src-mediated channel phosphorylation.

    Yang Y, Wu X, Gui P, Wu J, Sheng JZ, Ling S, Braun AP, Davis GE and Davis MJ

    Department of Medical Pharmacology and Physiology, University of Missouri School of Medicine, Columbia, Missouri 65212, USA.

    Large conductance, calcium-activated K(+) (BK) channels are important regulators of cell excitability and recognized targets of intracellular kinases. BK channel modulation by tyrosine kinases, including focal adhesion kinase and c-src, suggests their potential involvement in integrin signaling. Recently, we found that fibronectin, an endogenous alpha5beta1 integrin ligand, enhances BK channel current through both Ca(2+)- and phosphorylation-dependent mechanisms in vascular smooth muscle. Here, we show that macroscopic currents from HEK 293 cells expressing murine BK channel alpha-subunits (mSlo) are acutely potentiated following alpha5beta1 integrin activation. The effect occurs in a Ca(2+)-dependent manner, 1-3 min after integrin engagement. After integrin activation, normalized conductance-voltage relations for mSlo are left-shifted at free Ca(2+) concentrations >or=1 microm. Overexpression of human c-src with mSlo, in the absence of integrin activation, leads to similar shifts in mSlo Ca(2+) sensitivity, whereas overexpression of catalytically inactive c-src blocks integrin-induced potentiation. However, neither integrin activation nor c-src overexpression potentiates current in BK channels containing a point mutation at Tyr-766. Biochemical tests confirmed the critical importance of residue Tyr-766 in integrin-induced channel phosphorylation. Thus, BK channel activity is enhanced by alpha5beta1 integrin activation, likely through an intracellular signaling pathway involving c-src phosphorylation of the channel alpha-subunit at Tyr-766. The net result is increased current amplitude, enhanced Ca(2+) sensitivity, and rate of activation of the BK channel, which would collectively promote smooth muscle hyperpolarization in response to integrin-extracellular matrix interactions.

    Funded by: Canadian Institutes of Health Research; NCRR NIH HHS: C06 RR017353, RR-017353; NHLBI NIH HHS: HL-71796, HL-72989, R01 HL071796, R01 HL072989

    The Journal of biological chemistry 2010;285;1;131-41

  • Src kinase-mediated phosphorylation stabilizes inducible nitric-oxide synthase in normal cells and cancer cells.

    Tyryshkin A, Gorgun FM, Abdel Fattah E, Mazumdar T, Pandit L, Zeng S and Eissa NT

    Department of Medicine, Baylor College of Medicine, Houston, Texas 77030, USA.

    Src kinases are key regulators of cellular proliferation, survival, motility, and invasiveness. They play important roles in the regulation of inflammation and cancer. Overexpression or hyperactivity of c-Src has been implicated in the development of various types of cancer, including lung cancer. Src inhibition is currently being investigated as a potential therapy for non-small cell lung cancer in Phase I and II clinical trials. The mechanisms of Src implication in cancer and inflammation are linked to the ability of activated Src to phosphorylate multiple downstream targets that mediate its cellular effector functions. In this study, we reveal that inducible nitric-oxide synthase (iNOS), an enzyme also implicated in cancer and inflammation, is a downstream mediator of activated Src. We elucidate the molecular mechanisms of the association between Src and iNOS in models of inflammation induced by lipopolysaccharide and/or cytokines and in cancer cells and tissues. We identify human iNOS residue Tyr(1055) as a target for Src-mediated phosphorylation. These results are shown in normal cells and cancer cells as well as in vivo in mice. Importantly, such posttranslational modification serves to stabilize iNOS half-life. The data also demonstrate interactions and co-localization of iNOS and activated Src under inflammatory conditions and in cancer cells. This study demonstrates that phosphorylation of iNOS by Src plays an important role in the regulation of iNOS and nitric oxide production and hence could account for some Src-related roles in inflammation and cancer.

    The Journal of biological chemistry 2010;285;1;784-92

  • Diesel particle-induced transcriptional expression of p21 involves activation of EGFR, Src, and Stat3.

    Cao D, Bromberg PA and Samet JM

    Human Studies Division, National Health and Environmental Effects Research Laboratory, Research Triangle Park, NC 27711, USA.

    Exposure to diesel exhaust particles (DEP) has been associated with adverse health outcomes such as inflammation, adjuvancy, and mutagenesis. However, the molecular mechanisms by which DEP inhalation exerts these effects are still largely unknown. We previously reported that exposure to DEP activates the transcription factor Stat3 in airway epithelial cells, a primary target cell of inhaled DEP. To elucidate the functional role of Stat3 activation in these cells, we investigated the function of Stat3 in DEP-induced expression of the p21 gene in the human bronchial epithelial cell line BEAS-2B. We report that DEP exposure induces increased levels of p21 mRNA and protein in a manner that is independent of p53 and Sp1 expression or DNA binding to the p21 gene. Using chromatin immunoprecipitation assays and expression of a dominant-negative Stat3 mutant, we show that activation of Stat3 and its binding to the p21 promoter are required for DEP-induced expression of p21, suggesting that Stat3 plays an essential role in the induction of p21 by DEP. Additional experiments demonstrated that activation of p21 gene expression is dependent on the activation of epidermal growth factor receptor and Src kinase activities. Finally, we provide evidence suggesting that DEP exposure can inhibit the proliferation of human bronchial epithelial cells, suggesting a functional role of p21 activation airway epithelial cells exposed to DEP.

    American journal of respiratory cell and molecular biology 2010;42;1;88-95

  • Epidermal growth factor receptor translocation to the mitochondria: regulation and effect.

    Demory ML, Boerner JL, Davidson R, Faust W, Miyake T, Lee I, Hüttemann M, Douglas R, Haddad G and Parsons SJ

    Department of Microbiology and the Cancer Center, University of Virginia, Charlottesville, Virginia 22908, USA.

    Co-overexpression of the epidermal growth factor (EGF) receptor (EGFR) and c-Src frequently occurs in human tumors and is linked to enhanced tumor growth. In experimental systems this synergistic growth requires EGF-dependent association of c-Src with the EGFR and phosphorylation of Tyr-845 of the receptor by c-Src. A search for signaling mediators of Tyr(P)-845 revealed that mitochondrial cytochrome c oxidase subunit II (CoxII) binds EGFR in a Tyr(P)-845- and EGF-dependent manner. In cells this association involves translocation of EGFR to the mitochondria, but regulation of this process is ill-defined. The current study demonstrates that c-Src translocates to the mitochondria with similar kinetics as EGFR and that the catalytic activity of EGFR and c-Src as well as endocytosis and a mitochondrial localization signal are required for these events. CoxII can be phosphorylated by EGFR and c-Src, and EGF stimulation reduces Cox activity and cellular ATP, an event that is dependent in large part on EGFR localized to the mitochondria. These findings suggest EGFR plays a novel role in modulating mitochondrial function via its association with, and modification of CoxII.

    Funded by: NCI NIH HHS: CA123037, CA71449, R01 CA071449, R01 CA123037; NICHD NIH HHS: P01 HD 32473

    The Journal of biological chemistry 2009;284;52;36592-604

  • Primary human colonic epithelial cells are transiently protected from anoikis by a Src-dependent mechanism.

    Hofmann C, Lippert E, Falk W, Schölmerich J, Rogler G and Obermeier F

    Department of Internal Medicine I, Regensburg University Medical Center, 93042 Regensburg, Germany. claudia.hofmann@klinik.uni-regensburg.de

    Complete loss of cell anchorage triggers apoptosis in primary human colonic epithelial cells (CEC), a phenomenon known as anoikis. Besides the induction of pro-apoptotic events, activation of survival pathways was observed in detached intestinal epithelial cell lines, providing a transient apoptosis protection. However, nothing is known about molecular mechanisms protecting primary CEC from anoikis. In this study intact CEC crypts were isolated and kept in suspension, a condition which leads to the loss of cell-cell anchorage and induces anoikis. To reconstitute cell-cell contacts, cells were centrifuged to form cell aggregates. Induction of apoptosis was assessed by caspase-3 activity assay; activation of survival pathways was analyzed by Western blot. Immediately after loss of cell anchorage a rapid activation of survival proteins was observed before active caspase-3 could be detected. Src hyperactivation significantly contributed to transient protection from anoikis in CEC because its inhibition reversed the protecting effect of re-establishment of cell contacts. Basal levels of active Src in CEC from patients with inflammatory bowel disease were markedly reduced compared to control patients. These results demonstrate that loss of cell anchorage activates survival pathways in primary human CEC providing transient anoikis protection. Src is an important mediator of this mechanism and therefore constitutes a key regulatory molecule coordinating survival signals mediated by cell adhesion in primary human CEC.

    Biochemical and biophysical research communications 2009;390;3;908-14

  • A novel pro-apoptotic function of RACK1: suppression of Src activity in the intrinsic and Akt pathways.

    Mamidipudi V and Cartwright CA

    Department of Medicine, Stanford University, Stanford, CA 94305-5187, USA.

    Earlier we showed that RACK1 regulates growth of human colon cells by suppressing Src activity at G(1) and mitotic checkpoints. Here, we show that RACK1 also induces apoptosis of the cells, partly by inhibiting Src. In the intrinsic pathway, RACK1 inhibits expression of anti-apoptotic Bcl-2 and Bcl-X(L), induces expression of pro-apoptotic Bim, targets Bim and Bax to the mitochondria, induces oligomerization of Bax (which requires Bim and inhibition of Src), depolarizes mitochondria membranes, releases cytochrome c, and activates caspases-9 and -3 and death substrates. Bax and Bim are required for RACK1-mediated mitochondrial cell death. RACK1-induced oligomerization of Bax is required for staurosporine-mediated cell death. RACK1 also induces apoptosis by blocking Src activation of the Akt cell survival pathway. This leads to activation of the transcription factor FOXO3, a potent inducer of apoptosis and G(1) arrest. Collectively, our results show that RACK1, partly by inhibiting Src, promotes mitochondrial cell death and blocks Akt-mediated cell survival. Thus, RACK1 inhibits growth and induces death of colon cells. Exploitation of these dual functions could lead to novel colon cancer therapies that mimic RACK1 function.

    Funded by: NIDDK NIH HHS: R01 DK043743

    Oncogene 2009;28;50;4421-33

  • Migfilin interacts with Src and contributes to cell-matrix adhesion-mediated survival signaling.

    Zhao J, Zhang Y, Ithychanda SS, Tu Y, Chen K, Qin J and Wu C

    Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.

    Integrin-mediated cell-extracellular matrix (ECM) adhesion is essential for protection of epithelial cells against apoptosis, but the underlying mechanism is incompletely understood. Here we show that migfilin, an integrin-proximal adaptor protein, interacts with Src and contributes to cell-ECM-mediated survival signaling. Loss of cell-ECM adhesion markedly reduces the migfilin level in untransformed epithelial cells and concomitantly induces apoptosis. Overexpression of migfilin substantially desensitizes cell detachment-induced apoptosis. Conversely, depletion of migfilin promotes apoptosis despite the presence of cell-ECM adhesion. At the molecular level migfilin directly interacts with Src, and the migfilin binding surface overlaps with the inhibitory intramolecular interaction sites in Src. Consequently, the binding of migfilin activates Src, resulting in suppression of apoptosis. Our results reveal a novel mechanism by which cell-ECM adhesion regulates Src activation and survival signaling. This migfilin-mediated signaling pathway is dysfunctional in multiple types of carcinoma cells, which likely contributes to aberrant Src activation and anoikis resistance in the cancerous cells.

    Funded by: NIDDK NIH HHS: DK54639, R01 DK054639, R01 DK054639-13; NIGMS NIH HHS: GM62823, GM65188, R01 GM062823, R01 GM065188, R01 GM065188-08

    The Journal of biological chemistry 2009;284;49;34308-20

  • Abl interactor 1 regulates Src-Id1-matrix metalloproteinase 9 axis and is required for invadopodia formation, extracellular matrix degradation and tumor growth of human breast cancer cells.

    Sun X, Li C, Zhuang C, Gilmore WC, Cobos E, Tao Y and Dai Z

    Department of Internal Medicine, Texas Tech University Health Sciences Center, Amarillo, TX 79106, USA.

    Abl interactor 1 (Abi1) is a key regulator of actin polymerization/depolymerization. The involvement of Abi1 in the development of abnormal cytoskeletal functions of cancer cells has recently been reported. It remains unclear, however, how Abi1 exerts its effects in tumor cells and whether it contributes to tumor progression in vivo. We report here a novel function for Abi1 in the regulation of invadopodia formation and Src-inhibitor of differentiation protein 1 (Id1)-matrix metalloproteinase (MMP)-9 pathway in MDA-MB-231 human breast cancer cells. Abi1 is found in the invadopodia of MDA-MB-231 cells. Epigenetic silencing of the Abi1 gene by short hairpin RNA in MDA-MB-231 cells impaired the formation of invadopodia and resulted in downregulation of the Src activation and Id1/MMP-9 expression. The decreased invadopodia formation and MMP-9 expression correlate with a reduction in the ability of these cells to degrade extracellular matrix. Remarkably, the knockdown of Abi1 expression inhibited tumor cell proliferation and migration in vitro and slowed tumor growth in vivo. Taken together, these results indicate that the Abi1 signaling plays a critical role in breast cancer progression and suggest that this pathway may serve as a therapeutic target for the treatment of human breast cancer.

    Funded by: NCI NIH HHS: R01 CA094921, R01 CA094921-06, R21 CA133597, R21 CA133597-01A1; NIDDK NIH HHS: K01 DK067191

    Carcinogenesis 2009;30;12;2109-16

  • Cdk5-dependent regulation of Rho activity, cytoskeletal contraction, and epithelial cell migration via suppression of Src and p190RhoGAP.

    Tripathi BK and Zelenka PS

    Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, 5635 Fishers Lane, Room 1127, Bethesda, MD 20892, USA.

    Cdk5 regulates adhesion and migration in a variety of cell types. We previously showed that Cdk5 is strongly activated during stress fiber formation and contraction in spreading cells. Here we determine the mechanism linking Cdk5 to stress fiber contractility and its relevance to cell migration. Immunofluorescence showed that Cdk5 colocalized with phosphorylated myosin regulatory light chain (pMRLC) on contracting stress fibers. Inhibiting Cdk5 activity by various means significantly reduced pMRLC level and cytoskeletal contraction, with loss of central stress fibers. Blocking Cdk5 activity also reduced Rho-Rho kinase (ROCK) signaling, which is the principal pathway of myosin phosphorylation under these conditions. Next, we examined the effect of Cdk5 activity on Src, a known regulator of Rho. Inhibiting Cdk5 activity increased Src activation and phosphorylation of its substrate, p190RhoGAP, an upstream inhibitor of Rho. Inhibiting both Cdk5 and Src activity completely reversed the effect of Cdk5 inhibition on Rho and prevented the loss of central stress fibers, demonstrating that Cdk5 exerts its effects on Rho-ROCK signaling by suppressing Src activity. Moreover, inhibiting either Cdk5 or ROCK activity increased cell migration to an equal extent, while inhibiting both kinases produced no additional effect, demonstrating that Cdk5-dependent regulation of ROCK activity is a physiological determinant of migration rate.

    Funded by: Intramural NIH HHS; NEI NIH HHS: Z01 EY000238

    Molecular and cellular biology 2009;29;24;6488-99

  • High-density association study of 383 candidate genes for volumetric BMD at the femoral neck and lumbar spine among older men.

    Yerges LM, Klei L, Cauley JA, Roeder K, Kammerer CM, Moffett SP, Ensrud KE, Nestlerode CS, Marshall LM, Hoffman AR, Lewis C, Lang TF, Barrett-Connor E, Ferrell RE, Orwoll ES, Zmuda JM and MrOS Research Group

    Department of Epidemiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.

    Genetics is a well-established but poorly understood determinant of BMD. Whereas some genetic variants may influence BMD throughout the body, others may be skeletal site specific. We initially screened for associations between 4608 tagging and potentially functional single nucleotide polymorphisms (SNPs) in 383 candidate genes and femoral neck and lumbar spine volumetric BMD (vBMD) measured from QCT scans among 862 community-dwelling white men >or=65 yr of age in the Osteoporotic Fractures in Men Study (MrOS). The most promising SNP associations (p < 0.01) were validated by genotyping an additional 1156 white men from MrOS. This analysis identified 8 SNPs in 6 genes (APC, DMP1, FGFR2, FLT1, HOXA, and PTN) that were associated with femoral neck vBMD and 13 SNPs in 7 genes (APC, BMPR1B, FOXC2, HOXA, IGFBP2, NFATC1, and SOST) that were associated with lumbar spine vBMD in both genotyping samples (p < 0.05). Although most associations were specific to one skeletal site, SNPs in the APC and HOXA gene regions were associated with both femoral neck and lumbar spine BMD. This analysis identifies several novel and robust genetic associations for volumetric BMD, and these findings in combination with other data suggest the presence of genetic loci for volumetric BMD that are at least to some extent skeletal-site specific.

    Funded by: NCRR NIH HHS: UL1 RR024140, UL1 RR024153; NIA NIH HHS: T32 AG000181, T32-AG00181, U01 AG018197, U01 AG027810, U01 AG042140, U01 AG18197, U01-AG027810; NIAMS NIH HHS: R01 AR051124, R01-AR051124, U01 AR045580, U01 AR045583, U01 AR045614, U01 AR045632, U01 AR045647, U01 AR045654, U01 AR45580, U01 AR45583, U01 AR45614, U01 AR45632, U01 AR45647, U01 AR45654

    Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 2009;24;12;2039-49

  • Clostridium difficile toxin A binds colonocyte Src causing dephosphorylation of focal adhesion kinase and paxillin.

    Kim H, Rhee SH, Pothoulakis C and LaMont JT

    Department of Life Science, College of Natural Science, Daejin University, Pochen, Kyungkido, Republic of Korea.

    Clostridium difficile toxin A impairs tight junction function of colonocytes by glucosylation of Rho family proteins causing actin filament disaggregation and cell rounding. We investigated the effect of toxin A on focal contact formation by assessing its action on focal adhesion kinase (FAK) and the adapter protein paxillin. Exposure of NCM460 human colonocytes to toxin A for 1 h resulted in complete dephosphorylation of FAK and paxillin, while protein tyrosine phosphatase activity was reduced. Blockage of toxin A-associated glucosyltransferase activity by co-incubation with UDP-2'3' dialdehyde did not reduce toxin A-induced FAK and paxillin dephosphorylation. GST-pull down and in vitro kinase activity experiments demonstrated toxin A binding directly to the catalytic domain of Src with suppression of its kinase activity. Direct binding of toxin A to Src, independent of any effect on protein tyrosine phosphatase or Rho glucosylation, inhibits Src kinase activity followed by FAK/paxillin inactivation. These mechanisms may contribute to toxin A inhibition of colonocyte focal adhesion that occurs in human colonic epithelium exposed to toxin A.

    Funded by: NIDDK NIH HHS: DK R37-03458, P0-1 DK 33506, P01 DK033506, R37 DK034583-24

    Experimental cell research 2009;315;19;3336-44

  • An epigenetic switch involving NF-kappaB, Lin28, Let-7 MicroRNA, and IL6 links inflammation to cell transformation.

    Iliopoulos D, Hirsch HA and Struhl K

    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.

    Inflammation is linked clinically and epidemiologically to cancer, and NF-kappaB appears to play a causative role, but the mechanisms are poorly understood. We show that transient activation of Src oncoprotein can mediate an epigenetic switch from immortalized breast cells to a stably transformed line that forms self-renewing mammospheres that contain cancer stem cells. Src activation triggers an inflammatory response mediated by NF-kappaB that directly activates Lin28 transcription and rapidly reduces let-7 microRNA levels. Let-7 directly inhibits IL6 expression, resulting in higher levels of IL6 than achieved by NF-kappaB activation. IL6-mediated activation of the STAT3 transcription factor is necessary for transformation, and IL6 activates NF-kappaB, thereby completing a positive feedback loop. This regulatory circuit operates in other cancer cells lines, and its transcriptional signature is found in human cancer tissues. Thus, inflammation activates a positive feedback loop that maintains the epigenetic transformed state for many generations in the absence of the inducing signal.

    Funded by: NCI NIH HHS: CA 107486, R01 CA107486-03, R01 CA107486-05

    Cell 2009;139;4;693-706

  • An acidic extracellular pH disrupts adherens junctions in HepG2 cells by Src kinases-dependent modification of E-cadherin.

    Chen Y, Chen CH, Tung PY, Huang SH and Wang SM

    Department of Anatomy, College of Medicine, National Taiwan University, Taipei 10051, Taiwan.

    We have previously shown that culturing HepG2 cells in pH 6.6 culture medium increases the c-Src-dependent tyrosine phosphorylation of beta-catenin and induces disassembly of adherens junctions (AJs). Here, we investigated the upstream mechanism leading to this pH 6.6-induced modification of E-cadherin. In control cells cultured at pH 7.4, E-cadherin staining was linear and continuous at cell-cell contact sites. Culturing cells at pH 6.6 was not cytotoxic, and resulted in weak and discontinuous junctional E-cadherin staining, consistent with the decreased levels of E-cadherin in membrane fractions. pH 6.6 treatment activated c-Src and Fyn kinase and induced tyrosine phosphorylation of p120 catenin (p120ctn) and E-cadherin. Inhibition of Src family kinases by PP2 attenuated the pH 6.6-induced tyrosine phosphorylation of E-cadherin and p120ctn, and prevented the loss of these proteins from AJs. In addition, E-cadherin was bound to Hakai and ubiquitinated. Furthermore, pH 6.6-induced detachment of E-cadherin from AJs was blocked by pretreatment with MG132 or NH(4)Cl, indicating the involvement of ubiquitin-proteasomal/lysosomal degradation of E-cadherin. An early loss of p120ctn prior to E-cadherin detachment from AJs was noted, concomitant with a decreased association between p120ctn and E-cadherin at pH 6.6. PP2 pretreatment prevented the dissociation of these two proteins. In conclusion, pH 6.6 activated Src kinases, resulting in tyrosine phosphorylation of E-cadherin and p120ctn and a weakening of the association of E-cadherin with p120ctn and contributing to the instability of E-cadherin at AJs.

    Journal of cellular biochemistry 2009;108;4;851-9

  • The PDZ protein MPP2 interacts with c-Src in epithelial cells.

    Baumgartner M, Weiss A, Fritzius T, Heinrich J and Moelling K

    Institute of Medical Virology, University of Zürich, Zürich, Switzerland. Martin.Baumgartner@mopa.unibe.ch

    c-Src is a non-receptor tyrosine kinase involved in regulating cell proliferation, cell migration and cell invasion and is tightly controlled by reversible phosphorylation on regulatory sites and through protein-protein interactions. The interaction of c-Src with PDZ proteins was recently identified as novel mechanism to restrict c-Src function. The objective of this study was to identify and characterise PDZ proteins that interact with c-Src to control its activity. By PDZ domain array screen, we identified the interaction of c-Src with the PDZ protein Membrane Protein Palmitoylated 2 (MPP2), a member of the Membrane-Associated Guanylate Kinase (MAGUK) family, to which also the Discs large (Dlg) tumour suppressor protein belongs. The function of MPP2 has not been established and the functional significance of the MPP2 c-Src interaction is not known. We found that in non-transformed breast epithelial MCF-10A cells, endogenous MPP2 associated with the cytoskeleton in filamentous structures, which partially co-localised with microtubules and c-Src. MPP2 and c-Src interacted in cells, where c-Src kinase activity promoted increased interaction of c-Src with MPP2. We furthermore found that MPP2 was able to negatively regulate c-Src kinase activity in cells, suggesting that the functional significance of the MPP2-c-Src interaction is to restrict Src activity. Consequently, the c-Src-dependent disorganisation of the cortical actin cytoskeleton of epithelial cells expressing c-Src was suppressed by MPP2. In conclusion we demonstrate here that MPP2 interacts with c-Src in cells to control c-Src activity and morphological function.

    Experimental cell research 2009;315;17;2888-98

  • Tyrosine phosphorylation of nuclear-membrane protein emerin by Src, Abl and other kinases.

    Tifft KE, Bradbury KA and Wilson KL

    Department of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

    X-linked recessive Emery-Dreifuss muscular dystrophy (EDMD) is caused by loss of emerin, a nuclear-membrane protein with roles in nuclear architecture, gene regulation and signaling. Phosphoproteomic studies have identified 13 sites of tyrosine phosphorylation in emerin. We validated one study, confirming that emerin is hyper-tyrosine-phosphorylated in Her2-overexpressing cells. We discovered that non-receptor tyrosine kinases Src and Abl each phosphorylate emerin and a related protein, LAP2beta, directly. Src phosphorylated emerin specifically at Y59, Y74 and Y95; the corresponding triple Y-to-F (;FFF') mutation reduced tyrosine phosphorylation by approximately 70% in vitro and in vivo. Substitutions that removed a single hydroxyl moiety either decreased (Y19F, Y34, Y161F) or increased (Y4F) emerin binding to BAF in cells. Y19F, Y34F, Y161F and the FFF mutant also reduced recombinant emerin binding to BAF from HeLa lysates, demonstrating the involvement of both LEM-domain and distal phosphorylatable tyrosines in binding BAF. We conclude that emerin function is regulated by multiple tyrosine kinases, including Her2, Src and Abl, two of which (Her2, Src) regulate striated muscle. These findings suggest roles for emerin as a downstream effector and ;signal integrator' for tyrosine kinase signaling pathway(s) at the nuclear envelope.

    Funded by: NIGMS NIH HHS: GM48646

    Journal of cell science 2009;122;Pt 20;3780-90

  • Identification of SH3 domain interaction partners of human FasL (CD178) by phage display screening.

    Voss M, Lettau M and Janssen O

    Institute of Immunology, Christian-Albrechts-University of Kiel, D-24105 Kiel, Germany. matthias.voss@med.uni-muenchen.de

    Background: Fas ligand is a cytotoxic effector molecule of T and NK cells which is characterized by an intracellular N-terminal polyproline region that serves as a docking site for SH3 and WW domain proteins. Several previously described Fas ligand-interacting SH3 domain proteins turned out to be crucial for the regulation of storage, expression and function of the death factor. Recent observations, however, indicate that Fas ligand is also subject to posttranslational modifications including shedding and intramembrane proteolysis. This results in the generation of short intracellular fragments that might either be degraded or translocate to the nucleus to influence transcription. So far, protein-protein interactions that specifically regulate the fate of the intracellular fragments have not been identified.

    Results: In order to further define the SH3 domain interactome of the intracellular region of Fas ligand, we now screened a human SH3 domain phage display library. In addition to known SH3 domains mediating binding to the Fas ligand proline-rich domain, we were able to identify a number of additional SH3 domains that might also associate with FasL. Potential functional implications of the new binding proteins for the death factor's biology are discussed. For Tec kinases and sorting nexins, the observed interactions were verified in cellular systems by pulldown experiments.

    Conclusion: We provide an extended list of putative Fas ligand interaction partners, confirming previously identified interactions, but also introducing several novel SH3 domain proteins that might be important regulators of Fas ligand function.

    BMC immunology 2009;10;53

  • Src tyrosine kinase regulates adhesion and chemotaxis in Waldenstrom macroglobulinemia.

    Ngo HT, Azab AK, Farag M, Jia X, Melhem MM, Runnels J, Roccaro AM, Azab F, Sacco A, Leleu X, Anderson KC and Ghobrial IM

    Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts 02115, USA.

    Purpose: Waldenstrom macroglobulinemia is a lymphoplasmacytic lymphoma characterized by widespread involvement of the bone marrow. Despite different options of therapy, Waldenstrom macroglobulinemia is still incurable. Src tyrosine kinase has been shown to play a central role in the regulation of a variety of biological processes, such as cell proliferation, migration, adhesion, and survival in solid tumors. We sought to determine whether the protein tyrosine kinase Src regulates adhesion, migration, and survival in Waldenstrom macroglobulinemia.

    We tested the expression of Src tyrosine kinase in Waldenstrom macroglobulinemia and normal cells, and the effect of the specific Src inhibitor AZD0530 on the adhesion, migration, cell cycle, and survival of a Waldenstrom macroglobulinemia cell line and patient samples. Moreover, we tested the effect of AZD0530 on cytoskeletal and cell cycle signaling in Waldenstrom macroglobulinemia.

    Results: We show that Src is overexpressed in Waldenstrom macroglobulinemia cells compared with control B cells, and that the use of the Src inhibitor AZD0530 led to significant inhibition of adhesion, migration, and cytoskeletal signaling induced by SDF1. Moreover, inhibition of Src activity induced G(1) cell cycle arrest; however, it had minimal effect on survival of Waldenstrom macroglobulinemia cells, and no significant effect on survival of normal cells.

    Conclusions: Taken together, these results delineate the role of Src kinase activity in Waldenstrom macroglobulinemia and provide the framework for future clinical trials using Src inhibitors in combination with other drugs to improve the outcome of patients with Waldenstrom macroglobulinemia.

    Funded by: NCI NIH HHS: R01 CA125690, R01CA125690-01, R21 CA126119, R21 CA126119-01A1, R21CA126119-01

    Clinical cancer research : an official journal of the American Association for Cancer Research 2009;15;19;6035-41

  • Cross talk between receptor guanylyl cyclase C and c-src tyrosine kinase regulates colon cancer cell cytostasis.

    Basu N, Bhandari R, Natarajan VT and Visweswariah SS

    Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India.

    Increased activation of c-src seen in colorectal cancer is an indicator of a poor clinical prognosis, suggesting that identification of downstream effectors of c-src may lead to new avenues of therapy. Guanylyl cyclase C (GC-C) is a receptor for the gastrointestinal hormones guanylin and uroguanylin and the bacterial heat-stable enterotoxin. Though activation of GC-C by its ligands elevates intracellular cyclic GMP (cGMP) levels and inhibits cell proliferation, its persistent expression in colorectal carcinomas and occult metastases makes it a marker for malignancy. We show here that GC-C is a substrate for inhibitory phosphorylation by c-src, resulting in reduced ligand-mediated cGMP production. Consequently, active c-src in colonic cells can overcome GC-C-mediated control of the cell cycle. Furthermore, docking of the c-src SH2 domain to phosphorylated GC-C results in colocalization and further activation of c-src. We therefore propose a novel feed-forward mechanism of activation of c-src that is induced by cross talk between a receptor GC and a tyrosine kinase. Our findings have important implications in understanding the molecular mechanisms involved in the progression and treatment of colorectal cancer.

    Molecular and cellular biology 2009;29;19;5277-89

  • Integrin (alpha6beta4) signals through Src to increase expression of S100A4, a metastasis-promoting factor: implications for cancer cell invasion.

    Kim TH, Kim HI, Soung YH, Shaw LA and Chung J

    Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana, USA.

    Integrin alpha6beta4 is linked to cancer cell motility and invasion in aggressive and metastatic cancer cells. In this study, we showed that expression of the beta4 integrin in MDA-MB-435 cancer cells (MDA-MB-435/beta4) leads to a dramatic increase in expression of a metastasis-promoting factor, S100A4, as determined by affymetrix gene chip microarray, quantitative real-time PCR, and Western blot analysis. Alternatively, knocking down beta4 integrin expression in MDA-MB-231 breast carcinoma cells by shRNA reduced the level of S100A4 expression. The mechanism by which alpha6beta4 enhances S100A4 expression involves Src, Akt, and NFAT. We have further shown that Y1494, a tyrosine residue of the ITIM motif in the cytoplasmic domain of the beta4 integrin subunit, is essential for alpha6beta4-dependent S100A4 expression. Reduction of S100A4 expression by shRNA blocked migration, invasion, and anchorage-independent growth of MDA-MB-435/beta4, SUM-159, and MDA-MB-231 cells. These studies define a novel mechanism by which integrin alpha6beta4 promotes cancer cell motility and invasion, and provides insight into how S100A4 expression is regulated in cancer cells.

    Molecular cancer research : MCR 2009;7;10;1605-12

  • Is expression or activation of Src kinase associated with cancer-specific survival in ER-, PR- and HER2-negative breast cancer patients?

    Elsberger B, Tan BA, Mitchell TJ, Brown SB, Mallon EA, Tovey SM, Cooke TG, Brunton VG and Edwards J

    Section of Surgery, Division of Cancer Sciences and Molecular Pathology, Glasgow Royal Infirmary, Glasgow G31 2ER, UK.

    The aim of the current study was to assess the expression levels of c-Src and phosphorylated Src kinase in human breast cancers and to establish if these are linked to oestrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 status or patient survival. Tissue microarray technology was used to analyze 314 breast cancer specimens. Immunohistochemistry was performed using antibodies to c-Src, Y419Src, and Y215Src, and expression was assessed using the weighted histoscore method. High cytoplasmic c-Src kinase and high membrane phosphorylated activated Y419Src kinase was associated with decreased disease-specific survival. In contrast, phosphorylated activated nuclear and cytoplasmic Y215Src kinase expression levels were significantly associated with improved disease-specific survival. When the cohort was subdivided according to ER/PR/HER2 status, the ER-negative subgroup (105 patients) was associated with improved disease-specific survival and was found to be independent by multivariate analysis with a hazard ratio of 0.4 (interquartile range 0.2-0.8). High cytoplasmic c-Src expression was associated with decreased survival; high expression of activated c-Src (Y215) was associated with improved survival. This was potentiated in the ER/HER2-negative subgroup. Hence, administration of Src kinase inhibitors aiming to decrease phosphorylation should be approached with caution, especially in ER-negative patients. It is therefore essential to appropriately identify with the correct biomarkers which patients are most likely to respond to Src inhibitors.

    The American journal of pathology 2009;175;4;1389-97

  • Phosphorylation of histone H3 at Ser10: its role in cell transformation by v-Src.

    Tange S, Ito S, Senga T and Hamaguchi M

    Division of Cancer Biology, Department of Oncology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.

    We found that transformation by v-src constitutively activated phosphorylation of histone H3 at Ser10 in a transformation-specific manner. While nontransforming mutant of v-src did not activate H3 phosphorylation, H3 phosphorylation in cells expressing temperature-sensitive mutant of v-src was temperature-dependent. Inhibition of Ras signaling by Gap1m, a GTPase-activation protein for Ras, or S17N Ras, a dominant negative form of Ras, substantially suppressed the Ser10 phosphorylation of H3. Similarly, treatment of cells with manumycin A, a potent inhibitor of Ras-falnesyl transferase, clearly suppressed the H3 phosphorylation. In contrast, inhibition of STAT3 signaling or PI3K signaling did not perturb H3 phosphorylation. We found, however, inhibition of MEK or MSK1 markedly suppressed H3 phosphorylation. In addition, inhibition of MSK1 expression by its siRNA substantially suppressed H3 phosphorylation and anchorage-independent growth of transformed cells. Taken together, our results strongly suggest the importance of MSK1 and H3 phosphorylation in cell transformation by v-Src.

    Biochemical and biophysical research communications 2009;386;4;588-92

  • Fyn and SRC are effectors of oncogenic epidermal growth factor receptor signaling in glioblastoma patients.

    Lu KV, Zhu S, Cvrljevic A, Huang TT, Sarkaria S, Ahkavan D, Dang J, Dinca EB, Plaisier SB, Oderberg I, Lee Y, Chen Z, Caldwell JS, Xie Y, Loo JA, Seligson D, Chakravari A, Lee FY, Weinmann R, Cloughesy TF, Nelson SF, Bergers G, Graeber T, Furnari FB, James CD, Cavenee WK, Johns TG and Mischel PS

    Department of Pathology and Laboratory Medicine, University of California-Los Angeles David Geffen School of Medicine, USA.

    Activating epidermal growth factor receptor (EGFR) mutations are common in many cancers including glioblastoma. However, clinical responses to EGFR inhibitors are infrequent and short-lived. We show that the Src family kinases (SFK) Fyn and Src are effectors of oncogenic EGFR signaling, enhancing invasion and tumor cell survival in vivo. Expression of a constitutively active EGFR mutant, EGFRvIII, resulted in activating phosphorylation and physical association with Src and Fyn, promoting tumor growth and motility. Gene silencing of Fyn and Src limited EGFR- and EGFRvIII-dependent tumor cell motility. The SFK inhibitor dasatinib inhibited invasion, promoted tumor regression, and induced apoptosis in vivo, significantly prolonging survival of an orthotopic glioblastoma model expressing endogenous EGFRvIII. Dasatinib enhanced the efficacy of an anti-EGFR monoclonal antibody (mAb 806) in vivo, further limiting tumor growth and extending survival. Examination of a large cohort of clinical samples showed frequent coactivation of EGFR and SFKs in glioblastoma patients. These results establish a mechanism linking EGFR signaling with Fyn and Src activation to promote tumor progression and invasion in vivo and provide rationale for combined anti-EGFR and anti-SFK targeted therapies.

    Funded by: NCI NIH HHS: 5T32CA09056, CA097257, CA108633, CA119347; NINDS NIH HHS: NS049720, NS050151, R01 NS049720-06, R01 NS050151-03, R01 NS050151-05

    Cancer research 2009;69;17;6889-98

  • The variant rs1867277 in FOXE1 gene confers thyroid cancer susceptibility through the recruitment of USF1/USF2 transcription factors.

    Landa I, Ruiz-Llorente S, Montero-Conde C, Inglada-Pérez L, Schiavi F, Leskelä S, Pita G, Milne R, Maravall J, Ramos I, Andía V, Rodríguez-Poyo P, Jara-Albarrán A, Meoro A, del Peso C, Arribas L, Iglesias P, Caballero J, Serrano J, Picó A, Pomares F, Giménez G, López-Mondéjar P, Castello R, Merante-Boschin I, Pelizzo MR, Mauricio D, Opocher G, Rodríguez-Antona C, González-Neira A, Matías-Guiu X, Santisteban P and Robledo M

    Hereditary Endocrine Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.

    In order to identify genetic factors related to thyroid cancer susceptibility, we adopted a candidate gene approach. We studied tag- and putative functional SNPs in genes involved in thyroid cell differentiation and proliferation, and in genes found to be differentially expressed in thyroid carcinoma. A total of 768 SNPs in 97 genes were genotyped in a Spanish series of 615 cases and 525 controls, the former comprising the largest collection of patients with this pathology from a single population studied to date. SNPs in an LD block spanning the entire FOXE1 gene showed the strongest evidence of association with papillary thyroid carcinoma susceptibility. This association was validated in a second stage of the study that included an independent Italian series of 482 patients and 532 controls. The strongest association results were observed for rs1867277 (OR[per-allele] = 1.49; 95%CI = 1.30-1.70; P = 5.9x10(-9)). Functional assays of rs1867277 (NM_004473.3:c.-283G>A) within the FOXE1 5' UTR suggested that this variant affects FOXE1 transcription. DNA-binding assays demonstrated that, exclusively, the sequence containing the A allele recruited the USF1/USF2 transcription factors, while both alleles formed a complex in which DREAM/CREB/alphaCREM participated. Transfection studies showed an allele-dependent transcriptional regulation of FOXE1. We propose a FOXE1 regulation model dependent on the rs1867277 genotype, indicating that this SNP is a causal variant in thyroid cancer susceptibility. Our results constitute the first functional explanation for an association identified by a GWAS and thereby elucidate a mechanism of thyroid cancer susceptibility. They also attest to the efficacy of candidate gene approaches in the GWAS era.

    PLoS genetics 2009;5;9;e1000637

  • Integrin-dependent translocation of LASP-1 to the cytoskeleton of activated platelets correlates with LASP-1 phosphorylation at tyrosine 171 by Src-kinase.

    Traenka J, Hauck CR, Lewandrowski U, Sickmann A, Gambaryan S, Thalheimer P and Butt E

    Institute of Clinical Biochemistry and Pathobiochemistry, University of Wuerzburg, D-97080 Wuerzburg, Germany.

    During platelet adhesion, the complex cytoskeletal structure is rearranged resulting in the formation of F-actin-based filopodia and lamellipodia. Stimulatory platelet signalling pathways include binding of integrin alpha(IIb)beta(3) to fibrinogen followed by activation of protein tyrosine kinases (PTK) and phosphorylation of downstream signalling proteins. In this study, we demonstrate that the scaffolding and F-actin binding protein LASP-1 undergoes tyrosine phosphorylation in thrombin-stimulated human platelets. By means of specific inhibitors we identified Src-kinase as the primary enzyme phosphorylating LASP-1 in intact cells. These data were confirmed in platelet model cells (A5-CHO cells), constitutively expressing integrin alpha(IIb)beta(3). Fibrinogen-mediated cell stimulation resulted in a similar tyrosine phosphorylation of transiently transfected LASP-1. Site directed mutagenesis identified tyrosine 171 as the Src-kinase phosphorylation site. Immunofluorescence microscopic analysis of these cells revealed a relocation of LASP-1 to focal contacts and the leading edge of the membrane upon fibrinogen activation and tyrosine 171 phosphorylation. This translocation was also seen in adherent platelets. Concomitant with adhesion, LASP-1 translocated from the cytosol along the arms of the pseudopodia into the leading lamellae of the spreading platelets, indicating a crucial role of the protein in platelet cytoskeleton rearrangement.

    Thrombosis and haemostasis 2009;102;3;520-8

  • Osteopetrosis with micro-lacunar resorption because of defective integrin organization.

    Blair HC, Yaroslavskiy BB, Robinson LJ, Mapara MY, Pangrazio A, Guo L, Chen K, Vezzoni P, Tolar J and Orchard PJ

    Department of Pathology, University of Pittsburgh, and Veteran's Affairs Medical Center, Pittsburgh, PA 15261, USA. hcblair@imap.pitt.edu

    In vitro differentiated monocytes were used to characterize the cellular defect in a type of osteopetrosis with minimally functional osteoclasts, in which defects associated with common causes of osteopetrosis were excluded by gene sequencing. Monocytes from the blood of a 28-year-old patient were differentiated in media with RANKL and CSF-1. Cell fusion, acid compartments within cells, and tartrate resistant acid phosphatase (TRAP) activity were normal. However, the osteoclasts made abnormally small pits on the dentine. Phalloidin labeling showed that the cell attachments lacked the peripheral ring structure that supports lacunar resorption. Instead, the osteoclasts had clusters of podosomes near the center of cell attachments. Antibody to the alphavbeta3 integrin pair or to the C-terminal of beta3 did not label podosomes, but antibody to alphav labeled them. Western blots using antibody to the N-terminal of beta3 showed a protein of reduced size. Integrins beta1 and beta5 were upregulated, but, in contrast to observations in beta3 defects, alpha2 had not increased. The rho-GTP exchange protein Vav3, a key attachment organizing protein, did not localize normally with peripheral attachment structures. Vav3 forms of 70 kD and 90 kD were identified on western blots. However, the proteins beta3 integrin, Vav3, Plekhm1, and Src, implicated in attachment defects, had normal exon sequences. In this new type of osteopetrosis, the integrin-organizing complex is dysfunctional, and at least two attachment proteins may be partially degraded.

    Funded by: NIAMS NIH HHS: AR053566, AR053976, AR055208, K08 AR053566, K08 AR053566-03, R01 AR053976, R01 AR053976-04, R01 AR055208

    Laboratory investigation; a journal of technical methods and pathology 2009;89;9;1007-17

  • E-cadherin surface levels in epithelial growth factor-stimulated cells depend on adherens junction protein shrew-1.

    Gross JC, Schreiner A, Engels K and Starzinski-Powitz A

    Institute for Cell Biology and Neuroscience, Johann Wolfgang Goethe University of Frankfurt, 60323 Frankfurt, Germany.

    Gain- and loss-of-function studies indicate that the adherens junction protein shrew-1 acts as a novel modulator of E-cadherin internalization induced by epithelial growth factor (EGF) or E-cadherin function-blocking antibody during epithelial cell dynamics. Knocking down shrew-1 in MCF-7 carcinoma cells preserves E-cadherin surface levels upon EGF stimulation. Overexpression of shrew-1 leads to preformation of an E-cadherin/EGF receptor (EGFR) HER2/src-kinase/shrew-1 signaling complex and accelerated E-cadherin internalization. Shrew-1 is not sufficient to stimulate E-cadherin internalization, but facilitates the actions of EGFR and thus may promote malignant progression in breast cancer cells with constitutive EGFR stimulation by reducing surface E-cadherin expression.

    Molecular biology of the cell 2009;20;15;3598-607

  • Protein-tyrosine phosphatase-alpha and Src functionally link focal adhesions to the endoplasmic reticulum to mediate interleukin-1-induced Ca2+ signaling.

    Wang Q, Rajshankar D, Branch DR, Siminovitch KA, Herrera Abreu MT, Downey GP and McCulloch CA

    Canadian Institutes of Health Research Group in Matrix Dynamics, Faculty of Dentistry, University of Toronto, Toronto, Ontario M5S 3E2, Canada.

    Calcium (Ca2+) signaling by the pro-inflammatory cytokine interleukin-1 (IL-1) is dependent on focal adhesions, which contain diverse structural and signaling proteins including protein phosphatases. We examined here the role of protein-tyrosine phosphatase (PTP) alpha in regulating IL-1-induced Ca2+ signaling in fibroblasts. IL-1 promoted recruitment of PTPalpha to focal adhesions and endoplasmic reticulum (ER) fractions, as well as tyrosine phosphorylation of the ER Ca2+ release channel IP3R. In response to IL-1, catalytically active PTPalpha was required for Ca2+ release from the ER, Src-dependent phosphorylation of IP3R1 and accumulation of IP3R1 in focal adhesions. In pulldown assays and immunoprecipitations PTPalpha was required for the association of PTPalpha with IP3R1 and c-Src, and this association was increased by IL-1. Collectively, these data indicate that PTPalpha acts as an adaptor to mediate functional links between focal adhesions and the ER that enable IL-1-induced Ca2+ signaling.

    Funded by: NHLBI NIH HHS: HL090669, R01 HL090669-03; NIGMS NIH HHS: F32 GM19739, R01 GM54899

    The Journal of biological chemistry 2009;284;31;20763-72

  • The tyrosine kinase c-Src enhances RIG-I (retinoic acid-inducible gene I)-elicited antiviral signaling.

    Johnsen IB, Nguyen TT, Bergstroem B, Fitzgerald KA and Anthonsen MW

    Department of Laboratory Medicine, Children's and Women's Health, Faculty of Medicine, Norwegian University of Science and Technology, Trondheim N-7006, Norway.

    Antiviral immune responses are initiated through Toll-like receptors (TLRs) and RIG-I (retinoic acid-inducible gene-I)-like RNA helicases that recognize nucleic acids from distinct viruses. In this study, we show that the tyrosine kinase c-Src participates in antiviral responses induced by the cytoplasmic RNA helicase RIG-I. Sendai virus (SV), which is recognized by RIG-I, induced c-Src phosphorylation. Functional impairment of c-Src through chemical inhibition or transient expression of a c-Src kinase-inactive mutant attenuated production of endogenous antiviral proteins after SV infection or after expression of RIG-I or its adapter protein MAVS. Importantly, SV-stimulated synthesis of antiviral proteins was significantly impaired in cells treated with c-Src small interfering RNA and in cells from c-Src-deficient mice. In addition, we found that c-Src interacted with components of the RIG-I pathway: RIG-I, MAVS, and TRAF3 (tumor necrosis factor receptor-associated factor-3). The interaction between c-Src and TRAF3 was found to occur within the RING domain of TRAF3. Taken together, our results suggest that c-Src enhances RIG-I-mediated signaling, acting at the level of TRAF3.

    Funded by: NIAID NIH HHS: R01 AI067497, R01 AI067497-01, R01 AI067497-05

    The Journal of biological chemistry 2009;284;28;19122-31

  • Cortactin is a functional target of E-cadherin-activated Src family kinases in MCF7 epithelial monolayers.

    Ren G, Helwani FM, Verma S, McLachlan RW, Weed SA and Yap AS

    Division of Molecular Cell Biology, Institute for Molecular Bioscience, University of Queensland, St. Lucia, Brisbane 4072, Australia.

    Src family kinases (SFKs) signal in response to E-cadherin to support cadherin adhesion and the integrity of cell-cell contacts (McLachlan, R. W., Kraemer, A., Helwani, F. M., Kovacs, E. M., and Yap, A. S. (2007) Mol. Biol. Cell 18, 3214-3223). We now identify the actin-regulatory protein, cortactin, as a target of E-cadherin-activated SFK signaling. Tyr-phosphorylated cortactin was found at cell-cell contacts in established epithelial monolayers, and cortactin became acutely tyrosine-phosphorylated when E-cadherin adhesion was engaged. In all circumstances, cortactin tyrosine phosphorylation was blocked by inhibiting SFK signaling. Importantly, Tyr-phosphorylated cortactin was necessary to preserve the integrity of cadherin contacts and the perijunctional actin cytoskeleton. Moreover, expression of a phosphomimetic cortactin mutant could prevent SFK blockade from disrupting cadherin organization, thereby placing cortactin functionally downstream of SFK signaling at cadherin adhesions. We conclude that SFK and cortactin constitute an important signaling pathway that functionally links E-cadherin adhesion and the actin cytoskeleton.

    Funded by: NCRR NIH HHS: P20 RR16440; NIDCR NIH HHS: R01 DE014578

    The Journal of biological chemistry 2009;284;28;18913-22

  • Intestinal epithelial cancer cell anoikis resistance: EGFR-mediated sustained activation of Src overrides Fak-dependent signaling to MEK/Erk and/or PI3-K/Akt-1.

    Demers MJ, Thibodeau S, Noël D, Fujita N, Tsuruo T, Gauthier R, Arguin M and Vachon PH

    Département d'Anatomie et de Biologie Cellulaire, Université de Sherbrooke, Québec, Canada.

    Herein, we investigated the survival roles of Fak, Src, MEK/Erk, and PI3-K/Akt-1 in intestinal epithelial cancer cells (HCT116, HT29, and T84), in comparison to undifferentiated and differentiated intestinal epithelial cells (IECs). We report that: (1) cancer cells display striking anoikis resistance, as opposed to undifferentiated/differentiated IECs; (2) under anoikis conditions and consequent Fak down-activation, cancer cells nevertheless exhibit sustained Fak-Src interactions and Src/MEK/Erk activation, unlike undifferentiated/differentiated IECs; however, HCT116 and HT29 cells exhibit a PI3-K/Akt-1 down-activation, as undifferentiated/differentiated IECs, whereas T84 cells do not; (3) cancer cells require MEK/Erk for survival, as differentiated (but not undifferentiated) IECs; however, T84 cells do not require Fak and HCT116 cells do not require PI3-K/Akt-1, in contrast to the other cells studied; (4) Src acts as a cornerstone in Fak-mediated signaling to MEK/Erk and PI3-K/Akt-1 in T84 cells, as in undifferentiated IECs, whereas PI3-K/Akt-1 is Src-independent in HCT116, HT29 cells, as in differentiated IECs; and (5) EGFR activity inhibition abrogates anoikis resistance in cancer cells through a loss of Fak-Src interactions and down-activation of Src/MEK/Erk (T84, HCT116, HT29 cells) and PI3-K/Akt-1 (T84 cells). Hence, despite distinctions in signaling behavior not necessarily related to undifferentiated or differentiated IECs, intestinal epithelial cancer cells commonly display an EGFR-mediated sustained activation of Src under anoikis conditions. Furthermore, such sustained Src activation confers anoikis resistance at least in part through a consequent sustenance of Fak-Src interactions and MEK/Erk activation, thus not only overriding Fak-mediated signaling to MEK/Erk and/or PI3-K/Akt-1, but also the requirement of Fak and/or PI3-K/Akt-1 for survival.

    Journal of cellular biochemistry 2009;107;4;639-54

  • Activation of Src by protein tyrosine phosphatase 1B Is required for ErbB2 transformation of human breast epithelial cells.

    Arias-Romero LE, Saha S, Villamar-Cruz O, Yip SC, Ethier SP, Zhang ZY and Chernoff J

    Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA.

    Protein tyrosine phosphatase (PTP) 1B plays a major role in inhibiting signaling from the insulin and leptin receptors. Recently, PTP1B was found to have an unexpected positive role in ErbB2 signaling in a mouse model of breast cancer, but the mechanism underlying this effect has been unclear. Using human breast epithelial cells grown in a three-dimensional matrix, we found that PTP1B, but not the closely related enzyme T-cell PTP, is required for ErbB2 transformation in vitro. Activation of ErbB2, but not ErbB1, increases PTP1B expression, and increased expression of PTP1B activates Src and induces a Src-dependent transformed phenotype. These findings identify a molecular mechanism by which PTP1B links an important oncogenic receptor tyrosine kinase to signaling pathways that promote aberrant cell division and survival in human breast epithelial cells.

    Funded by: NCI NIH HHS: P30 CA006927, P30 CA006927-46, R01 CA058836, R01 CA058836-15, R01 CA069202, R01 CA069202-13, R01 CA100724, R01 CA100724-06, R01 CA58836, R01 CA69202; NIDDK NIH HHS: F32 DK079474, F32 DK079474-01A1

    Cancer research 2009;69;11;4582-8

  • Formation of endothelial lumens requires a coordinated PKCepsilon-, Src-, Pak- and Raf-kinase-dependent signaling cascade downstream of Cdc42 activation.

    Koh W, Sachidanandam K, Stratman AN, Sacharidou A, Mayo AM, Murphy EA, Cheresh DA and Davis GE

    Department of Medical Pharmacology and Physiology, School of Medicine, University of Missouri, Columbia, MO 65212, USA.

    In this study, we present data showing that Cdc42-dependent lumen formation by endothelial cells (ECs) in three-dimensional (3D) collagen matrices involves coordinated signaling by PKCepsilon in conjunction with the Src-family kinases (SFKs) Src and Yes. Activated SFKs interact with Cdc42 in multiprotein signaling complexes that require PKCepsilon during this process. Src and Yes are differentially expressed during EC lumen formation and siRNA suppression of either kinase, but not Fyn or Lyn, results in significant inhibition of EC lumen formation. Concurrent with Cdc42 activation, PKCepsilon- and SFK-dependent signaling converge to activate p21-activated kinase (Pak)2 and Pak4 in steps that are also required for EC lumen formation. Pak2 and Pak4 further activate two Raf kinases, B-Raf and C-Raf, leading to ERK1 and ERK2 (ERK1/2) activation, which all seem to be necessary for EC lumen formation. This work reveals a multicomponent kinase signaling pathway downstream of integrin-matrix interactions and Cdc42 activation involving PKCepsilon, Src, Yes, Pak2, Pak4, B-Raf, C-Raf and ERK1/2 to control EC lumen formation in 3D collagen matrices.

    Funded by: NHLBI NIH HHS: HL59373, HL79460, R01 HL059373

    Journal of cell science 2009;122;Pt 11;1812-22

  • Src stimulates fibroblast growth factor receptor-2 shedding by an ADAM15 splice variant linked to breast cancer.

    Maretzky T, Le Gall SM, Worpenberg-Pietruk S, Eder J, Overall CM, Huang XY, Poghosyan Z, Edwards DR and Blobel CP

    Arthritis and Tissue Degeneration Program, Hospital for Special Surgery, and Department of Physiology, Weill Medical College of Cornell University, New York, New York 10021, USA.

    ADAMs (a disintegrin and metalloproteinase) have important roles in development and diseases such as cancer. Previously, an ADAM15 splice variant (ADAM15B), which contains an inserted cytoplasmic Src-binding site, was linked to clinical aggressiveness in breast cancer, yet little was known about how this splice variant affects the function of ADAM15. Here, we show that ADAM15B has enhanced catalytic activity in cell-based assays compared with ADAM15A, which lacks a Src-binding site, using shedding of fibroblast growth factor receptor 2iiib variant as an assay for catalytic activity. Moreover, the enhanced activity of ADAM15B compared with ADAM15A depends on Src because it is abolished by Src-kinase inhibitors and in Src(-/-) cells, but not in Src(-/-) cells rescued with Src. These findings provide insights into the mechanism of how a splice variant linked to clinical agressiveness in breast cancer causes increased activity of ADAM15B, and suggest that inhibitors of the ADAM15 protease activity or of the interaction of ADAM15B with Src could be useful to treat breast cancer in patients with dysregulated ADAM15B.

    Funded by: Biotechnology and Biological Sciences Research Council: C09636, C20059; Medical Research Council: G0100250, G0100250(57209); Wellcome Trust: 068239

    Cancer research 2009;69;11;4573-6

  • Rho signaling, ROCK and mDia1, in transformation, metastasis and invasion.

    Narumiya S, Tanji M and Ishizaki T

    Department of Pharmacology, Kyoto University Faculty of Medicine, Kyoto, 606-8501, Japan. snaru@mfour.med.kyoto-u.ac.jp

    The Rho subgroup of the Rho GTPases consisting of RhoA, RhoB and RhoC induces a specific type of actin cytoskeleton and carry out a variety of functions in the cell. mDia and ROCK are downstream effectors of Rho mediating Rho action on the actin cytoskeleton; mDia produces actin filaments by nucleation and polymerization and ROCK activate myosin to cross-link them for induction of actomyosin bundles and contractility. mDia is potentially linked to Rac activation and membrane ruffle formation through c-Src-induced phosphorylation of focal adhesion proteins, and ROCK antagonizes this mDia action. Thus, cell morphogenesis, adhesion, and motility can be determined by the balance between mDia and ROCK activities. Though they are not oncogenes by themselves, overexpression of RhoA and RhoC are often found in clinical cancers, and RhoC has been repeatedly identified as a gene associated with metastasis. The Rho-ROCK pathway is implicated in Ras-mediated transformation, the amoeboid movement of tumor cells in the three-dimensional matrix, and transmigration of tumor cells through the mesothelial monolayer. On the other hand, the Rho-mDia1 pathway is implicated in Src-mediated remodeling of focal adhesions and migration of tumor cells. There is also an indication that the Rho pathway other than ROCK is involved in Src-mediated induction of podosome and regulation of matrix metalloproteases. Thus, Rho mediates various phenotypes of malignant transformation by Ras and Src through its effectors, ROCK and mDia.

    Cancer metastasis reviews 2009;28;1-2;65-76

  • Activation of ROS/NF-kappaB and Ca2+/CaM kinase II are necessary for VCAM-1 induction in IL-1beta-treated human tracheal smooth muscle cells.

    Luo SF, Chang CC, Lee IT, Lee CW, Lin WN, Lin CC and Yang CM

    Department of Internal Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan.

    Histone acetylation regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs) plays a critical role in the expression of inflammatory genes, such as vascular cell adhesion molecule-1 (VCAM-1). Oxidative processes have been shown to induce VCAM-1 expression. Here, we investigated the mechanisms underlying IL-1beta-induced VCAM-1 expression in human tracheal smooth muscle cells (HTSMCs). Our results showed that IL-1beta enhanced HTSMCs-monocyte adhesion through up-regulation of VCAM-1, which was inhibited by pretreatment with selective inhibitors of PKCalpha (Gö6976), c-Src (PP1), NADPH oxidase [diphenylene iodonium (DPI) and apocynin (APO)], intracellular calcium chelator (BAPTA/AM), PI-PLC (U73122), CaM (calmidazolium chloride), CaM kinase II (KN62), p300 (garcinol), NF-kappaB (Bay11-7082), HDAC (trichostatin A), and ROS scavenger [N-acetyl-L-cysteine (NAC)] or transfection with siRNAs of MyD88, PKCalpha, Src, p47(phox), p300, and HDAC4. Moreover, IL-1beta stimulated NF-kappaB and CaMKII phosphorylation through MyD88-dependent PI-PLC/PKCalpha/c-Src/ROS and PI-PLC/Ca2+/CaM pathways, respectively. Activation of NF-kappaB and CaMKII may eventually lead to the acetylation of histone residues and phosphorylation of histone deacetylases. These findings suggested that IL-1beta induced VCAM-1 expression via these multiple signaling pathways in HTSMCs. Blockade of these pathways may reduce monocyte adhesion via VCAM-1 suppression and attenuation of the inflammatory responses in airway diseases.

    Toxicology and applied pharmacology 2009;237;1;8-21

  • SRC family kinase activity is up-regulated in hormone-refractory prostate cancer.

    Tatarov O, Mitchell TJ, Seywright M, Leung HY, Brunton VG and Edwards J

    Division of Cancer Sciences and Molecular Pathology, Faculty of Medicine, Glasgow Royal Infirmary, Glasgow, UK.

    Purpose: Although Src family kinase (SFK) inhibitors are now in clinical trials for the treatment of androgen-independent prostate cancer (AIPC), there are no studies relating SFK activation to patient survival. This study was designed to determine if SFK activation was up-regulated with the development of AIPC and if patients could be selected who were more likely to respond to therapy.

    A unique cohort of matched prostate tumor samples, taken before hormone deprivation therapy and following hormone relapse, was used to determine by immunohistochemistry on an individual patient basis if SFK activity changed with progression to AIPC and whether this related to patient outcome measures. Using matched, hormone-sensitive and hormone-refractory cell lines, we determined if hormone status affected the way prostate cancer cells respond to suppression of SFK activity by the small-molecule inhibitor dasatinib.

    Results: In the current study, 28% of patients with AIPC exhibited an increase in SFK activity in prostate cancer tissue, these patients had significantly shorter overall survival (P<0.0001), and activated SFK expression correlated with the presence of distant metastases. Dasatinib inhibited phosphorylation of Src and Lyn and the downstream substrate FAK in hormone-sensitive and hormone-refractory cell lines. Although migration was reduced by dasatinib in both cell lines, proliferation of hormone-refractory cells only was inhibited.

    Conclusion: Appropriate patient selection may allow better targeting of prostate cancer patients who are likely to respond to the treatment with SFK inhibitors at the same time improving the outcome of clinical trials.

    Funded by: Cancer Research UK; Medical Research Council: G0500966

    Clinical cancer research : an official journal of the American Association for Cancer Research 2009;15;10;3540-9

  • HBx protein induces EMT through c-Src activation in SMMC-7721 hepatoma cell line.

    Yang SZ, Zhang LD, Zhang Y, Xiong Y, Zhang YJ, Li HL, Li XW and Dong JH

    Department of Hepatobiliary Surgery, Southwest Hospital, Third Military Medical University, ChongQing 400038, China.

    The relationships between epithelial-to-mesenchymal transition (EMT), hepatitis B virus X protein (HBx), and the non-receptor tyrosine kinase c-Src were investigated. The HBx gene transfected SMMC-7721 cells underwent morphological changes from a classic epithelial morphology to a spindle-like shape. The HBx transfection increased the invasive potential of these cells. When the transfected cells were exposed to the c-Src kinase inhibitor PP2, the cells recovered their original epithelial morphology and the mRNA and protein expression of epithelial and mesenchymal markers returned to the parental cell levels. Our data suggest that activated c-Src played a critical role in the HBx-induced EMT of SMMC-7721 cells.

    Biochemical and biophysical research communications 2009;382;3;555-60

  • Dual targeting of Src and ER prevents acquired antihormone resistance in breast cancer cells.

    Hiscox S, Jordan NJ, Smith C, James M, Morgan L, Taylor KM, Green TP and Nicholson RI

    Tenovus Centre for Cancer Research, Welsh School of Pharmacy, Cardiff, Wales, UK. hiscoxse1@cf.ac.uk

    Acquired resistance to endocrine therapies presents a major obstacle to the successful treatment of breast cancer patients. Previously, we have shown that acquisition of resistance to tamoxifen in breast cancer cells is accompanied by an elevation in Src kinase activity which promotes an aggressive, invasive phenotype in vitro. Here, we have explored the potential therapeutic effects of combining Src inhibition with anti-oestrogen treatment on the development of endocrine insensitivity in breast cancer cells. Treatment of MCF7 and T47D cells with tamoxifen alone resulted in an initial growth inhibitory phase followed by the eventual development of tamoxifen resistance together with an elevation of Src kinase activity, which was central to their increased invasive capacity. Chronic exposure of both cell types to the Src inhibitor, AZD0530, as a monotherapy resulted in outgrowth of AZD0530-resistant cells, in which Src kinase activity remained suppressed as did their in vitro invasive nature. Treatment of both MCF7 and T47D cells with AZD0530 in combination with tamoxifen resulted in a reduction of Src activity together with inhibition of focal adhesion kinase phosphorylation and a complete abrogation of their in vitro invasive behaviour. Furthermore, combination therapy significantly suppressed expression of cyclinD1 and c-myc and prevented cell proliferation and the subsequent emergence of a resistant phenotype, with total cell loss occurring by 12 weeks. These data demonstrate that pharmacological targeting of Src kinase, in conjunction with antihormone therapies, effectively prevents antihormone resistance in breast cancer cells in vitro and suggests a potential novel therapeutic benefit of Src kinase inhibitors clinically.

    Breast cancer research and treatment 2009;115;1;57-67

  • UNC80 functions as a scaffold for Src kinases in NALCN channel function.

    Wang H and Ren D

    Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA.

    Ion channels can be regulated by a wide spectrum of neurotransmitters and hormones, largely through G-protein-coupled receptors (GPCRs). G-protein-independent activation of ion channel currents by GPCRs has also been recorded, although the molecular identity of the channels and the activation mechanisms remain largely unknown. UNC80 is a protein that is associated with the NALCN Na(+) leak cation channel, and is required for the activation of this channel by the neuropeptide substance P through GPCRs in a G-protein-independent fashion. Here, we show that UNC80 binds Src kinases and recruits Src into the channel complex. This finding is consistent with the known requirement for Src kinases in the activation of NALCN, and may lead to new insights into the molecular mechanisms underlying G-protein-independent activation of the channel.

    Funded by: NINDS NIH HHS: 1 R01 NS055293, R01 NS055293, R01 NS055293-01A1, R01 NS074257

    Channels (Austin, Tex.) 2009;3;3;161-3

  • The D816V mutation of c-Kit circumvents a requirement for Src family kinases in c-Kit signal transduction.

    Sun J, Pedersen M and Rönnstrand L

    Experimental Clinical Chemistry, the Department of Laboratory Medicine, Lund University, Malmö University Hospital, SE-205 02 Malmö, Sweden.

    The receptor tyrosine kinase c-Kit plays a critical role in hematopoiesis, and gain-of-function mutations of the receptor are frequently seen in several malignancies, including acute myeloid leukemia, gastrointestinal stromal tumors, and testicular carcinoma. The most common mutation of c-Kit in these disorders is a substitution of the aspartic acid residue in position 816 to a valine (D816V), leading to constitutive activation of the receptor. In this study, we aimed to investigate the role of Src family kinases in c-Kit/D816V signaling. Src family kinases are necessary for the phosphorylation of wild-type c-Kit as well as of activation of downstream signaling pathways including receptor ubiquitination and the Ras/Mek/Erk pathway. Our data demonstrate that, unlike wild-type c-Kit, the phosphorylation of c-Kit/D816V is not dependent on Src family kinases. In addition, we found that neither receptor ubiquitination nor Erk activation by c-Kit/D816V required activation of Src family kinases. In vitro kinase assay using synthetic peptides revealed that c-Kit/D816V had an altered substrate specificity resembling Src and Abl tyrosine kinases. We further present evidence that, in contrast to wild-type c-Kit, Src family kinases are dispensable for c-Kit/D816V cell survival, proliferation, and colony formation. Taken together, we demonstrate that the signal transduction pathways mediated by c-Kit/D816V are markedly different from those activated by wild-type c-Kit and that altered substrate specificity of c-Kit circumvents a need for Src family kinases in signaling of growth and survival, thereby contributing to the transforming potential of c-Kit/D816V.

    The Journal of biological chemistry 2009;284;17;11039-47

  • The role of cooperativity with Src in oncogenic transformation mediated by non-small cell lung cancer-associated EGF receptor mutants.

    Chung BM, Dimri M, George M, Reddi AL, Chen G, Band V and Band H

    Eppley Institute for Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, 68198-6805, USA.

    Non-small cell lung cancer (NSCLC)-associated epidermal growth factor receptor (EGFR) mutants are constitutively active and induce ligand-independent transformation in non-malignant cell lines. We investigated the possibility that the ability of mutant EGFRs to transform cells reflects a constitutive cooperativity with Src using a system in which the overexpression of mutant, but not wild-type, EGFR induced anchorage-independent cell growth. Src was constitutively activated and showed enhanced interaction with mutant EGFRs, suggesting that constitutive EGFR-Src cooperativity may contribute to mutant EGFR-mediated oncogenesis. Indeed, the mutant EGFR-mediated cell transformation was inhibited by Src- as well as EGFR-directed inhibitors. Importantly, a tyrosine to phenylalanine mutation of the major Src phosphorylation site on EGFR, Y845, reduced the constitutive phosphorylation of NSCLC-EGFR mutants, as well as that of STAT3, Akt, Erk and Src, and reduced the mutant EGFR-Src association as well as proliferation, migration and anchorage-independent growth. Reduced anchorage-independent growth and migration were also observed when dominant-negative-Src was expressed in mutant EGFR-expressing cells. Overall, our findings show that mutant EGFR-Src interaction and cooperativity play critical roles in constitutive engagement of the downstream signaling pathways that allow NSCLC-associated EGFR mutants to mediate oncogenesis, and support the rationale to target Src-dependent signaling pathways in mutant EGFR-mediated malignancies.

    Funded by: NCI NIH HHS: 1U54 CA119341-01, CA105489,, CA116552, CA81076, CA87986,, CA94143,, CA96844, CA99163, CA99900,, R01 CA076118-02, R01 CA076118-03, R01 CA076118-04, R01 CA076118-05, R01 CA081076-01, R01 CA081076-02, R01 CA081076-03, R01 CA081076-04, R01 CA081076-05, R01 CA087986-01, R01 CA087986-02, R01 CA087986-03, R01 CA087986-04, R01 CA087986-05, R01 CA087986-06, R01 CA087986-07, R01 CA087986-08, R01 CA087986-09, R01 CA087986-10, R01 CA087986-11, R01 CA087986-12, R01 CA094143-01A1, R01 CA094143-02, R01 CA094143-03, R01 CA094143-04, R01 CA094143-05, R01 CA094143-06, R01 CA096844-01, R01 CA096844-02, R01 CA096844-03, R01 CA096844-04, R01 CA096844-05, R01 CA096844-06, R01 CA096844-07, R01 CA096844-08, R01 CA099163-01, R01 CA099163-02, R01 CA099163-03, R01 CA099163-03S1, R01 CA099163-04, R01 CA099163-05, R01 CA099163-06, R01 CA099163-07, R01 CA099163-08, R01 CA099163-09, R01 CA099900-01, R01 CA099900-02, R01 CA099900-03, R01 CA099900-04, R01 CA099900-05, R01 CA099900-06, R01 CA099900-07, R01 CA105489-08, R01 CA116552-01A2, R01 CA116552-02, R01 CA116552-03, R01 CA116552-04

    Oncogene 2009;28;16;1821-32

  • Presenilin 1 affects focal adhesion site formation and cell force generation via c-Src transcriptional and posttranslational regulation.

    Waschbüsch D, Born S, Niediek V, Kirchgessner N, Tamboli IY, Walter J, Merkel R and Hoffmann B

    Institute of Bio- and Nanosystems 4: Biomechanics, Research Centre Jülich GmbH, 52425 Jülich, Germany.

    Presenilin 1 and 2 (PS) are critical components of the gamma-secretase complex that cleaves type I transmembrane proteins within their transmembrane domains. This process leads to release of proteolytically processed products from cellular membranes and plays an essential role in signal transduction or vital functions as cell adhesion. Here we studied the function of presenilins in cell-matrix interaction of wild-type and PS knock-out mouse embryonic fibroblasts. We found for PS1(-/-) cells an altered morphology with significantly reduced sizes of focal adhesion sites compared with wild type. Cell force analyses on micropatterned elastomer films revealed PS1(-/-) cell forces to be reduced by 50%. Pharmacological inhibition confirmed this function of gamma-secretase in adhesion site and cell force formation. On the regulatory level, PS1 deficiency was associated with strongly decreased phosphotyrosine levels of focal adhesion site-specific proteins. The reduced tyrosine phosphorylation was caused by a down-regulation of c-Src kinase activity primarily at the level of c-Src transcription. The direct regulatory connection between PS1 and c-Src could be identified with ephrinB2 as PS1 target protein. Overexpression of ephrinB2 cytoplasmic domain resulted in its nuclear translocation with increased levels of c-Src and a full complementation of the PS1(-/-) adhesion and phosphorylation phenotype. Cleavage of full-length EB2 and subsequent intracellular domain translocation depended on PS1 as these processes were only found in WT cells. Therefore, we conclude that gamma-secretase is vital for controlling cell adhesion and force formation by transcriptional regulation of c-Src via ephrinB2 cleavage.

    The Journal of biological chemistry 2009;284;15;10138-49

  • Src phosphorylation of RhoGDI2 regulates its metastasis suppressor function.

    Wu Y, Moissoglu K, Wang H, Wang X, Frierson HF, Schwartz MA and Theodorescu D

    Department of Molecular Physiology, University of Virginia, Charlottesville, VA 22908, USA.

    RhoGDI2 is a suppressor of metastasis in human bladder cancer. Although diminished RhoGDI2 expression in tumors is associated with decreased patient survival, normal expression in some metastatic tumors led us to wonder whether other mechanisms regulate RhoGDI2 function. Protein interaction analysis identified Src as a novel RhoGDI2 interaction partner. Gene expression profiling and immunohistochemistry of human tumors revealed that Src levels diminish as a function of bladder cancer stage. In addition, diminished Src levels and RhoGDI2 levels appear mutually exclusive in individual tumors, indicating that both genes are likely involved in the same signaling pathway leading to metastasis suppression. Studies confirmed that activated Src kinase binds and phosphorylates RhoGDI2 in vitro and vivo. Mutagenesis revealed that Tyr-153 and, to a lesser degree, Tyr-24 were the primary Src phosphorylation sites. Phosphorylation decreased the amount of Rac1 in RhoGDI2 complexes and increased RhoGDI2 association with cell membranes. Stable expression of phosphomimetic Tyr-153 RhoGDI2 in metastatic human bladder cancer cell lines had no effect on primary tumor growth but suppressed metastasis more potently than WT RhoGDI2. These data suggest that phosphorylation by Src enhances RhoGDI2 metastasis suppression and that loss of Src relieves metastasis suppression in tumor cells that maintain RhoGDI2 expression. Our findings also suggest caution in using Src inhibitors in the hope of delaying progression in patients with bladder cancer.

    Funded by: NCI NIH HHS: CA075115, R01 CA075115, R01 CA075115-12, R29 CA075115

    Proceedings of the National Academy of Sciences of the United States of America 2009;106;14;5807-12

  • Differential trafficking of Src, Lyn, Yes and Fyn is specified by the state of palmitoylation in the SH4 domain.

    Sato I, Obata Y, Kasahara K, Nakayama Y, Fukumoto Y, Yamasaki T, Yokoyama KK, Saito T and Yamaguchi N

    Department of Molecular Cell Biology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan.

    Src-family tyrosine kinases (SFKs), which participate in a variety of signal transduction events, are known to localize to the cytoplasmic face of the plasma membrane through lipid modification. Recently, we showed that Lyn, an SFK member, is exocytosed to the plasma membrane via the Golgi region along the secretory pathway. We show here that SFK trafficking is specified by the palmitoylation state. Yes is also a monopalmitoylated SFK and is biosynthetically transported from the Golgi pool of caveolin to the plasma membrane. This pathway can be inhibited in the trans-Golgi network (TGN)-to-cell surface delivery by temperature block at 19 degrees C or dominant-negative Rab11 GTPase. A large fraction of Fyn, a dually palmitoylated SFK, is directly targeted to the plasma membrane irrespective of temperature block of TGN exit. Fyn(C6S), which lacks the second palmitoylation site, is able to traffic in the same way as Lyn and Yes. Moreover, construction of Yes(S6C) and chimeric Lyn or Yes with the Fyn N-terminus further substantiates the importance of the dual palmitoylation site for plasma membrane targeting. Taken together with our recent finding that Src, a nonpalmitoylated SFK, is rapidly exchanged between the plasma membrane and late endosomes/lysosomes, these results suggest that SFK trafficking is specified by the palmitoylation state in the SH4 domain.

    Journal of cell science 2009;122;Pt 7;965-75

  • HIV-1 Nef induces p47(phox) phosphorylation leading to a rapid superoxide anion release from the U937 human monoblastic cell line.

    Olivetta E, Mallozzi C, Ruggieri V, Pietraforte D, Federico M and Sanchez M

    National AIDS Centre, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Roma, Italy.

    The Nef protein of the human immunodeficiency virus type 1 (HIV-1) plays a crucial role in AIDS pathogenesis by modifying host cell signaling pathways. We investigated the effects of Nef on the NADPH oxidase complex, a key enzyme involved in the generation of reactive oxygen species during the respiratory burst in human monocyte/macrophages. We have recently shown that the inducible expression of HIV-1 Nef in human macrophages cell line modulates in bi-phasic mode the superoxide anion release by NADPH oxidase, inducing a fast increase of the superoxide production, followed by a delayed strong inhibition mediated by Nef-induced soluble factor(s). Our study is focused on the molecular mechanisms involved in Nef-mediated activation of NADPH oxidase and superoxide anion release. Using U937 cells stably transfected with different Nef alleles, we found that both Nef membrane localization and intact SH3-binding domain are needed to induce superoxide release. The lack of effect during treatment with a specific MAPK pathway inhibitor, PD98059, demonstrated that Nef-induced superoxide release is independent of Erk1/2 phosphorylation. Furthermore, Nef induced the phosphorylation and then the translocation of the cytosolic subunit of NADPH oxidase complex p47(phox) to the plasma membrane. Adding the inhibitor PP2 prevented this process, evidencing the involvement of the Src family kinases on Nef-mediated NADPH oxidase activation. In addition, LY294002, a specific inhibitor of phosphoinositide 3-kinase (PI3K) inhibited both the Nef-induced p47(phox) phosphorylation and the superoxide anion release. These data indicate that Nef regulates the NADPH oxidase activity through the activation of the Src kinases and PI3K.

    Journal of cellular biochemistry 2009;106;5;812-22

  • S100B Protein Regulates Astrocyte Shape and Migration via Interaction with Src Kinase: IMPLICATIONS FOR ASTROCYTE DEVELOPMENT, ACTIVATION, AND TUMOR GROWTH.

    Brozzi F, Arcuri C, Giambanco I and Donato R

    Department of Experimental Medicine and Biochemical Sciences, University of Perugia, C.P. 81 Succ. 3, 06122 Perugia, Italy.

    S100B is a Ca(2+)-binding protein of the EF-hand type that is abundantly expressed in astrocytes and has been implicated in the regulation of several intracellular activities, including proliferation and differentiation. We show here that reducing S100B levels in the astrocytoma cell line GL15 and the Müller cell line MIO-M1 by small interference RNA technique results in a rapid disassembly of stress fibers, collapse of F-actin onto the plasma membrane and reduced migration, and acquisition of a stellate shape. Also, S100B-silenced GL15 and MIO-M1 Müller cells show a higher abundance of glial fibrillary acidic protein filaments, which mark differentiated astrocytes, compared with control cells. These effects are dependent on reduced activation of the phosphatidylinositol 3-kinase (PI3K) downstream effectors, Akt and RhoA, and consequently elevated activity of GSK3beta and Rac1 and decreased activity of the RhoA-associated kinase. Also, rat primary astrocytes transiently down-regulate S100B expression when exposed to the differentiating agent dibutyryl cyclic AMP and re-express S100B at later stages of dibutyryl cyclic AMP-induced differentiation. Moreover, reducing S100B levels results in a remarkably slow resumption of S100B expression, suggesting the S100B might regulate its own expression. Finally, we show that S100B interacts with Src kinase, thereby stimulating the PI3K/Akt and PI3K/RhoA pathways. These results suggest that S100B might contribute to reduce the differentiation potential of cells of the astrocytic lineage and participate in the astrocyte activation process in the case of brain insult and in invasive properties of glioma cells.

    The Journal of biological chemistry 2009;284;13;8797-811

  • Role of Src signal transduction pathways in scatter factor-mediated cellular protection.

    Fan S, Meng Q, Laterra JJ and Rosen EM

    Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057, USA.

    Scatter factor (SF) (hepatocyte growth factor) is a pleiotrophic cytokine that accumulates in tumors, where it may induce invasion, angiogenesis, and chemoresistance. We have studied the mechanisms by which SF and its receptor (c-Met) protect cells against the DNA-damaging agent adriamycin (ADR) as a model for chemoresistance of SF/c-Met-overexpressing tumors. Previous studies identified a phosphatidylinositol 3-kinase/c-Akt/Pak1/NF-kappaB cell survival pathway in DU-145 prostate cancer and Madin-Darby canine kidney epithelial cells. Here we studied Src signaling pathways involved in SF cell protection. Src enhanced basal and SF stimulated NF-kappaB activity and SF protection against ADR, in a manner dependent upon its kinase and Src homology 3 domains; and endogenous Src was required for SF stimulation of NF-kappaB activity and cell protection. The ability of Src to enhance SF stimulation of NF-kappaB activity was due, in part, to its ability to stimulate Akt and IkappaB kinase activity; and Src-mediated stimulation of NF-kappaB was due, in part, to a Rac1/MKK3/6/p38 pathway and was Akt-dependent. SF caused the activation of Src and the Rac1 effector Pak1. Furthermore, SF induced activating phosphorylations of MKK3, MKK6, and p38 within the c-Met signalsome in an Src-dependent manner. The NF-kappaB-inducing kinase was found to act downstream of TAK1 (transforming growth factor-beta-activated kinase 1) as a mediator of SF- and Src-stimulated NF-kappaB activity. Finally, the Src/Rac1/MKK3/6/p38 and Src/TAK1/NF-kappaB-inducing kinase pathways exhibited cross-talk at the level of MKK3. These findings delineate some novel signaling pathways for SF-mediated resistance to ADR.

    Funded by: NIEHS NIH HHS: R01-ES09169; NINDS NIH HHS: R01 NS032148, R01-NS43987

    The Journal of biological chemistry 2009;284;12;7561-77

  • Sustained Src inhibition results in signal transducer and activator of transcription 3 (STAT3) activation and cancer cell survival via altered Janus-activated kinase-STAT3 binding.

    Sen B, Saigal B, Parikh N, Gallick G and Johnson FM

    Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030-4009, USA.

    Locoregional and distant recurrence remains common and usually fatal for patients with advanced head and neck squamous cell carcinoma (HNSCC). One promising molecular target in HNSCC is the Src family kinases (SFK). SFKs can affect cellular proliferation and survival by activating the signal transducer and activator of transcription (STAT) family of transcription factors, especially STAT3. Surprisingly, sustained SFK inhibition resulted in only transient inhibition of STAT3. We investigated the mechanism underlying STAT3 activation and its biological importance. Specific c-Src knockdown with small interfering RNA (siRNA) resulted in STAT3 activation showing specificity, which was inhibited by Janus-activated kinase (JAK; TYK2 and JAK2) depletion with siRNA. Sustained SFK inhibition also resulted in recovered JAK-STAT3 binding and JAK kinase activity after an initial reduction, although JAK phosphorylation paradoxically decreased. To determine the biological significance of STAT3 activation, we combined specific STAT3 depletion with a pharmacologic SFK inhibitor and observed increased cell cycle arrest and apoptosis. Likewise, the addition of STAT3- or JAK-specific siRNA to c-Src-depleted cells enhanced cytotoxicity relative to cells incubated with c-Src siRNA alone. These results show that reactivation of STAT3 after sustained, specific c-Src inhibition is mediated through altered JAK-STAT3 binding and JAK kinase activity and that this compensatory pathway allows for cancer cell survival and proliferation despite durable c-Src inhibition. To our knowledge, this novel feedback pathway has never been described previously. Given that pharmacologic SFK inhibitors are currently being evaluated in clinical trials, these results have potential clinical implications for cancer therapy.

    Funded by: NCI NIH HHS: P50 CA097007, P50 CA097007-07

    Cancer research 2009;69;5;1958-65

  • An in vitro analysis of mechanical wounding-induced ligand-independent KGFR activation.

    Li M, Firth JD and Putnins EE

    Laboratory of Periodontal Biology, Department of Oral Biological and Medical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada.

    Background: KGFR (keratinocyte growth factor receptor), exclusively expressed in epithelial cells, plays an important role in wound healing. However, mechanisms of KGFR activation and signaling in wound healing are not clearly understood.

    Objectives: We utilized an in vitro mechanical wounding model to examine ligand-independent KGFR activation, its regulation by reactive oxygen species (ROS) and the functional significance of this activation mechanism.

    Methods: Confluent HaCaT cell line cultures were mechanically wounded and KGFR internalization and phosphorylation were examined using immunostaining with confocal microscopy and immunoprecipitation with Western blotting. Wounding-induced generation of reactive oxygen species and ligand-independent activation of KGFR were examined. In addition, phosphorylation of its associated molecules FRS2 and c-Src were examined in the presence and absence of the ROS and pathway specific inhibitors. The importance of this activation process on cell migration was also examined in the presence and absence of these inhibitors.

    Results: Mechanical wounding induced ligand-independent KGFR activation and internalization. KGFR internalization and phosphorylation was associated with ROS generation along the wound edge and scavenging of ROS with NAC inhibited KGFR phosphorylation. Intracellularly, c-Src was phosphorylated by wounding but its inhibitor, PP1, significantly inhibited KGFR activation and associated FRS2 phosphorylation. Mechanical wounding induced wound edge migration, which was significantly reduced by the selective receptor and pathway inhibitors PP1 (82.7%), KGFR inhibitor SU5402 (70%) and MAPK inhibitor PD98059 (57%).

    Conclusion: Mechanical wounding induces significant ROS generation at the wound edge which, in turn, induced ligand-independent KGFR and FRS2 activation via c-Src kinase signaling. Functionally, downstream MAPK signaling induced wound edge cell migration.

    Journal of dermatological science 2009;53;3;182-91

  • Histidine triad nucleotide-binding protein 1 up-regulates cellular levels of p27KIP1 by targeting ScfSKP2 ubiquitin ligase and Src.

    Cen B, Li H and Weinstein IB

    Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, New York 10032, USA. cen@musc.edu

    The one or more underlying mechanisms of the tumor suppressing activity of the histidine triad nucleotide-binding protein 1 (HINT1) are not well defined. In this study we found that HINT1 regulates cellular levels of the cyclin-dependent kinase inhibitor p27(KIP1) through multiple mechanisms. Increased expression of HINT1 increases cellular levels of p27(KIP1), and HINT1 knockdown with small hairpin RNA leads to decreased cellular levels of p27(KIP1). HINT1 does not affect the transcription of p27(KIP1), but it does inhibit proteasomal degradation of the p27(KIP1) protein. HINT1 directly interacts with the SCF(SKP2) ubiquitin ligase complex and inhibits the ubiquitylation of p27(KIP1). Src has been shown to phosphorylate p27(KIP1) and thus decrease its stability. We found that HINT1 is a negative regulator of Src transcription apparently by forming a complex with the transcription factor Sp1 on the promoter of Src. Taken together, our findings indicate that HINT1 up-regulates cellular levels of p27(KIP1) by two mechanisms: 1) it inhibits its ubiquitylation by targeting the SCF(SKP2) ubiquitin ligase complex, and 2) it inhibits the phosphorylation of p27(KIP1) by Src via inhibiting Src expression.

    The Journal of biological chemistry 2009;284;8;5265-76

  • A novel peptide from human apolipoprotein(a) inhibits angiogenesis and tumor growth by targeting c-Src phosphorylation in VEGF-induced human umbilical endothelial cells.

    Yi ZF, Cho SG, Zhao H, Wu YY, Luo J, Li D, Yi T, Xu X, Wu Z and Liu M

    Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China.

    Many angiogenesis inhibitors are derived from large plasma proteins. Previous studies showed that the Kringle5-like domain (termed KV) in human apolipoprotein (a) is a potential antiangiogenic factor. However, its active region and the underling molecular mechanism remain elusive. Here, we identified an 11-amino acid peptide (named KV11) as the key region for the antiangiogenic function of the KV domain of apolipoprotein (a). We demonstrate that KV11 inhibits angiogenesis in vitro by suppressing human umbilical vein endothelial cell migration and microtubule formation. KV11 inhibits angiogenesis in chicken chorioallantoic membrane assays and mouse corneal micropocket angiogenesis assays in vivo. KV11 peptide shows no effect on tumor cell growth or proliferation, but significantly inhibits tumor growth in SCID mouse xenograft tumor model (p < 0.01) by preventing tumor angiogenesis. We elucidate that KV11 peptide suppresses angiogenesis and tumor progression by targeting the c-Src/ERK signaling pathways. Together, these studies provide the first evidence that KV11 from apolipoprotein KV domain has anti-angiogenesis functions and may be an anti-tumor drug candidate.

    Funded by: NCI NIH HHS: R01 CA106479, R01 CA106479-04

    International journal of cancer 2009;124;4;843-52

  • Activation of Src and Src-associated signaling pathways in relation to hypoxia in human cancer xenograft models.

    Pham NA, Magalhaes JM, Do T, Schwock J, Dhani N, Cao PJ, Hill RP and Hedley DW

    Ontario Cancer Institute, Princess Margaret Hospital, and Department of Laboratory Medicine and Pathology, University of Toronto, Toronto, Ontario, Canada.

    The hypoxic response in vitro involves alterations in signaling proteins, including Src, STAT3 and AKT that are considered to be broadly pro-survival. The involvement of these signaling proteins in the hypoxic microenviroments that occur in solid tumors was investigated by the use of multicolor fluorescence image analysis to colocalize signaling proteins and regions of hypoxia in 4 human tumor xenografts, pancreatic carcinoma BxPC3 and PANC1 and cervical squamous cell carcinoma ME180 and SiHa. Expression levels of total Src protein (mean intensity x labeled region fraction) were higher in hypoxic regions, identified using the nitroimidazole probe EF5, relative to non-EF5 regions in all 4 tumor models. This was associated with higher levels of phosphorylated (p-) Y419p-Src and its substrate Y861p-FAK in EF5 positive regions of BxPC3 tumors. This effect was also seen in tumor-bearing mice continuously breathing 7% oxygen for 3 hr which markedly increased the extent of EF5 positive labeling. In contrast, the hypoxia treatment resulted in a significant decrease in S727p-STAT3 in BxPC3 xenografts and suggested that STAT3 activity is responsive to acute hypoxia, whereas Src-FAK signaling is associated with predominantly chronically hypoxic EF5 positive regions. Src activity in both hypoxic and nonhypoxic BxPC3 tumor regions was suppressed when mice were treated with the Src inhibitor AZD0530 (25 mg/kg/day, 5 days), suggesting that both hypoxic and normoxic tumor regions are accessible to pharmacological Src inhibition. These results show that signaling pathways are responsive to tumor hypoxia in vivo, although the effects appear to differ between individual tumor types.

    International journal of cancer 2009;124;2;280-6

  • Src activation and translocation from focal adhesions to membrane ruffles contribute to formation of new adhesion sites.

    Hamadi A, Deramaudt TB, Takeda K and Rondé P

    UMR CNRS 7175, Dépt. Pharmacologie et Physicochimie, Université Louis Pasteur - Strasbourg I, Faculté de Pharmacie, 74 rte du Rhin, BP 60024, 67401, Illkirch, France.

    Cell migration requires the coordinated turnover of focal adhesions, a process that involves FAK phosphorylation. Since Src is the major kinase implicated in FAK phosphorylation, we focus here on the role of Src activation on adhesion remodelling. In astrocytoma cells, constitutively activated Src induces both FAK phosphorylation and adhesion rearrangement. To evaluate how Src controls these processes, we used a recently described Src reporter to monitor the dynamics of Src phosphorylation. Upon Src activation, focal adhesions started to disassemble while Src appeared highly expressed at newly formed membrane ruffles. Kinetic analysis of time-lapse movies showed that loss of phospho-Src at focal adhesions was time-correlated with the appearance of membrane ruffles containing phospho-Src. Moreover, FLIP analysis revealed a dynamic equilibrium of Src between focal adhesions and membrane ruffles. We conclude that upon phosphorylation, Src is directly translocated from focal adhesions to membrane ruffles, thereby promoting formation of new adhesion complexes.

    Cellular and molecular life sciences : CMLS 2009;66;2;324-38

  • Aberrant trafficking of NSCLC-associated EGFR mutants through the endocytic recycling pathway promotes interaction with Src.

    Chung BM, Raja SM, Clubb RJ, Tu C, George M, Band V and Band H

    Eppley Institute for Cancer and Allied Diseases, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, NE 68198-5950, USA. chungj@unmc.edu

    Background: Epidermal growth factor receptor (EGFR) controls a wide range of cellular processes, and altered EGFR signaling contributes to human cancer. EGFR kinase domain mutants found in non-small cell lung cancer (NSCLC) are constitutively active, a trait critical for cell transformation through activation of downstream pathways. Endocytic trafficking of EGFR is a major regulatory mechanism as ligand-induced lysosomal degradation results in termination of signaling. While numerous studies have examined mutant EGFR signaling, the endocytic traffic of mutant EGFR within the NSCLC milieu remains less clear.

    Results: This study shows that mutant EGFRs in NSCLC cell lines are constitutively endocytosed as shown by their colocalization with the early/recycling endosomal marker transferrin and the late endosomal/lysosomal marker LAMP1. Notably, mutant EGFRs, but not the wild-type EGFR, show a perinuclear accumulation and colocalization with recycling endosomal markers such as Rab11 and EHD1 upon treatment of cells with endocytic recycling inhibitor monensin, suggesting that mutant EGFRs preferentially traffic through the endocytic recycling compartments. Importantly, monensin treatment enhanced the mutant EGFR association and colocalization with Src, indicating that aberrant transit through the endocytic recycling compartment promotes mutant EGFR-Src association.

    Conclusion: The findings presented in this study show that mutant EGFRs undergo aberrant traffic into the endocytic recycling compartment which allows mutant EGFRs to engage in a preferential interaction with Src, a critical partner for EGFR-mediated oncogenesis.

    Funded by: NCI NIH HHS: CA10548, CA116552, CA81076, CA87986, CA94143, CA96844, CA99163, CA99900, R01 CA099163-09, R01 CA099163-10, R01 CA099163-11, R01 CA105489-08

    BMC cell biology 2009;10;84

  • c-Src regulates Akt signaling in response to ghrelin via beta-arrestin signaling-independent and -dependent mechanisms.

    Lodeiro M, Theodoropoulou M, Pardo M, Casanueva FF and Camiña JP

    Laboratory of Molecular and Cellular Endocrinology, Instituto de Investigaciones Sanitarias, Complejo Hospitalario Universitario de Santiago (CHUS), Santiago de Compostela, Spain.

    The aim of the present study was to identify the signaling mechanisms to ghrelin-stimulated activation of the serine/threonine kinase Akt. In human embryonic kidney 293 (HEK293) cells transfected with GHS-R1a, ghrelin leads to the activation of Akt through the interplay of distinct signaling mechanisms: an early G(i/o) protein-dependent pathway and a late pathway mediated by beta-arrestins. The starting point is the G(i/o)-protein dependent PI3K activation that leads to the membrane recruitment of Akt, which is phosphorylated at Y by c-Src with the subsequent phosphorylation at A-loop (T308) and HM (S473) by PDK1 and mTORC2, respectively. Once the receptor is activated, a second signaling pathway is mediated by beta-arrestins 1 and 2, involving the recruitment of at least beta-arrestins, c-Src and Akt. This beta-arrestin-scaffolded complex leads to full activation of Akt through PDK1 and mTORC2, which are not associated to the complex. In agreement with these results, assays performed in 3T3-L1 preadipocyte cells indicate that beta-arrestins and c-Src are implicated in the activation of Akt in response to ghrelin through the GHS-R1a. In summary this work reveals that c-Src is crucially involved in the ghrelin-mediated Akt activation. Furthermore, the results support the view that beta-arrestins act as both scaffolding proteins and signal transducers on Akt activation.

    PloS one 2009;4;3;e4686

  • NC2213: a novel methionine aminopeptidase 2 inhibitor in human colon cancer HT29 cells.

    Selvakumar P, Lakshmikuttyamma A, Das U, Pati HN, Dimmock JR and Sharma RK

    Department of Pathology and Laboratory Medicine, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S7N4H4, Canada. ponniah.selvakumar@saskcancer.ca

    Methionine aminopeptidase 2 (MetAP2) is a bifunctional protein that plays a critical role in the regulation of post-translational processing and protein synthesis. MetAP2 is overexpressed in human colon cancer. In this report we screened various MetAP2 inhibitors and treated HT29 cells with various concentrations of compounds. We evaluated the expression of MetAP2 and pp60c-src expressions in HT29 cells. In addition we also carried out the cell proliferation and cell cycle analysis in the MetAP2 inhibitor-treated HT29 cells. The cell cycle analysis of HT29 treated with 1.0 microM of NC2213 showed an arrest in the G2 phase followed by an induction in the percentage of cells undergoing apoptosis in the sub-G1 phase. Western blot analysis revealed that the MetAP2 expression was dose-dependently decreased when the HT29 cells were treated with the 3,5-bis(benzylidene)-4-piperidone derivative (NC2213). In addition, phosphorylation of Src, a myristoylated oncoprotein was significantly decreased by 1.0 microM of NC2213 as revealed by Western blot analysis. Furthermore, NC2213 also inhibits the expression of pp60c-src in HT29 cells. Interestingly, this compound also inhibits the phosphorylation at Tyr416 of pp60c-src while increasing the phosphorylation at Tyr527 of pp60c-src. NC2213 inhibits the growth of HT29 cells by inducing apoptosis and might be useful for the treatment of human colon cancer.

    Funded by: Canadian Institutes of Health Research: 53171

    Molecular cancer 2009;8;65

  • Src activity alters alpha3 integrin expression in colon tumor cells.

    Kline CL, Olson TL and Irby RB

    Department of Medicine, Penn State Hershey Cancer Institute, H072, The Pennsylvania State University College of Medicine, 500 University Drive, Box 850, Hershey, PA 17033, USA.

    Src kinase has been linked to increased motility in the progression and metastasis of human colon cancer, although the mechanisms are not fully understood. Integrins are involved in metastasis by mediating attachment and migration of cells, as well as through transducing signals. This study examines the link between Src and integrin activity in the metastatic process in colon cancer cells. To determine Src involvement in integrin expression, the human colon cancer cell line, HCT116, was transfected with an activated Src construct and assayed for its ability to attach to and migrate across collagen and laminin. These cells attached more readily and migrated less rapidly on the extracellular matrix (ECM) than did cells transfected with empty vector. Examination of integrin levels showed a decrease in the alpha3 subunit in Src transfected cells as well as decreased cell surface localization of alpha3 integrin. The downregulation of alpha3 integrin was reversed by inhibition of Src and by inhibition of MAP kinase. Inhibition of alpha3 integrin using shRNA resulted in decreased MMP7 secretion, a possible cause of decreased invasion with low alpha3 integrin expression. This study shows that Src overexpression downregulates alpha3 integrin total protein expression and localization to the cell surface of HCT116 colon cancer cells. This indicates that Src activity may enhance metastasis by altering alpha3 integrin expression.

    Clinical & experimental metastasis 2009;26;2;77-87

  • c-Src-p38 mitogen-activated protein kinase signaling is required for Akt activation in response to ionizing radiation.

    Kim MJ, Byun JY, Yun CH, Park IC, Lee KH and Lee SJ

    Laboratory of Molecular Biochemistry, Department of Chemistry, Hanyang University, 17 Haengdang-Dong, Seongdong-Gu, Seoul, Korea.

    The Akt and mitogen-activated protein kinase (MAPK) pathways have been implicated in tumor cell survival and contribute to radiation resistance. However, the molecular basis for link between MAPK and Akt in cell survival response to radiation is unclear. Here, we show that c-Src-Rac1-p38 MAPK pathway signals Akt activation and cell survival in response to radiation. Ionizing radiation triggered Thr(308) and Ser(473) phosphorylation of Akt. Exposure of cells to radiation also induced p38 MAPK and c-Jun NH(2)-terminal kinase activations. Inhibition of c-Jun NH(2)-terminal kinase suppressed radiation-induced cell death, whereas inhibition of p38 MAPK effectively increased sensitivity to radiation. Interestingly, inhibition of p38 MAPK completely attenuated radiation-induced Ser(473) phosphorylation of Akt but did not affect Thr(308) phosphorylation. Conversely, overexpression of p38 MAPK enhanced Ser(473) phosphorylation of Akt in response to radiation. In addition, inhibition of p38 MAPK failed to alter phosphoinositide 3-kinase and phosphoinositide-dependent protein kinase activities. Ectopic expression of RacN17, dominant-negative form of Rac1, inhibited p38 MAPK activation and Ser(473) phosphorylation of Akt. Following exposure to radiation, c-Src was selectively activated among Src family tyrosine kinases. Inhibition of c-Src attenuated Rac1 and p38 MAPK activations and Ser(473) phosphorylation of Akt. Our results support the notion that the c-Src-Rac1-p38 MAPK pathway is required for activation of Akt in response to radiation and plays a cytoprotective role against radiation in human cancer cells.

    Molecular cancer research : MCR 2008;6;12;1872-80

  • Src family kinases mediate betel quid-induced oral cancer cell motility and could be a biomarker for early invasion in oral squamous cell carcinoma.

    Chen JY, Hung CC, Huang KL, Chen YT, Liu SY, Chiang WF, Chen HR, Yen CY, Wu YJ, Ko JY and Jou YS

    Department of Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan.

    Betel quid (BQ)-chewing oral cancer is a prevalent disease in many countries of Southeast Asia. Yet, the precise disease mechanism remains largely unknown. Here, we show that BQ extract-induced cell motility in three oral cancer cells (Ca9-22, SAS, and SCC9) presumably involves the Src family kinases (SFKs). Besides, BQ extract can markedly induce cell migration of wild type mouse embryonic fibroblasts (MEFs) but not MEFs lacking three SFK members, namely, Src, Yes, and Fyn, indicating the requirement of SFKs for BQ-induced cell motility. Betel quid extract can also elevate cellular SFK activities because phosphorylation of tyrosine 416 at the catalytic domain is increased, which in turn promotes phosphorylation of an in vitro substrate, enolase. Furthermore, we identified that areca nut, a major component of BQ, is the key factor accounting for BQ-induced cell migration and invasion through SFKs-mediated signaling pathways. Immunohistochemistry revealed that, particularly in BQ-chewing cases, the activity of SFKs was significantly higher in tumor-adjacent mucosa than that in solid tumor areas (P < .01). These results suggest a possible role of SFKs in tumor-host interface and thus in early tumor invasion in vivo. Consistent with this is the observation that activation of SFKs is colocalized with invasive tumor fronts in oral squamous cell carcinoma. Together, we conclude that SFKs may represent a potential biomarker of invasion and therapeutic target in BQ-induced oral cancer.

    Neoplasia (New York, N.Y.) 2008;10;12;1393-401

  • Cell cycle-dependent regulation of SFK, JAK1 and STAT3 signalling by the protein tyrosine phosphatase TCPTP.

    Shields BJ, Court NW, Hauser C, Bukczynska PE and Tiganis T

    Department of Biochemistry and Molecular Biology, Monash University, Victoria, Australia.

    Janus-activated kinases (JAKs) and Src family kinases (SFKs) and their common substrate signal transducer and activator of transcription (STAT)-3 are frequently hyperactivated in human cancer contributing to the proliferative drive by promoting G(1)/S and G(2)/M progression. Previous studies have established that the protein tyrosine phosphatase TCPTP can dephosphorylate and inactivate the SFK and JAK protein tyrosine kinases (PTKs) to attenuate cytokine signalling in vivo. In this study we determined whether TCPTP regulates SFK and JAK signalling during the cell cycle. We used primary mouse embryonic fibroblasts (MEFs) isolated from TCPTP(-/-) versus +/+ mice, immortalised TCPTP(-/-) MEFs versus those reconstituted with physiological levels of TCPTP and HeLa cells in which TCPTP protein levels had been suppressed by RNA interference, to establish TCPTP as a negative regulator of SFK, JAK1 and STAT3 signalling during the cell cycle. We found that the progression of TCPTP-deficient MEFs after the G(1) restriction point into S-phase was enhanced. We used RNA interference and pharmacological inhibitors to demonstrate that elevated SFK and downstream phosphatidylinositol 3-kinase signalling but not JAK1 or STAT3 signalling were required for the enhanced G(1)/S transition. These results identify TCPTP as a negative regulator of the cell cycle.

    Cell cycle (Georgetown, Tex.) 2008;7;21;3405-16

  • Ephrin-B2-induced cleavage of EphB2 receptor is mediated by matrix metalloproteinases to trigger cell repulsion.

    Lin KT, Sloniowski S, Ethell DW and Ethell IM

    Division of Biomedical Sciences, University of California, Riverside, California 92521-0121, USA.

    EphB receptors provide crucial adhesive and repulsive signals during cell migration and axon guidance, but it is unclear how they switch between these opposing responses. Here we provide evidence of an important role for matrix metalloproteinases (MMPs) in repulsive EphB2 signaling. We found that EphB2 is cleaved by MMPs both in vitro and in vivo, and that this cleavage is induced by interaction with its ligand ephrin-B2. Our findings demonstrate that MMP-2/MMP-9-specific inhibition or cleavage-resistant mutations in the ectodomain of EphB2 can prevent EphB2-mediated cell-cell repulsion in HEK293 cells, and block ephrin-B1-induced growth cone withdrawal in cultured hippocampal neurons. Transient expression of wtEphB2, but not noncleavable EphB2-4/5 mutant, restored ephrin-B1-induced growth cone collapse and withdrawal in EphB-deficient neurons. The inhibition of EphB2 cleavage also had potent regulatory effects on EphB2 activity. This study provides the first evidence that MMP-mediated cleavage of EphB2 is induced by receptor-ligand interactions at the cell surface and that this event triggers cell-repulsive responses.

    Funded by: NIMH NIH HHS: MH67121, R01 MH067121-05

    The Journal of biological chemistry 2008;283;43;28969-79

  • Src and ADAM-17-mediated shedding of transforming growth factor-alpha is a mechanism of acute resistance to TRAIL.

    Van Schaeybroeck S, Kelly DM, Kyula J, Stokesberry S, Fennell DA, Johnston PG and Longley DB

    Drug Resistance Group, Center for Cancer Research and Cell Biology, Queen's University Belfast, Belfast, Northern Ireland.

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo-2L) has emerged as a promising anticancer agent. However, resistance to TRAIL is likely to be a major problem, and sensitization of cancer cells to TRAIL may therefore be an important anticancer strategy. In this study, we examined the effect of the epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) gefitinib and a human epidermal receptor 2 (HER2)-TKI (M578440) on the sensitivity of human colorectal cancer (CRC) cell lines to recombinant human TRAIL (rhTRAIL). A synergistic interaction between rhTRAIL and gefitinib and rhTRAIL and M578440 was observed in both rhTRAIL-sensitive and resistant CRC cells. This synergy correlated with an increase in EGFR and HER2 activation after rhTRAIL treatment. Furthermore, treatment of CRC cells with rhTRAIL resulted in activation of the Src family kinases (SFK). Importantly, we found that rhTRAIL treatment induced shedding of transforming growth factor-alpha (TGF-alpha) that was dependent on SFK activity and the protease ADAM-17. Moreover, this shedding of TGF-alpha was critical for rhTRAIL-induced activation of EGFR. In support of this, SFK inhibitors and small interfering RNAs targeting ADAM-17 and TGF-alpha also sensitized CRC cells to rhTRAIL-mediated apoptosis. Taken together, our findings indicate that both rhTRAIL-sensitive and resistant CRC cells respond to rhTRAIL treatment by activating an EGFR/HER2-mediated survival response and that these cells can be sensitized to rhTRAIL using EGFR/HER2-targeted therapies. Furthermore, this acute response to rhTRAIL is regulated by SFK-mediated and ADAM-17-mediated shedding of TGF-alpha, such that targeting SFKs or inhibiting ADAM-17, in combination with rhTRAIL, may enhance the response of CRC tumors to rhTRAIL.

    Funded by: Cancer Research UK: A5785, A7261; Medical Research Council: G0400302

    Cancer research 2008;68;20;8312-21

  • mda-9/Syntenin promotes metastasis in human melanoma cells by activating c-Src.

    Boukerche H, Su ZZ, Prévot C, Sarkar D and Fisher PB

    Department of Human and Molecular Genetics, Virginia Commonwealth University, School of Medicine, Richmond, VA 23298, USA.

    The scaffold PDZ-domain containing protein mda-9/syntenin functions as a positive regulator of cancer cell progression in human melanoma and other tumors. mda-9/Syntenin regulates cell motility and invasion by altering defined biochemical and signaling pathways, including focal adhesion kinase (FAK), p38 mitogen-activated protein kinase (MAPK) and NF-kappaB, but precisely how mda-9/syntenin organizes these multiprotein signaling complexes is not well understood. Using a clinically relevant human melanoma model, we demonstrate that mda-9/syntenin physically interacts with c-Src and this communication correlates with an increase in FAK/c-Src complex formation and c-Src activation. Inhibiting mda-9/syntenin, using an adenovirus expressing antisense mda-9/syntenin or addition of c-Src siRNA, suppresses melanoma cell migration, anchorage-independent growth, and spontaneous tumor cell dissemination in vivo in a human melanoma animal metastasis model. These data are compatible with a model wherein interaction of MDA-9/syntenin with c-Src promotes the formation of an active FAK/c-Src signaling complex, leading to enhanced tumor cell invasion and metastatic spread. These provocative findings highlight mda-9/syntenin and its interacting partners as promising therapeutic targets for intervention of metastasis.

    Funded by: NCI NIH HHS: R01 CA035675

    Proceedings of the National Academy of Sciences of the United States of America 2008;105;41;15914-9

  • The Diaphanous-related Formin FHOD1 associates with ROCK1 and promotes Src-dependent plasma membrane blebbing.

    Hannemann S, Madrid R, Stastna J, Kitzing T, Gasteier J, Schönichen A, Bouchet J, Jimenez A, Geyer M, Grosse R, Benichou S and Fackler OT

    Department of Virology, University of Heidelberg, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany.

    Diaphanous-related formins (DRFs) mediate GTPase-triggered actin rearrangements to regulate central cellular processes, such as cell motility and cytokinesis. The DRF FHOD1 interacts with the Rho-GTPase Rac1 and mediates formation of actin stress fibers in its deregulated form; the physiologically relevant activities and molecular mechanisms of endogenous FHOD1, however, are still unknown. Here we report that FHOD1 physically associates via the N-terminal part of its FH2 domain with the central domain of ROCK1. Although FHOD1 does not affect the kinase activity of ROCK1, the DRF is an efficient substrate for phosphorylation by ROCK1. Co-expression of FHOD1 and ROCK1 results in the generation of nonapoptotic plasma membrane (PM) blebs, to which the DRF is efficiently recruited. Blebbing induced by FHOD1 and ROCK1 depends on F-actin integrity, the Rho-ROCK cascade, and Src activity and is reminiscent of the recently described PM blebs triggered by expression of Src homology 4 (SH4) domain PM targeting signals. Consistently, endogenous FHOD1 is required in SH4 domain expressing cells for efficient PM blebbing and rounded cell morphology in two-dimensional cultures or three-dimensional matrices, respectively. Efficient association of FHOD1 with ROCK1, as well as recruitment of the DRF to blebs, depends on Src activity, suggesting that the functional interaction between both proteins is regulated by Src. These results define a role for endogenous FHOD1 in SH4 domain-induced blebbing and suggest that its activity is regulated by ROCK1 in a Src-dependent manner.

    The Journal of biological chemistry 2008;283;41;27891-903

  • The transactivated epidermal growth factor receptor recruits Pyk2 to regulate Src kinase activity.

    Schauwienold D, Sastre AP, Genzel N, Schaefer M and Reusch HP

    Abteilung Klinische Pharmakologie, Ruhr-Universität Bochum, 44780 Bochum, Germany.

    G protein-coupled receptors such as proteinase-activated receptor 1 induce phosphorylation of mitogen-activated protein kinases through multiple pathways including transactivation of receptor tyrosine kinases. In vascular smooth muscle cells, both matrix-metalloproteinase-dependent extracellular shedding of membrane-bound epidermal growth factor (EGF) receptor ligands and activation of the nonreceptor tyrosine kinases Pyk2 and Src contributed to the thrombin-induced ERK1/2 phosphorylation. Surprisingly, disruption of the HB-EGF-mediated extracellular mode of EGF receptor transactivation also prevented the phosphorylation of the nonreceptor tyrosine kinases Pyk2 and Src, locating these kinases downstream of the transactivated EGF receptor. The ionomycin-induced Pyk2 phosphorylation was partially sensitive to AG1478, heparin, or the matrix-metalloproteinase inhibitor BB2116, and the ionomycin-induced EGF receptor phosphorylation was almost completely blocked by these inhibitors of extracellular transactivation. Coimmunoprecipitation experiments revealed that, upon thrombin stimulation, a signaling complex consisting of Pyk2 and Src assembles at the EGF receptor. Reconstitution of the signaling molecules in HEK293 or vascular smooth muscle cells and subsequent determination of the EGF-induced Src kinase activity applying fluorescent sensor proteins demonstrated that a Ca(2+)-independent mode of Pyk2 activation is critical for the activation of Src downstream of the EGF receptor.

    The Journal of biological chemistry 2008;283;41;27748-56

  • Lipoteichoic acid induces HO-1 expression via the TLR2/MyD88/c-Src/NADPH oxidase pathway and Nrf2 in human tracheal smooth muscle cells.

    Lee IT, Wang SW, Lee CW, Chang CC, Lin CC, Luo SF and Yang CM

    Department of Physiology and Pharmacology.

    Heme oxygenase (HO)-1 is a stress-inducible rate-limiting enzyme in heme degradation that confers cytoprotection against oxidative injury and provides a vital function in maintaining tissue homeostasis. Increasing reports have indicated that lipoteichoic acid (LTA) exerts as LPS as an immune system-stimulating agent and plays a role in the pathogenesis of severe inflammatory responses induced by Gram-positive bacterial infection. We report that LTA is an inducer of HO-1 expression mediated through the signaling pathways in human tracheal smooth muscle cells (HTSMCs). LTA-induced HO-1 protein levels, mRNA expression, and promoter activity were attenuated by transfection with dominant negative mutants of TLR2 and MyD88, by pretreatment with the inhibitors of c-Src (PP1), NADPH oxidase (diphenylene iodonium chloride (DPI) and apocynin (APO)), and reactive oxygen species (ROS) scavenger (N-acetyl-l-cysteine) or by transfection with small interfering RNAs of Src and NF-E2-related factor 2 (Nrf2). LTA-stimulated translocation of p47(phox) and Nrf2 or ROS production was attenuated by transfection with dominant negative mutants of TLR2, MyD88, and c-Src and by pretreatment with DPI or APO. Furthermore, LTA-induced TLR2, MyD88, TNFR-associated factor (TRAF)6, c-Src, and p47(phox) complex formation was revealed by immunoprecipitation using an anti-TLR2 or anti-c-Src Ab followed by Western blot analysis against an anti-TLR2, anti-MyD88, anti-TRAF6, anti-c-Src, or anti-p47(phox) Ab. These results demonstrated that LTA-induced ROS generation was mediated through the TLR2/MyD88/TRAF6/c-Src/NADPH oxidase pathway, in turn initiates the activation of Nrf2, and ultimately induces HO-1 expression in HTSMCs.

    Journal of immunology (Baltimore, Md. : 1950) 2008;181;7;5098-110

  • Both EGFR kinase and Src-related tyrosine kinases regulate human ether-à-go-go-related gene potassium channels.

    Zhang DY, Wang Y, Lau CP, Tse HF and Li GR

    Department of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China.

    Human ether-à-go-go-related gene (hERG or Kv11.1) encodes the rapidly activated delayed rectifier K(+) current (I(Kr)) in the human heart. Potential regulation of hERG channel by protein tyrosine kinases (PTKs) is not understood. The present study was designed to investigate whether this channel is modulated by PTKs using whole-cell patch clamp technique, and immunoprecipitation and Western blot analysis in HEK 293 cells stably expressing hERG gene. We found that the broad-spectrum PTK inhibitor genistein (30 microM), the selective EGFR (epidermal growth factor receptor) kinase inhibitor AG556 (10 microM) and the Src-family kinase inhibitor PP2 (10 microM) remarkably inhibited hERG channel current (I(hERG)), and the effects were significantly countered by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate (1 mM). Immunoprecipitation and Western blot analysis demonstrated that membrane protein tyrosine phosphorylation of hERG channels was reduced by genistein, AG556, and PP2. The reduction of hERG channel phosphorylation level by genistein, AG556 or PP2 was antagonized by orthovanadate. Single point mutation(s) of Y475A and/or Y611A dramatically attenuated the inhibitory effect of I(hERG) by PP2 and/or AG556. Our results demonstrate the novel information that I(hERG) is modulated not only by Src-family kinases, but also by EGFR kinases. Y475 and/or Y611 are likely the preferred phosphorylation sites. Regulation of hERG channels by PTKs modifies the channel activity and thus likely alters electrophysiological properties including action potential duration and cell excitability in human heart and neurons.

    Cellular signalling 2008;20;10;1815-21

  • Parallel phosphatidylinositol 3-kinase (PI3K)-dependent and Src-dependent pathways lead to CXCL8-mediated Rac2 activation and chemotaxis.

    Sai J, Raman D, Liu Y, Wikswo J and Richmond A

    Department of Cancer Biology, School of Medicine, Vanderbilt University, Nashville, Tennessee 37232, USA.

    The requirement for phosphatidylinositol 3-kinase (PI3K) in the establishment of cell polarity and motility in a number of cell types has recently come into question. In this study, we demonstrate that inhibition of PI3K by wortmannin in neutrophil-like differentiated HL60 cells expressing CXCR2 resulted in reduced cell motility but normal chemotaxis in response to a gradient of CXCL8. However, wortmannin inhibition of PI3K did impair the ability of cells to re-orient their polarity and respond quickly to a change in the direction of the CXCL8 gradient. We hypothesized that Src-regulated ELMO-Dock2-Rac2 activation mediates chemotaxis in the absence of PI3K activity. Inhibition of Src with the small molecule inhibitor, PP2, or inhibition of Dock2 by shRNA knockdown confirmed the functional role of Src and Dock2 in regulating chemotaxis when PI3K was inhibited. Moreover, neutrophils isolated from bone marrow of hck(-/-)fgr(-/-)lyn(-/-) mice exhibited much more severe inhibition of chemotaxis when PI3K was blocked with wortmannin as compared with neutrophils isolated from bone marrow of wild-type mice. Thus, PI3K and Src-ELMO-Dock2 pathways work in parallel to activate Rac2 and modulate chemotaxis in response to a CXCL8 gradient in neutrophils.

    Funded by: NCI NIH HHS: CA34590, CA68485, R01 CA034590, U54CA113007

    The Journal of biological chemistry 2008;283;39;26538-47

  • CD44 engagement promotes matrix-derived survival through the CD44-SRC-integrin axis in lipid rafts.

    Lee JL, Wang MJ, Sudhir PR and Chen JY

    Institute of Biomedical Sciences, Academia Sinica, 128 Section 2 Academia Road, Taipei 11529, Taiwan, Republic of China.

    CD44 is present in detergent-resistant, cholesterol-rich microdomains, called lipid rafts, in many types of cells. However, the functional significance of CD44 in lipid rafts is still unknown. We have previously demonstrated that osteopontin-mediated engagement of CD44 spliced variant isoforms promotes an extracellular matrix-derived survival signal through integrin activation. By using a series of CD44 mutants and pharmacological inhibitors selectively targeted to various cellular pathways, we show in this study that engagement of CD44 induces lipid raft coalescence to facilitate a CD44-Src-integrin signaling axis in lipid rafts, leading to increased matrix-derived survival. Palmitoylation of the membrane-proximal cysteine residues and carboxyl-terminal linkage to the actin cytoskeleton both contribute to raft targeting of CD44. The enrichment of integrin beta1 in lipid rafts is tightly coupled to CD44 ligation-elicited lipid raft reorganization and associated with temporally delayed endocytosis. Through the interaction with the CD44 carboxyl-terminal ankyrin domain, Src is cotranslocated to lipid rafts, where it induces integrin activation via an inside-out mechanism. Collectively, this study demonstrates an important role of the dynamic raft reorganization induced by CD44 clustering in eliciting the matrix-derived survival signal.

    Molecular and cellular biology 2008;28;18;5710-23

  • Focal adhesion kinase is not required for Src-induced formation of invadopodia in KM12C colon cancer cells and can interfere with their assembly.

    Vitale S, Avizienyte E, Brunton VG and Frame MC

    The Beatson Institute for Cancer Research, Garscube Estate, Bearsden, Glasgow, UK.

    Overexpression of active Src induces invadopodia formation and associated matrix degradation in KM12C colon cancer cells. FAK is present with active Src at sites of matrix-degrading activity (invadopodia), specifically residing in rings surrounding the cortactin-containing invadopodia cores. Since FAK is a key effector protein in many aspects of Src function, we addressed whether FAK is necessary for Src-induced invadopodia formation and matrix degradation in KM12C colon cancer cells. We found that efficient knockdown of FAK expression by siRNA had no effect on invadopodia formation or matrix degradation. However, overexpression of FAK could actually suppress invadopodia formation and matrix degradation. FAK phosphorylation on the putative auto-phosphorylation tyrosine 397 and the Src-specific sites are all required for overexpressed FAK to inhibit invadopodia formation, while the kinase activity of exogenous FAK is apparently not required. These data imply that kinase activities other than FAK auto-phosphorylation may contribute to the phosphorylation of FAK tyrosine 397 in some contexts to promote an activity of FAK that can counteract invadopodia formation. Further work is required to determine how the strength of signalling through FAK suppresses invadopodia, but we propose that FAK controls the balance of adhesion types in cells, and that this is one of the determinants of whether a cancer cell can make stable matrix-degrading invadopodia.

    European journal of cell biology 2008;87;8-9;569-79

  • TUSC4/NPRL2, a novel PDK1-interacting protein, inhibits PDK1 tyrosine phosphorylation and its downstream signaling.

    Kurata A, Katayama R, Watanabe T, Tsuruo T and Fujita N

    Division of Experimental Chemotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-10-6 Ariake, Koto-ku, Tokyo 135-8550, Japan.

    3-Phosphoinositide-dependent protein kinase-1 (PDK1) is a key regulator of cell proliferation and survival signal transduction. PDK1 is known to be constitutively active and is further activated by Src-mediated phosphorylation at the tyrosine-9, -373, and -376 residues. To identify novel regulators of PDK1, we performed E. coli-based two-hybrid screening and revealed that tumor suppressor candidate 4 (TUSC4), also known as nitrogen permease regulator-like 2 (NPRL2), formed a complex with PDK1 and suppressed Src-dependent tyrosine phosphorylation and activation of PDK1 in vitro and in cells. The NH(2)-terminal 133 amino acid residues of TUSC4 were involved in binding to PDK1. The deletion mutant of TUSC4 that lacked the NH(2)-terminal domain showed no inhibitory effects on PDK1 tyrosine phosphorylation or activation. Thus, complex formation is indispensable for TUSC4-mediated PDK1 inactivation. The siRNA-mediated down-regulation of TUSC4 induced cell proliferation, while ectopic TUSC4 expression inactivated the PDK1 downstream signaling pathway, including Akt and p70 ribosomal protein S6 kinase, and increased cancer cell sensitivity to several anticancer drugs. Our results suggest that TUSC4/NPRL2, a novel PDK1-interacting protein, plays a role in regulating the Src/PDK1 signaling pathway and cell sensitivity to multiple cancer chemotherapeutic drugs.

    Cancer science 2008;99;9;1827-34

  • Validation of PDGFRbeta and c-Src tyrosine kinases as tumor/vessel targets in patients with multiple myeloma: preclinical efficacy of the novel, orally available inhibitor dasatinib.

    Coluccia AM, Cirulli T, Neri P, Mangieri D, Colanardi MC, Gnoni A, Di Renzo N, Dammacco F, Tassone P, Ribatti D, Gambacorti-Passerini C and Vacca A

    Department of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy. malu.coluccia@libero.it

    Inhibition of multiple myeloma (MM) plasma cells in their permissive bone marrow microenvironment represents an attractive strategy for blocking the tumor/vessel growth associated with the disease progression. However, target specificity is an essential aim of this approach. Here, we identified platelet-derived growth factor (PDGF)-receptor beta (PDGFRbeta) and pp60c-Src as shared constitutively activated tyrosine-kinases (TKs) in plasma cells and endothelial cells (ECs) isolated from MM patients (MMECs). Our cellular and molecular dissection showed that the PDGF-BB/PDGFRbeta kinase axis promoted MM tumor growth and vessel sprouting by activating ERK1/2, AKT, and the transcription of MMEC-released proangiogenic factors, such as vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8). Interestingly, pp60c-Src TK-activity was selectively induced by VEGF in MM tumor and ECs, and the use of small-interfering (si)RNAs validated pp60c-Src as a key signaling effector of VEGF loop required for MMEC survival, migration, and angiogenesis. We also assessed the antitumor/vessel activity of dasatinib, a novel orally bioactive PDGFRbeta/Src TK-inhibitor that significantly delayed MM tumor growth and angiogenesis in vivo, showing a synergistic cytotoxicity with conventional and novel antimyeloma drugs (ie, melphalan, prednisone, bor-tezomib, and thalidomide). Overall data highlight the biologic and therapeutic relevance of the combined targeting of PDGFRbeta/c-Src TKs in MM, providing a framework for future clinical trials.

    Blood 2008;112;4;1346-56

  • Signal transducer and activator of transcription 5b, c-Src, and epidermal growth factor receptor signaling play integral roles in estrogen-stimulated proliferation of estrogen receptor-positive breast cancer cells.

    Fox EM, Bernaciak TM, Wen J, Weaver AM, Shupnik MA and Silva CM

    Department of Pharmacology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.

    17beta-Estradiol (E2) acts through the estrogen receptor alpha (ERalpha) to stimulate breast cancer proliferation. Here, we investigated the functional relationship between ERalpha and signal transducer and activator of transcription (STAT)5b activity in ER+ MCF-7 and T47D human breast cancer cells after specific knockdown of STAT5b. STAT5b small interfering RNA (siRNA) inhibited E2-induced bromodeoxyuridine (BrdU) incorporation in both cell lines, as well as the E2-induced increase in MCF-7 cell number, cyclin D1 and c-myc mRNA, and cyclin D1 protein expression, indicating that STAT5b is required for E2-stimulated breast cancer proliferation. E2 treatment stimulated STAT5b tyrosine phosphorylation at the activating tyrosine Y699, resulting in increased STAT5-mediated transcriptional activity, which was inhibited by a Y669F STAT5b mutant. E2-induced STAT5-mediated transcriptional activity was inhibited by overexpressing a kinase-defective epidermal growth factor receptor (EGFR), or the EGFR tyrosine kinase inhibitor tyrphostin AG1478, indicating a requirement for EGFR kinase activity. Both E2-induced STAT5b tyrosine phosphorylation and STAT5-mediated transcription were also inhibited by the ER antagonist ICI 182,780 and the c-Src inhibitor PP2, indicating additional requirements for the ER and c-Src kinase activity. EGFR and c-Src kinase activities were also required for E2-induced cyclin D1 and c-myc mRNA. Together, these studies demonstrate positive cross talk between ER, c-Src, EGFR, and STAT5b in ER+ breast cancer cells. Increased EGFR and c-Src signaling is associated with tamoxifen resistance in ER+ breast cancer cells. Here we show that constitutively active STAT5b not only increased basal DNA synthesis, but also conferred tamoxifen resistance. Because STAT5b plays an integral role in E2-stimulated proliferation and tamoxifen resistance, it may be an effective therapeutic target in ER+ breast tumors.

    Funded by: NCI NIH HHS: NCI-P30 CA44579, P30 CA044579; NIDDK NIH HHS: DK57082, R01 DK057082

    Molecular endocrinology (Baltimore, Md.) 2008;22;8;1781-96

  • Activation of platelet-activating factor receptor and pleiotropic effects on tyrosine phospho-EGFR/Src/FAK/paxillin in ovarian cancer.

    Aponte M, Jiang W, Lakkis M, Li MJ, Edwards D, Albitar L, Vitonis A, Mok SC, Cramer DW and Ye B

    Laboratory of Gynecologic Oncology and Epidemiology, Department of Obstetrics and Gynecology and Reproductive Biology, Brigham and Women's Hospital, Dana-Farber Cancer Center, Harvard Medical School, Boston, Massachusetts 02115, USA.

    Among the proinflammatory mediators, platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) is a major primary and secondary messenger involved in intracellular and extracellular communication. Evidence suggests that PAF plays a significant role in oncogenic transformation, tumor growth, angiogenesis, and metastasis. However, PAF, with its receptor (PAFR) and their downstream signaling targets, has not been thoroughly studied in cancer. Here, we characterized the PAFR expression pattern in 4 normal human ovarian surface epithelial (HOSE) cell lines, 13 ovarian cancer cell lines, paraffin blocks (n = 84), and tissue microarrays (n = 230) from patients with ovarian cancer. Overexpression of PAFR was found in most nonmucinous types of ovarian cancer but not in HOSE and mucinous cancer cells. Correspondingly, PAF significantly induced cell proliferation and invasion only in PAFR-positive cells (i.e., OVCA429 and OVCA432), but not in PAFR-negative ovarian cells (HOSE and mucinous RMUG-L). The dependency of cell proliferation and invasion on PAFR was further confirmed using PAFR-specific small interfering RNA gene silencing probes, antibodies against PAFR and PAFR antagonist, ginkgolide B. Using quantitative multiplex phospho-antibody array technology, we found that tyrosine phosphorylation of EGFR/Src/FAK/paxillin was coordinately activated by PAF treatment, which was correlated with the activation of phosphatidylinositol 3-kinase and cyclin D1 as markers for cell proliferation, as well as matrix metalloproteinase 2 and 9 for invasion. Specific tyrosine Src inhibitor (PP2) reversibly blocked PAF-activated cancer cell proliferation and invasion. We suggest that PAFR is an essential upstream target of Src and other signal pathways to control the PAF-mediated cancer progression.

    Funded by: NCI NIH HHS: 1P50-CA105009-01, P50 CA105009, P50 CA105009-01, R01 CA054419, R01 CA054419-13, R01 CA54419-13, R21 CA111949-01, R21 CA111949-01A1, R21 CA111949-02

    Cancer research 2008;68;14;5839-48

  • Type 1 TNF receptor forms a complex with and uses Jak2 and c-Src to selectively engage signaling pathways that regulate transcription factor activity.

    Pincheira R, Castro AF, Ozes ON, Idumalla PS and Donner DB

    Department of Surgery and Comprehensive Cancer Center, University of California, San Francisco, CA 94115, USA. pincheirar@surgery.ucsf.edu

    The type 1 TNFR (TNFR1) contains a death domain through which it interacts with other death-domain proteins to promote cellular responses. However, signaling through death-domain proteins does not explain how TNFR1 induces the tyrosine phosphorylation of intracellular proteins, which are important to cellular responses induced by TNFR1. In this study, we show that TNFR1 associates with Jak2, c-Src, and PI3K in various cell types. Jak2 and c-Src constitutively associate with and are constitutively active in the TNFR1 complex. Stimulation with TNF induces a time-dependent change in the level of Jak2, c-Src, and PI3K associated with TNFR1. The tyrosine kinase activity of the complex varies with the level of tyrosine kinase associated with TNFR1. TNFR1/c-Src plays a role in activating Akt, but not JNK or p38 MAPK, whereas TNFR1/Jak2 plays a role in activating p38 MAPK, JNK, and Akt. TNFR1/c-Src, but not TNFR1/Jak2, plays an obligate role in the activation of NF-kappaB by TNF, whereas TNFR1/Jak2, but not TNFR1/c-Src, plays an obligate role in the activation of STAT3. Activation of TNFR1 increased the expression of vascular endothelial growth factor, p21(WAF1/CIP1), and manganese superoxide dismutase in MCF7 breast cancer cells, and increased the expression of CCl2/MCP-1 and IL-1beta in THP-1 macrophages. Inhibitors of Jak2 and c-Src impaired the induction of each of these target proteins. These observations show that TNFR1 associates with and uses nonreceptor tyrosine kinases to engage signaling pathways, activate transcription factors, and modulate gene expression in cells.

    Funded by: NCI NIH HHS: CA67891

    Journal of immunology (Baltimore, Md. : 1950) 2008;181;2;1288-98

  • Endothelial cell annexin A2 regulates polyubiquitination and degradation of its binding partner S100A10/p11.

    He KL, Deora AB, Xiong H, Ling Q, Weksler BB, Niesvizky R and Hajjar KA

    Department of Cell and Developmental Biology, Weill Cornell Medical College, New York, New York 10065, USA.

    The annexin A2 (A2) heterotetramer, consisting of two copies of A2 and two copies of S100A10/p11, promotes fibrinolytic activity on the surface of vascular endothelial cells by assembling plasminogen and tissue plasminogen activator (tPA) and accelerating the generation of plasmin. In humans, overexpression of A2 by acute promyelocytic leukemia cells is associated with excessive fibrinolysis and hemorrhage, whereas anti-A2 autoantibodies appear to accentuate the risk of thrombosis in patients with anti-phospholipid syndrome. Complete deficiency of A2 in mice leads to a lack of tPA cofactor activity, accumulation of intravascular fibrin, and failure to clear arterial thrombi. Within the endothelial cell, p11 is required for Src kinase-mediated tyrosine phosphorylation of A2, which signals translocation of both proteins to the cell surface. Here we show that p11 is expressed at very low levels in the absence of A2 both in vitro and in vivo. We demonstrate further that unpartnered p11 becomes polyubiquitinated and degraded via a proteasome-dependent mechanism. A2 stabilizes intracellular p11 through direct binding, thus masking an autonomous p11 polyubiquitination signal that triggers proteasomal degradation. This interaction requires both the p11-binding N-terminal domain of A2 and the C-terminal domain of p11. This mechanism prevents accumulation of free p11 in the endothelial cell and suggests that regulation of tPA-dependent cell surface fibrinolytic activity is precisely tuned to the intracellular level of p11.

    Funded by: NHLBI NIH HHS: HL42493, HL46403, HL67839

    The Journal of biological chemistry 2008;283;28;19192-200

  • SRC directly phosphorylates Bif-1 and prevents its interaction with Bax and the initiation of anoikis.

    Yamaguchi H, Woods NT, Dorsey JF, Takahashi Y, Gjertsen NR, Yeatman T, Wu J and Wang HG

    H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33612, USA.

    Bif-1 interacts with Bax and enhances its conformational rearrangement, resulting in apoptosis. However, the molecular mechanism governing the interaction between Bif-1 and Bax is poorly defined. Here we provide evidence that Bif-1 is phosphorylated, an event that can be repressed by apoptotic stimuli. The protein kinase c-Src binds to and directly phosphorylates Bif-1 on tyrosine 80. Moreover, Src phosphorylation of Bif-1 suppresses the interaction between Bif-1 and Bax, resulting in the inhibition of Bax activation during anoikis. Together, these results suggest that phosphorylation of Bif-1 impairs its binding to Bax and represses apoptosis, providing another mechanism by which Src oncogenic signaling can prevent cell death.

    Funded by: NCI NIH HHS: P01 CA118210, R01 CA077467, R01 CA082197, R01 CA090315

    The Journal of biological chemistry 2008;283;27;19112-8

  • Combined inhibition of c-Src and epidermal growth factor receptor abrogates growth and invasion of head and neck squamous cell carcinoma.

    Koppikar P, Choi SH, Egloff AM, Cai Q, Suzuki S, Freilino M, Nozawa H, Thomas SM, Gooding WE, Siegfried JM and Grandis JR

    Department of Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

    Purpose: Increased expression and/or activation of epidermal growth factor receptor (EGFR) is associated with tumor progression and poor prognosis in many cancers, including head and neck squamous cell carcinoma (HNSCC). Src family kinases, including c-Src, mediate a variety of intracellular or extracellular signals that contribute to tumor formation and progression. This study was undertaken to elucidate the role of c-Src in the growth and invasion of HNSCC and to determine the effects of combined targeting of EGFR and Src kinases in HNSCC cell lines.

    HNSCC cells were engineered to stably express a dominant-active form of c-Src and investigated in cell growth and invasion assays. The biochemical effects of combined treatment with the Src inhibitor AZD0530, a potent, orally active Src inhibitor with Bcr/Abl activity, and the EGFR kinase inhibitor gefitinib were examined, as well as the consequences of dual Src/EGFR targeting on the growth and invasion of a panel of HNSCC cell lines.

    Results: HNSCC cells expressing dominant-active c-Src showed increased growth and invasion compared with vector-transfected controls. Combined treatment with AZD0530 and gefitinib resulted in greater inhibition of HNSCC cell growth and invasion compared with either agent alone.

    Conclusions: These results suggest that increased expression and activation of c-Src promotes HNSCC progression where combined targeting of EGFR and c-Src may be an efficacious treatment approach.

    Funded by: NCI NIH HHS: CA097190, CA098372, CA77308, P50 CA097190, R01 CA077308, R01 CA098372

    Clinical cancer research : an official journal of the American Association for Cancer Research 2008;14;13;4284-91

  • Combined inhibition of PLC{gamma}-1 and c-Src abrogates epidermal growth factor receptor-mediated head and neck squamous cell carcinoma invasion.

    Nozawa H, Howell G, Suzuki S, Zhang Q, Qi Y, Klein-Seetharaman J, Wells A, Grandis JR and Thomas SM

    Department of Oral and Maxillofacial Surgery, National Defense Medical College, Tokorozawa, Saitama, Japan.

    Purpose: Mortality from head and neck squamous cell carcinoma (HNSCC) is usually associated with locoregional invasion of the tumor into vital organs, including the airway. Understanding the signaling mechanisms that abrogate HNSCC invasion may reveal novel therapeutic targets for intervention. The purpose of this study was to investigate the efficacy of combined inhibition of c-Src and PLCgamma-1 in the abrogation of HNSCC invasion.

    PLCgamma-1 and c-Src inhibition was achieved by a combination of small molecule inhibitors and dominant negative approaches. The effect of inhibition of PLCgamma-1 and c-Src on invasion of HNSCC cells was assessed in an in vitro Matrigel-coated transwell invasion assay. In addition, the immunoprecipitation reactions and in silico database mining was used to examine the interactions between PLCgamma-1 and c-Src.

    Results: Here, we show that inhibition of PLCgamma-1 or c-Src with the PLC inhibitor U73122 or the Src family inhibitor AZD0530 or using dominant-negative constructs attenuated epidermal growth factor (EGF)-stimulated HNSCC invasion. Furthermore, EGF stimulation increased the association between PLCgamma-1 and c-Src in HNSCC cells. Combined inhibition of PLCgamma-1 and c-Src resulted in further attenuation of HNSCC cell invasion in vitro.

    Conclusions: These cumulative results suggest that PLCgamma-1 and c-Src activation contribute to HNSCC invasion downstream of EGF receptor and that targeting these pathways may be a novel strategy to prevent tumor invasion in HNSCC.

    Funded by: NCI NIH HHS: P50 CA097190, P50CA097190, P50CA097190-01A1, R01 CA077308, R01 CA77308

    Clinical cancer research : an official journal of the American Association for Cancer Research 2008;14;13;4336-44

  • The involvement of the tyrosine kinase c-Src in the regulation of reactive oxygen species generation mediated by NADPH oxidase-1.

    Gianni D, Bohl B, Courtneidge SA and Bokoch GM

    Departments of Immunology and Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

    NADPH oxidase (Nox) family enzymes are one of the main sources of cellular reactive oxygen species (ROS), which have been shown to function as second messenger molecules. To date, seven members of this family have been reported, including Nox1-5 and Duox1 and -2. With the exception of Nox2, the regulation of the Nox enzymes is still poorly understood. Nox1 is highly expressed in the colon, and it requires two cytosolic regulators, NoxO1 and NoxA1, as well as the binding of Rac1 GTPase, for its activity. In this study, we investigate the role of the tyrosine kinase c-Src in the regulation of ROS formation by Nox1. We show that c-Src induces Nox1-mediated ROS generation in the HT29 human colon carcinoma cell line through a Rac-dependent mechanism. Treatment of HT29 cells with the Src inhibitor PP2, expression of a kinase-inactive form of c-Src, and c-Src depletion by small interfering RNA (siRNA) reduce both ROS generation and the levels of active Rac1. This is associated with decreased Src-mediated phosphorylation and activation of the Rac1-guanine nucleotide exchange factor Vav2. Consistent with this, Vav2 siRNA that specifically reduces endogenous Vav2 protein is able to dramatically decrease Nox1-dependent ROS generation and abolish c-Src-induced Nox1 activity. Together, these results establish c-Src as an important regulator of Nox1 activity, and they may provide insight into the mechanisms of tumor formation in colon cancers.

    Funded by: NHLBI NIH HHS: HL-48008, R01 HL048008

    Molecular biology of the cell 2008;19;7;2984-94

  • Roles of cytosolic phospholipase A2 and Src kinase in the early action of 2,3,7,8-tetrachlorodibenzo-p-dioxin through a nongenomic pathway in MCF10A cells.

    Dong B and Matsumura F

    Department of Environmental Toxicology, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA.

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, or dioxin) is known to induce rapid inflammatory cellular responses through the mechanism that has not yet been fully elucidated. In this report, we show that in MCF10A cells, an immortalized, normal mammary epithelial cell line, TCDD rapidly activates the enzymatic activity of cytosolic phospholipase A2 (cPLA2) as at-tested to by arachidonic acid release within 15 min, followed by activation of Src kinase and induction of several inflammation markers. Such an action of TCDD is clearly blocked by methylarachidonyl fluorophosphonate, a specific inhibitor of cPLA2, short interfering RNA against cPLA2, and several calcium signaling blockers, indicating that this action of TCDD is mediated by calcium-triggered activation of cPLA2. This action of TCDD is quite different from the classic action of TCDD to induce cytochrome P450 1A1 (CYP1A1) because blocking this newly identified pathway did not affect the induction of CYP1A1. Moreover, this newly identified pathway was found to depend only on aryl hydrocarbon receptor but not on aryl hydrocarbon receptor nuclear translocator. Together, these findings support the model that the early action of TCDD to induce rapid inflammatory responses is carried out through a characteristic "nongenomic" pathway, which is clearly different from the conventional model of action of TCDD through the "genomic" pathway.

    Funded by: NIEHS NIH HHS: P30-ES05707, R01-ES05233, R01-ES07284

    Molecular pharmacology 2008;74;1;255-63

  • mRNA silencing in human erythroid cell maturation: heterogeneous nuclear ribonucleoprotein K controls the expression of its regulator c-Src.

    Naarmann IS, Harnisch C, Flach N, Kremmer E, Kühn H, Ostareck DH and Ostareck-Lederer A

    Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Halle (Saale), Germany.

    Erythroid precursor cells undergo nuclear extrusion and degradation of mitochondria when they mature to erythrocytes. It has been suggested before that the reticulocyte 15-lipoxygenase (r15-LOX) plays an important role in initiating the breakdown of mitochondria in rabbit reticulocytes. The expression of rabbit r15-LOX is regulated by the heterogeneous nuclear ribonucleoproteins (hnRNP) K and E1 at the translational level. However, this mechanism has never been confirmed in human erythropoiesis. Based on K562 cells we have set up an inducible human erythroid cell system. We show that, during induction, K562 cells exhibit changes in morphology and protein expression that are characteristic for terminal erythroid maturation: nuclear exclusion, expression of endogenous human r15-LOX regulated by hnRNP K and hnRNP E1, and loss of mitochondria. Importantly, induction of terminal erythroid maturation in primary human CD34(+) cells recapitulated the results obtained in K562 cells. Employing the physiologically relevant K562 cell system we uncovered a new mechanism of interdependent post-transcriptional regulation of gene expression. The timely expression of the tyrosine kinase c-Src, which phosphorylates hnRNP K in later stages, is controlled by hnRNP K in early stages of erythroid maturation. hnRNP K binds to the 3'-untranslated region of the c-Src mRNA and inhibits its translation by blocking 80 S ribosome formation. In premature erythroid cells, small interfering RNA-mediated knockdown of hnRNP K, but not of hnRNP E1, leads to the de-repression of c-Src synthesis.

    The Journal of biological chemistry 2008;283;26;18461-72

  • Phosphorylation regulates tau interactions with Src homology 3 domains of phosphatidylinositol 3-kinase, phospholipase Cgamma1, Grb2, and Src family kinases.

    Reynolds CH, Garwood CJ, Wray S, Price C, Kellie S, Perera T, Zvelebil M, Yang A, Sheppard PW, Varndell IM, Hanger DP and Anderton BH

    The MRC Centre for Neurodegeneration Research, King's College London, Institute of Psychiatry, London, UK. h.reynolds@iop.kcl.ac.uk

    The microtubule-associated protein tau can associate with various other proteins in addition to tubulin, including the SH3 domains of Src family tyrosine kinases. Tau is well known to aggregate to form hyperphosphorylated filamentous deposits in several neurodegenerative diseases (tauopathies) including Alzheimer disease. We now report that tau can bind to SH3 domains derived from the p85alpha subunit of phosphatidylinositol 3-kinase, phospholipase Cgamma1, and the N-terminal (but not the C-terminal) SH3 of Grb2 as well as to the kinases Fyn, cSrc, and Fgr. However, the short inserts found in neuron-specific isoforms of Src prevented the binding of tau. The experimentally determined binding of tau peptides is well accounted for when modeled into the peptide binding cleft in the SH3 domain of Fyn. After phosphorylation in vitro or in transfected cells, tau showed reduced binding to SH3 domains; no binding was detected with hyperphosphorylated tau isolated from Alzheimer brain, but SH3 binding was restored by phosphatase treatment. Tau mutants with serines and threonines replaced by glutamate, to mimic phosphorylation, showed reduced SH3 binding. These results strongly suggest that tau has a potential role in cell signaling in addition to its accepted role in cytoskeletal assembly, with regulation by phosphorylation that may be disrupted in the tauopathies including Alzheimer disease.

    Funded by: Medical Research Council: G0300408

    The Journal of biological chemistry 2008;283;26;18177-86

  • Control of excitatory synaptic transmission by C-terminal Src kinase.

    Xu J, Weerapura M, Ali MK, Jackson MF, Li H, Lei G, Xue S, Kwan CL, Manolson MF, Yang K, Macdonald JF and Yu XM

    Faculty of Dentistry, University of Toronto, Toronto, Ontario M5G 1G6, Canada.

    The induction of long-term potentiation at CA3-CA1 synapses is caused by an N-methyl-d-aspartate (NMDA) receptordependent accumulation of intracellular Ca(2+), followed by Src family kinase activation and a positive feedback enhancement of NMDA receptors (NMDARs). Nevertheless, the amplitude of baseline transmission remains remarkably constant even though low frequency stimulation is also associated with an NMDAR-dependent influx of Ca(2+) into dendritic spines. We show here that an interaction between C-terminal Src kinase (Csk) and NMDARs controls the Src-dependent regulation of NMDAR activity. Csk associates with the NMDAR signaling complex in the adult brain, inhibiting the Src-dependent potentiation of NMDARs in CA1 neurons and attenuating the Src-dependent induction of long-term potentiation. Csk associates directly with Src-phosphorylated NR2 subunits in vitro. An inhibitory antibody for Csk disrupts this physical association, potentiates NMDAR mediated excitatory postsynaptic currents, and induces long-term potentiation at CA3-CA1 synapses. Thus, Csk serves to maintain the constancy of baseline excitatory synaptic transmission by inhibiting Src kinase-dependent synaptic plasticity in the hippocampus.

    Funded by: NINDS NIH HHS: 5R01 NS053567-01

    The Journal of biological chemistry 2008;283;25;17503-14

  • Role of Src-family kinases in formation of the cortical actin cap at the dorsal cell surface.

    Kuga T, Hoshino M, Nakayama Y, Kasahara K, Ikeda K, Obata Y, Takahashi A, Higashiyama Y, Fukumoto Y and Yamaguchi N

    Department of Molecular Cell Biology, Graduate School of Pharmaceutical Sciences, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba 260-8675, Japan.

    Protein-tyrosine phosphorylation is regulated by protein-tyrosine kinases and protein-tyrosine phosphatases (PTPs). Src-family tyrosine kinases (SFKs) participate in the regulation of the actin cytoskeleton. Actin filaments can be accumulated in a cap at the dorsal cell surface, which is called the cortical actin cap. Here, we show that SFKs play an important role in formation of the cortical actin cap. HeLa cells normally exhibit the cortical actin cap, one of the major sites of tyrosine phosphorylation. The cortical actin cap is disrupted by SFK inhibitors or overexpression of the Lyn SH3 domain. Csk-knockout cells form the cortical actin cap when the level of tyrosine phosphorylation is increased by Na(3)VO(4), a PTP inhibitor, and the formation of the cortical actin cap is inhibited by SFK inactivation with re-introduction of Csk. SYF cells lacking SFKs minimally exhibit the cortical actin cap even in the presence of Na(3)VO(4), and transfection with Lyn restores the cortical actin cap in the presence of Na(3)VO(4). Disruption of the cortical actin cap by dominant-negative Cdc42 causes loss of tyrosine phosphorylation at the cell top. These results suggest that SFK(s) is involved in formation of the cortical actin cap, which may serve as a platform of tyrosine phosphorylation signaling.

    Experimental cell research 2008;314;10;2040-54

  • Src kinase potentiates androgen receptor transactivation function and invasion of androgen-independent prostate cancer C4-2 cells.

    Asim M, Siddiqui IA, Hafeez BB, Baniahmad A and Mukhtar H

    Department of Dermatology, University of Wisconsin, Madison, WI 53706, USA.

    Prostate cancer is one of the most prominent malignancies of elderly men in many Western countries including Europe and the United States with increasing trend worldwide. The growth of normal prostate as well as of prostate carcinoma cells depends on functional androgen receptor (AR) signaling. AR manifests the biological actions of androgens and its transcriptional activity is known to be influenced by signal transduction pathways. Here we show that Src, a nonreceptor tyrosine kinase, is overexpressed in androgen-independent prostate carcinoma C4-2 cells. Interestingly, the expression of Src was found to progressively increase (up to threefold) in transgenic adenocarcinoma of mouse prostate mice as a function of age and cancer progression. Blocking Src kinase function by a specific inhibitor, PP2, resulted in decreased AR transactivation function on two different reporters, mouse mammary tumor virus (MMTV) and prostate-specific antigen (PSA). Consistent with this, overexpression of a functional Src mutant also led to a dramatic decrease in AR transactivation potential in a hormone-dependent manner. Interference with Src function in C4-2 cells led to decreased recruitment of AR on the target gene PSA enhancer and also resulted in the abrogation of hormone-dependent PSA transcript induction. Src inhibition also led to a dramatic decrease in the cell invasion in addition to decreasing the cellular growth. We suggest that targeting Src kinase could be an effective strategy to inhibit prostate cancer growth and metastasis.

    Funded by: NCI NIH HHS: R01CA101039, R01CA120451, R01CA78809; NIDDK NIH HHS: P50DK065303-01

    Oncogene 2008;27;25;3596-604

  • The small peptide OGP(10-14) acts through Src kinases and RhoA pathways in Mo-7e cells: morphologic and immunologic evaluation.

    Mattii L, Battolla B, Moscato S, Fazzi R, Galimberti S, Bernardini N, Dolfi A and Petrini M

    Department of Human Morphology and Applied Biology, Section of Histology and General Embryology, University of Pisa, Via Roma 67, Pisa, Italy.

    Background: Osteogenic growth peptide (OGP) is an endogenous tetradecapeptide present in micromolar concentrations in mammalian serum; its carboxy-terminal pentapeptide, OGP(10-14), represents its physiologically active fragment. OGP(10-14) induces proliferation and differentiation in fibroblast and osteoblast cell lines, and it enhances hematopoiesis in vitro and in vivo. The signaling pathways triggered by OGP(10-14) are not yet fully known. In the present report, we evaluated the effect of OGP(10-14) on differentiation of a cancer megakaryoblast cell line and its involvement on RhoA and Src family kinases signaling pathway.

    Cell proliferation of the Mo-7e line was evaluated using the MTT test. Mo-7e differentiation was evaluated by microscopic observation of cell morphology and by expression of the factor VIII-related antigen. Involvement of RhoA and Src kinases on signaling pathways triggered by OGP(10-14) was analyzed using RhoA and Src family kinase (SFK) inhibitors (C3 and PP2) and an immunoperoxidase technique.

    Results: OGP(10-14) induces expression of the factor VIII-related antigen, morphologic changes indicative of megakaryocytic differentiation, and a down-regulation of the Fyn Src kinase. These OGP(10-14) effects were prevented by C3 and enhanced by PP2.

    Conclusions: The anti-proliferative and pro-differentiating activities of OGP(10-14) on thrombopoietin (TPO)-primed Mo-7e cells are mediated by RhoA and Src kinase pathways as demonstrated by the use of C3 and PP2.

    Medical science monitor : international medical journal of experimental and clinical research 2008;14;6;BR103-108

  • alpha-Catenin overrides Src-dependent activation of beta-catenin oncogenic signaling.

    Inge LJ, Rajasekaran SA, Wolle D, Barwe SP, Ryazantsev S, Ewing CM, Isaacs WB and Rajasekaran AK

    Nemours Center for Childhood Cancer Research, Alfred I. DuPont Hospital for Children, 1701 Rockland Road, Wilmington, DE 19803, USA.

    Loss of alpha-catenin is one of the characteristics of prostate cancer. The catenins (alpha and beta) associated with E-cadherin play a critical role in the regulation of cell-cell adhesion. Tyrosine phosphorylation of beta-catenin dissociates it from E-cadherin and facilitates its entry into the nucleus, where beta-catenin acts as a transcriptional activator inducing genes involved in cell proliferation. Thus, beta-catenin regulates cell-cell adhesion and cell proliferation. Mechanisms controlling the balance between these functions of beta-catenin invariably are altered in cancer. Although a wealth of information is available about beta-catenin deregulation during oncogenesis, much less is known about how or whether alpha-catenin regulates beta-catenin functions. In this study, we show that alpha-catenin acts as a switch regulating the cell-cell adhesion and proliferation functions of beta-catenin. In alpha-catenin-null prostate cancer cells, reexpression of alpha-catenin increased cell-cell adhesion and decreased beta-catenin transcriptional activity, cyclin D1 levels, and cell proliferation. Further, Src-mediated tyrosine phosphorylation of beta-catenin is a major mechanism for decreased beta-catenin interaction with E-cadherin in alpha-catenin-null cells. alpha-Catenin attenuated the effect of Src phosphorylation by increasing beta-catenin association with E-cadherin. We also show that alpha-catenin increases the sensitivity of prostate cancer cells to a Src inhibitor in suppressing cell proliferation. This study reveals for the first time that alpha-catenin is a key regulator of beta-catenin transcriptional activity and that the status of alpha-catenin expression in tumor tissues might have prognostic value for Src targeted therapy.

    Funded by: NIDDK NIH HHS: DK 56216, R01 DK056216, R01 DK056216-06, R01 DK056216-07, R01 DK056216-08; NIGMS NIH HHS: F31 GM068985, F31-GM068985

    Molecular cancer therapeutics 2008;7;6;1386-97

  • Two modes of ERK activation by TNF in keratinocytes: different cellular outcomes and bi-directional modulation by vitamin D.

    Ziv E, Rotem C, Miodovnik M, Ravid A and Koren R

    The Basil and Gerald Felsenstein Medical Research Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

    Inflammation, elicited in the skin following tissue damage or pathogen invasion, may become chronic with deleterious consequences. Tumor necrosis factor (TNF) is a key mediator of cutaneous inflammation and the keratinocyte an important protagonist of skin immunity. Calcitriol, the hormonally active vitamin D metabolite, and its analogs attenuate epidermal inflammation and inhibit the hyperproliferation of keratinocytes associated with the inflammatory disorder, psoriasis. Since activation of extracellular signal-regulated kinase (ERK) promotes keratinocyte proliferation and mediates epidermal inflammation, we studied the effect of calcitriol on ERK activation in HaCaT keratinocytes exposed to the ubiquitous inflammatory cytokine TNF. By using the EGF receptor (EGFR) tyrosine kinase inhibitor, AG1487 and the Src family inhibitor, PP-1, we established that TNF activated ERK in an EGFR and Src dependent and an EGFR and Src independent modes. EGFR dependent activation resulted in the upregulation of the transcription factor, c-Fos, while the EGFR independent activation mode was of a shorter duration, did not affect c-Fos expression but induced IL-8 mRNA expression. Pretreatment with calcitriol, enhanced TNF-induced EGFR-Src dependent ERK activation and tyrosine phosphorylation of the EGFR, but abolished the EGFR-Src independent ERK activation. These effects were mirrored by enhancement of c-Fos and inhibition of IL-8 induction by TNF. Treatment with calcitriol increased the rate of the de-phosphorylation of activated ERK, accounting for the inhibition of EGFR-Src independent ERK activation by TNF. It is possible that effects on the ERK cascade contribute to the effects of calcitriol and its synthetic analogs on cutaneous inflammation and keratinocyte proliferation.

    Journal of cellular biochemistry 2008;104;2;606-19

  • Transforming growth factor beta1 induces alphavbeta3 integrin expression in human lung fibroblasts via a beta3 integrin-, c-Src-, and p38 MAPK-dependent pathway.

    Pechkovsky DV, Scaffidi AK, Hackett TL, Ballard J, Shaheen F, Thompson PJ, Thannickal VJ and Knight DA

    University of British Columbia, Vancouver, British Columbia V6Z1Y6, Canada.

    In response to transforming growth factor beta1 (TGFbeta) stimulation, fibroblasts modify their integrin repertoire and adhesive capabilities to certain extracellular matrix proteins. Although TGFbeta has been shown to increase the expression of specific alphav integrins, the mechanisms underlying this are unknown. In this study we demonstrate that TGFbeta1 increased both beta3 integrin subunit mRNA and protein levels as well as surface expression of alphavbeta3 in human lung fibroblasts. TGFbeta1-induced alphavbeta3 expression was strongly adhesion-dependent and associated with increased focal adhesion kinase and c-Src kinase phosphorylation. Inhibition of beta3 integrin activation by the Arg-Gly-Asp tripeptide motif-specific disintegrin echistatin or alphavbeta3 blocking antibody prevented the increase in beta3 but not beta5 integrin expression. In addition, echistatin inhibited TGFbeta1-induced p38 MAPK but not Smad3 activation. Furthermore, inhibition of the Src family kinases, but not focal adhesion kinase, completely abrogated TGFbeta1-induced expression of alphavbeta3 and p38 MAPK phosphorylation but not beta5 integrin expression and Smad3 activation. The TGFbeta1-induced alphavbeta3 expression was blocked by pharmacologic and genetic inhibition of p38 MAPK- but not Smad2/3-, Sp1-, ERK-, phosphatidylinositol 3-kinase, and NF-kappaB-dependent pathways. Our results demonstrate that TGFbeta1 induces alphavbeta3 integrin expression via a beta3 integrin-, c-Src-, and p38 MAPK-dependent pathway. These data identify a novel mechanism for TGFbeta1 signaling in human lung fibroblasts by which they may contribute to normal and pathological wound healing.

    The Journal of biological chemistry 2008;283;19;12898-908

  • Endosomal NADPH oxidase regulates c-Src activation following hypoxia/reoxygenation injury.

    Li Q, Zhang Y, Marden JJ, Banfi B and Engelhardt JF

    Department of Anatomy and Cell Biology and the Center for Gene Therapy, The College of Medicine, University of Iowa, Iowa City, IA 52242, USA.

    c-Src has been shown to activate NF-kappaB (nuclear factor kappaB) following H/R (hypoxia/reoxygenation) by acting as a redox-dependent IkappaBalpha (inhibitory kappaB) tyrosine kinase. In the present study, we have investigated the redox-dependent mechanism of c-Src activation following H/R injury and found that ROS (reactive oxygen species) generated by endosomal Noxs (NADPH oxidases) are critical for this process. Endocytosis following H/R was required for the activation of endosomal Noxs, c-Src activation, and the ability of c-Src to tyrosine-phosphorylate IkappaBalpha. Quenching intra-endosomal ROS during reoxygenation inhibited c-Src activation without affecting c-Src recruitment from the plasma membrane to endosomes. However, siRNA (small interfering RNA)-mediated knockdown of Rac1 prevented c-Src recruitment into the endosomal compartment following H/R. Given that Rac1 is a known activator of Nox1 and Nox2, we investigated whether these two proteins were required for c-Src activation in Nox-deficient primary fibroblasts. Findings from these studies suggest that both Nox1 and Nox2 participate in the initial redox activation of c-Src following H/R. In summary, our results suggest that Rac1-dependent Noxs play a critical role in activating c-Src following H/R injury. This signalling pathway may be a useful therapeutic target for ischaemia/reperfusion-related diseases.

    Funded by: NIDDK NIH HHS: DK067928, DK51315, P30 DK054759, P30 DK54759, R01 DK051315, R01 DK067928

    The Biochemical journal 2008;411;3;531-41

  • Epithelial to mesenchymal transition in head and neck squamous carcinoma: association of Src activation with E-cadherin down-regulation, vimentin expression, and aggressive tumor features.

    Mandal M, Myers JN, Lippman SM, Johnson FM, Williams MD, Rayala S, Ohshiro K, Rosenthal DI, Weber RS, Gallick GE and El-Naggar AK

    Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.

    Background: Epithelial-mesenchymal transformations (EMT) are critical for the invasion, progression, and metastasis of epithelial carcinogenesis. The role of EMT in head and neck squamous carcinoma (HNSC) tumorigenesis remains unexplored. In the current study, the expressions of several factors associated with the induction of EMT in HNSC cell lines and tumor specimens were investigated to define their functional and pathologic role in HNSC.

    Methods: Eleven HNSC cell lines and 50 primary tumor tissue specimens formed the materials of this study. Western blot analysis as well as immunohistochemical, and functional techniques were used to assess the status of activated Src (p-Src), E-cadherin, and vimentin in both cell lines and tumor tissues and the results were correlated with patients' clinicopathologic parameters.

    Results: The results demonstrated the inverse expression of p-Src and E-cadherin in the majority of cell lines and in primary tumor tissues compared with normal squamous mucosa. Elevated levels of p-Src were accompanied by down-regulation of E-cadherin and the expression of vimentin in epithelial tumor cells. In vitro inhibition of Src led to E-cadherin reexpression and increased cell contact in squamous carcinoma cell lines. Immunophenotypic analysis of these markers in primary tumor tissues demonstrated a significant correlation between increased p-Src, decreased E-cadherin, and vimentin expression and aggressive tumor features including penetrating invasive fronts, high-grade sarcomatoid transformation, and lymph node metastasis.

    Conclusions: The results of the current study indicate that Src and E-cadherin may play an important role in EMT, invasion, and aggressive clinicopathologic features of HNSC. These proteins may be targeted for the therapeutic intervention of patients with HNSC.

    Funded by: NCI NIH HHS: CA 16672

    Cancer 2008;112;9;2088-100

  • Met and c-Src cooperate to compensate for loss of epidermal growth factor receptor kinase activity in breast cancer cells.

    Mueller KL, Hunter LA, Ethier SP and Boerner JL

    Karmanos Cancer Institute, Wayne State University, Detroit, MI 48201, USA.

    Breast cancers are not responsive to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), although 30% of breast cancers overexpress EGFR. The mechanism of intrinsic resistance to EGFR TKIs in breast cancer is the focus of current studies. Here, we observed that EGFR remains tyrosine phosphorylated in breast cancer cells that proliferate in the presence of EGFR TKIs. In one such cell line, SUM229, inhibiting c-Src kinase activity with either a dominant-negative c-Src or a c-Src TKI decreased EGFR phosphorylation on Tyr(845), Tyr(992), and Tyr(1086) in the presence of EGFR TKIs. Conversely, overexpressing wild-type (wt) c-Src in the EGFR TKI-sensitive breast cancer cell line SUM149 increased EGFR kinase-independent EGFR tyrosine phosphorylation. In addition, in the presence of EGFR TKIs, inhibiting c-Src kinase activity decreased cell growth in SUM229 cells, and overexpressing wt-c-Src increased cell growth in SUM149 cells. We identified the receptor tyrosine kinase Met to be responsible for activating c-Src in SUM229 cells. Inhibiting Met kinase activity with a small molecule inhibitor decreased c-Src phosphorylation and kinase activation. In addition, inhibiting Met kinase activity in SUM229 cells decreased EGFR tyrosine phosphorylation and growth in the presence of EGFR TKIs. Stimulating Met kinase activity in SUM149 cells with hepatocyte growth factor increased EGFR tyrosine phosphorylation and cell growth in the presence of EGFR TKIs. These data suggest a Met/c-Src-mediated signaling pathway as a mediator of EGFR tyrosine phosphorylation and cell growth in the presence of EGFR TKIs.

    Funded by: NCI NIH HHS: P30 CA022453

    Cancer research 2008;68;9;3314-22

  • Cell adhesion-dependent cofilin serine 3 phosphorylation by the integrin-linked kinase.c-Src complex.

    Kim YB, Choi S, Choi MC, Oh MA, Lee SA, Cho M, Mizuno K, Kim SH and Lee JW

    Department of Tumor Biology, Cancer Research Institute, Cell Dynamics Research Center, College of Medicine, Seoul National University, Seoul 110-799, Korea.

    Integrin-linked kinase (ILK) is involved in signal transduction by integrin-mediated cell adhesion that leads to dynamic actin reorganization. Actin (de)polymerization is regulated by cofilin, the Ser(3) phosphorylation (pS(3)cofilin) of which inhibits its actin-severing activity. To determine how ILK regulates pS(3)cofilin, we examined the effects of ILK on pS(3)cofilin using normal RIE1 cells. Compared with suspended cells, fibronectin-adherent cells showed enhanced pS(3)cofilin, depending on ILK expression and c-Src activity. The ILK-mediated pS(3)cofilin in RIE1 cells did not involve Rho-associated kinase, LIM kinase, or testicular protein kinases, which are known to be upstream of cofilin. The kinase domain of ILK, including proline-rich regions, appeared to interact physically with the Src homology 3 domain of c-Src. In vitro kinase assay revealed that ILK immunoprecipitates phosphorylated the recombinant glutathione S-transferase-cofilin, which was abolished by c-Src inhibition. Interestingly, epidermal growth factor treatment abolished the ILK effects, indicating that the linkage from ILK to cofilin is biologically responsive to extracellular cues. Altogether, this study provides evidence for a new signaling connection from ILK to cofilin for dynamic actin polymerization during cell adhesion, depending on the activity of ILK-associated c-Src.

    The Journal of biological chemistry 2008;283;15;10089-96

  • B1 integrin/Fak/Src signaling in intestinal epithelial crypt cell survival: integration of complex regulatory mechanisms.

    Bouchard V, Harnois C, Demers MJ, Thibodeau S, Laquerre V, Gauthier R, Vézina A, Noël D, Fujita N, Tsuruo T, Arguin M and Vachon PH

    Département d'Anatomie et de Biologie Cellulaire, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, QC, Canada.

    The molecular determinants which dictate survival and apoptosis/anoikis in human intestinal crypt cells remain to be fully understood. To this effect, the roles of beta1 integrin/Fak/Src signaling to the PI3-K/Akt-1, MEK/Erk, and p38 pathways, were investigated. The regulation of six Bcl-2 homologs (Bcl-2, Mcl-1, Bcl-X(L), Bax, Bak, Bad) was likewise analyzed. We report that: (1) Anoikis causes a down-activation of Fak, Src, Akt-1 and Erk1/2, a loss of Fak-Src association, and a sustained/enhanced activation of p38beta, which is required as apoptosis/anoikis driver; (2) PI3-K/Akt-1 up-regulates the expression of Bcl-X(L) and Mcl-1, down-regulates Bax and Bak, drives Bad phosphorylation (both serine112/136 residues) and antagonizes p38beta activation; (3) MEK/Erk up-regulates Bcl-2, drives Bad phosphorylation (serine112 residue), but does not antagonize p38bactivation; (4) PI3-K/Akt-1 is required for survival, whereas MEK/Erk is not; (5) Src acts as a cornerstone in the engagement of both pathways by beta1 integrins/Fak, and is crucial for survival; and (6) beta1 integrins/Fak and/or Src regulate Bcl-2 homologs as both PI3-K/Atk-1 and MEK/Erk combined. Hence, beta1 integrin/Fak/Src signaling translates into integrated mediating functions of p38beta activation and regulation of Bcl-2 homologs by PI3-K/Akt-1 and MEK/Erk, consequently determining their requirement (or not) for survival.

    Apoptosis : an international journal on programmed cell death 2008;13;4;531-42

  • MUC1 initiates Src-CrkL-Rac1/Cdc42-mediated actin cytoskeletal protrusive motility after ligating intercellular adhesion molecule-1.

    Shen Q, Rahn JJ, Zhang J, Gunasekera N, Sun X, Shaw AR, Hendzel MJ, Hoffman P, Bernier A and Hugh JC

    Department of Laboratory Medicine and Pathology, 5B4.21 Mackenzie Health Sciences Centre, University of Alberta Hospital, Edmonton, AB, Canada T6G 2R7.

    MUC1, a transmembrane glycoprotein of the mucin family, when aberrantly expressed on breast cancer cells is correlated with increased lymph node metastases. We have previously shown that MUC1 binds intercellular adhesion molecule-1 (ICAM-1) on surrounding accessory cells and facilitates transendothelial migration of MUC1-bearing cells. Nevertheless, the underlying molecular mechanism is still obscure. In the present study, we used a novel assay of actin cytoskeletal reorganization to show that by ligating ICAM-1, MUC1 triggers Rac1- and Cdc42-dependent actin cytoskeletal protrusive activity preferentially at the heterotypic cell-cell contact sites. Further, we show that these MUC1/ICAM-1 interaction-initiated lamellipodial and filopodial protrusions require Src family kinase and CT10 regulator of kinase like (CrkL) accompanied by the rapid formation of a Src-CrkL signaling complex at the MUC1 cytoplasmic domain. Through inhibition of Src kinase activity, we further revealed that Src is required for recruiting CrkL to the MUC1 cytoplasmic domain as well as mediating the observed actin cytoskeleton dynamics. These findings suggest a novel MUC1-Src-CrkL-Rac1/Cdc42 signaling cascade following ICAM-1 ligation, through which MUC1 regulates cytoskeletal reorganization and directed cell motility during cell migration.

    Molecular cancer research : MCR 2008;6;4;555-67

  • Src kinase regulates metalloproteinase-9 secretion induced by type IV collagen in MCF-7 human breast cancer cells.

    Cortes-Reynosa P, Robledo T, Macias-Silva M, Wu SV and Salazar EP

    Departamento de Biologia Celular. Cinvestav-IPN, Mexico, DF. 07360, Mexico.

    Matrix metalloproteinases (MMPs) are a family of endopeptidases that collectively are capable to degrading all components of the extracellular matrix (ECM) and they have been implicated in several aspects of tumor progression, such as invasion through basement membrane (BM) and insterstitial matrices, angiogenesis and tumor cell growth. In particular, MMP-2 and MMP-9 have been associated with the ability of tumor cells to metastasize due to their capacity to degrade type IV collagen (Col-IV), the main component of BM, and to their elevated expression in malignant tumors. However, nothing is known about the regulation of MMP-9 secretion and expression in breast cancer cells stimulated with Col-IV. Our results demonstrate that stimulation of MCF-7 cells with Col-IV promoted the secretion of MMP-9, as revealed by gelatin zymography and Western blotting using specific antibodies that recognized MMP-9. In addition, inhibition of Src and FAK kinase activity prevented MMP-9 secretion. In contrast, MMP-9 expression was not up-regulated by treatment with Col-IV. These results demonstrate that Col-IV regulates the secretion of MMP-9 via a Src and FAK dependent pathway in MCF-7 cells.

    Matrix biology : journal of the International Society for Matrix Biology 2008;27;3;220-31

  • Decreased CHK protein levels are associated with Src activation in colon cancer cells.

    Zhu S, Bjorge JD, Cheng HC and Fujita DJ

    Department of Biochemistry and Molecular Biology, and Southern Alberta Cancer Research Institute, University of Calgary, Calgary, Alberta, Canada.

    Src activation has been associated with colon cancers but the mechanism underlying Src activation is largely unknown. Csk-homologous kinase (CHK) can inhibit the kinase activity of certain Src kinase family members in vitro by phosphorylating the C-terminal tyrosine and by a non-catalytic mechanism. CHK was previously reported to be expressed primarily in brain and hematopoietic cells. We report herein that CHK is also expressed in normal colon cell lines. Furthermore, CHK protein levels are significantly decreased in various colon cancer cell lines and the decrease correlates with the increased specific activity of Src in these cell lines, while the level of the other Src inhibitory kinase, C-terminal Src kinase, is not significantly changed. CHK is also expressed in normal colon tissues but its expression level is decreased in colon cancer tissues collected from the same patients. Immunofluorescence microscopy shows that CHK colocalizes with Src in normal colon FHC cells. Overexpression of CHK in colon cancer cells results in inactivation of Src without phosphorylating Y530 at its C-terminus. In addition, CHK suppresses anchorage-independent cell growth and cell invasion of colon cancer cells. These results reveal a potentially important role for CHK in Src activation and tumorigenicity in colon cancer cells.

    Oncogene 2008;27;14;2027-34

  • Expression of tetraspan protein CD63 activates protein-tyrosine kinase (PTK) and enhances the PTK-induced inhibition of ROMK channels.

    Lin D, Kamsteeg EJ, Zhang Y, Jin Y, Sterling H, Yue P, Roos M, Duffield A, Spencer J, Caplan M and Wang WH

    Department of Pharmacology, New York Medical College, Valhalla, New York 10595, USA.

    In the present study, we tested the role of CD63 in regulating ROMK1 channels by protein-tyrosine kinase (PTK). Immunocytochemical staining shows that CD63 and receptor-linked tyrosine phosphatase alpha (RPTPalpha) are expressed in the cortical collecting duct and outer medulla collecting duct. Immunoprecipitation of tissue lysates from renal cortex and outer medulla or 293T cells transfected with CD63 reveals that CD63 was associated with RPTPalpha both in situ and in transfected cells. Expression of CD63 in 293T cells stimulated the phosphorylation of tyrosine residue 416 of c-Src but decreased the phosphorylation of tyrosine residue 527, indicating that expression of CD63 stimulates the activity of c-Src. Furthermore, c-Src was coimmunoprecipitated with RPTPalpha and CD63 both in 293T cells transfected with CD63 and in lysates prepared from native rat kidney. Potassium restriction had no effect on the expression of RPTPalpha, but it increased the association between c-Src and RPTPalpha in the renal cortex and outer medulla. We also used two-electrode voltage clamp to study the effect of CD63 on ROMK channels in Xenopus oocytes. Expression of CD63 had no significant effect on potassium currents in oocytes injected with ROMK1; however, it significantly enhanced the c-Src-induced inhibition of ROMK channels in oocytes injected with ROMK1+c-Src. The effect of CD63 on the c-Src-induced inhibition was not due to a decreased expression of ROMK1 channels, because blocking PTK with herbimycin A abolished the inhibitory effect of c-Src on ROMK channels in oocytes injected with ROMK1+c-Src+CD63. Furthermore, coexpression of CD63 enhanced tyrosine phosphorylation of ROMK1. We conclude that CD63 plays a role in the regulation of ROMK channels through its association with RPTPalpha, which in turn interacts with and activates Src family PTK, thus reducing ROMK activity.

    Funded by: NHLBI NIH HHS: P01 HL034300; NIDDK NIH HHS: DK17433, DK54983, R01 DK054983

    The Journal of biological chemistry 2008;283;12;7674-81

  • A novel role of vascular endothelial cadherin in modulating c-Src activation and downstream signaling of vascular endothelial growth factor.

    Ha CH, Bennett AM and Jin ZG

    Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, Rochester, New York 14586, USA.

    Vascular endothelial growth factor (VEGF) is a potent mediator of angiogenesis and vascular permeability, in which c-Src tyrosine kinase plays an essential role. However, the mechanisms by which VEGF stimulates c-Src activation have remained unclear. Here, we demonstrate that vascular endothelial cadherin (VE-cadherin) plays a critical role in regulating c-Src activation in response to VEGF. In vascular endothelial cells, VE-cadherin was basally associated with c-Src and Csk (C-terminal Src kinase), a negative regulator of Src activation. VEGF stimulated Csk release from VE-cadherin by recruiting the protein tyrosine phosphatase SHP2 to VE-cadherin signaling complex, leading to an increase in c-Src activation. Silencing VE-cadherin with small interference RNA significantly reduced VEGF-stimulated c-Src activation. Disrupting the association of VE-cadherin and Csk through the reconstitution of Csk binding-defective mutant of VE-cadherin also diminished Src activation. Moreover, inhibiting SHP2 by small interference RNA and adenovirus-mediated expression of a catalytically inactive mutant of SHP2 attenuated c-Src activation by blocking the disassociation of Csk from VE-cadherin. Furthermore, VE-cadherin and SHP2 differentially regulates VEGF downstream signaling. The inhibition of c-Src, VE-cadherin, and SHP2 diminished VEGF-mediated activation of Akt and endothelial nitric-oxide synthase. In contrast, inhibiting VE-cadherin and SHP2 enhanced ERK1/2 activation in response to VEGF. These findings reveal a novel role for VE-cadherin in modulating c-Src activation in VEGF signaling, thus providing new insights into the importance of VE-cadherin in VEGF signaling and vascular function.

    Funded by: NHLBI NIH HHS: R01 HL080611, R01 HL080611-05; NIAMS NIH HHS: R01 AR046524, R01 AR046524-01

    The Journal of biological chemistry 2008;283;11;7261-70

  • Activation of Src-family tyrosine kinases in hyperproliferative epidermal disorders.

    Ayli EE, Li W, Brown TT, Witkiewicz A, Elenitsas R and Seykora JT

    Department of Dermatology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

    Background: Src-family tyrosine kinases (SFKs) are important regulators of keratinocyte growth and differentiation. In a broad range of cell types, persistent activation of SFKs correlates with increased cell proliferation. In this study, we determined if SFK activity is increased in cutaneous neoplasia and psoriasis, common hyperproliferative epidermal disorders.

    Methods: Formalin-fixed tissue sections of unremarkable epidermis, psoriasis, actinic keratoses (AKs), squamous cell carcinoma in situ (SCIS) and squamous cell carcinoma (SCC) were subjected to immunohistochemical staining for activated SFKs.

    Results: All psoriasis specimens displayed significantly greater staining for activated SFKs than sections of unremarkable skin. In the psoriasis biopsies, the degree of epidermal hyperplasia was proportional to the level of activated SFK staining. All AKs, SCISs and SCCs exhibited more prominent staining than sections of unremarkable epidermis. No discernable difference in activated SFK staining was seen between AKs, SCIS and SCC specimens.

    Conclusions: This study shows increased staining of activated SFKs in human biopsy specimens of psoriasis and cutaneous neoplasia. These data provide direct evidence for increased activation of SFKs in the pathogenesis of hyperproliferative epidermal disorders.

    Funded by: NIAMS NIH HHS: K08 AR047597, R01 AR051380, R01 AR051380-02, R01 AR051380-03

    Journal of cutaneous pathology 2008;35;3;273-7

  • Activation of SRC kinase and phosphorylation of signal transducer and activator of transcription-5 are required for decidual transformation of human endometrial stromal cells.

    Nagashima T, Maruyama T, Uchida H, Kajitani T, Arase T, Ono M, Oda H, Kagami M, Masuda H, Nishikawa S, Asada H and Yoshimura Y

    Department of Obstetrics and Gynecology, Keio University School of Medicine, Shinanomachi, Shinjyuku-ku, Tokyo 160-8582, Japan.

    Progesterone induces decidual transformation of estrogen-primed human endometrial stromal cells (hESCs), critical for implantation and maintenance of pregnancy, through activation of many signaling pathways involving protein kinase A and signal transducer and activator of transcription (STAT)-5. We have previously shown that kinase activation of v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (SRC) kinase is closely associated with decidualization and that SRC is indispensable for maximal decidualization in mice. To address whether SRC kinase activity is essential for decidualization in humans, hESCs were infected with adenoviruses carrying enhanced green fluorescent protein alone (Ad-EGFP), a kinase-inactive dominant-negative mutant (Ad-SRC/K295R), or an inactive autophosphorylation site mutant (Ad-SRC/Y416F). The cells were cultured in the presence of estradiol and progesterone (EP) to induce decidualization and subjected to RT-PCR, immunoblot, and ELISA analyses. Ad-EGFP-infected hESCs exhibited decidual transformation and up-regulation of decidualization markers including IGF binding protein 1 and prolactin in response to 12-d treatment with EP. In contrast, hESCs infected with Ad-SRC/K295R remained morphologically fibroblastoid without production of IGF binding protein 1 and prolactin even after EP treatment. Ad-SRC/Y416F displayed similar but less inhibitory effects on decidualization, compared with Ad-SRC/K295R. During decidualization, STAT5 was phosphorylated on tyrosine 694, a well-known SRC phosphorylation site. Phosphorylation was markedly attenuated by Ad-SRC/K295R but not Ad-EGFP. These results indicate that the SRC-STAT5 pathway is essential for decidualization of hESCs.

    Endocrinology 2008;149;3;1227-34

  • TAE226-induced apoptosis in breast cancer cells with overexpressed Src or EGFR.

    Golubovskaya VM, Virnig C and Cance WG

    Department of Surgery, University of Florida, Gainesville, Florida, USA.

    Focal adhesion kinase, FAK is a 125 kDa nonreceptor tyrosine kinase that localizes to focal adhesions. FAK is overexpressed in human tumors and regulates cellular adhesion and survival signaling. We have shown previously that the dominant-negative FAK, C-terminal FAK-CD, caused detachment and apoptosis in human breast cancer cells, and that overexpression of an activated form of Src tyrosine kinase or epidermal growth factor receptor, EGFR, suppressed FAK-CD induced apoptotic effects in breast cancer cells. In the present study, we studied the effect of a novel FAK inhibitor, TAE226 (Novartis, Inc.), on the breast cancer cell lines. We used stable breast cancer cell lines overexpressing Src (MCF-7-Src and BT474-Src) or overexpressing EGFR (BT474-EGFR), and control breast cancer cell lines for the treatment with different doses of TAE226 drug. The detachment and apoptosis caused by TAE226 was analyzed and compared with the effect of the dominant-negative adenoviral FAK-CD. The TAE226 drug caused a dose-dependent increase of detachment and apoptosis in both BT474 and MCF-7-Vector and Src cells and in BT474-EGFR and BT474-pcDNA3 cells. Additionally, TAE226 caused downregulation of Y397-FAK, FAK and activation of PARP or caspase-3 proteins. Both Src and EGFR-overexpressing cells were not resistant to the TAE226 treatment compared to FAK-CD treatment. In addition, normal breast MCF-10A cell line was resistant to both TAE226 drug and to the Ad-FAK-CD inhibitor. Thus, inhibition of autophosphorylation activity of FAK with the TAE226 inhibitor at 10-20 microM is effective in causing apoptosis in breast cancer cells, resistant to the Ad-FAK-CD inhibitor that can be used effectively in therapy.

    Funded by: NCI NIH HHS: CA65910

    Molecular carcinogenesis 2008;47;3;222-34

  • Yes and PI3K bind CD95 to signal invasion of glioblastoma.

    Kleber S, Sancho-Martinez I, Wiestler B, Beisel A, Gieffers C, Hill O, Thiemann M, Mueller W, Sykora J, Kuhn A, Schreglmann N, Letellier E, Zuliani C, Klussmann S, Teodorczyk M, Gröne HJ, Ganten TM, Sültmann H, Tüttenberg J, von Deimling A, Regnier-Vigouroux A, Herold-Mende C and Martin-Villalba A

    Molecular Neurobiology Group, German Cancer Research Center (DKFZ), INF 581, 69120 Heidelberg, Germany.

    Invasion of surrounding brain tissue by isolated tumor cells represents one of the main obstacles to a curative therapy of glioblastoma multiforme. Here we unravel a mechanism regulating glioma infiltration. Tumor interaction with the surrounding brain tissue induces CD95 Ligand expression. Binding of CD95 Ligand to CD95 on glioblastoma cells recruits the Src family member Yes and the p85 subunit of phosphatidylinositol 3-kinase to CD95, which signal invasion via the glycogen synthase kinase 3-beta pathway and subsequent expression of matrix metalloproteinases. In a murine syngeneic model of intracranial GBM, neutralization of CD95 activity dramatically reduced the number of invading cells. Our results uncover CD95 as an activator of PI3K and, most importantly, as a crucial trigger of basal invasion of glioblastoma in vivo.

    Cancer cell 2008;13;3;235-48

  • Cdc42 regulates E-cadherin ubiquitination and degradation through an epidermal growth factor receptor to Src-mediated pathway.

    Shen Y, Hirsch DS, Sasiela CA and Wu WJ

    Division of Monoclonal Antibodies, Office of Biotechnology Products, Center for Drug Evaluation and Research, United States Food and Drug Administration, Bethesda, Maryland 20892-4555, USA.

    E-cadherins play an essential role in maintaining epithelial polarity by forming Ca2+-dependent adherens junctions between epithelial cells. Here, we report that Ca2+ depletion induces E-cadherin ubiquitination and lysosomal degradation and that Cdc42 plays an important role in regulating this process. We demonstrate that Ca2+ depletion induces activation of Cdc42. This in turn up-regulates epidermal growth factor receptor (EGFR) signaling to mediate Src activation, leading to E-cadherin ubiquitination and lysosomal degradation. Silencing Cdc42 blocks activation of EGFR and Src induced by Ca2+ depletion, resulting in a reduction in E-cadherin degradation. The role of Cdc42 in regulating E-cadherin ubiquitination and degradation is underscored by the fact that constitutively active Cdc42(F28L) increases the activity of EGFR and Src and significantly enhances E-cadherin ubiquitination and lysosomal degradation. Furthermore, we found that GTP-dependent binding of Cdc42 to E-cadherin is critical for Cdc42 to induce the dissolution of adherens junctions. Our data support a model that activation of Cdc42 contributes to mesenchyme-like phenotype by targeting of E-cadherin for lysosomal degradation.

    The Journal of biological chemistry 2008;283;8;5127-37

  • Epidermal growth factor stimulates RSK2 activation through activation of the MEK/ERK pathway and src-dependent tyrosine phosphorylation of RSK2 at Tyr-529.

    Kang S, Dong S, Guo A, Ruan H, Lonial S, Khoury HJ, Gu TL and Chen J

    Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

    The Ser/Thr kinase ribosomal S6 kinase 2 (RSK2) has been demonstrated to phosphorylate transcription factor CREB (cyclic AMP-responsive-binding protein) and histone H3 in response to mitogenic stimulation by epidermal growth factor (EGF). EGF activates the MEK/ERK pathway to activate RSK2. We recently reported that receptor tyrosine kinase fibroblast growth factor receptor 3 (FGFR3) directly tyrosine phosphorylates RSK2 at Tyr-529, which consequently regulates RSK2 activation by facilitating inactive ERK binding to RSK2 that is required for ERK-dependent phosphorylation and activation of RSK2 (Kang, S., Dong, S., Gu, T. L., Guo, A., Cohen, M. S., Lonial, S., Khoury, H. J., Fabbro, D., Gilliland, D. G., Bergsagel, P. L., Taunton, J., Polakiewicz, R. D., and Chen, J. (2007) Cancer Cell 12, 201-214). Here we report that upon treatment of EGF, RSK2 was tyrosine-phosphorylated at Tyr-529 and activated in 293T and COS7 cells that do not express FGFR3. In contrast to FGFR3, the receptor tyrosine kinase EGF receptor did not directly phosphorylate RSK2 at Tyr-529 in an in vitro kinase assay using recombinant RSK2 and active EGF receptor or FGFR3. By mass spectroscopy-based studies, we identified Src tyrosine kinase family members Src and Fyn as upstream kinases of RSK2 Tyr-529. Treatment of Src inhibitor PP2 effectively attenuated EGF-dependent activation and Tyr-529 phosphorylation of RSK2, suggesting that Src family members are the kinases that phosphorylate RSK2 at Tyr-529 in response to EGF. Src and Fyn were able to directly phosphorylate RSK2 at Tyr-529 in the in vitro kinase assay. In vitro reconstitution of Tyr-529 phosphorylation by Src in glutathione S-transferase-tagged RSK2 enhanced inactive ERK binding to RSK2 wild type, but not the Y529F mutant. Together, our findings suggest that Src-dependent phosphorylation at Tyr-529 facilitates inactive ERK binding to RSK2, which might be a general requirement for RSK2 activation by EGF through the MEK/ERK pathway.

    Funded by: NCI NIH HHS: CA120272, R01 CA120272, R01 CA120272-02

    The Journal of biological chemistry 2008;283;8;4652-7

  • Suppression of v-Src transformation by andrographolide via degradation of the v-Src protein and attenuation of the Erk signaling pathway.

    Liang FP, Lin CH, Kuo CD, Chao HP and Fu SL

    Institute of Traditional Medicine, Faculty of Life Sciences, National Yang-Ming University, 155, Sec. 2, Li-Nong St., Taipei 11221, and Department of Research and Education, Taipei City Hospital, Taiwan.

    Elevated expression and aberrant activation of the src oncogene are strongly associated with cancer initiation and progression, thereby making Src a promising molecular target for anti-cancer therapy. Through drug screening using a temperature-inducible v-Src-transformed epithelial cell line, we found that andrographolide could suppress v-Src-induced transformation and down-regulate v-Src protein expression. In addition, actin cable dissolution and E-cadherin down-regulation, features of transformed phenotype, are perturbed by andrographolide. Moreover, andrographolide promoted v-Src degradation via a ubiquitin-dependent manner. Although andrographolide treatment altered the tyrosine phosphorylation pattern in v-Src-expressing cells, it did not directly affect the kinase activity of v-Src. Both the Erk and phosphatidylinositol 3-kinase signaling pathways were strongly inhibited in andrographolide-treated v-Src cells. However, only MKK inhibitors (PD98059 and U0126) were able to cause a non-transformed morphology similar to that of andrographolide-treated v-Src cells. Moreover, overexpression of constitutively active MKK1 in v-Src cells blocked andrographolide-mediated morphological inhibition. Interestingly, andrographolide treatment could also reduce the protein level of the c-Src truncation mutant (Src531), an Src mutant originally identified from human colon cancer cells. In summary, we demonstrated that andrographolide antagonized v-Src action through promotion of v-Src protein degradation. Furthermore, attenuation of the Erk1/2 signaling pathway is essential for andrographolide-mediated inhibition of v-Src transformation. Our results demonstrate that andrographolide can act as a v-Src inhibitor and reveal a novel action mechanism of andrographolide.

    The Journal of biological chemistry 2008;283;8;5023-33

  • Functional conservation of the glutamine-rich domains of yeast Gal11 and human SRC-1 in the transactivation of glucocorticoid receptor Tau 1 in Saccharomyces cerevisiae.

    Kim DH, Kim GS, Yun CH and Lee YC

    Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, 300 Yongbong-dong, Buk-gu, Gwangju 500-757, South Korea.

    The yeast Gal11 protein, a component of the Mediator complex, is required for the transcriptional activation of many class II genes as a physiological target of various activator proteins in vivo. In this study, we identified the yeast (Saccharomyces cerevisiae) Mediator complex as a novel coactivator of the transcriptional activity of the glucocorticoid receptor (GR) tau 1 (tau1), the major transcriptional activation domain of the GR. GR tau1 directly interacted with the Mediator complex in vivo and in vitro in a Gal11 module-dependent manner, and the Gal11p subunit interacted directly with GR tau1. Specific amino acid residues within the glutamine-rich (Qr) domain of Gal11p (residues 116 to 277) were essential for its interaction with GR tau1 and GR tau1 transactivity in yeast, as demonstrated by mutational analysis of the Gal11 Qr domain, which is highly conserved among human steroid receptor coactivator (SRC) proteins. A Gal11p variant, mini-Gal11p, comprised of the Mediator association and Qr domains of Gal11p or chimeric mini-Gal11p containing the Qr domain of SRC-1 could potentiate the GR tau1 transactivity in a gal11Delta yeast strain. These results suggest that there is functional conservation between Qr domains of yeast Gal11p and mammalian SRC proteins as direct targets of activator proteins in yeast.

    Molecular and cellular biology 2008;28;3;913-25

  • Three-dimensional matrix induces sustained activation of ERK1/2 via Src/Ras/Raf signaling pathway.

    Damianova R, Stefanova N, Cukierman E, Momchilova A and Pankov R

    Department of Cytology, Histology and Embryology, Biological Faculty, Sofia University St. Kliment Ohridski, 1164 Sofia, Bulgaria.

    Research in cell signaling often depends on tissue culture, but the artificial substrates used to grow cells in vitro are likely to distort the conclusions, particularly when adhesion-mediated signaling events are investigated. Studies of signal transduction pathways operating in cells grown in three-dimensional (3D) matrices provide a better system, giving a closer insight of the cell signaling in vivo. We compared the steady-state levels of ERK1/2 activity in primary human fibroblasts, induced by cell-derived 3D fibronectin matrix or fibronectin, coated on flat surfaces. 3D environment caused ERK1/2 stimulation concomitant with a 2.5-fold increase in Ras GTP loading and Src activation. Under these conditions FAK autophosphorylation was suppressed. Treatment with Src inhibitor PP2 abolished these effects indicating that 3D fibronectin matrix activated ERK1/2 through Src/Ras/Raf pathway, bypassing FAK. These observations suggest that within in vivo-like conditions Src may have a leading role in the induction of sustained ERK1/2 activation.

    Cell biology international 2008;32;2;229-34

  • Regulation of 3-phosphoinositide-dependent protein kinase-1 (PDK1) by Src involves tyrosine phosphorylation of PDK1 and Src homology 2 domain binding.

    Yang KJ, Shin S, Piao L, Shin E, Li Y, Park KA, Byun HS, Won M, Hong J, Kweon GR, Hur GM, Seok JH, Chun T, Brazil DP, Hemmings BA and Park J

    Department of Pharmacology, Daejeon Regional Cancer Center, Cancer Research Institute, Chungnam National University, 6 Munhwa-Dong, Jung-Gu, Taejeon 301-131, South Korea.

    3-Phosphoinositide-dependent protein kinase-1 (PDK1) appears to play a central regulatory role in many cell signalings between phosphoinositide-3 kinase and various intracellular serine/threonine kinases. In resting cells, PDK1 is known to be constitutively active and is further activated by tyrosine phosphorylation (Tyr(9) and Tyr(373/376)) following the treatment of the cell with insulin or pervanadate. However, little is known about the mechanisms for this additional activation of PDK1. Here, we report that the SH2 domain of Src, Crk, and GAP recognized tyrosine-phosphorylated PDK1 in vitro. Destabilization of PDK1 induced by geldanamycin (a Hsp90 inhibitor) was partially blocked in HEK 293 cells expressing PDK1-Y9F. Co-expression of Hsp90 enhanced PDK1-Src complex formation and led to further increased PDK1 activity toward PKB and SGK. Immunohistochemical analysis with anti-phospho-Tyr(9) antibodies showed that the level of Tyr(9) phosphorylation was markedly increased in tumor samples compared with normal. Taken together, these data suggest that phosphorylation of PDK1 on Tyr(9), distinct from Tyr(373/376), is important for PDK1/Src complex formation, leading to PDK1 activation. Furthermore, Tyr(9) phosphorylation is critical for the stabilization of both PDK1 and the PDK1/Src complex via Hsp90-mediated protection of PDK1 degradation.

    The Journal of biological chemistry 2008;283;3;1480-91

  • Identification of a functional EGF-R/p60c-src/STAT3 pathway in colorectal carcinoma: analysis of its long-term prognostic value.

    Hbibi AT, Lagorce C, Wind P, Spano JP, Des Guetz G, Milano G, Benamouzig R, Rixe O, Morere JF, Breau JL, Martin A and Fagard R

    Université Paris 13, EA 3406, Bobigny, France.

    Aims: The Epidermal Growth Factor-Receptor (EGF-R) is frequently overexpressed in colorectal carcinoma (CRC) and patients can benefit from anti-EGF-R therapy. Yet, the relationship, within tumours, between EGF-R and the activity of downstream effectors such as the non-receptor tyrosine kinase p60c-src and the signal transducer and activator of transcription 3 (STAT3) has not been extensively analyzed.

    We evaluated EGF-R, tyrosine 416-phosphorylated p60c-src (P-p60c-src), STAT3 and tyrosine 705-phosphorylated STAT3 (P-STAT3) on Tissue Micro Array (TMA) from 126 patients with CRC. Composite immunohistochemistry scores based on the intensity of labelling and the percentage of positive cells were determined on TMA for EGF-R, P-p60c-src, STAT3 and P- STAT3. A high score was found in 56%, 61%, 62% and 27% of the cases for EGF-R, P-p60c-src, STAT3 and P-STAT3 respectively. There was a significant correlation between EGF-R and P-p60c-src (p=0.006) and between P-p60c-src and P-STAT3 (p=0.0009). STAT3 was significantly correlated with vascular emboli (p=0.03) and perineural invasion (p=0.02).

    Conclusions: The correlations between EGF-R, P-p60-src and P-STAT3 and some stage-related pathological features point to a critical role for a EGF-R-connected p60c-src-kinase-mediated pathway involving STAT3 and contributing to cell survival and proliferation within CRC tumours.

    Cancer biomarkers : section A of Disease markers 2008;4;2;83-91

  • Serotonin (5-HT) transport in human platelets is modulated by Src-catalysed Tyr-phosphorylation of the plasma membrane transporter SERT.

    Zarpellon A, Donella-Deana A, Folda A, Turetta L, Pavanetto M and Deana R

    Department of Biological Chemistry, University of Padova, Padova, Italy.

    platelets possess tightly regulated systems for serotonin (5-HT) transport. This study analysed whether the 5-HT transport mediated by the plasma-membrane transporter SERT is regulated by its Tyr-phosphorylation.

    Methods: 5-HT transport was determined by filtration techniques, while immunoblotting procedures were adopted for detecting the Tyr-phosphorylation of SERT in human platelet fractions.

    Results: 5-HT accumulation in platelets pre-treated with reserpine, which prevents the neurotransmitter transport into the dense granules, decreased upon cellular exposure to PP2 and SU6656, two structurally unrelated inhibitors of Src-kinases. By contrast, the protein Tyr-phosphatase inhibitor pervanadate increased the 5-HT accumulation. Anti-SERT immunostaining of the platelet fractions showed a major band displaying an apparent molecular mass of 50 kappaDa, indicating that, during the analytical procedure, SERT underwent proteolysis, which was counteracted by addition of 4 M urea in the cellular disrupting medium. The Tyr-phosphorylation degree of SERT immunoprecipitated from membrane extracts decreased by platelet treatment with SU6656 or PP2, and enhanced upon pervanadate treatment. The anti-SERT immunoprecipitates displayed anti-Src immunostaining and in vitro kinase activity towards a Src-specific peptide-substrate. Platelet treatment with PP2 or SU6656 also caused a decrease in the imipramine binding to platelets. It was concluded that the Src-mediated SERT Tyr-phosphorylation regulates the 5-HT transport by affecting the neurotransmitter binding sites.

    Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology 2008;21;1-3;87-94

  • Novel noncatalytic role for caspase-8 in promoting SRC-mediated adhesion and Erk signaling in neuroblastoma cells.

    Finlay D and Vuori K

    Cancer Center, Burnham Institute for Medical Research, La Jolla, California 92037, USA.

    Neuroblastomas are extremely aggressive, although heterogeneous, cancers with a poor prognosis upon metastasis. Some evidence has suggested a correlative silencing of caspase-8 with MYCN amplification in neuroblastoma. A prognostic effect of this silencing, however, has been disputed. We report here hitherto undescribed roles for caspase-8 in the modulation of cell adhesion and subsequent activation of the Erk signaling pathway. Re-expression of caspase-8 in neuroblastoma cells lacking endogenous caspase-8 expression was found to promote cell adhesion to extracellular matrix and to activate adhesion-dependent signaling pathways, such as the Erk kinase cascade. This function of caspase-8 occurred irrespective of its proteolytic activity. Additionally, a pool of caspase-8 was shown to co-localize with the Src tyrosine kinase at the cellular periphery. Furthermore, our studies showed that caspase-8 forms a physical protein complex with Src via its death effector domains (DED) and maintains the complex in a detergent-soluble fraction. We also show that the DEDs of caspase-8 alone are necessary and sufficient to recreate the adhesive and biochemical phenotypes observed with the full-length protein, suggesting that caspase-8 may exert these effects via its association with Src. This protein complex association of caspase-8 and Src, and concomitant downstream signaling events, may help reconcile why a potential tumor suppressor such as caspase-8 is rarely absent in cancers.

    Cancer research 2007;67;24;11704-11

  • A RNA interference screen identifies the protein phosphatase 2A subunit PR55gamma as a stress-sensitive inhibitor of c-SRC.

    Eichhorn PJ, Creyghton MP, Wilhelmsen K, van Dam H and Bernards R

    Division of Molecular Carcinogenesis, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

    Protein Phosphatase type 2A (PP2A) represents a family of holoenzyme complexes with diverse biological activities. Specific holoenzyme complexes are thought to be deregulated during oncogenic transformation and oncogene-induced signaling. Since most studies on the role of this phosphatase family have relied on the use of generic PP2A inhibitors, the contribution of individual PP2A holoenzyme complexes in PP2A-controlled signaling pathways is largely unclear. To gain insight into this, we have constructed a set of shRNA vectors targeting the individual PP2A regulatory subunits for suppression by RNA interference. Here, we identify PR55gamma and PR55delta as inhibitors of c-Jun NH(2)-terminal kinase (JNK) activation by UV irradiation. We show that PR55gamma binds c-SRC and modulates the phosphorylation of serine 12 of c-SRC, a residue we demonstrate to be required for JNK activation by c-SRC. We also find that the physical interaction between PR55gamma and c-SRC is sensitive to UV irradiation. Our data reveal a novel mechanism of c-SRC regulation whereby in response to stress c-SRC activity is regulated, at least in part, through loss of the interaction with its inhibitor, PR55gamma.

    PLoS genetics 2007;3;12;e218

  • Tissue factor induction by protease-activated receptor 1 requires intact caveolin-enriched membrane microdomains in human endothelial cells.

    Banfi C, Brioschi M, Barcella S, Pignieri A, Parolari A, Biglioli P, Tremoli E and Mussoni L

    Monzino Cardiologic Centre IRCCS, and Department of Pharmacological Sciences, University of Milan, via Parea 4, Milan, Italy. cristina.banfi@unimi.it

    Background: Protease-activated receptors (PARs) comprise a family of G-protein-coupled receptors with a unique mechanism of proteolytic activation. PARs regulate a broad range of cellular functions and are active in the pathogenesis of disorders characterized by chronic inflammation or activation of the coagulation cascade. Signaling through PAR1 and PAR2 shifts the endothelium towards a prothrombotic phenotype, thereby exacerbating the initial pathophysiologic condition.

    Objectives: This study aimed to analyze the localization of PARs in the cell membrane and how their compartmentalization affects tissue factor (TF) in human endothelial cells.

    Methods: TF expression was determined by quantitative real-time polymerase chain reaction analysis and by activity assays. The interaction of PARs with caveolin was investigated through: (i) caveolin-1 gene knockdown performed by transfection with specific small interfering RNA (siRNA); (ii) caveolin-enriched membrane microdomain disruption; and (iii) coimmunoprecipitation assay.

    Results: We have shown that PAR1, but not PAR2, is present in endothelial caveolin-enriched membrane microdomains, where it is bound to caveolin-1, and that these structures must be intact if PAR1-induced signaling is to increase TF activity. Cholesterol depletion of endothelial cells by cholesterol-sequestering agents caused the PAR1 to relocate to high-density membranes, and impaired the induction of TF (P < 0.01) without affecting the PAR2-mediated procoagulant effect. In addition, siRNA directed against caveolin-1 inhibited TF activation by PAR1 (P < 0.01 and P < 0.01, respectively).

    Conclusions: PAR1 localization in the caveolin-enriched membrane microdomain, bound to caveolin-1, represents a crucial requirement for TF induction in endothelial cells.

    Journal of thrombosis and haemostasis : JTH 2007;5;12;2437-44

  • Dynamic interaction between Src and C-terminal Src kinase in integrin alphaIIbbeta3-mediated signaling to the cytoskeleton.

    Vielreicher M, Harms G, Butt E, Walter U and Obergfell A

    Institute of Clinical Biochemistry and Pathobiochemistry, University of Wurzburg, D-97080 Wurzburg, Germany.

    Integrin-bound Src tyrosine kinase mediates alpha(IIb)beta(3) out-side-in signaling to the cytoskeleton required for platelet adhesion and thrombus formation. Src activation (signal initiation) by phosphorylation of Tyr-418 occurs at lamellipodia leading edges. However, little is known about Src inactivation mediated by C-terminal Src kinase (Csk) Tyr-529 phosphorylation. In an established platelet model cell line (A5-Chinese hamster ovary), we studied the inactivation of Src during alpha(IIb)beta(3)-mediated adhesion to fibrinogen with live cell fluorescence resonance energy transfer (FRET) microscopy. Imaging revealed highly dynamic Src-Csk interactions at the leading edges of active lamellipodia. The Src-Csk interaction followed a highly dynamic pattern. Every 2-3 min, Src-Csk complexes moved inward in the cell, reorganized, and formed stable focal adhesions. These accumulations were primarily seen during retraction of lamellipodia, whereas no interaction was observed during protrusions. Western blot analysis during the run time of FRET signaling revealed an increase in Csk-mediated SrcTyr-529 phosphorylation with a parallel decline of tyrosine 418 phosphorylation. Mutation analysis provided additional insights into the role of Src. Although inactivation of Csk (CskK222R) had no effect on cell adhesion and spreading efficiency, cells with constitutively active expressed Src (SrcY529F) exhibited hardly any adhesion and no spreading. The few adherent cells showed weak focal adhesions that were disorganized and oversized. The data clearly demonstrate the important role of tight Src control by Csk for functional cell adhesion and spreading. The novel experimental FRET approach reported here for the inactivation of Src can be readily applied to other integrin and signaling pathways, including closely related Src family kinase members.

    The Journal of biological chemistry 2007;282;46;33623-31

  • PTP1B contributes to the oncogenic properties of colon cancer cells through Src activation.

    Zhu S, Bjorge JD and Fujita DJ

    Department of Biochemistry and Molecular Biology, and Southern Alberta Cancer Research Institute, University of Calgary, Calgary, Alberta, Canada.

    Src-specific activity has been reported to be elevated in a high percentage of colon cancer cell lines and tumors, but the underlying mechanisms are largely unknown. In this study, we report that, in the seven cancer cell lines tested, Src-specific activity was elevated (5.2- to 18.7-fold) relative to normal colon cells (FHC). This activation of Src correlated with reduced phosphorylation at Y530 of Src, whereas there was no significant change in the level of phosphorylation at Y419. The membrane tyrosine phosphatase activity for a Src family-specific phosphopeptide substrate FCP (Fyn COOH-terminal peptide phosphorylated by Csk) was greatly increased in the cancer cells and was attributed to PTP1B in most of the cell lines. Membrane PTP1B protein levels were also greatly increased. Overexpression of PTP1B increased Src specific activity in colon cancer cells by reducing phosphorylation at Y530 of Src. It also increased anchorage-independent cell growth and this increase was blocked by the Src inhibitor PP2 and Src small interfering RNA (siRNA). Down-regulating PTP1B activity by PTP1B inhibitor CinnGEL 2Me or knocking down PTP1B using siRNA also reduced Src kinase activity and colony formation ability of colon cancer cells. PTP1B siRNA reduced tumor growth in nonobese diabetic/severe combined immunodeficient mice. This study suggests that (a) PTP1B can act as an important activator of Src in colon cancer cells via dephosphorylation at Y530 of Src and (b) elevated levels of PTP1B can increase tumorigenicity of colon cancer cells by activating Src.

    Cancer research 2007;67;21;10129-37

  • A novel activating role of SRC and STAT3 on HGF transcription in human breast cancer cells.

    Sam MR, Elliott BE and Mueller CR

    Department of Pathology and Molecular Medicine, Queen's University, Kingston, Canada. michellesam@hotmail.com

    We have previously determined that the HGF promoter can be transactivated by a combination of activated Src and wild-type Stat3 in the mouse breast cell lines HC11 and SP1. To determine if this pathway is of relevance for the human disease, a series of human breast and other human cells lines were examined, and the status of key proteins in these cells determined. All of the human breast cell lines exhibited strong transactivation by a combination of activated Src and Stat3. This activation was dependent on a Stat3 recognition element present at nt-95. The exception was the ErbB2 over-expressing cell line SK-BR-3 where Stat3 alone could transactivate HGF though Src augmented this effect. Increased phosphorylation of Stat3 tyrosine 705 was also observed in this line. Analysis of three ovarian cell lines revealed that Src/Stat3 expression was not able to activate the HGF promoter in two of these lines (SKOV3 and IOSE-80PC). Src/Stat3 expression did activate HGF transcription in OVCAR3 cells, but this effect was not mediated by the Stat3 site at nt-95. Stat3 phosphorylation at tyrosine 705 was observed in IOSE-80PC cells, but was insufficient to allow for activation of the HGF promoter. Human kidney (HEK293) and cervical carcinoma (HeLa) cells were also not Src/Stat3 permissive, despite high levels of Stat3 phospho-Y705. These results suggest that human breast cells are a uniquely permissive environment for HGF transactivation by Src/Stat3 which may allow for the inappropriate activation of HGF transcription during the early stages of breast transformation. This could lead to paracrine or autocrine activation of the Met receptor in breast carcinoma cells.

    Molecular cancer 2007;6;69

  • Stress hormones regulate interleukin-6 expression by human ovarian carcinoma cells through a Src-dependent mechanism.

    Nilsson MB, Armaiz-Pena G, Takahashi R, Lin YG, Trevino J, Li Y, Jennings N, Arevalo J, Lutgendorf SK, Gallick GE, Sanguino AM, Lopez-Berestein G, Cole SW and Sood AK

    Department of Cancer Biology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.

    Recent studies have demonstrated that chronic stress promotes tumor growth, angiogenesis, and metastasis. In ovarian cancer, levels of the pro-angiogenic cytokine, interleukin 6 (IL-6), are known to be elevated in individuals experiencing chronic stress, but the mechanism(s) by which this cytokine is regulated and its role in tumor growth remain under investigation. Here we show that stress hormones such as norepinephrine lead to increased expression of IL-6 mRNA and protein levels in ovarian carcinoma cells. Furthermore, we demonstrate that norepinephrine stimulation activates Src tyrosine kinase and this activation is required for increased IL-6 expression. These results demonstrate that stress hormones activate signaling pathways known to be critical in ovarian tumor progression.

    Funded by: NCI NIH HHS: CA109298, CA110793, P50 CA 083639

    The Journal of biological chemistry 2007;282;41;29919-26

  • CD99 isoforms dictate opposite functions in tumour malignancy and metastases by activating or repressing c-Src kinase activity.

    Scotlandi K, Zuntini M, Manara MC, Sciandra M, Rocchi A, Benini S, Nicoletti G, Bernard G, Nanni P, Lollini PL, Bernard A and Picci P

    Laboratorio di Ricerca Oncologica, Istituti Ortopedici Rizzoli, Bologna, Italy. katia.scotlandi@ior.it

    CD99 gene encodes two distinct proteins, produced by alternative splicing of CD99 gene transcript. Full-length CD99 isoform (CD99wt) is formed by an extracellular domain, followed by a transmembrane domain and a 36 amino-acid intracytoplasmic domain, which is partially deleted in the truncated, short form (CD99sh). A differential expression of these two CD99 molecules can lead to distinct functional outcomes in lymphocytes. To investigate the functional effects of CD99 molecules on malignancy, forced overexpression of the two CD99 isoforms was induced in osteosarcoma and prostate cancer cells. The two isoforms exhibited opposite functions: the major form dramatically inhibits anchorage-independent growth, anoikis resistance, migration and metastasis, whereas the CD99sh remarkably favours the phenomena. A mechanistic analysis of CD99-transfected osteosarcoma cells points to involvement of c-Src family kinase activity in regulating CD99 functions in malignancy. Ser168 residue of CD99 plays a pivotal role in the reversion of the malignant phenotype. Our findings highlight the involvement of CD99 in crucial processes of cancer malignancy, serving as a curtain raiser for this, so far neglected molecule. In addition, a dualistic role for the two CD99 isoforms was shown in agreement with what was observed for other cell adhesion molecules.

    Oncogene 2007;26;46;6604-18

  • ICAM-1-mediated, Src- and Pyk2-dependent vascular endothelial cadherin tyrosine phosphorylation is required for leukocyte transendothelial migration.

    Allingham MJ, van Buul JD and Burridge K

    Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, NC 27599, USA. michael_allingham@med.unc.edu

    Leukocyte transendothelial migration (TEM) has been modeled as a multistep process beginning with rolling adhesion, followed by firm adhesion, and ending with either transcellular or paracellular passage of the leukocyte across the endothelial monolayer. In the case of paracellular TEM, endothelial cell (EC) junctions are transiently disassembled to allow passage of leukocytes. Numerous lines of evidence demonstrate that tyrosine phosphorylation of adherens junction proteins, such as vascular endothelial cadherin (VE-cadherin) and beta-catenin, correlates with the disassembly of junctions. However, the role of tyrosine phosphorylation in the regulation of junctions during leukocyte TEM is not completely understood. Using human leukocytes and EC, we show that ICAM-1 engagement leads to activation of two tyrosine kinases, Src and Pyk2. Using phospho-specific Abs, we show that engagement of ICAM-1 induces phosphorylation of VE-cadherin on tyrosines 658 and 731, which correspond to the p120-catenin and beta-catenin binding sites, respectively. These phosphorylation events require the activity of both Src and Pyk2. We find that inhibition of endothelial Src with PP2 or SU6656 blocks neutrophil transmigration (71.1 +/- 3.8% and 48.6 +/- 3.8% reduction, respectively), whereas inhibition of endothelial Pyk2 also results in decreased neutrophil transmigration (25.5 +/- 6.0% reduction). Moreover, overexpression of the nonphosphorylatable Y658F or Y731F mutants of VE-cadherin impairs transmigration of neutrophils compared with overexpression of wild-type VE-cadherin (32.7 +/- 7.1% and 38.8 +/- 6.5% reduction, respectively). Our results demonstrate that engagement of ICAM-1 by leukocytes results in tyrosine phosphorylation of VE-cadherin, which is required for efficient neutrophil TEM.

    Funded by: NHLBI NIH HHS: HL 080166, HL 45100

    Journal of immunology (Baltimore, Md. : 1950) 2007;179;6;4053-64

  • Mechanisms of integrin-vascular endothelial growth factor receptor cross-activation in angiogenesis.

    Mahabeleshwar GH, Feng W, Reddy K, Plow EF and Byzova TV

    Department of Molecular Cardiology, The Cleveland Clinic, 9500 Euclid Ave, Cleveland, OH 44195, USA.

    The functional responses of endothelial cells are dependent on signaling from peptide growth factors and the cellular adhesion receptors, integrins. These include cell adhesion, migration, and proliferation, which, in turn, are essential for more complex processes such as formation of the endothelial tube network during angiogenesis. This study identifies the molecular requirements for the cross-activation between beta3 integrin and tyrosine kinase receptor 2 for vascular endothelial growth factor (VEGF) receptor (VEGFR-2) on endothelium. The relationship between VEGFR-2 and beta3 integrin appears to be synergistic, because VEGFR-2 activation induces beta3 integrin tyrosine phosphorylation, which, in turn, is crucial for VEGF-induced tyrosine phosphorylation of VEGFR-2. We demonstrate here that adhesion- and growth factor-induced beta3 integrin tyrosine phosphorylation are directly mediated by c-Src. VEGF-stimulated recruitment and activation of c-Src and subsequent beta3 integrin tyrosine phosphorylation are critical for interaction between VEGFR-2 and beta3 integrin. Moreover, c-Src mediates growth factor-induced beta3 integrin activation, ligand binding, beta3 integrin-dependent cell adhesion, directional migration of endothelial cells, and initiation of angiogenic programming in endothelial cells. Thus, the present study determines the molecular mechanisms and consequences of the synergism between 2 cell surface receptor systems, growth factor receptor and integrins, and opens new avenues for the development of pro- and antiangiogenic strategies.

    Funded by: NHLBI NIH HHS: HL071625, HL073311, P01 HL073311-050004, R01 HL071625-05

    Circulation research 2007;101;6;570-80

  • The tumor suppressor DAPK is reciprocally regulated by tyrosine kinase Src and phosphatase LAR.

    Wang WJ, Kuo JC, Ku W, Lee YR, Lin FC, Chang YL, Lin YM, Chen CH, Huang YP, Chiang MJ, Yeh SW, Wu PR, Shen CH, Wu CT and Chen RH

    Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan.

    Death-associated protein kinase (DAPK) is a calmodulin-regulated serine/threonine kinase and elicits tumor suppression function through inhibiting cell adhesion/migration and promoting apoptosis. Despite these biological functions, the signaling mechanisms through which DAPK is regulated remain largely elusive. Here, we show that the leukocyte common antigen-related (LAR) tyrosine phosphatase dephosphorylates DAPK at pY491/492 to stimulate the catalytic, proapoptotic, and antiadhesion/antimigration activities of DAPK. Conversely, Src phosphorylates DAPK at Y491/492, which induces DAPK intra-/intermolecular interaction and inactivation. Upon EGF stimulation, a rapid Src activation leads to subsequent LAR downregulation, and these two events act in synergism to inactivate DAPK, thereby facilitating tumor cell migration and invasion toward EGF. Finally, DAPK Y491/492 hyperphosphorylation is found in human cancers in which Src activity is aberrantly elevated. These results identify LAR and Src as a DAPK regulator through their reciprocal modification of DAPK Y491/492 residues and establish a functional link of this DAPK-regulatory circuit to tumor progression.

    Molecular cell 2007;27;5;701-16

  • Subdomain switching reveals regions that harbor substrate specificity and regulatory properties of protein tyrosine kinases.

    Wang YH, Huang K, Lin X and Sun G

    Department of Cell and Molecular Biology, University of Rhode Island, Kingston, Rhode Island 02881, USA.

    Csk and Src are two protein tyrosine kinases that share a similar overall multidomain structural organization and a high degree of sequence homology but have different substrate specificities and regulatory properties. In this study, we generated chimeric kinases of Csk and Src by switching the C-terminal lobes of their catalytic domains, and we characterized their substrate specificity and regulatory properties. First, both Csk and Src phosphorylate Src as a common substrate, but on different Tyr residues. The C-terminal lobes of the kinase catalytic domain determined the site of phosphorylation on Src. Furthermore, toward several physiological substrates of Src, the substrate specificity was also determined by the C-terminal lobe of the catalytic domain regardless of the regulatory domains and the N-terminal lobe of the catalytic domain. Second, Csk and Src represent two general regulatory strategies for protein tyrosine kinases. Csk catalytic domain is inactive and is positively regulated by the regulatory domains, while Src catalytic domain is active and suppressed by its interactions with the regulatory domains. The regulatory properties of the chimeric kinases were more complicated. The regulatory domains and the N-lobe did not fully determine the response to a regulatory ligand, suggesting that the C-lobe also contributes to such responses. On the other hand, the intrinsic kinase activity of the catalytic domain correlates with the identity of the N-lobe. These results demonstrate that the chimeric strategy is useful for detailed dissection of the mechanistic basis of substrate specificity and regulation of protein tyrosine kinases.

    Funded by: NCI NIH HHS: 1R01CA111687; NCRR NIH HHS: 1 P20RR16457

    Biochemistry 2007;46;35;10162-9

  • Epidermal growth factor receptor (EGFR) ubiquitination as a mechanism of acquired resistance escaping treatment by the anti-EGFR monoclonal antibody cetuximab.

    Lu Y, Li X, Liang K, Luwor R, Siddik ZH, Mills GB, Mendelsohn J and Fan Z

    Department of Experimental Therapeutics, The University of Texas M D Anderson Cancer Center, Houston, TX 77030, USA.

    Cetuximab is an epidermal growth factor receptor (EGFR)-blocking antibody that has been approved for treatment of patients with metastatic colorectal cancer. In this study, we investigated biochemical changes in signaling pathways of a cetuximab-resistant subline of DiFi colorectal cancer cells (DiFi5) that was developed by exposing the parental sensitive cells to subeffective doses of cetuximab over an extended period of time. Compared with parental DiFi cells that express high levels of EGFR and in which cetuximab induces apoptosis, the cetuximab-resistant DiFi5 cells showed markedly lower protein levels of EGFR, an increased association of EGFR with Cbl, and an increased ubiquitination of EGFR. DiFi5 cells also had a markedly higher level of Src-Y416 phosphorylation both at baseline and on EGF stimulation. Although EGFR levels were low, DiFi5 cells responded to EGF stimulation with robust phosphorylation of EGFR on Y845 and strong phosphorylation of Akt and extracellular signal-regulated kinase, comparable to those of parental cells. Most importantly, inhibition of Src kinase activity with PP2 reversed the resistance of DiFi5 cells to cetuximab-induced apoptosis without affecting the levels of EGFR in the cells. Our results indicate that colorectal cancer cells may develop acquired resistance to cetuximab via altering EGFR levels through promotion of EGFR ubiquitination and degradation and using Src kinase-mediated cell signaling to bypass their dependency on EGFR for cell growth and survival.

    Cancer research 2007;67;17;8240-7

  • Fak/Src signaling in human intestinal epithelial cell survival and anoikis: differentiation state-specific uncoupling with the PI3-K/Akt-1 and MEK/Erk pathways.

    Bouchard V, Demers MJ, Thibodeau S, Laquerre V, Fujita N, Tsuruo T, Beaulieu JF, Gauthier R, Vézina A, Villeneuve L and Vachon PH

    Département d'Anatomie et de Biologie Cellulaire, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec, Canada.

    Human intestinal epithelial cell survival and anoikis are distinctively regulated according to the state of differentiation. In the present study, we analyzed the roles of focal adhesion kinase (Fak)/Src signaling to the PI3-K/Akt-1 and mitogen-activated protein kinase (MEK)/extracellular regulated kinases (Erk) pathways, within the context of such differentiation-state distinctions. Anoikis was induced by inhibition of beta1 integrins (antibody blocking), inhibition of Fak (pharmacologic inhibition or overexpression of dominant negative mutants), or by maintaining cells in suspension. Activation parameters of Fak, Src, Akt-1, and Erk1/2 were analyzed. Activities of Src, Akt-1, or Erk1/2 were also blocked by pharmacological inhibition or by overexpression of dominant-negative mutants. We report that: (1) the loss or inhibition of beta1 integrin binding activity causes anoikis and results in a down-activation of Fak, Src, Akt-1, and Erk1/2 in both undifferentiated, and differentiated cells; (2) the inhibition of Fak likewise causes anoikis and a down-activation of Src, Akt-1, and Erk1/2, regardless of the differentiation state; (3) Src, PI3-K/Akt-1, and MEK/Erk contribute to the survival of differentiated cells, whereas MEK/Erk does not play a role in the survival of undifferentiated ones; (4) the inhibition/loss of beta1 integrin binding and/or Fak activity results in a loss of Src engagement with Fak, regardless of the state of differentiation; and (5) Src contributes to the activation of both the PI3-K/Akt-1 and MEK/Erk pathways in undifferentiated cells, but does not influence PI3-K/Akt-1 in differentiated ones. Hence, Fak/Src signaling to the PI3-K/Akt-1 and MEK/Erk pathways undergoes a differentiation state-specific uncoupling which ultimately reflects upon the selective engagement of these same pathways in the mediation of intestinal epithelial cell survival.

    Journal of cellular physiology 2007;212;3;717-28

  • CUTL1 promotes tumor cell migration by decreasing proteasome-mediated Src degradation.

    Aleksic T, Bechtel M, Krndija D, von Wichert G, Knobel B, Giehl K, Gress TM and Michl P

    Department of Internal Medicine I, University of Ulm, Ulm, Germany.

    Recently, we identified the homeodomain transcription factor CUTL1 as important mediator of cell migration and tumor invasion downstream of transforming growth factor beta (TGFbeta). The molecular mechanisms and effectors mediating the pro-migratory and pro-invasive phenotype induced by CUTL1 have not been elucidated so far. Therefore, the aim of this study was to identify signaling pathways downstream of CUTL1 which are responsible for its effects on tumor cell migration. We found that the reduced motility seen after knock down of CUTL1 by RNA interference is accompanied by a delay in tumor cell spreading. This spreading defect is paralleled by a marked reduction of Src protein levels. We show that CUTL1 leads to Src protein stabilization and activation of Src-regulated downstream signaling molecules such as RhoA, Rac1, Cdc42 and ROCK. In addition, we demonstrate that CUTL1 decreases proteasome-mediated Src protein degradation, possibly via transcriptionally upregulating C-terminal Src kinase (Csk). Based on experiments using Src knockout cells (SYF), we present evidence that Src plays a crucial role in CUTL1-induced tumor cell migration. In conclusion, our findings linking the pro-invasive transcription factor CUTL1 and the Src pathway provide important new insights in the molecular effector pathways mediating CUTL-induced migration and invasion.

    Oncogene 2007;26;40;5939-49

  • E-cadherin adhesion activates c-Src signaling at cell-cell contacts.

    McLachlan RW, Kraemer A, Helwani FM, Kovacs EM and Yap AS

    Division of Molecular Cell Biology, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Brisbane, Queensland, Australia 4072.

    Cadherin-based cell-cell contacts are prominent sites for phosphotyrosine signaling, being enriched in tyrosine-phosphorylated proteins and tyrosine kinases and phosphatases. The functional interplay between cadherin adhesion and tyrosine kinase signaling, however, is complex and incompletely understood. In this report we tested the hypothesis that cadherin adhesion activates c-Src signaling and sought to assess its impact on cadherin function. We identified c-Src as part of a cadherin-activated cell signaling pathway that is stimulated by ligation of the adhesion receptor. However, c-Src has a biphasic impact on cadherin function, exerting a positive supportive role at lower signal strengths, but inhibiting function at high signal strengths. Inhibiting c-Src under circumstances when it is activated by cadherin adhesion decreased several measures of cadherin function. This suggests that the cadherin-activated c-Src signaling pathway serves positively to support cadherin function. Finally, our data implicate PI3-kinase signaling as a target for cadherin-activated c-Src signaling that contributes to its positive impact on cadherin function. We conclude that E-cadherin signaling is an important activator of c-Src at cell-cell contacts, providing a key input into a signaling pathway where quantitative changes in signal strength may result in qualitative differences in functional outcome.

    Molecular biology of the cell 2007;18;8;3214-23

  • Pharmacological evidence for altered src kinase regulation of I (Ca,L) in patients with chronic atrial fibrillation.

    Greiser M, Halaszovich CR, Frechen D, Boknik P, Ravens U, Dobrev D, Lückhoff A and Schotten U

    Institut für Physiologie, Universitätsklinikum Aachen, Aachen, Germany. m.greiser@fys.unimaas.nl

    A reduction in L-type Ca(2+) current (I (Ca,L)) contributes to electrical remodeling in chronic atrial fibrillation (AF). Whether the decrease in I (Ca,L) is solely due to a reduction in channel proteins remains controversial. Protein tyrosine kinases (PTK) have been described as potent modulators of I (Ca,L) in cardiomyocytes. We studied alpha(1C) L-type Ca(2+) channel subunit expression and the regulation of I (Ca,L) by PTK in chronic AF using PTK inhibitors: genistein, a nonselective inhibitor of PTK, and 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-3,4-d-pyrimidine (PP1), a selective inhibitor of src kinases. Furthermore, type-1 and type-2A protein phosphatase activity was measured with phosphorylase as substrate in whole-cell lysates derived from atrial tissue of AF patients. Right atrial appendages were obtained from patients undergoing open-heart surgery. Protein levels of alpha(1C) L-type Ca(2+) channel subunit were determined using Western blot analysis and normalized to the protein amounts of calsequestrin as internal control. The protein concentrations of alpha(1C) did not differ between AF and sinus rhythm (SR; alpha(1C)/calsequestrin: 1.0 +/- 0.1 and 1.2 +/- 0.2, respectively, n = 8 patients). In cardiomyocytes from patients in SR (n = 20 patients), genistein and PP1 both evoked similar increases in I (Ca,L) from 3.0 +/- 0.3 to 6.1 +/- 0.8 pA/pF and from 2.8 +/- 0.4 to 6.1 +/- 0.6 pA/pF, respectively. In cells from AF patients (n = 10 patients), basal I (Ca,L) was significantly lower. In this case, genistein lead to the same relative increase in I (Ca,L) as in SR cells (from 1.46 +/- 0.30 to 3.2 +/- 1.0 pA/pF), whereas no increase was elicited by PP1 suggesting impaired regulation of I (Ca,L) by src kinases in AF. Total and type 1 and type 2A-related phosphatase activities were higher in tissue from patients with chronic AF compared to SR (4.8 +/- 0.4, 2.1 +/- 0.2, and 2.7 +/- 0.4 nmol/mg/min and 3.6 +/- 0.4, 1.3 +/- 0.2, and 2.4 +/- 0.3 nmol/mg/min, respectively, n = 7 patients per group). Downregulation of I (Ca,L) in AF is not due to a reduction in L-type Ca(2+) channel protein expression. Indirect evidence for an impaired src kinase regulation of I (Ca,L) together with an increased phosphatase activity suggests that a complex alteration in the kinase/phosphatase balance leads to I (Ca,L) dysregulation in chronic AF.

    Naunyn-Schmiedeberg's archives of pharmacology 2007;375;6;383-92

  • Palladin interacts with SH3 domains of SPIN90 and Src and is required for Src-induced cytoskeletal remodeling.

    Rönty M, Taivainen A, Heiska L, Otey C, Ehler E, Song WK and Carpen O

    Department of Pathology, Neuroscience Program, Biomedicum Helsinki, University of Helsinki and Helsinki University Hospital, Helsinki, Finland. mikko.ronty@helsinki.fi

    Palladin and SPIN90 are widely expressed proteins, which participate in modulation of actin cytoskeleton by binding to a variety of scaffold and signaling molecules. Cytoskeletal reorganization can be induced by activation of signaling pathways, including the PDGF receptor and Src tyrosine kinase pathways. In this study we have analyzed the interplay between palladin, SPIN90 and Src and characterized the role of palladin and SPIN90 in PDGF and Src-induced cytoskeletal remodeling. We show that the SH3 domains of SPIN90 and Src directly bind palladin's poly-proline sequence and the interaction controls intracellular targeting of SPIN90. In PDGF-treated cells, palladin and SPIN90 co-localize in actin-rich membrane ruffles and lamellipodia. The effect of PDGF on the cytoskeleton is at least partly mediated by the Src kinase since PP2, a selective Src kinase family inhibitor, blocked PDGF-induced changes. Furthermore, expression of active Src kinase resulted in coordinated translocation of both palladin and SPIN90 to membrane protrusions. Knock-down of endogenous SPIN90 did not inhibit Src-induced cytoskeletal rearrangement, whereas knock-down of palladin resulted in cytoskeletal disorganization and inhibition of remodeling. Further studies showed that palladin is tyrosine phosphorylated in cells expressing active Src indicating bidirectional interplay between palladin and Src. These results may have implications in understanding the invasive and metastatic phenotype of neoplastic cells induced by Src.

    Funded by: Medical Research Council: G0400153; NIGMS NIH HHS: GM61743, R01 GM061743, R01 GM061743-01A2, R01 GM061743-02, R01 GM061743-03, R01 GM061743-04; NINDS NIH HHS: NS43253, R01 NS043253, R01 NS043253-01A1, R01 NS043253-02, R01 NS043253-03, R01 NS043253-04, R01 NS043253-04S1, R01 NS043253-05

    Experimental cell research 2007;313;12;2575-85

  • RACK1 regulates Src activity and modulates paxillin dynamics during cell migration.

    Doan AT and Huttenlocher A

    Department of Pharmacology, University of Wisconsin, Medical Sciences Center, Madison, WI 53706, USA.

    Receptor for Activated C Kinase, RACK1, is an adaptor protein that regulates signaling via Src and PKC-dependent pathways, and has been implicated in cell migration. In this study we demonstrate novel functions for RACK1 in regulating adhesion dynamics during cell migration. We report that cells lacking RACK1 are less motile and show reduced dynamics of paxillin and talin at focal complexes. To investigate the role of the RACK1/Src interactions in adhesion dynamics, we used RACK1 in which the putative Src binding site has been mutated (RACK Y246F). RACK1-deficient cells showed enhanced c-Src activity that was rescued by expression of wild type RACK1, but not by RACK Y246F. Expression of wild type RACK1, but not RACK Y246F, was also able to rescue the adhesion and migration defects observed in the RACK1-deficient cells. Furthermore, our findings indicate that RACK1 functions to regulate paxillin phosphorylation and that its effects on paxillin dynamics require the Src-mediated phosphorylation of tyrosine 31/118 on paxillin. Taken together, these findings support a novel role for RACK1 as a key regulator of cell migration and adhesion dynamics through the regulation of Src activity, and the modulation of paxillin phosphorylation at early adhesions.

    Funded by: NCI NIH HHS: R01 CA085862, R01 CA085862-09, R01CA85862-06

    Experimental cell research 2007;313;12;2667-79

  • Rapid trafficking of c-Src, a non-palmitoylated Src-family kinase, between the plasma membrane and late endosomes/lysosomes.

    Kasahara K, Nakayama Y, Kihara A, Matsuda D, Ikeda K, Kuga T, Fukumoto Y, Igarashi Y and Yamaguchi N

    Department of Molecular Cell Biology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan.

    Src-family kinases (SFKs) are co-expressed with multiple combinations of each member in a single cell and involved in various signalings. Recently, we showed by sucrose-density gradient fractionation that the subcellular distribution of c-Src is distinct from that of Lyn. However, little is known about the trafficking of c-Src in living cells. Here, we show by time-lapse monitoring combined with photobleaching techniques that c-Src, a non-palmitoylated SFK, is rapidly exchanged between the plasma membrane and intracellular organelles representing late endosomes/lysosomes possibly through its cytosolic release. Although Lyn, a palmitoylated SFK, is exocytosed to the plasma membrane via the Golgi apparatus along the secretory pathway, lack of palmitoylation directs Lyn away from the exocytotic transport to the c-Src-type trafficking between the plasma membrane and late endosomes/lysosomes. Intriguingly, c-Src and a non-palmitoylated Lyn mutant are efficiently delivered and immobilized to focal adhesions when their SH2 domains are able to mediate protein-protein interactions in place of intramolecular bindings. However, palmitoylation of Lyn inhibits its recruitment to focal adhesions. These results suggest that palmitoylation of SFKs is critical for SFK localization and trafficking and implicate that two distinct trafficking pathways for SFKs may be involved in SFKs' specific functions.

    Experimental cell research 2007;313;12;2651-66

  • Inhibition of c-Src expression and activation in malignant pleural mesothelioma tissues leads to apoptosis, cell cycle arrest, and decreased migration and invasion.

    Tsao AS, He D, Saigal B, Liu S, Lee JJ, Bakkannagari S, Ordonez NG, Hong WK, Wistuba I and Johnson FM

    Department of Thoracic/Head and Neck Medical Oncology, M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Unit 432, Houston, TX 77030, USA. astsao@mdanderson.org

    Malignant pleural mesothelioma (MPM) is a deadly disease with few systemic treatment options. One potential therapeutic target, the non-receptor tyrosine kinase c-Src, causes changes in proliferation, motility, invasion, survival, and angiogenesis in cancer cells and may be a valid therapeutic target in MPM. To test this hypothesis, we determined the effects of c-Src inhibition in MPM cell lines and examined c-Src expression and activation in tissue samples. We analyzed four MPM cell lines and found that all expressed total and activated c-Src. Three of the four cell lines were sensitive by in vitro cytotoxicity assays to the c-Src inhibitor dasatinib, which led to cell cycle arrest and increased apoptosis. Dasatinib also inhibited migration and invasion independent of the cytotoxic effects, and led to the rapid and durable inhibition of c-Src and its downstream pathways. We used immunohistochemical analysis to determine the levels of c-Src expression and activation in 46 archived MPM tumor specimens. The Src protein was highly expressed in tumor cells, but expression did not correlate with survival. However, expression of activated Src (p-Src Y419) on the tumor cell membrane was higher in patients with advanced-stage disease; the presence of metastasis correlated with higher membrane (P = 0.03) and cytoplasmic (P = 0.04) expression of p-Src Y419. Lower levels of membrane expression of inactive c-Src (p-Src Y530) correlated with advanced N stage (P = 0.02). Activated c-Src may play a role in survival, metastasis, and invasion of MPM, and targeting c-Src may be an important therapeutic strategy.

    Molecular cancer therapeutics 2007;6;7;1962-72

  • Integrin-mediated host cell invasion by type 1-piliated uropathogenic Escherichia coli.

    Eto DS, Jones TA, Sundsbak JL and Mulvey MA

    Division of Cell Biology and Immunology, Pathology Department, University of Utah, Salt Lake City, Utah, United States of America.

    Uropathogenic Escherichia coli (UPEC), the primary causative agent of urinary tract infections, typically express filamentous adhesive organelles called type 1 pili that mediate both bacterial attachment to and invasion of bladder urothelial cells. Several host proteins have previously been identified as receptors for type 1 pili, but none have been conclusively shown to promote UPEC entry into host bladder cells. Using overlay assays with FimH, the purified type 1 pilus adhesin, and mass spectroscopy, we have identified beta1 and alpha3 integrins as key host receptors for UPEC. FimH recognizes N-linked oligosaccharides on these receptors, which are expressed throughout the urothelium. In a bladder cell culture system, beta1 and alpha3 integrin receptors co-localize with invading type 1-piliated bacteria and F-actin. FimH-mediated bacterial invasion of host bladder cells is inhibited by beta1 and alpha3 integrin-specific antibodies and by disruption of the beta1 integrin gene in the GD25 fibroblast cell line. Phosphorylation site mutations within the cytoplasmic tail of beta1 integrin that alter integrin signaling also variably affect UPEC entry into host cells, by either attenuating or boosting invasion frequencies. Furthermore, focal adhesion and Src family kinases, which propagate integrin-linked signaling and downstream cytoskeletal rearrangements, are shown to be required for FimH-dependent bacterial invasion of target host cells. Cumulatively, these results indicate that beta1 and alpha3 integrins are functionally important receptors for type 1 pili-expressing bacteria within the urinary tract and possibly at other sites within the host.

    Funded by: NIAID NIH HHS: T32 AI055434-01A1; NIDDK NIH HHS: DK068585, DK069526, R01 DK068585-04

    PLoS pathogens 2007;3;7;e100

  • P-selectin cross-links PSGL-1 and enhances neutrophil adhesion to fibrinogen and ICAM-1 in a Src kinase-dependent, but GPCR-independent mechanism.

    Xu T, Zhang L, Geng ZH, Wang HB, Wang JT, Chen M and Geng JG

    Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, The Graduate School of Chinese Academy of Sciences, Shanghai, China.

    Endothelial and platelet P-selectin (CD62P) and leukocyte integrin alpha(M)beta(2) (CD11bCD18, Mac-1) are cell adhesion molecules essential for host defense and innate immunity. Upon inflammatory challenges, P-selectin binds to PSGL-1 (P-selectin glycoprotein ligand-1, CD162) to mediate neutrophil rolling, during which integrins become activated by extracellular stimuli for their firm adhesion in a G-protein coupled receptor (GPCR)-dependent mechanism. Here we show that cross-linking of PSGL-1 by dimeric or multimeric forms of platelet P-selectin, P-selectin receptor-globulin, anti-PSGL-1 mAb and its F(ab')2 induced adhesion of human neutrophils to fibrinogen (Fg) and intercellular cell adhesion molecule-1 (ICAM-1, CD54) and triggered a moderate clustering of alpha(M)beta(2), but monomeric forms of soluble P-selectin and anti-PSGL-1 Fab did not. Interestingly, P-selectin did not induce a detectable interleukine-8 (IL-8) secretion (<0.1 ng/ml) in 30 minutes, whereas a high concentration of IL-8 (>50 ng/ml) was required to increase neutrophil adhesion to Fg. P-selectin-induced neutrophil adhesion was significantly inhibited by PP2 (a Src kinase inhibitor), but not by pertussis toxin (PTX; a GPCR inhibitor). Activated platelets also increased neutrophil binding to fibrinogen and triggered tyrosine phosphorylation of cellular proteins. Our results indicate that P-selectin-induced integrin activation (Src kinase-dependent) is distinct from that elicited by cytokines, chemokines, chemoattractants (GPCR-dependent), suggesting that these two signal transduction pathways may cooperate for maximal activation of leukocyte integrins.

    Cell adhesion & migration 2007;1;3;115-23

  • XB130, a novel adaptor protein for signal transduction.

    Xu J, Bai XH, Lodyga M, Han B, Xiao H, Keshavjee S, Hu J, Zhang H, Yang BB and Liu M

    Division of Cellular and Molecular Biology, University Health Network Toronto General Research Institute, and Hospital for Sick Children, Toronto, Ontario M5G 1L7, Canada.

    Adaptor proteins are important mediators in signal transduction. In the present study, we report the cloning and characterization of a novel adaptor protein, XB130. This gene is located on human chromosome 10q25.3 and encodes a protein of 818 amino acids. It contains several Src homology (SH)2- and SH3-binding motifs, two pleckstrin homology domains, a coiled-coil region, and a number of potential tyrosine or serine/threonine phosphorylation sites. Endogenous XB130 interacts with c-Src tyrosine kinase. Their co-expression in COS-7 cells resulted in activation of c-Src and elevated tyrosine phosphorylation of multiple proteins, including XB130 itself. XB130 expression in HEK293 cells enhanced serum response element- and AP-1-dependent transcriptional activation mediated by c-Src. XB130DeltaN, an N-terminal deletion mutant lacking a putative SH3-binding motif and several putative SH2-binding sites, reduced its ability to mediate Src signal transduction. Down-regulation of endogenous XB130 with siRNA reduced c-Src activity, IL-8 production, EGF-induced phosphorylation of Akt and GSK3beta, and altered cell cycles in human lung epithelial cells. These data suggest that XB130 as an adaptor may play an important role in the regulation of signal transduction and cellular functions.

    The Journal of biological chemistry 2007;282;22;16401-12

  • The lipid raft proteins flotillins/reggies interact with Galphaq and are involved in Gq-mediated p38 mitogen-activated protein kinase activation through tyrosine kinase.

    Sugawara Y, Nishii H, Takahashi T, Yamauchi J, Mizuno N, Tago K and Itoh H

    Laboratory of Signal Transduction, Department of Cell Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.

    The heterotrimeric G protein alpha q subunit (Galphaq) mediates a variety of cell functions by activating the effector molecule phospholipase Cbeta. Galphaq activity is regulated by G protein betagamma subunits, G protein-coupled receptors, RGS proteins, and Ric-8. In this study, we identified the lipid raft resident proteins, flotillin-1/reggie-2 and flotillin-2/reggie-1, as Galphaq-binding proteins. The interactions of Galphaq and flotillins were independent of the nucleotide-binding state of Galphaq, and the N-terminal portion of flotillins was critical for the interaction. A short interfering RNA-mediated knockdown of flotillins, particularly flotillin-2, attenuated the UTP-induced activation of p38 mitogen-activated protein kinase (MAPK) but not that of ERK1/2. The activation of p38 MAPK was inhibited by the Src family tyrosine kinase inhibitor PP2 and the cholesterol-depleting agent methyl-beta-cyclodextrin, which is generally used for the disruption of lipid rafts. In contrast, the activation of ERK1/2 was not inhibited by these compounds. These lines of evidence suggested that a Gq-coupled receptor activates specifically p38 MAPK through lipid rafts and Src kinase activation, in which flotillins positively modulate the Gq signaling.

    Cellular signalling 2007;19;6;1301-8

  • CSK controls retinoic acid receptor (RAR) signaling: a RAR-c-SRC signaling axis is required for neuritogenic differentiation.

    Dey N, De PK, Wang M, Zhang H, Dobrota EA, Robertson KA and Durden DL

    Section of Pediatric Hematology/Oncology, Department of Pediatrics, Aflac Cancer Center and Blood Disorders Services, Children's Healthcare of Atlanta, Emory University School of Medicine, Atlanta, GA 30022, USA.

    Herein, we report the first evidence that c-SRC is required for retinoic acid (RA) receptor (RAR) signaling, an observation that suggests a new paradigm for this family of nuclear hormone receptors. We observed that CSK negatively regulates RAR functions required for neuritogenic differentiation. CSK overexpression inhibited RA-mediated neurite outgrowth, a result which correlated with the inhibition of the SFK c-SRC. Consistent with an extranuclear effect of CSK on RAR signaling and neurite outgrowth, CSK overexpression blocked the downstream activation of RAC1. The conversion of GDP-RAC1 to GTP-RAC1 parallels the activation of c-SRC as early as 15 min following all-trans-retinoic acid treatment in LA-N-5 cells. The cytoplasmic colocalization of c-SRC and RARgamma was confirmed by immunofluorescence staining and confocal microscopy. A direct and ligand-dependent binding of RAR with SRC was observed by surface plasmon resonance, and coimmunoprecipitation studies confirmed the in vivo binding of RARgamma to c-SRC. Deletion of a proline-rich domain within RARgamma abrogated this interaction in vivo. CSK blocked the RAR-RA-dependent activation of SRC and neurite outgrowth in LA-N-5 cells. The results suggest that transcriptional signaling events mediated by RA-RAR are necessary but not sufficient to mediate complex differentiation in neuronal cells. We have elucidated a nongenomic extranuclear signal mediated by the RAR-SRC interaction that is negatively regulated by CSK and is required for RA-induced neuronal differentiation.

    Funded by: NCI NIH HHS: CA 94233

    Molecular and cellular biology 2007;27;11;4179-97

  • FAK association with multiple signal proteins mediates pressure-induced colon cancer cell adhesion via a Src-dependent PI3K/Akt pathway.

    Thamilselvan V, Craig DH and Basson MD

    Department of Surgery, John D. Dingell VA Medical Center, 4646 John R. St., Detroit, Michigan 48201-1932, USA.

    Cancer cell adhesion is traditionally viewed as random, occurring if the cell's receptors match the substrate. Cancer cells are subjected to pressure and shear during growth against a constraining stroma, surgical manipulation, and passage through the venous and lymphatic system. Cells shed into a cavity such as the abdomen postoperatively also experience increased pressure from postoperative edema. Increased extracellular pressure stimulates integrin-mediated cancer cell adhesion via FAK and Src. PI 3-kinase (PI3K) inhibitors (LY294002 or wortmannin), Akt inhibitors, or Akt1 siRNA blocked adhesion stimulated by 15 mmHg pressure in SW620 or primary human malignant colonocytes. Pressure activated PI3K, tyrosine-phosphorylated and membrane-translocated the p85 subunit, and phosphorylated Akt. PI3K inhibitor (LY294002) prevented pressure-stimulated Akt Ser473 and FAK Tyr397, but not FAK576 or Src416 phosphorylation. PP2 inhibited PI3K activity and Akt phosphorylation. FAK siRNA did not affect pressure-induced PI3K activation but blocked Akt phosphorylation. Pressure also stimulated FAK or FAKY397F mutant translocation to the membrane. Akt inhibitor IV blocked pressure-induced Akt and FAK translocation. Pressure activated Src- and PI3K-dependently induced p85 interaction with FAK, and FAK with beta1 integrin. These results delineate a novel force-activated inside-out Src/PI3K/FAK/Akt pathway by which cancer cells regulate their own adhesion. These signals may be potential targets for inhibition of metastatic adhesion.

    Funded by: NIDDK NIH HHS: R01 DK60771

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2007;21;8;1730-41

  • Systematic identification of SH3 domain-mediated human protein-protein interactions by peptide array target screening.

    Wu C, Ma MH, Brown KR, Geisler M, Li L, Tzeng E, Jia CY, Jurisica I and Li SS

    Department of Biochemistry and the Siebens-Drake Research Institute, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada.

    Systematic identification of direct protein-protein interactions is often hampered by difficulties in expressing and purifying the corresponding full-length proteins. By taking advantage of the modular nature of many regulatory proteins, we attempted to simplify protein-protein interactions to the corresponding domain-ligand recognition and employed peptide arrays to identify such binding events. A group of 12 Src homology (SH) 3 domains from eight human proteins (Swiss-Prot ID: SRC, PLCG1, P85A, NCK1, GRB2, FYN, CRK) were used to screen a peptide target array composed of 1536 potential ligands, which led to the identification of 921 binary interactions between these proteins and 284 targets. To assess the efficiency of the peptide array target screening (PATS) method in identifying authentic protein-protein interactions, we examined a set of interactions mediated by the PLCgamma1 SH3 domain by coimmunoprecipitation and/or affinity pull-downs using full-length proteins and achieved a 75% success rate. Furthermore, we characterized a novel interaction between PLCgamma1 and hematopoietic progenitor kinase 1 (HPK1) identified by PATS and demonstrated that the PLCgamma1 SH3 domain negatively regulated HPK1 kinase activity. Compared to protein interactions listed in the online predicted human interaction protein database (OPHID), the majority of interactions identified by PATS are novel, suggesting that, when extended to the large number of peptide interaction domains encoded by the human genome, PATS should aid in the mapping of the human interactome.

    Proteomics 2007;7;11;1775-85

  • Src and focal adhesion kinase mediate mechanical strain-induced proliferation and ERK1/2 phosphorylation in human H441 pulmonary epithelial cells.

    Chaturvedi LS, Marsh HM and Basson MD

    John D. Dingell Veterans Affairs Medical Center, 4646 John R. St., Detroit, MI 48201, USA.

    Pulmonary epithelial cells are exposed to repetitive deformation during physiological breathing and mechanical ventilation. Such deformation may influence pulmonary growth, development, and barotrauma. Although deformation stimulates proliferation and activates extracellular signal-regulated kinases (ERK1/2) in human pulmonary epithelial H441 cells, the upstream mechanosensors that induce ERK activation are poorly understood. We investigated whether c-Src or focal adhesion kinase (FAK) mediates cyclic mechanical strain-induced ERK1/2 activation and proliferation in human pulmonary epithelial (NCI-H441) cells. The H441 and A549 cells were grown on collagen I-precoated membranes and were subjected to an average 10% cyclic mechanical strain at 20 cycles/min. Cyclic strain activated Src within 2 min by increasing phosphorylation at Tyr(418), followed by rapid phosphorylation of FAK at Tyr(397) and Tyr(576) and ERK1/2 at Thr(202)/Tyr(204) (n = 5, P < 0.05). Twenty-four (A549 cells) and 24-72 h (H441 cells) of cyclic mechanical strain increased cell numbers compared with static culture. Twenty-four hours of cyclic strain also increased H441 FAK, Src, and ERK phosphorylation without affecting total FAK, Src, or ERK protein. The mitogenic effect was blocked by Src (10 micromol/l PP2 or short interfering RNA targeted to Src) or MEK (50 micromol/l PD-98059) inhibition. PP2 also blocked strain-induced phosphorylation of FAK-Tyr(576) and ERK-Thr(202)/Tyr(204) but not FAK-Tyr(397). Reducing FAK by FAK-targeted short interfering RNA blocked mechanical strain-induced mitogenicity and significantly attenuated strain-induced ERK activation but not strain-induced Src phosphorylation. Together, these results suggest that repetitive mechanical deformation induced by ventilation supports pulmonary epithelial proliferation by a pathway involving Src, FAK, and then ERK signaling.

    Funded by: NIDDK NIH HHS: R01 DK067257

    American journal of physiology. Cell physiology 2007;292;5;C1701-13

  • Src induces urokinase receptor gene expression and invasion/intravasation via activator protein-1/p-c-Jun in colorectal cancer.

    Leupold JH, Asangani I, Maurer GD, Lengyel E, Post S and Allgayer H

    Department of Experimental Surgery Mannheim Faculty, University of Heidelberg, Heidelberg, Germany.

    The urokinase receptor [urokinase plasminogen activator receptor (u-PAR)] promotes invasion and metastasis and is associated with poor patient survival. Recently, it was shown that Src induces u-PAR gene expression via Sp1 bound to the u-PAR promoter region -152/-135. However, u-PAR is regulated by diverse promoter motifs, among them being an essential activator protein-1 (AP-1) motif at -190/-171. Moreover, an in vivo relevance of Src-induced transcriptional regulators of u-PAR-mediated invasion, in particular intravasation, and a relevance in resected patient tumors have not sufficiently been shown. The present study was conducted (a) to investigate if, in particular, AP-1-related transcriptional mediators are required for Src-induced u-PAR-gene expression, (b) to show in vivo relevance of AP-1-mediated Src-induced u-PAR gene expression for invasion/intravasation and for resected tissues from colorectal cancer patients. Src stimulation of the u-PAR promoter deleted for AP-1 region -190/-171 was reduced as compared with the wild-type promoter in cultured colon cancer cells. In gelshifts/chromatin immunoprecipitation, Src-transfected SW480 cells showed an increase of phospho-c-Jun, in addition to JunD and Fra-1, bound to region -190/-171. Src-transfected cells showed a significant increase in c-Jun phosphorylated at Ser(73) and also Ser(63), which was paralleled by increased phospho-c-jun-NH(2)-kinase. Significant decreases of invasion/in vivo intravasation (chorionallantoic membrane model) were observed in Src-overexpressing cells treated with Src inhibitors, u-PAR-small interfering RNA, and dominant negative c-Jun (TAM67). In resected tissues of 20 colorectal cancer patients, a significant correlation between Src activity, AP-1 complexes bound to u-PAR region -190/-171, and advanced pN stage were observed. These data suggest that Src-induced u-PAR gene expression and invasion/intravasation in vivo is also mediated via AP-1 region -190/-171, especially bound with c-Jun phosphorylated at Ser(73/63), and that this pathway is biologically relevant for colorectal cancer patients, suggesting therapeutic potential.

    Funded by: NCI NIH HHS: R01 CA111882, R01 CA111882-01A1

    Molecular cancer research : MCR 2007;5;5;485-96

  • P60-c-src suppresses apoptosis through inhibition of caspase 8 activation in hepatoma cells, but not in primary hepatocytes.

    De Toni EN, Kuntzen C, Gerbes AL, Thasler WE, Sonuc N, Mucha SR, Camaj P, Bruns C, Göke B and Eichhorst ST

    Ludwig-Maximilians-University, University Hospital Grosshadern, Department of Medicine 2, Research Lab B 5 E01 308, Marchioninistrasse 15, D-81377 Munich, Germany. enrico.detoni@med.uni-muenchen.de

    Failure to induce apoptosis triggered by members of the death receptor family has been described in hepatocellular carcinoma (HCC) and sensitization of malignant cells to pro-apoptotic molecules such as TRAIL has been proposed as an alternative cancer therapy. Limiting to this approach are the resistance of many tumor cells to TRAIL and safety concerns about the toxicity of TRAIL in normal hepatocytes.

    Methods: We here explored the possibility that the protooncogene c-Src, known to be overexpressed in a variety of tumors, could be specifically responsible for the loss of response to receptor-mediated apoptosis.

    Results: Cotreatment of several hepatoma cell lines with the Src inhibitor PP2 potently sensitized these cells to TRAIL and CD95, dramatically decreasing effective doses of TRAIL to as low as 1 ng/ml. Remarkably, Src-inhibition did not synergize with TRAIL signaling in primary hepatocytes. Specific siRNAs showed that the effect was due to blockade of p60(c-Src) and occurred through increased recruitment of caspase 8.

    Conclusions: We provide evidence that p60(c-Src) is an important and effective suppressor of receptor-mediated apoptosis in hepatoma cells but not in primary human hepatocytes. Inhibition of Src sensitizes tumor cells to apoptosis and decreases effective doses of TRAIL to therapeutic concentrations.

    Journal of hepatology 2007;46;4;682-91

  • TGF-beta-regulated collagen type I accumulation: role of Src-based signals.

    Mishra R, Zhu L, Eckert RL and Simonson MS

    Division of Nephrology, Department of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA.

    Transforming growth factor-beta (TGF-beta) stimulates myofibroblast transdifferentiation, leading to type I collagen accumulation and fibrosis. We investigated the function of Src in TGF-beta-induced collagen I accumulation. In human mesangial cells, PTyr416 Src (activated Src) was 3.3-fold higher in TGF-beta-treated cells than in controls. Src activation by TGF-beta was blocked by rottlerin and by a dominant negative mutant of protein kinase Cdelta (PKCdelta), showing that TGF-beta activates Src by a PKCdelta-based mechanism. Pharmacological inhibitors and a dominant negative Src mutant prevented the increase in collagen type I secretion in cells exposed to TGF-beta. Similarly, on-target Src small interference RNA (siRNA) prevented type I collagen secretion in response to TGF-beta, but off-target siRNA complexes had no effect. It is well established in mesangial cells that upregulation of type I collagen by TGF-beta requires extracellular signal-regulated kinase 1/2 (ERK1/2), and we found that activation of ERK1/2 by TGF-beta requires Src. In conclusion, these results suggest that stimulation of collagen type I secretion by TGF-beta requires a PKCdelta-Src-ERK1/2 signaling motif.

    Funded by: NIAMS NIH HHS: AR-046494, AR-41456

    American journal of physiology. Cell physiology 2007;292;4;C1361-9

  • Proteomics analysis of protein kinases by target class-selective prefractionation and tandem mass spectrometry.

    Wissing J, Jänsch L, Nimtz M, Dieterich G, Hornberger R, Kéri G, Wehland J and Daub H

    Department of Cell Biology, Helmholtz Centre for Infection Research (HZI), Inhoffenstrasse 7, 38124 Braunschweig, Germany.

    Protein kinases constitute a large superfamily of enzymes with key regulatory functions in nearly all signal transmission processes of eukaryotic cells. However, due to their relatively low abundance compared with the vast majority of cellular proteins, currently available proteomics techniques do not permit the comprehensive biochemical characterization of protein kinases. To address these limitations, we have developed a prefractionation strategy that uses a combination of immobilized low molecular weight inhibitors for the selective affinity capture of protein kinases. This approach resulted in the direct purification of cell type-specific sets of expressed protein kinases, and more than 140 different members of this enzyme family could be detected by LC-MS/MS. Furthermore the enrichment technique combined with phosphopeptide fractionation led to the identification of more than 200 different phosphorylation sites on protein kinases, which often remain occluded in global phosphoproteome analysis. As the phosphorylation states of protein kinases can provide a readout for the signaling activities within a cellular system, kinase-selective phosphoproteomics based on the procedures described here has the potential to become an important tool in signal transduction analysis.

    Molecular & cellular proteomics : MCP 2007;6;3;537-47

  • Late signaling in the activated platelets upregulates tyrosine phosphatase SHP1 and impairs platelet adhesive functions: Regulation by calcium and Src kinase.

    Gupta R, Chakrabarti P, Dikshit M and Dash D

    Department of Biochemistry, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005, India.

    Sustained stimulation of platelets with protease-activated receptor agonists in presence of extracellular calcium was associated with tyrosine dephosphorylation of specific proteins of relative mobilities 35, 67, and 75 kDa. From phosphatase assays and inhibitor studies SHP1, a Src homology 2 (SH2) domain-containing tyrosine phosphatase expressed abundantly in hemopoietic cells, was found to be upregulated in platelets between 25 and 30 min following thrombin stimulation. Concomitantly, SHP1 was tyrosine phosphorylated by, and coprecipitated with, Src tyrosine kinase. SHP1 activation, association with Src and dephosphorylation of specific proteins were dependent on extracellular calcium and maintenance of a higher cytosolic calcium plateau. There was progressive impairment of platelet functions like aggregability and clot retraction, associated with downregulation of fibrinogen-binding affinity of integrin alpha(IIb)beta(3), in the platelets exposed to thrombin for 45 min. This could reflect the late physiological changes in platelets when the cells are consistently exposed to stimulatory signals under thrombogenic environment in vivo.

    Biochimica et biophysica acta 2007;1773;2;131-40

  • p27 phosphorylation by Src regulates inhibition of cyclin E-Cdk2.

    Chu I, Sun J, Arnaout A, Kahn H, Hanna W, Narod S, Sun P, Tan CK, Hengst L and Slingerland J

    Braman Family Breast Cancer Institute of the University of Miami Sylvester Comprehensive Cancer Center and Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, FL 33136, USA.

    The kinase inhibitor p27Kip1 regulates the G1 cell cycle phase. Here, we present data indicating that the oncogenic kinase Src regulates p27 stability through phosphorylation of p27 at tyrosine 74 and tyrosine 88. Src inhibitors increase cellular p27 stability, and Src overexpression accelerates p27 proteolysis. Src-phosphorylated p27 is shown to inhibit cyclin E-Cdk2 poorly in vitro, and Src transfection reduces p27-cyclin E-Cdk2 complexes. Our data indicate that phosphorylation by Src impairs the Cdk2 inhibitory action of p27 and reduces its steady-state binding to cyclin E-Cdk2 to facilitate cyclin E-Cdk2-dependent p27 proteolysis. Furthermore, we find that Src-activated breast cancer lines show reduced p27 and observe a correlation between Src activation and reduced nuclear p27 in 482 primary human breast cancers. Importantly, we report that in tamoxifen-resistant breast cancer cell lines, Src inhibition can increase p27 levels and restore tamoxifen sensitivity. These data provide a new rationale for Src inhibitors in cancer therapy.

    Funded by: NCI NIH HHS: R01 CA105118, R01 CA123415, R01-CA105118

    Cell 2007;128;2;281-94

  • Dosage-dependent switch from G protein-coupled to G protein-independent signaling by a GPCR.

    Sun Y, Huang J, Xiang Y, Bastepe M, Jüppner H, Kobilka BK, Zhang JJ and Huang XY

    Department of Physiology, Weill Medical College, Cornell University, New York, NY 10021, USA.

    G-protein-coupled receptors (GPCRs) mostly signal through heterotrimeric G proteins. Increasing evidence suggests that GPCRs could function in a G-protein-independent manner. Here, we show that at low concentrations of an agonist, beta(2)-adrenergic receptors (beta(2)-ARs) signal through Galpha(s) to activate the mitogen-activated protein kinase pathway in mouse embryonic fibroblast cells. At high agonist concentrations, signals are also transduced through beta(2)-ARs via an additional pathway that is G-protein-independent but tyrosine kinase Src-dependent. This new dosage-dependent switch of signaling modes of GPCRs has significant implications for GPCR intrinsic properties and desensitization.

    Funded by: NIA NIH HHS: AG23202, R01 AG023202; NIDDK NIH HHS: P30 DK040561, P30 DK040561-11

    The EMBO journal 2007;26;1;53-64

  • Repetitive deformation activates focal adhesion kinase and ERK mitogenic signals in human Caco-2 intestinal epithelial cells through Src and Rac1.

    Chaturvedi LS, Marsh HM, Shang X, Zheng Y and Basson MD

    Surgical Service, John D. Dingell Veterans Affairs Medical Center, Wayne State University, Detroit, Michigan 48201, USA.

    Intestinal epithelial cells are subject to repetitive deformation during peristalsis and villous motility, whereas the mucosa atrophies during sepsis or ileus when such stimuli are abnormal. Such repetitive deformation stimulates intestinal epithelial proliferation via focal adhesion kinase (FAK) and extracellular signal-regulated kinases (ERK). However, the upstream mediators of these effects are unknown. We investigated whether Src and Rac1 mediate deformation-induced FAK and ERK phosphorylation and proliferation in human Caco-2 and rat IEC-6 intestinal epithelial cells. Cells cultured on collagen-I were subjected to an average 10% cyclic strain at 10 cycles/min. Cyclic strain activated Rac1 and induced Rac1 translocation to cell membranes. Mechanical strain also induced rapid sustained phosphorylation of c-Src at Tyr(418), Rac1 at Ser(71), FAK at Tyr(397) and Tyr(576), and ERK1/2 at Thr(202)/Tyr(204). The mitogenic effect of cyclic strain was blocked by inhibition of Src (PP2 or short interfering RNA) or Rac1 (NSC23766). Src or Rac1 inhibition also prevented strain-induced FAK phosphorylation at Tyr(576) and ERK phosphorylation but not FAK phosphorylation at Tyr(397). Reducing FAK using short interfering RNA blocked strain-induced mitogenicity and attenuated ERK phosphorylation but not Src or Rac1 phosphorylation. Src inhibition blocked strain-induced Rac1 phosphorylation, but Rac inhibition did not alter Src phosphorylation. Transfection of a two-tyrosine phosphorylation-deficient FAK mutant Y576F/Y577F prevented activation of cotransfected myc-ERK2 by cyclic strain. Repetitive deformation induced by peristalsis or villus motility may support the gut mucosa by a pathway involving Src, Rac1, FAK, and ERK. This pathway may present important targets for interventions to prevent mucosal atrophy during prolonged ileus or fasting.

    Funded by: NIDDK NIH HHS: R01 DK067257

    The Journal of biological chemistry 2007;282;1;14-28

  • Loss of Hsp90 association up-regulates Src-dependent ErbB2 activity.

    Xu W, Yuan X, Beebe K, Xiang Z and Neckers L

    Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, 9000 Rockville Pike, Bethesda, MD 20892-1107, USA.

    The receptor tyrosine kinase ErbB2 plays a crucial role in tumorigenesis. We showed previously that the molecular chaperone Hsp90 protects ErbB2 from proteasome-mediated degradation by binding to a short loop structure in the N-lobe of the kinase domain. Here we show that loss of Hsp90 binding correlates with enhanced ErbB2 kinase activity and its transactivating potential, concomitant with constitutively increased phosphorylation of Tyr877, located in the activation loop of the kinase domain. We show further that Tyr877 phosphorylation is mediated by Src and that it is necessary for the enhanced kinase activity of ErbB2. Finally, computer modeling of the kinase domain suggests a phosphorylation-dependent reorientation of the activation loop, denoting the importance of Tyr877 phosphorylation for ErbB2 activity. These findings suggest that Hsp90 binding to ErbB2 participates in regulation of kinase activity as well as kinase stability.

    Molecular and cellular biology 2007;27;1;220-8

  • Signal transducer and activator of transcription 5b: a new target of breast tumor kinase/protein tyrosine kinase 6.

    Weaver AM and Silva CM

    Department of Microbiology, University of Virginia, Charlottesville, VA 22908, USA.

    Introduction: Signal transducers and activators of transcription (STATs) are mediators of cytokine and growth factor signaling. In recent years, STAT5b has emerged as a key regulator of tumorigenesis. STAT5b phosphorylation and activation is mediated by several kinases known to be overexpressed in breast cancer, such as epidermal growth factor receptor, HER2, and c-Src. Breast tumor kinase (Brk), also known as protein tyrosine kinase 6, is a nonreceptor tyrosine kinase expressed in more than 60% of breast cancers. Only a few substrates of the Brk tyrosine kinase have been identified, the most recent being STAT3. In the present article we investigate the potential role of Brk in the phosphorylation and activation STAT5b.

    Methods: To determine whether Brk can phosphorylate STAT5b, transient transfection and in vitro kinase assays were performed. Luciferase reporter assays were used to measure Brk-induced STAT5b transcriptional activity. siRNA technology was utilized to investigate the biological significance of Brk-induced activation of STAT5b in breast cancer cell models.

    Results: Phosphospecific antibodies, mutational analysis, and in vitro kinase assays demonstrated that Brk specifically mediated STAT5b phosphorylation at the activating tyrosine, Y699. Transient transfection of Brk into the Brk-negative BT-549 breast cancer cell line enhanced STAT5b transcriptional activity, as measured by a STAT5-specific luciferase reporter. Furthermore, overexpression of kinase active c-Src enhanced Brk-induced STAT5b transcriptional activity. In Brk-positive breast cancer cell lines BT-20 and SKBr3, knockdown of Brk protein or of STAT5b protein using siRNA methodology resulted in a decrease in DNA synthesis. Knockdown of Brk and STAT5b together did not further decrease DNA synthesis compared with each alone, suggesting that Brk and STAT5b converge on the same pathway, ultimately leading to cellular proliferation.

    Conclusion: Our studies demonstrate that Brk phosphorylates STAT5b on Y699, leading to increased STAT5b transcriptional activity. Furthermore, analysis of DNA synthesis suggests that STAT5b and Brk are converging upon the same proproliferative signaling pathway in breast cancer cells. We propose that Brk, like other tyrosine kinases, signals downstream to STAT5b to mediate proliferation of breast cancer cells. These results further establish STAT5b as well as Brk as potential targets for breast cancer therapy.

    Funded by: NCI NIH HHS: R01 CA085462, R01-CA085462

    Breast cancer research : BCR 2007;9;6;R79

  • PI3-kinase/Akt-dependent antiapoptotic signaling by the PDGF alpha receptor is negatively regulated by Src family kinases.

    Vantler M, Huntgeburth M, Caglayan E, Ten Freyhaus H, Schnabel P and Rosenkranz S

    Klinik III für Innere Medizin, Universität zu Köln, Kerpener Strasse 62, D-50924 Köln, Germany.

    Regulation of growth factor dependent cell survival is crucial for development and disease progression. Here, we report a novel function of Src kinases as a negative regulator of platelet-derived growth factor (PDGF) dependent cell survival. We characterized a series of PDGF alpha receptor (PDGFRA) mutants, which lack the binding sites for Src, phosphatidylinositol 3'-kinase (PI3K), SHP-2 or phospholipase C-gamma. We found that PDGFRA-dependent cell survival was mainly mediated through activation of PI3K, and was negatively regulated by Src. Characterization of the downstream signaling events revealed that PI3K activates the protein kinase Akt, which in turn phosphorylates and thus inactivates proapoptotic Forkhead transcription factors. Src phosphorylates the ubiquitin-ligase c-Cbl, which is required for degradation of the activated receptor. Consequently, overexpression of c-Cbl prevented PDGFRA-mediated cell survival, whereas it did not affect this response, when Src was unable to associate with the receptor. This novel function of Src in antiapoptotic signaling introduces Src kinases as an interesting therapeutic target in apoptosis related diseases.

    FEBS letters 2006;580;30;6769-76

  • Proline-rich motifs in the parathyroid hormone (PTH)/PTH-related protein receptor C terminus mediate scaffolding of c-Src with beta-arrestin2 for ERK1/2 activation.

    Rey A, Manen D, Rizzoli R, Caverzasio J and Ferrari SL

    Service of Bone Diseases, Department of Rehabilitation and Geriatrics, University Hospital, 1211 Geneva 14, Switzerland.

    Parathyroid hormone (PTH) stimulates ERK1/2 through both G-protein signaling and beta-arrestin2-mediated internalization. Beta-arrestin may serve as a scaffold for c-Src. However, the molecular mechanisms for ERK1/2 activation by PTH remain unclear. By using a targeted mutagenesis approach, we investigated the PTH/PTH-related protein receptor (PTH1R) structural determinants for ERK1/2 activation and transcriptional activity in HEK-293 cells. First, ERK1/2 activation was inhibited by PTH1R mutations that specifically abrogate G(q)-protein kinase C signaling without a decrease in cAMP-protein kinase A. Second, PTH1R C-terminal mutations and/or deletions that prevent interaction with beta-arrestin inhibited ERK1/2 activation. Similar results were obtained in HEK-293 cells co-expressing wild-type PTH1R and a dominant-negative beta-arrestin2. Third, the c-Src inhibitor PP2 and a kinase-dead c-SrcK295M mutant co-expressed with wild-type PTH1R both inhibited ERK1/2 activation. Furthermore, c-Src co-precipitated with both PTH1R and beta-arrestin2 in response to PTH. Deleting the PTH1R-proximal C terminus abolished these interactions. However, the need for receptor interaction with beta-arrestin to co-precipitate Src and activate ERK1/2 was obviated by expressing a constitutively active c-SrcY527A mutant, suggesting direct binding of activated Src to PTH1R. Subsequently, we identified and mutated to alanine four proline-rich motifs in the PTH1R distal C terminus, which resulted in loss of both c-Src and arrestin co-precipitation and significantly decreased ERK1/2 activation. These data delineate the multiple PTH1R structural determinants for ERK1/2 activation and newly identify a unique mechanism involving proline-rich motifs in the receptor C terminus for reciprocal scaffolding of c-Src and beta-arrestin2 with a class II G-protein-coupled receptor.

    The Journal of biological chemistry 2006;281;50;38181-8

  • SRC tail phosphorylation is limited by structural changes in the regulatory tyrosine kinase Csk.

    Lieser SA, Shaffer J and Adams JA

    Department of Chemistry and Biochemistry and Pharmacology, University of California, San Diego, La Jolla, California 92093, USA.

    Src family tyrosine kinases are down-regulated through phosphorylation of a single C-terminal tyrosine by the nonreceptor tyrosine kinase Csk. Despite the fundamental role of Csk in controlling cell growth and differentiation, it is unclear what limits this key signaling reaction and controls the production of catalytically repressed Src. To investigate this issue, stopped-flow fluorescence experiments were performed to determine which steps modulate catalysis. Both Src binding and phosphorylation can be monitored by changes in intrinsic tryptophan fluorescence. Association kinetics are biphasic with the initial phase corresponding to the bimolecular interaction of both proteins and the second phase representing a slow conformational change that coincides with the rate of maximum turnover. The kinetic transients for the phosphorylation reaction are also biphasic with the initial phase corresponding to the rapid phosphorylation and the release of phospho-Src. These data, along with equilibrium sedimentation and product inhibition experiments, suggest that steps involving Src association, phosphorylation, and product release are fast and that a structural change in Csk participates in limiting the catalytic cycle.

    Funded by: NIGMS NIH HHS: GM68168

    The Journal of biological chemistry 2006;281;49;38004-12

  • Force sensing by mechanical extension of the Src family kinase substrate p130Cas.

    Sawada Y, Tamada M, Dubin-Thaler BJ, Cherniavskaya O, Sakai R, Tanaka S and Sheetz MP

    Department of Biological Sciences, Columbia University, Sherman Fairchild Center Room 715, MC-2416, 1212 Amsterdam Avenue, New York, NY 10027, USA. ys454-ind@umin.ac.jp

    How physical force is sensed by cells and transduced into cellular signaling pathways is poorly understood. Previously, we showed that tyrosine phosphorylation of p130Cas (Cas) in a cytoskeletal complex is involved in force-dependent activation of the small GTPase Rap1. Here, we mechanically extended bacterially expressed Cas substrate domain protein (CasSD) in vitro and found a remarkable enhancement of phosphorylation by Src family kinases with no apparent change in kinase activity. Using an antibody that recognized extended CasSD in vitro, we observed Cas extension in intact cells in the peripheral regions of spreading cells, where higher traction forces are expected and where phosphorylated Cas was detected, suggesting that the in vitro extension and phosphorylation of CasSD are relevant to physiological force transduction. Thus, we propose that Cas acts as a primary force sensor, transducing force into mechanical extension and thereby priming phosphorylation and activation of downstream signaling.

    Funded by: NIBIB NIH HHS: R01 EB001480, R01 EB001480-03

    Cell 2006;127;5;1015-26

  • Curcumin regulates signal transducer and activator of transcription (STAT) expression in K562 cells.

    Blasius R, Reuter S, Henry E, Dicato M and Diederich M

    Laboratoire de Biologie Moléculaire et Cellulaire du Cancer, Hôpital Kirchberg, 9 Rue Edward Steichen, L-2540 Luxembourg, Luxembourg.

    Signal transducers and activators of transcription (STATs) play important roles in numerous cellular events as for example differentiation, inflammation or immune response. Furthermore, constitutive STAT activation can be observed in a high number of tumors. In our hands, curcumin treatment induced a decrease of nuclear STAT3, -5a and -5b, without affecting neither STAT1, nor the phosphorylation state of STAT1, -3 or -5 in the K562 cell line. Most interestingly, the decrease of nuclear STAT5a and -5b after curcumin treatment was accompanied by an increase of truncated STAT5 isoforms, indicating that curcumin is able to induce the cleavage of STAT5 into its dominant negative variants lacking the STAT5 C-terminal region. Interferon (IFN)-beta and -gamma treatment induced IFN-stimulated responsive element (ISRE) transcriptional activity, which was efficiently inhibited by curcumin pre-treatment. In parallel, IFN-gamma treatment induced an increase of the amount of nuclear STAT1 and -3, as well as their phosphorylated isoforms. Again, curcumin pre-treatment inhibited these increases. Finally, curcumin treatment inhibited Jak2 mRNA expression as well as cyclin D1 and v-src gene expression in K562 chronic leukaemia cells.

    Biochemical pharmacology 2006;72;11;1547-54

  • Regulation of RKIP binding to the N-region of the Raf-1 kinase.

    Park S, Rath O, Beach S, Xiang X, Kelly SM, Luo Z, Kolch W and Yeung KC

    Medical University of Ohio, Department of Biochemistry and Cancer Biology, 3035 Arlington Avenue, Toledo, OH 43614-5804, USA.

    The Raf kinase inhibitory protein (RKIP) binds to Raf-1 interfering with binding of the MEK substrate and potentially also Raf-1 activation. In response to mitogen stimulation RKIP dissociates from Raf-1 and later re-associates. Here, using a combination of mutational approaches, biochemical studies, peptide arrays and plasmon surface resonance (BIAcore), we fine map and characterize a minimal 24 amino acid long RKIP binding domain in the Raf-1 N-region, which consists of constitutive elements at both flanks and a center element that is regulated by phosphorylation and enhances the re-binding of RKIP to Raf-1 in the later phase of mitogen stimulation.

    Funded by: NIGMS NIH HHS: R01 GM057959, R01 GM064767, R01 GM64767

    FEBS letters 2006;580;27;6405-12

  • Regulation of c-Src activity by the expression of wild-type v-Src and its kinase-dead double Y416F-K295N mutant.

    Vojtechová M, Senigl F, Sloncová E and Tuhácková Z

    Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo n. 2, 166 37 Praha 6, Czech Republic. vojtech@img.cas.cz

    Active, wild-type v-Src and its kinase-dead double Y416F-K295N mutant were expressed in hamster fibroblasts. Expression of the active v-Src induced activation of endogenous c-Src and increased general protein-tyrosine phosphorylation in the infected cells. Expression of the kinase-dead mutant induced hypophosphorylation of Tyr416 of the endogenous c-Src. The inactivation of c-Src was reversible, as confirmed by in vitro kinase activity of c-Src immunoprecipitated from the kinase-dead v-Src-expressing cells. Both activation and inactivation of c-Src may be explained by direct interaction of the v-Src and c-Src that may either facilitate transphosphorylation of the regulatory Tyr416 in the activation loop, or prevent it by formation of transient dead-end complexes of the Y416F-K295N mutant with c-Src. The interaction was also indicated by co-localization of v- and c-Src proteins in immunofluorescent images of the infected cells. These results suggest that dimerization of Src plays an important role in the regulation of Src tyrosine kinase activity.

    Archives of biochemistry and biophysics 2006;455;2;136-43

  • Tyrosine phosphorylation of p145met mediated by EGFR and Src is required for serum-independent survival of human bladder carcinoma cells.

    Yamamoto N, Mammadova G, Song RX, Fukami Y and Sato K

    Graduate School of Science and Technology, Kobe University, Nada, Kobe, Japan.

    Here we address the molecular mechanism of serum-independent survival and growth of human bladder carcinoma cell line 5637. Serum starvation promoted tyrosine phosphorylation of a 145-kDa protein and activation of the tyrosine kinase Src and the receptor for epidermal growth factor (EGFR) over a slow time course (>8 hours). The phosphorylated 145-kDa protein was identified as the beta-subunit of c-Met/hepatocyte growth factor (HGF) receptor, p145(met), in which tyrosine residues 1003, 1234, and 1235 were phosphorylated. Inhibitors of Src (PP2, SU6656) or EGFR (AG99), but not p145(met) (K252a), effectively blocked tyrosine phosphorylation of p145(met) and promoted cell death accompanied by activation of caspase-like proteases. Conditioned medium from the serum-starved 5637 cells or purified EGF readily promoted the activation of Src and EGFR, and tyrosine phosphorylation of p145(met) in normally grown 5637 cells, suggesting that autocrine signaling of EGFR ligands is responsible for signal transduction events in serum-starved cells. Consistent with this idea, a monoclonal antibody against EGFR that would interfere with the ligand binding to EGFR blocked tyrosine phosphorylation events and promoted the caspase activation and cell death in serum-free conditions. Such apoptotic cell death was also induced by pretreatment of cells with a high concentration of HGF that downregulated endogenous p145(met). Nevertheless, Cu2+ ions, competitive inhibitors for HGF-binding to p145(met), did not show any effect on cellular functions in serum-free conditions. These results suggest that the serum-independent growth of 5637 cells involves the transmembrane signaling cascade via EGFR ligand(s) (but not HGF), EGFR, Src and p145(met).

    Journal of cell science 2006;119;Pt 22;4623-33

  • Phosphorylation of Tyr1214 within VEGFR-2 triggers the recruitment of Nck and activation of Fyn leading to SAPK2/p38 activation and endothelial cell migration in response to VEGF.

    Lamalice L, Houle F and Huot J

    Centre de Recherche en Cancérologie de l'Université Laval, 9 rue McMahon, Québec G1R 2J6, Canada.

    VEGFR-2 is the major receptor that regulates the different functions of VEGF in adults. We have previously reported that following VEGF treatment of endothelial cells, VEGFR-2 is phosphorylated on Tyr1214 upstream of the Cdc42-SAPK2/p38-MAPKAP K2 pathway. However, little is known of the earliest molecular events that compose the SAPK2/p38 pathway following VEGFR-2 activation. In this study, we address this question using HA-tagged constructs of either wild-type VEGFR-2 or Y1214F VEGFR-2 mutant in immunoprecipitation assays. We show that the Src family kinase member Fyn, but not c-Src itself, is recruited to VEGFR-2 and is activated in a p-Tyr1214-dependent manner. We also report that the SH2 domain-containing adapter molecule Nck, but not Grb2, is recruited to VEGFR-2 in a p-Tyr1214-dependent manner and that it associates with Fyn. Moreover, PAK-2 is phosphorylated in a Fyn-dependent manner. Using chemical and genetic inhibitors, we show that Fyn activity is required for SAPK2/p38 but not for FAK activation in response to VEGF. In contrast, c-Src permits activation of FAK, but not that of SAPK2/p38. In addition, Fyn is required for stress fiber formation and endothelial cell migration. We propose a model in which Fyn forms a molecular complex with Nck and PAK-2 and suggest that this complex assembles in a p-Tyr1214-dependent manner within VEGFR-2 following VEGF treatment. In turn, this triggers the activation of the SAPK2/p38 MAP kinase module, and promotes stress fiber formation and endothelial cell migration.

    The Journal of biological chemistry 2006;281;45;34009-20

  • PSD-95 is a negative regulator of the tyrosine kinase Src in the NMDA receptor complex.

    Kalia LV, Pitcher GM, Pelkey KA and Salter MW

    Division of Neurology, Department of Medicine, University of Toronto, Toronto, Ontario, Canada.

    The tyrosine kinase Src upregulates the activity of the N-methyl-D-aspartate subtype of glutamate receptor (NMDAR) and tyrosine phosphorylation of this receptor is critical for induction of NMDAR-dependent plasticity of synaptic transmission. A binding partner for Src within the NMDAR complex is the protein PSD-95. Here we demonstrate an interaction of PSD-95 with Src that does not require the well-characterized domains of PSD-95. Rather, we show binding to Src through a 12-amino-acid sequence in the N-terminal region of PSD-95, a region not previously known to participate in protein-protein interactions. This region interacts directly with the Src SH2 domain. Contrary to typical SH2 domain binding, the PSD-95-Src SH2 domain interaction is phosphotyrosine-independent. Binding of the Src-interacting region of PSD-95 inhibits Src kinase activity and reduces NMDAR phosphorylation. Intracellularly administering a peptide matching the Src SH2 domain-interacting region of PSD-95 depresses NMDAR currents in cultured neurons and inhibits induction of long-term potentiation in hippocampus. Thus, the PSD-95-Src SH2 domain interaction suppresses Src-mediated NMDAR upregulation, a finding that may be of broad importance for synaptic transmission and plasticity.

    The EMBO journal 2006;25;20;4971-82

  • Perlecan proteolysis induces an alpha2beta1 integrin- and Src family kinase-dependent anti-apoptotic pathway in fibroblasts in the absence of focal adhesion kinase activation.

    Laplante P, Raymond MA, Labelle A, Abe J, Iozzo RV and Hébert MJ

    Centre de Recherche du Centre Hospitalier de l'Université de Montreal, University of Montreal, 1560 Sherbrooke East, Montreal, Quebec H2L 4M1, Canada.

    Dysregulation of apoptosis in endothelial cells (EC) and fibroblasts contributes to fibrosis. We have shown previously that apoptosis of EC triggers the proteolysis of extracellular matrix components and the release of a C-terminal fragment of perlecan, which in turn inhibits apoptosis of fibroblasts. Here we have defined the receptors and pathways implicated in this anti-apoptotic response in fibroblasts. Neutralizing alpha2beta1 integrin activity in fibroblasts exposed to either medium conditioned by apoptotic EC (SSC) or a recombinant perlecan C-terminal fragment (LG3) prevented resistance to apoptosis and is associated with decreased levels of Akt phosphorylation. Co-incubation of fibroblasts for 24 h with SSC or LG3 in the presence of PP2 (AG1879), a biochemical inhibitor of Src family kinases (SFKs) and focal adhesion kinase, showed a significantly decreased anti-apoptotic response. However, focal adhesion kinase gene silencing with RNA interference did not inhibit the anti-apoptotic response in fibroblasts. Src phosphorylation was increased in fibroblasts exposed to SSC, and transfection of fibroblasts with constitutively active Src mutants induced an anti-apoptotic response that was not further increased by SSC. Also, Src(-/-)Fyn(-/-) fibroblasts failed to mount an anti-apoptotic response in presence of SSC for 24 h but developed a complete anti-apoptotic response when exposed to SSC for 7 days. These results suggest that extracellular matrix fragments produced by apoptotic EC initiate a state of resistance to apoptosis in fibroblasts via an alpha2beta1 integrin/SFK (Src and Fyn)/phosphatidylinositol 3-kinase (PI3K)-dependent pathway. In the long term, additional SFK members are recruited for sustaining the anti-apoptotic response, which could play crucial roles in abnormal fibrogenic healing.

    The Journal of biological chemistry 2006;281;41;30383-92

  • Differential effects of Stat3 inhibition in sparse vs confluent normal and breast cancer cells.

    Anagnostopoulou A, Vultur A, Arulanandam R, Cao J, Turkson J, Jove R, Kim JS, Glenn M, Hamilton AD and Raptis L

    Department of Microbiology, Queen's University, Kingston, Ont., Canada K7L3N6.

    The signal transducer and activator of transcription-3 (Stat3) is persistently activated in many cancers such as cancer of the breast and is required for transformation by a number of oncogenes. Signaling through Stat3 is determined by a key phosphorylation at tyr-705. We previously demonstrated that cell-to-cell adhesion brought about through cell aggregation or confluence of cultured cells causes a dramatic increase in Stat3 tyr705 phosphorylation and consequently Stat3 activity in both normal and tumor cells. To examine the role of Stat3 at specific time-points relative to confluence, we used two different approaches of Stat3 inhibition: (1). Introduction of high levels of peptide analogues, which block the Stat3-SH2 domain, to inhibit Stat3 binding to and phosphorylation by growth factor receptors. (2). Treatment with two platinum compounds which bind the Stat3 protein and inhibit its activity without affecting its phosphorylation directly. The results demonstrate that Stat3 downregulation in vSrc transformed NIH3T3 cells or in breast cancer lines harboring activated Src induces apoptosis, which is evident at all densities but is more pronounced at post-confluence. In normal cells on the other hand, Stat3 inhibition at post-confluence caused apoptosis while in sparsely growing cells it induced merely a growth retardation.

    Cancer letters 2006;242;1;120-32

  • Underexpressed coactivators PGC1alpha and SRC1 impair hepatocyte nuclear factor 4 alpha function and promote dedifferentiation in human hepatoma cells.

    Martínez-Jiménez CP, Gómez-Lechón MJ, Castell JV and Jover R

    Unidad de Hepatología Experimental, Centro de Investigación, Hospital Universitario La Fe, 46009 Valencia, Spain.

    Hepatocyte nuclear factor 4alpha (HNF4alpha) plays critical roles during liver development and in the transcriptional regulation of many hepatic genes in adult liver. Here we have demonstrated that in human hepatoma HepG2 cells, HNF4alpha is expressed at levels as high as in human liver but its activity on target genes is very low or absent. We have discovered that the low expression of key coactivators (PGC1alpha, SRC1, SRC2, and PCAF) might account for the lack of function of HNF4alpha in HepG2 cells. Among them, PGC1alpha and SRC1 are the two most important HNF4alpha coactivators as revealed by reporter assays with an Apo-CIII promoter construct. Moreover, the expression of these two coactivators was found to be down-regulated in all human hepatomas investigated. Overexpression of SRC1 and PGC1alpha by recombinant adenoviruses led to a significant up-regulation of well characterized HNF4alpha-dependent genes (ApoCIII, ApoAV, PEPCK, AldoB, OTC, and CYP7A1) and forced HepG2 cells toward a more differentiated phenotype as demonstrated by increased ureogenic rate. The positive effect of PGC1alpha was seen to be dependent on HNF4alpha. Finally, insulin treatment of human hepatocytes and HepG2 cells caused repression of PGC1alpha and a concomitant down-regulation of ApoCIII, PEPCK, AldoB, and OTC. Altogether, our results suggest that SRC1, and notably PGC1alpha, are key coactivators for the proper function of HNF4alpha in human liver and for an integrative control of multiple hepatic genes involved in metabolism and homeostasis. The down-regulation of key HNF4alpha coactivators could be a determinant factor for the dedifferentiation of human hepatomas.

    The Journal of biological chemistry 2006;281;40;29840-9

  • Magicin associates with the Src-family kinases and is phosphorylated upon CD3 stimulation.

    Lee MF, Beauchamp RL, Beyer KS, Gusella JF and Ramesh V

    Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA 02114, USA.

    We recently identified a novel actin cytoskeleton-associated protein magicin, for merlin and Grb2 interacting cytoskeletal protein. To unravel the cellular functions of magicin, we used a yeast two-hybrid system and identified Fyn tyrosine kinase as a specific binding partner for magicin. Fyn phosphorylates magicin in vitro. In addition to Fyn, Src and Lck also interact with magicin. Upon stimulation with anti-CD3 antibody, magicin is phosphorylated in the T lymphocyte leukemia Jurkat cell line. Magicin phosphorylation is not observed in an Lck-deficient line, J.CaM1.6, indicating that Lck is the major Src family kinase for phosphorylating magicin in Jurkat cells. Employing site-directed mutagenesis along with in vitro kinase assays, we found that Y64 of magicin is phosphorylated by Lck creating a SH2-Grb2 binding motif. Magicin has also been identified as a Mediator subunit (MED28) in the nucleus involved in transcriptional regulation, therefore we propose that magicin may serve as a multi-faceted adaptor/scaffold to relay cellular signaling to the cytoskeleton and from the cytoskeleton to the nucleus.

    Funded by: NINDS NIH HHS: NS24279

    Biochemical and biophysical research communications 2006;348;3;826-31

  • v-Src-dependent down-regulation of the Ste20-like kinase SLK by casein kinase II.

    Chaar Z, O'reilly P, Gelman I and Sabourin LA

    Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario K1H8L6, Canada.

    We have previously shown that the Ste20-like kinase SLK is a microtubule-associated protein inducing actin stress fiber disassembly. Here, we show that v-Src expression can down-regulate SLK activity. This down-regulation is independent of focal adhesion kinase but requires v-Src kinase activity and membrane translocation. SLK down-regulation by v-Src is indirect and is accompanied by SLK hyperphosphorylation on serine residues. Deletion analysis revealed that casein kinase II (CK2) sites at position 347/348 are critical for v-Src-dependent modulation of SLK activity. Further studies show that CK2 can directly phosphorylate SLK at these positions and that inhibition of CK2 in v-Src-transformed cells results in normal kinase activity. Finally, CK2 and SLK can be co-localized in fibroblasts spreading on fibronectin-coated substrates, suggesting a mechanism whereby SLK may be regulated at sites of actin remodeling, such as membrane lamellipodia and ruffles, through CK2.

    The Journal of biological chemistry 2006;281;38;28193-9

  • HIV-1 Nef selectively activates Src family kinases Hck, Lyn, and c-Src through direct SH3 domain interaction.

    Trible RP, Emert-Sedlak L and Smithgall TE

    Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.

    Nef is an HIV-1 virulence factor that promotes viral pathogenicity by altering host cell signaling pathways. Nef binds several members of the Src kinase family, and these interactions have been implicated in the pathogenesis of HIV/AIDS. However, the direct effect of Nef interaction on Src family kinase (SFK) regulation and activity has not been systematically addressed. We explored this issue using Saccharomyces cerevisiae, a well defined model system for the study of SFK regulation. Previous studies have shown that ectopic expression of c-Src arrests yeast cell growth in a kinase-dependent manner. We expressed Fgr, Fyn, Hck, Lck, Lyn, and Yes as well as c-Src in yeast and found that each kinase was active and induced growth suppression. Co-expression of the negative regulatory kinase Csk suppressed SFK activity and reversed the growth-inhibitory effect. We then co-expressed each SFK with HIV-1 Nef in the presence of Csk. Nef strongly activated Hck, Lyn, and c-Src but did not detectably affect Fgr, Fyn, Lck, or Yes. Mutagenesis of the Nef PXXP motif essential for SH3 domain binding greatly reduced the effect of Nef on Hck, Lyn, and c-Src, suggesting that Nef activates these Src family members through allosteric displacement of intramolecular SH3-linker interactions. These data show that Nef selectively activates Hck, Lyn, and c-Src among SFKs, identifying these kinases as proximal effectors of Nef signaling and potential targets for anti-HIV drug discovery.

    Funded by: NCI NIH HHS: CA81398, R01 CA081398, R01 CA081398-07; NIAID NIH HHS: AI057083, R01 AI057083

    The Journal of biological chemistry 2006;281;37;27029-38

  • Screening of genetic and expression alterations of SRC1 gene in prostate cancer.

    Mäki HE, Waltering KK, Wallén MJ, Martikainen PM, Tammela TL, van Weerden WM, Vessella RL and Visakorpi T

    Cancer Genetics, Institute of Medical Technology, University of Tampere and Tampere University Hospital, Tampere, Finland.

    Background: Genetic alterations of the SRC1 gene have not been thoroughly studied in prostate cancer.

    Five prostate cancer cell lines and 32 xenografts were screened for mutations and gene copy number alterations. Subsequently, frequencies of detected sequence variations were further analyzed in 44 clinical prostate cancers, 6 benign prostate hyperplasias, and 48 normal controls. Finally, the protein expression of SRC1 in 254 clinical prostate tumors was investigated.

    Results: Three non-recurrent sequence variations, and one single nucleotide polymorphism in the coding region of SRC1, as well as one case of SRC1 gene amplification were found. The protein expression of SRC1 was higher in androgen ablation resistant than untreated prostate carcinomas, but the difference was not statistically significant (P = 0.0796).

    Conclusions: Genetic alterations of SRC1 are rare in prostate cancer. The nuclear protein accumulation of SRC1 seems to be mildly increased in androgen ablation resistant prostate cancers. .

    The Prostate 2006;66;13;1391-8

  • Csk-binding protein (Cbp) negatively regulates epidermal growth factor-induced cell transformation by controlling Src activation.

    Jiang LQ, Feng X, Zhou W, Knyazev PG, Ullrich A and Chen Z

    Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.

    Epidermal growth factor receptor (EGFR) and Src tyrosine kinase cooperate in regulating EGFR-mediated cell signaling and promoting cell transformation and tumorigenesis in pathological conditions. Activation of Src is tightly regulated by the C-terminal Src kinase (Csk). The Csk-binding protein (Cbp) is a ubiquitously expressed transmembrane protein. Its functions include suppression of T-cell receptor activation through recruiting Csk and inhibiting Src family kinase (SFK). However, a potential role of Cbp in EGF-induced cell activities has not been investigated. Here, we report that EGF-stimulation-induced Cbp tyrosine phosphorylation followed by Cbp-Csk association, in a SFK-dependent manner. Expression of wild-type (wt) Cbp remarkably suppressed EGF-induced activation of Src, ERK1/2, and Akt-1 enzymes, and NIH3T3 cell transformation, as well as colony formation of a breast cancer cell line (MDA-MB-468) in soft agar. In contrast, expression of CbpY317F or knockdown endogenous Cbp in NIH3T3 cells by RNA interference significantly enhanced EGF-induced activation of these enzymes and cell transformation. In addition, overexpression of multiple receptor tyrosine kinases (RTKs)-induced Cbp tyrosine phosphorylation. These results demonstrate that Cbp functions as a negative regulator of cell transformation and tumor cell growth through downregulation of Src activation, suggesting that Cbp might be broadly involved in RTKs-activated signaling pathways and tumorigenesis.

    Oncogene 2006;25;40;5495-506

  • The role of Src kinase in insulin-like growth factor-dependent mitogenic signaling in vascular smooth muscle cells.

    Lieskovska J, Ling Y, Badley-Clarke J and Clemmons DR

    Department of Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7170, USA.

    Activation of the MAPK pathway mediates insulin-like growth factor-I (IGF-I)-dependent proliferation in vascular smooth muscle cells (SMC). Our previous studies have shown that IGF-I-induced Shc phosphorylation is necessary for sustained activation of MAPK and increased cell proliferation of SMCs, and both Shc and the tyrosine phosphatase SHP-2 must be recruited to the membrane protein SHPS-1 in order for Shc to be phosphorylated. These studies were undertaken to determine whether Src kinase activity is required to phosphorylate Shc in response to IGF-I in SMC and because SHP-2 binds to Src whether their interaction was also required for IGF-I-stimulated mitogenesis. Our results show that IGF-I induces activation of Src kinase and that is required for Shc phosphorylation and for optimal MAPK activation. We tested whether Shc is a substrate of c-Src in SMC by disrupting Src/Shc association using a peptide containing a YXXL (Tyr328) motif sequence derived from Src. The peptide blocked the binding of Src and Shc in vitro and in vivo. Cells expressing a mutant Src (Src-FF) that had Tyr328/Tyr358 substituted with phenylalanines (Src-FF) showed defective Src/Shc binding, impaired IGF-I-dependent Shc phorylation, and impaired mitogenesis. This supports the conclusion that Src phosphorylates Shc. IGF-I induced both Src/SHP-2 and Src/SHPS-1 association. SMCs expressing an SHP-2 mutant that had the polyproline-rich region of SH2 deleted (SHP-2Delta10) had disrupted SHP-2/Src association, impaired IGF-I-dependent Shc phosphorylation, and an attenuated mitogenic response. IGF-I-induced association of Src and SHPS-1 was also impaired in SHP-2Delata10-expressing cells, although SHP-2/SHPS-1 association was unaffected. Upon IGF-I stimulation, a complex assembles on SHPS-1 that contains SHP-2, c-Src, and Shc wherein Src phosphorylates Shc, a signaling step that is necessary for an optimal mitogenic response.

    Funded by: NIA NIH HHS: AG-02331

    The Journal of biological chemistry 2006;281;35;25041-53

  • Antiangiogenic and antitumor effects of SRC inhibition in ovarian carcinoma.

    Han LY, Landen CN, Trevino JG, Halder J, Lin YG, Kamat AA, Kim TJ, Merritt WM, Coleman RL, Gershenson DM, Shakespeare WC, Wang Y, Sundaramoorth R, Metcalf CA, Dalgarno DC, Sawyer TK, Gallick GE and Sood AK

    Department of Gynecologic Oncology, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA.

    Src, a nonreceptor tyrosine kinase, is a key mediator for multiple signaling pathways that regulate critical cellular functions and is often aberrantly activated in a number of solid tumors, including ovarian carcinoma. The purpose of this study was to determine the role of activated Src inhibition on tumor growth in an orthotopic murine model of ovarian carcinoma. In vitro studies on HeyA8 and SKOV3ip1 cell lines revealed that Src inhibition by the Src-selective inhibitor, AP23846, occurred within 1 hour and responded in a dose-dependent manner. Furthermore, Src inhibition enhanced the cytotoxicity of docetaxel in both chemosensitive and chemoresistant ovarian cancer cell lines, HeyA8 and HeyA8-MDR, respectively. In vivo, Src inhibition by AP23994, an orally bioavailable analogue of AP23846, significantly decreased tumor burden in HeyA8 (P = 0.02), SKOV3ip1 (P = 0.01), as well as HeyA8-MDR (P < 0.03) relative to the untreated controls. However, the greatest effect on tumor reduction was observed in combination therapy with docetaxel (P < 0.001, P = 0.002, and P = 0.01, for the above models, respectively). Proliferating cell nuclear antigen staining showed that Src inhibition alone (P = 0.02) and in combination with docetaxel (P = 0.007) significantly reduced tumor proliferation. In addition, Src inhibition alone and in combination with docetaxel significantly down-regulated tumoral production of vascular endothelial growth factor and interleukin 8, whereas combination therapy decreased the microvessel density (P = 0.02) and significantly affected vascular permeability (P < 0.05). In summary, Src inhibition with AP23994 has potent antiangiogenic effects and significantly reduces tumor burden in preclinical ovarian cancer models. Thus, Src inhibition may be an attractive therapeutic approach for patients with ovarian carcinoma.

    Funded by: NCI NIH HHS: 2P50CA083639-06A1, CA10929801, CA11079301, P50 CA083639, R01 CA109298, R01 CA110793, U54 CA090810

    Cancer research 2006;66;17;8633-9

  • Nicotine induces cell proliferation by beta-arrestin-mediated activation of Src and Rb-Raf-1 pathways.

    Dasgupta P, Rastogi S, Pillai S, Ordonez-Ercan D, Morris M, Haura E and Chellappan S

    Department of Interdisciplinary Oncology, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa 33612, USA.

    Recent studies have shown that nicotine, a component of cigarette smoke, can stimulate the proliferation of non-neuronal cells. While nicotine is not carcinogenic by itself, it has been shown to induce cell proliferation and angiogenesis. Here we find that mitogenic effects of nicotine in non-small cell lung cancers (NSCLCs) are analogous to those of growth factors and involve activation of Src, induction of Rb-Raf-1 interaction, and phosphorylation of Rb. Analysis of human NSCLC tumors show enhanced levels of Rb-Raf-1 complexes compared with adjacent normal tissue. The mitogenic effects of nicotine were mediated via the alpha7-nAChR subunit and resulted in enhanced recruitment of E2F1 and Raf-1 on proliferative promoters in NSCLC cell lines and human lung tumors. Nicotine stimulation of NSCLC cells caused dissociation of Rb from these promoters. Proliferative signaling via nicotinic acetylcholine receptors (nAChRs) required the scaffolding protein beta-arrestin; ablation of beta-arrestin or disruption of the Rb-Raf-1 interaction blocked nicotine-induced proliferation of NSCLCs. Additionally, suppression of beta-arrestin also blocked activation of Src, suppressed levels of phosphorylated ERK, and abrogated Rb-Raf-1 binding in response to nicotine. It appears that nicotine induces cell proliferation by beta-arrestin-mediated activation of the Src and Rb-Raf-1 pathways.

    Funded by: NCI NIH HHS: CA63136, R01 CA063136

    The Journal of clinical investigation 2006;116;8;2208-2217

  • Role of c-Src in human MCF7 breast cancer cell tumorigenesis.

    González L, Agulló-Ortuño MT, García-Martínez JM, Calcabrini A, Gamallo C, Palacios J, Aranda A and Martín-Pérez J

    Instituto de Investigaciones Biomédicas Alberto Sols (CSIC/UAM), Madrid, Spain.

    To study the role of c-Src in breast cancer tumorigenesis, we generated a cell line derived from MCF7 carrying an inducible dominant negative c-Src (c-SrcDN: K295M/Y527F) under tetracycline control (Tet-On system). c-SrcDN expression caused phenotypic changes, relocation of c-Src, Fak, and paxillin, and loss of correct actin fiber assembly. These alterations were coupled to increased Fak-Tyr(397) autophosphorylation and to inhibition of Fak-Tyr(925), p130(CAS), and paxillin phosphorylation. An increased association of total Src with Fak and a decreased interaction of p130(CAS) and p85-PI3K with Fak were also observed. SrcDN inhibited cell attachment, spreading, and migration. Serum and EGF-induced stimulation of cell proliferation and Akt phosphorylation were also significantly reduced by SrcDN, whereas p27(Kip1) expression was increased. Consistently, silencing c-Src expression by siRNA in MCF7 cells significantly reduced cell migration, attachment, spreading and proliferation. Inoculation of MCF7 cells carrying inducible SrcDN to nude mice generated tumors. However, doxycycline administration to mice significantly reduced tumorigenesis, and when doxycycline treatment was installed after tumor development, a significant tumor regression was observed. In both situations, inhibition of tumorigenesis was associated with decreased Ki67 staining and increased apoptosis in tumors. These data undoubtedly demonstrate the relevance of the Src/Fak complex in breast cancer tumorigenesis.

    The Journal of biological chemistry 2006;281;30;20851-64

  • Hsp90 inhibition transiently activates Src kinase and promotes Src-dependent Akt and Erk activation.

    Koga F, Xu W, Karpova TS, McNally JG, Baron R and Neckers L

    Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, Building 10, Room 1-5940, Bethesda, MD 20892-1107, USA.

    Hsp90 plays an essential role in maintaining stability and activity of its clients, including oncogenic signaling proteins that regulate key signal transduction nodes. Hsp90 inhibitors interfere with diverse signaling pathways by destabilizing and attenuating activity of such proteins, and thus they exhibit antitumor activity. However, Hsp90 inhibition has recently been reported to activate Akt and Erk and potentiate Akt activation induced by insulin-like growth factor 1 and insulin, raising the concern that clinical use of Hsp90 inhibitors might promote tumor progression under certain circumstances. Here, we show that the prototypical Hsp90 inhibitor geldanamycin induces Akt and Erk activation that is independent of PTEN status and is mediated by transient activation of Src kinase. Activated Src phosphorylates Cbl, which recruits the p85 subunit of phosphatidylinositol 3-kinase, resulting in phosphatidylinositol 3-kinase activation and eventually the activation of Akt and Erk. We show that geldanamycin rapidly disrupts Src association with Hsp90, suggesting that Src activation results directly from dissociation of the chaperone. These data suggest that, under certain circumstances, dual inhibition of Hsp90 and Src may be warranted.

    Proceedings of the National Academy of Sciences of the United States of America 2006;103;30;11318-22

  • Regulation of neuronal nitric-oxide synthase activity by somatostatin analogs following SST5 somatostatin receptor activation.

    Cordelier P, Estève JP, Najib S, Moroder L, Vaysse N, Pradayrol L, Susini C and Buscail L

    INSERM U531, IFR31, Centre Hospitalier Universitaire Rangueil, 31432 Toulouse Cedex 4, France. pierre.cordelier@toulouse.inserm.fr

    Somatostatin receptor SST5 is an inhibitory G protein-coupled receptor that exerts a strong cytostatic effect on various cell types. We reported previously that the SST5 anti-proliferative effect results in the inhibition of mitogen-induced increases in intracellular cGMP levels and MAPK activity. This study was conducted to define the early molecular events accountable for the SST5-mediated anti-proliferative effect. Here, we demonstrate that, in Chinese hamster ovary cells expressing SST5 (CHO/SST5 cells), somatostatin inhibited cell proliferation induced by nitric oxide donors and overexpression of the neuronal nitric-oxide synthase (nNOS) protein isoform. Accordingly, nNOS activity and dimerization were strongly inhibited following SST5 activation by the somatostatin analog RC-160. In CHO/SST5 cells, nNOS was dynamically recruited by the SST5 receptor and phosphorylated at tyrosyl residues following RC-160 treatment. RC-160 induced SST5-p60(src) kinase complex formation and subsequent p60(src) kinase activation. Coexpression of an inactive p60(src) kinase mutant with SST5 blocked RC-160-induced nNOS phosphorylation and inactivation and prevented the SST5-mediated anti-proliferative effect. In CHO/SST5 cells, p60(src) kinase associated with nNOS to induce its inactivation by phosphorylation at tyrosyl residues following RC-160 treatment. Using recombinant proteins, we demonstrated that such phosphorylation prevented nNOS homodimerization. Next, surface plasmon resonance and mutation analysis revealed that p60(src) directly associated with nNOS phosphorylated Tyr604. SST5-mediated inhibition of nNOS activity was demonstrated to be essential to the RC-160 anti-proliferative effect on pancreatic endocrine tumor-derived cells. We therefore identified nNOS as a new p60(src) kinase substrate essential for SST5-mediated anti-proliferative action.

    The Journal of biological chemistry 2006;281;28;19156-71

  • Collagen type I selectively activates ectodomain shedding of the discoidin domain receptor 1: involvement of Src tyrosine kinase.

    Slack BE, Siniaia MS and Blusztajn JK

    Department of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, Massachusetts 02118, USA. bslack@bu.edu

    The discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that is highly expressed in breast carcinoma cells. Upon binding to collagen, DDR1 undergoes autophosphorylation followed by limited proteolysis to generate a tyrosine phosphorylated C-terminal fragment (CTF). Although it was postulated that this fragment is formed as a result of shedding of the N-terminal ectodomain, collagen-dependent release of the DDR1 extracellular domain has not been demonstrated. We now report that, in conjunction with CTF formation, collagen type I stimulates concentration-dependent, saturable shedding of the DDR1 ectodomain from two carcinoma cell lines, and from transfected cells. In contrast, collagen did not promote cleavage of other transmembrane proteins including the amyloid precursor protein (APP), ErbB2, and E-cadherin. Collagen-dependent tyrosine phosphorylation and proteolysis of DDR1 in carcinoma cells were reduced by a pharmacologic Src inhibitor. Moreover, expression of a dominant negative Src mutant protein in human embryonic kidney cells inhibited collagen-dependent phosphorylation and shedding of co-transfected DDR1. The hydroxamate-based metalloproteinase inhibitor TAPI-1 (tumor necrosis factor-alpha protease inhibitor-1), and tissue inhibitor of metalloproteinase (TIMP)-3, also blocked collagen-evoked DDR1 shedding, but did not reduce levels of the phosphorylated CTF. Neither shedding nor CTF formation were affected by the gamma-secretase inhibitor, L-685,458. The results demonstrate that collagen-evoked ectodomain cleavage of DDR1 is mediated in part by Src-dependent activation or recruitment of a matrix- or disintegrin metalloproteinase, and that CTF formation can occur independently of ectodomain shedding. Delayed shedding of the DDR1 ectodomain may represent a mechanism that limits DDR1-dependent cell adhesion and migration on collagen matrices.

    Funded by: NIMH NIH HHS: R01 MH059775, R01 MH59775; NINDS NIH HHS: R01 NS030791-07, R01 NS042793, R01 NS30791

    Journal of cellular biochemistry 2006;98;3;672-84

  • Epidermal growth factor receptor exposed to oxidative stress undergoes Src- and caveolin-1-dependent perinuclear trafficking.

    Khan EM, Heidinger JM, Levy M, Lisanti MP, Ravid T and Goldkorn T

    Signal Transduction Laboratory, Department of Internal Medicine, University of California, School of Medicine, Davis, California 95616, USA.

    The epidermal growth factor (EGF) receptor (EGFR) has been found to be overexpressed in several types of cancer cells, and the regulation of its oncogenic potential has been widely studied. The paradigm for EGFR down-regulation involves the trafficking of activated receptor molecules from the plasma membrane, through clathrin-coated pits, and into the cell for lysosomal degradation. We have previously shown that oxidative stress generated by H2O2 results in aberrant phosphorylation of the EGFR. This leads to the loss of c-Cbl-mediated ubiquitination of the EGFR and, consequently, prevents its degradation. However, we have found that c-Cbl-mediated ubiquitination is required solely for degradation but not for internalization of the EGFR under oxidative stress. To further examine the fate of the EGFR under oxidative stress, we used confocal analysis to show that the receptor not only remains co-localized with caveolin-1 at the plasma membrane, but at longer time points, is also sorted to a perinuclear compartment via a clathrin-independent, caveolae-mediated pathway. Our findings indicate that although the EGFR associates with caveolin-1 constitutively, caveolin-1 is hyperphosphorylated only under oxidative stress, which is essential in transporting the EGFR to a perinuclear location, where it is not degraded and remains active. Thus, oxidative stress may have a role in tumorigenesis by not only activating the EGFR but also by promoting prolonged activation of the receptor both at the plasma membrane and within the cell.

    Funded by: NHLBI NIH HHS: HL-66189, HL-71871

    The Journal of biological chemistry 2006;281;20;14486-93

  • Src kinase phosphorylates Caspase-8 on Tyr380: a novel mechanism of apoptosis suppression.

    Cursi S, Rufini A, Stagni V, Condò I, Matafora V, Bachi A, Bonifazi AP, Coppola L, Superti-Furga G, Testi R and Barilà D

    Department of Experimental Medicine and Biochemical Sciences, Dulbecco Telethon Institute, University of Rome Tor Vergata, Rome, Italy.

    We identified Caspase-8 as a new substrate for Src kinase. Phosphorylation occurs on Tyr380, situated in the linker region between the large and the small subunits of human Procaspase-8, and results in downregulation of Caspase-8 proapoptotic function. Src activation triggers Caspase-8 phosphorylation on Tyr380 and impairs Fas-induced apoptosis. Accordingly, Src failed to protect Caspase-8-defective human cells in which a Caspase-8-Y380F mutant is expressed from Fas-induced cell death. Remarkably, Src activation upon EGF-receptor stimulation triggers endogenous Caspase-8 phosphorylation and prevents Fas-induced apoptosis. Tyr380 is phosphorylated also in human colon cancers where Src is aberrantly activated. These data provide the first evidence for a direct role of tyrosine phosphorylation in the control of caspases and reveal a new mechanism through which tyrosine kinases inhibit apoptosis and participate in tumor progression.

    Funded by: Telethon: TCP00061

    The EMBO journal 2006;25;9;1895-905

  • Nephrin ectodomain engagement results in Src kinase activation, nephrin phosphorylation, Nck recruitment, and actin polymerization.

    Verma R, Kovari I, Soofi A, Nihalani D, Patrie K and Holzman LB

    Division of Nephrology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0676, USA.

    A properly established and maintained podocyte intercellular junction, or slit diaphragm, is a necessary component of the selective permeability barrier of the kidney glomerulus. The observation that mutation or deletion of the slit diaphragm transmembrane protein nephrin results in failure of podocyte foot process morphogenesis and concomitant proteinuria first suggested the hypothesis that nephrin serves as a component of a signaling complex that directly integrates podocyte junctional integrity with cytoskeletal dynamics. The observations made herein provide the first direct evidence to our knowledge for a phosphorylation-mediated signaling mechanism by which this integrative function is derived. Our data support the model that during podocyte intercellular junction formation, engagement of the nephrin ectodomain induces transient Fyn catalytic activity that results in nephrin phosphorylation on specific nephrin cytoplasmic domain tyrosine residues. We found that this nephrin phosphorylation event resulted in recruitment of the SH2-SH3 domain-containing adapter protein Nck and assembly of actin filaments in an Nck-dependent fashion. Considered in the context of the role of nephrin family proteins in other organisms and the integral relationship of actin dynamics and junction formation, these observations establish a function for nephrin in regulating actin cytoskeletal dynamics.

    The Journal of clinical investigation 2006;116;5;1346-59

  • p130Cas: a versatile scaffold in signaling networks.

    Defilippi P, Di Stefano P and Cabodi S

    Department of Genetics, Biology and Biochemistry, University of Turin, Via Santena 5 bis 10126 Turin, Italy. paola.defilippi@unito.it

    The Cas family of multiadaptor and scaffold molecules has an essential role in intracellular signaling events. Although these proteins do not have enzymatic or transcriptional activity, they spatially and temporally control signaling events through their ability to undergo changes in phosphorylation and to associate with effectors proteins in multimolecular complexes. The involvement of p130Cas in cell motility as a component of the integrin signaling machinery is well established. Here, we discuss recent developments that highlight a fundamental role in cell transformation and microbial pathogenesis and the implications of these developments on p130Cas function under normal and pathological conditions.

    Trends in cell biology 2006;16;5;257-63

  • Receptor association and tyrosine phosphorylation of S6 kinases.

    Rebholz H, Panasyuk G, Fenton T, Nemazanyy I, Valovka T, Flajolet M, Ronnstrand L, Stephens L, West A and Gout IT

    Ludwig Institute for Cancer Research, London, UK. hrebholz@rockefeller.edu

    Ribosomal protein S6 kinase (S6K) is activated by an array of mitogenic stimuli and is a key player in the regulation of cell growth. The activation process of S6 kinase involves a complex and sequential series of multiple Ser/Thr phosphorylations and is mainly mediated via phosphatidylinositol 3-kinase (PI3K)-3-phosphoinositide-dependent protein kinase-1 (PDK1) and mTor-dependent pathways. Upstream regulators of S6K, such as PDK1 and protein kinase B (PKB/Akt), are recruited to the membrane via their pleckstrin homology (PH) or protein-protein interaction domains. However, the mechanism of integration of S6K into a multi-enzyme complex around activated receptor tyrosine kinases is not clear. In the present study, we describe a specific interaction between S6K with receptor tyrosine kinases, such as platelet-derived growth factor receptor (PDGFR). The interaction with PDGFR is mediated via the kinase or the kinase extension domain of S6K. Complex formation is inducible by growth factors and leads to S6K tyrosine phosphorylation. Using PDGFR mutants, we have shown that the phosphorylation is exerted via a PDGFR-src pathway. Furthermore, src kinase phosphorylates and coimmunoprecipitates with S6K in vivo. Inhibitors towards tyrosine kinases, such as genistein and PP1, or src-specific SU6656, but not PI3K and mTor inhibitors, lead to a reduction in tyrosine phosphorylation of S6K. In addition, we mapped the sites of tyrosine phosphorylation in S6K1 and S6K2 to Y39 and Y45, respectively. Mutational and immunofluorescent analysis indicated that phosphorylation of S6Ks at these sites does not affect their activity or subcellular localization. Our data indicate that S6 kinase is recruited into a complex with RTKs and src and becomes phosphorylated on tyrosine/s in response to PDGF or serum.

    The FEBS journal 2006;273;9;2023-36

  • Asymmetric arginine dimethylation of heterogeneous nuclear ribonucleoprotein K by protein-arginine methyltransferase 1 inhibits its interaction with c-Src.

    Ostareck-Lederer A, Ostareck DH, Rucknagel KP, Schierhorn A, Moritz B, Huttelmaier S, Flach N, Handoko L and Wahle E

    Institute of Biochemistry, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06120 Halle (Saale), Germany. aostareck@biochemtech.uni-halle.de

    Arginine methylation is a post-translational modification found in many RNA-binding proteins. Heterogeneous nuclear ribonucleoprotein K (hnRNP K) from HeLa cells was shown, by mass spectrometry and Edman degradation, to contain asymmetric N(G),N(G)-dimethylarginine at five positions in its amino acid sequence (Arg256, Arg258, Arg268, Arg296, and Arg299). Whereas these five residues were quantitatively modified, Arg303 was asymmetrically dimethylated in <33% of hnRNP K and Arg287 was monomethylated in <10% of the protein. All other arginine residues were unmethylated. Protein-arginine methyltransferase 1 was identified as the only enzyme methylating hnRNP K in vitro and in vivo. An hnRNP K variant in which the five quantitatively modified arginine residues had been substituted was not methylated. Methylation of arginine residues by protein-arginine methyltransferase 1 did not influence the RNA-binding activity, the translation inhibitory function, or the cellular localization of hnRNP K but reduced the interaction of hnRNP K with the tyrosine kinase c-Src. This led to an inhibition of c-Src activation and hnRNP K phosphorylation. These findings support the role of arginine methylation in the regulation of protein-protein interactions.

    The Journal of biological chemistry 2006;281;16;11115-25

  • TRAF6 activation of PI 3-kinase-dependent cytoskeletal changes is cooperative with Ras and is mediated by an interaction with cytoplasmic Src.

    Wang KZ, Wara-Aswapati N, Boch JA, Yoshida Y, Hu CD, Galson DL and Auron PE

    Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.

    Interleukin 1 (IL-1) has been implicated in the reorganization of the actin cytoskeleton. An expression vector encoding a PKB/Akt pleckstrin-homology domain fused to a fluorescent protein was used to detect phosphoinositide 3-kinase (PI 3-kinase) products. It was observed that PI 3-kinase was activated either by treatment with IL-1 or by expression of either TRAF6, Src, MyD88 or dominant-positive PI 3-kinase, and resulted in the formation of long filopodia-like cellular protrusions that appeared to branch at membrane sites consisting of clusters of phosphoinositide. This depended upon a TRAF6 polyproline motif and Src catalytic activity, and was blocked by inhibitors of PI 3-kinase, Src and Ras. Using both conventional and split fluorescent protein probes fused to expressed TRAF6 and Src in living cells, the polyproline sequence of TRAF6 and the Src-homology 3 (SH3) domain of Src were shown to be required for interaction between these two proteins. Interaction occurred within the cytoplasm, and not at either the cell membrane or cytoplasmic sequestosomes. In addition, co-transfection of vectors expressing fluorescent-protein-fused TRAF6 and non-fluorescent MyD88, IRAK1 and IRAK2 revealed an inverse correlation between increased sequestosome formation and activation of both PI 3-kinase and NF-kappaB. Although a key factor in TRAF6-dependent activation of PI 3-kinase, ectopic expression of Src was insufficient for NF-kappaB activation and, in contrast to NF-kappaB, was not inhibited by IRAK2.

    Funded by: NCI NIH HHS: CA68544; NIAID NIH HHS: AI44122; NIDCR NIH HHS: DE14197

    Journal of cell science 2006;119;Pt 8;1579-91

  • Migration inhibitory factor up-regulates vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 via Src, PI3 kinase, and NFkappaB.

    Amin MA, Haas CS, Zhu K, Mansfield PJ, Kim MJ, Lackowski NP and Koch AE

    Department of Medicine, University of Michigan Medical School, Ann Arbor, MI, USA.

    Cell adhesion molecules are critical in monocyte (MN) recruitment in immune-mediated and hematologic diseases. We investigated the novel role of recombinant human migration inhibitory factor (rhMIF) in up-regulating vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) and their signaling pathways in human MNs. rhMIF-induced expression of VCAM-1 and ICAM-1 was significantly higher compared with nonstimulated MNs. rhMIF induced MN VCAM-1 and ICAM-1 expression in a concentration-dependent manner (P < .05). Antisense oligodeoxynucleotides (ODNs) and inhibitors of Src, PI3K, p38, and NFkappaB significantly reduced rhMIF-induced MN VCAM-1 and ICAM-1 expression (P < .05). However, Erk1/2 and Jak2 were not involved. Silencing RNA directed against MIF, and inhibitors of Src, PI3K, NFkappaB, anti-VCAM-1, and anti-ICAM-1 significantly inhibited rhMIF-induced adhesion of HL-60 cells to human dermal microvascular endothelial cells (HMVECs) or an endothelial cell line, HMEC-1, in cell adhesion assays, suggesting the functional significance of MIF-induced adhesion molecules (P < .05). rhMIF also activated MN phospho-Src, -Akt, and -NFkappaB in a time-dependent manner. rhMIF induced VCAM-1 and ICAM-1 up-regulation in 12 hours via Src, PI3K, and NFkappaB as shown by Western blotting and immunofluorescence. MIF and MIF-dependent signaling pathways may be a potential target for treating diseases characterized by up-regulation of cell adhesion molecules.

    Funded by: NHLBI NIH HHS: HL58695, R01 HL058695; NIAID NIH HHS: AI40987, R01 AI040987; NIAMS NIH HHS: AR48267, R01 AR048267

    Blood 2006;107;6;2252-61

  • Src tyrosine kinase inhibitor PP2 suppresses ERK1/2 activation and epidermal growth factor receptor transactivation by X-irradiation.

    Li Z, Hosoi Y, Cai K, Tanno Y, Matsumoto Y, Enomoto A, Morita A, Nakagawa K and Miyagawa K

    Department of Radiation Research, Faculty of Medicine, University of Tokyo, Tokyo 113-0033, Japan.

    Exposure of MDA-MB-468 cells to ionizing radiation (IR) caused biphasic activation of ERK as indicated by its phosphorylation at Thr202/Tyr204. Specific epidermal growth factor receptor (EGFR) inhibitor AG1478 and specific Src inhibitor PP2 inhibited IR-induced ERK1/2 activation but phosphatidylinositol-3 kinase inhibitor wortmannin did not. IR caused EGFR tyrosine phosphorylation, whereas it did not induce EGFR autophosphorylation at Tyr992, Tyr1045, and Tyr1068 or Src-dependent EGFR phosphorylation at Tyr845. SHP-2, which positively regulates EGFR/Ras/ERK signaling cascade, became activated by IR as indicated by its phosphorylation at Tyr542. This activation was inhibited by PP2 not by AG1478, which suggests Src-dependent activation of SHP-2. Src and PTPalpha, which positively regulates Src, became activated as indicated by phosphorylation at Tyr416 and Tyr789, respectively. These data suggest that IR-induced ERK1/2 activation involves EGFR through a Src-dependent pathway that is distinct from EGFR ligand activation.

    Biochemical and biophysical research communications 2006;341;2;363-8

  • Role of c-Src and focal adhesion kinase in progression and metastasis of estrogen receptor-positive breast cancer.

    Planas-Silva MD, Bruggeman RD, Grenko RT and Stanley Smith J

    Department of Pharmacology, Penn State College of Medicine, Hershey, PA 17033, USA. mcplanas@psu.edu

    The non-receptor tyrosine kinases c-Src and focal adhesion kinase (Fak) mediate signal transduction pathways that regulate cell proliferation, survival, invasion, and metastasis. Here, we investigated whether c-Src and Fak are activated during progression of hormone-dependent breast cancer. Maximally active c-Src was overexpressed in a subset of tamoxifen-resistant variants and in metastases of recurrent hormone-treated breast cancer. Active Fak was also frequently observed in these tumors. We also show that estrogen receptor (ER) can bind to Fak and that estrogen can modulate Fak autophosphorylation supporting a cross-talk between these two pathways. Inhibition of c-Src activity blocked proliferation of all tamoxifen-resistant variants, suggesting that inhibitors of c-Src-Fak activity may delay or prevent progression and metastasis of ER-positive tumors. These studies also raise the possibility that fully active forms of c-Src and Fak in breast tumors may be biomarkers to predict tamoxifen resistance and/or risk of recurrence in ER-positive breast cancer.

    Biochemical and biophysical research communications 2006;341;1;73-81

  • The adaptor protein Tom1L1 is a negative regulator of Src mitogenic signaling induced by growth factors.

    Franco M, Furstoss O, Simon V, Benistant C, Hong WJ and Roche S

    CRBM, CNRS FRE2593, 1919 Route de Mende, 34293 Montpellier Cedex 05, France. Serge.Roche@crbm.cnrs.fr.

    The Src family of protein-tyrosine kinases (SFK) play important roles in mitogenesis and morphological changes induced by growth factors. The involved substrates are, however, ill defined. Using an antiphosphotyrosine antibody to screen tyrosine-phosphorylated cDNA expression library, we have identified Tom1L1, an adaptor protein of the Tom1 family and a novel substrate and activator of the SFK. Surprisingly, we found that Tom1L1 does not promote DNA synthesis induced by Src. Furthermore, we report that Tom1L1 negatively regulates SFK mitogenic signaling induced by platelet-derived growth factor (PDGF) through modulation of SFK-receptor association: (i) Tom1L1 inhibits DNA synthesis induced by PDGF; (ii) inhibition is overcome by c-myc expression or p53 inactivation, two regulators of SFK mitogenic function; (iii) Src or Fyn coexpression overrides Tom1L1 mitogenic activity; (iv) overexpression of the adaptor reduces Src association with the receptor; and (v) protein inactivation potentiates receptor complex formation, allowing increased SFK activation and DNA synthesis. However, Tom1L1 affects neither DNA synthesis induced by the constitutively active allele SrcY527F nor SFK-regulated actin assembly induced by PDGF. Finally, overexpressed Tom1 and Tom1L2 also associate with Src and affected mitogenic signaling in agreement with some redundancy among members of the Tom1 family. We concluded that Tom1L1 defines a novel mechanism for regulation of SFK mitogenic signaling induced by growth factors.

    Molecular and cellular biology 2006;26;5;1932-47

  • Expression of c-Src and comparison of cytologic features in cherubism, central giant cell granuloma and giant cell tumors.

    Wang C, Song Y, Peng B, Fan M, Li J, Zhu S and Bian Z

    Key Laboratory of Oral Biomedical Engineering, Ministry of Education of China, Wuhan University, Wuhan 430079, P.R. China.

    Cherubism (CBM) and central giant cell granuloma (CGCG) of the jaw and giant cell tumor (GCT) of the long bone are clinically different diseases. Histologically, they are all multinucleated giant cell (MGC)-containing lesions. This study aims to evaluate the expression of c-Src and cytologic features in CBM, CGCG and GCT and to clarify whether there is a common mechanism underlying the formation of multi-nucleated giant cells (MGCs) in these lesions. Specimens and paraffin blocks were collected from patients with CBM (12 cases), CGCG (24 cases) and GCT (37 cases). Histomorpho-metric differences in MGCs were compared among the three types of lesions. The expression of c-Src by immunohistochemistry and in situ hybridization and the expression of TRAP by enzyme histochemical staining were examined. Expression of c-Src mRNA and protein, as well as TRAP staining, was detected in both MGCs and a fraction of mononuclear cells in all investigated lesions. There are no quantitative differences for cytologic features and c-Src expression among the lesions. The results suggested that CBM, CGCG and GCT have overlapping cytological features at the histological level, and c-Src may be involved in the formation of MGCs in the three different diseases.

    Oncology reports 2006;15;3;589-94

  • Role of Src-specific phosphorylation site on focal adhesion kinase for senescence-associated apoptosis resistance.

    Ryu SJ, Cho KA, Oh YS and Park SC

    Department of Biochemistry and Molecular Biology, The Aging and Apoptosis Research Center, Seoul National University College of Medicine, Seoul, S. Korea.

    A decreased apoptotic response toward noxious stress is an issuing characteristic of the aging phenotype. Hydrogen peroxide or staurosporine induced apoptosis readily in young cells but not in senescent cells. We showed that focal adhesion kinase (FAK) expression and its phosphorylation at Tyr397, autophosphorylation site for focal adhesion formation, and Tyr577, Src-dependent phosphorylation site, were both increased in senescent cells. Moreover, FAK was inactivated proteolytically by apoptotic stimuli in young cells, but not in senescent cells. In addition, senescent cells whose FAK expression was downregulated by siRNA showed the increased level of apoptosis by staurosporine treatment via caspase-3 activation but not by hydrogen peroxide treatment. Interestingly dephosphorylation at Tyr577 of FAK by PP2 treatment, Src-family kinase inhibitor, induced the apoptosis by staurosporine in senescent cells but dephosphorylation at Tyr397 by downregulation of caveolin-1 was not affected. These data suggest that FAK might differently regulate apoptosis and focal adhesion formation through site-specific tyrosine phosphorylation in senescent cells.

    Apoptosis : an international journal on programmed cell death 2006;11;3;303-13

  • Transactivation of fetal liver kinase-1/kinase-insert domain-containing receptor by lysophosphatidylcholine induces vascular endothelial cell proliferation.

    Fujita Y, Yoshizumi M, Izawa Y, Ali N, Ohnishi H, Kanematsu Y, Ishizawa K, Tsuchiya K and Tamaki T

    Department of Pharmacology, The University of Tokushima Graduate School of Medical Sciences, 3-18-15 Kuramoto, Tokushima 770-8503, Japan. fujita56@ri.ncvc.go.jp

    Lysophosphatidylcholine (LPC), a major lipid component of oxidized low-density lipoprotein, is a bioactive lipid molecule involved in numerous biological processes including the progression of atherosclerosis. Recently orphan G protein-coupled receptors were identified as high-affinity receptors for LPC. Although several G protein-coupled receptor ligands transactivate receptor tyrosine kinases, LPC-stimulated transactivation of receptor tyrosine kinase has not yet been reported. Here we observed for the first time that LPC treatment of human umbilical vein endothelial cells (HUVECs) induces tyrosyl phosphorylation of vascular endothelial growth factor receptor 2 [fetal liver kinase-1/kinase-insert domain-containing receptor, Flk-1/KDR)]. Flk-1/KDR transactivation by LPC was inhibited by vascular endothelial growth factor receptor tyrosine kinase inhibitors, SU1498 and 4-[(4'-chloro-2'-fluoro) phenylamino]6,7-dimethoxyquinazoline (VTKi) in immunoprecipitation. Furthermore, we examined the effects of the Src family kinases inhibitors, herbimycin A and 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP2), on LPC-induced Flk-1/KDR transactivation. Results from Western blots, c-Src is involved in LPC-induced Flk-1/KDR transactivation because herbimycin A and PP2 inhibited this transactivation. Kinase-inactive (KI) Src transfection also inhibited LPC-induced Flk-1/KDR transactivation. In addition, results from Western blots, ERK1/2 and Akt, which are downstream effectors of Flk-1/KDR, were also activated by LPC, and this was inhibited by SU1498, VTKi, herbimycin A, PP2, and KI Src transfection in HUVECs. LPC-induced stimulation of HUVEC proliferation was shown to be secondary to transactivation because it was suppressed by SU1498, VTKi, herbimycin A, PP2, and KI Src transfection in dimethylthiazoldiphenyltetra-zoliumbromide assay. These findings suggest that LPC-induced Flk-1/KDR transactivation via c-Src may have important implications for the progression of atherosclerosis.

    Endocrinology 2006;147;3;1377-85

  • Crosstalk between Src and major vault protein in epidermal growth factor-dependent cell signalling.

    Kim E, Lee S, Mian MF, Yun SU, Song M, Yi KS, Ryu SH and Suh PG

    Institue of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Jinju, Korea.

    Vaults are highly conserved, ubiquitous ribonucleoprotein (RNP) particles with an unidentified function. For the three protein species (TEP1, VPARP, and MVP) and a small RNA that comprises vault, expression of the unique 100-kDa major vault protein (MVP) is sufficient to form the basic vault structure. To identify and characterize proteins that interact with the Src homology 2 (SH2) domain of Src and potentially regulate Src activity, we used a pull-down assay using GST-Src-SH2 fusion proteins. We found MVP as a Src-SH2 binding protein in human stomach tissue. Interaction of Src and MVP was also observed in 253J stomach cancer cells. A subcellular localization study using immunofluorescence microscopy shows that epidermal growth factor (EGF) stimulation triggers MVP translocation from the nucleus to the cytosol and perinuclear region where it colocalizes with Src. We found that the interaction between Src and MVP is critically dependent on Src activity and protein (MVP) tyrosyl phosphorylation, which are induced by EGF stimulation. Our results also indicate MVP to be a novel substrate of Src and phosphorylated in an EGF-dependent manner. Interestingly, purified MVP inhibited the in vitro tyrosine kinase activity of Src in a concentration-dependent manner. MVP overexpression downregulates EGF-dependent ERK activation in Src overexpressing cells. To our knowledge, this is the first report of MVP interacting with a protein tyrosine kinase involved in a distinct cell signalling pathway. It appears that MVP is a novel regulator of Src-mediated signalling cascades.

    The FEBS journal 2006;273;4;793-804

  • Identification of preferred protein interactions by phage-display of the human Src homology-3 proteome.

    Kärkkäinen S, Hiipakka M, Wang JH, Kleino I, Vähä-Jaakkola M, Renkema GH, Liss M, Wagner R and Saksela K

    Institute of Medical Technology, University of Tampere and Tampere University Hospital, Biokatu 8, Tampere 33014, Finland.

    We have determined the human genome to contain 296 different Src homology-3 (SH3) domains and cloned them into a phage-display vector. This provided a powerful and unbiased system for simultaneous assaying of the complete human SH3 proteome for the strongest binding to target proteins of interest, without the limitations posed by short linear peptide ligands or confounding variables of more indirect methods for protein interaction screening. Studies involving three ligand proteins, human immunodeficiency virus-1 Nef, p21-activated kinase (PAK)2 and ADAM15, showed previously reported as well as novel SH3 partners with nanomolar affinities specific for them. This argues that SH3 domains may have a more dominant role in directing cellular protein interactions than has been assumed. Besides showing potentially important new SH3-directed interactions, these studies also led to the discovery of novel signalling proteins, such as the PAK2-binding adaptor protein POSH2 and the ADAM15-binding sorting nexin family member SNX30.

    EMBO reports 2006;7;2;186-91

  • Phosphorylated alpha-actinin and protein-tyrosine phosphatase 1B coregulate the disassembly of the focal adhesion kinase x Src complex and promote cell migration.

    Zhang Z, Lin SY, Neel BG and Haimovich B

    Department of Surgery, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, New Brunswick 08903, USA.

    The focal adhesion kinase (FAK) is a key regulator of cell migration. Phosphorylation at Tyr-397 activates FAK and creates a binding site for Src family kinases. FAK phosphorylates the cytoskeletal protein alpha-actinin at Tyr-12. Here we report that protein-tyrosine phosphatase 1B (PTP 1B) is an alpha-actinin phosphatase. PTP 1B-dependent dephosphorylation of alpha-actinin was seen in COS-7 cells and PTP 1B-null fibroblasts reconstituted with PTP 1B. Furthermore, we show that coexpression of wild-type alpha-actinin and PTP 1B causes dephosphorylation at Tyr-397 in FAK. No dephosphorylation was observed in cells coexpressing the alpha-actinin phosphorylation mutant Y12F and PTP 1B. Furthermore, the phosphorylation at four other sites in FAK was not altered by PTP 1B. In addition, we found that phosphorylated alpha-actinin bound to Src and reduced the binding of FAK to Src. The dephosphorylation at Tyr-397 in FAK triggered by wild-type alpha-actinin and PTP 1B caused a significant increase in cell migration. We propose that phosphorylated alpha-actinin disrupts the FAK x Src complex exposing Tyr-397 in FAK to PTP 1B. These findings uncover a novel feedback loop involving phosphorylated alpha-actinin and PTP 1B that regulates FAK x Src interaction and cell migration.

    Funded by: NHLBI NIH HHS: HL54104

    The Journal of biological chemistry 2006;281;3;1746-54

  • Src-mediated phosphorylation regulates subcellular distribution and activity of human inducible nitric oxide synthase.

    Hausel P, Latado H, Courjault-Gautier F and Felley-Bosco E

    Department of Pharmacology and Toxicology, Rue du Bugnon 27, Lausanne 1005, Switzerland.

    Inducible nitric oxide synthase (iNOS) expression is regulated at both the transcriptional and post-transcriptional level in epithelial cells. The aim of this study was to characterize the effects of tyrosine phosphorylation on iNOS activity. In a human intestinal epithelial cell line stimulated with cytokines, tyrosine phosphorylation of human iNOS protein was observed after 30 min exposure to pervanadate (PV), an inhibitor of protein tyrosine phosphatases. 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, a specific inhibitor of Src tyrosine kinases, abolished the PV-induced iNOS tyrosine phosphorylation. Cotransfection of Src with iNOS cDNA in human embryonic kidney (HEK) 293 cells resulted in a threefold (P<0.001) increase of iNOS protein levels and tyrosine phosphorylation of iNOS. In the presence of Src, 76% of wild-type (wt) iNOS was redistributed to detergent-insoluble domains and iNOS activity was decreased by 28% (P<0.05) despite increased iNOS protein levels. Analysis of iNOS tyrosine mutants revealed decreased Src-induced effects in Y151F iNOS mutant. Using a GST-fusion protein containing a domain encompassing Y151, we show that Y151 is a direct substrate for active Src in vitro. These findings indicate a role for iNOS tyrosine phosphorylation in the regulation of iNOS activity and the implication of Src tyrosine kinases in this pathway.

    Oncogene 2006;25;2;198-206

  • MUC1 oncoprotein is targeted to mitochondria by heregulin-induced activation of c-Src and the molecular chaperone HSP90.

    Ren J, Bharti A, Raina D, Chen W, Ahmad R and Kufe D

    Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.

    The MUC1 heterodimeric transmembrane glycoprotein is aberrantly overexpressed by most human carcinomas. The MUC1 C-terminal subunit localizes to mitochondria and blocks stress-induced activation of the intrinsic apoptotic pathway. How MUC1 is delivered to mitochondria is not known. The present studies demonstrate that MUC1 forms intracellular complexes with HSP70 and HSP90. We show that the MUC1 cytoplasmic domain binds directly to HSP70 in vitro. By contrast, binding of MUC1 to HSP90 in vitro is induced by c-Src-mediated phosphorylation of the MUC1 cytoplasmic domain. c-Src also increases binding of MUC1 to HSP90 in cells. In concert with these results, we show that heregulin (HRG), a ligand for ErbB receptors, activates c-Src and, in turn, stimulates binding of MUC1 to HSP90. We also show that inhibitors of c-Src or HSP90 block HRG-induced targeting of MUC1 to mitochondria and integration of MUC1 into the mitochondrial outer membrane. These findings indicate that MUC1 is delivered to mitochondria by a mechanism involving activation of the ErbB receptor-->c-Src pathway and transport by the molecular chaperone HSP70/HSP90 complex.

    Funded by: NCI NIH HHS: CA97098

    Oncogene 2006;25;1;20-31

  • FAK and Src kinases are required for netrin-induced tyrosine phosphorylation of UNC5.

    Li W, Aurandt J, Jürgensen C, Jürgense C, Rao Y and Guan KL

    Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA. wqli@umich.edu

    During neuronal development, netrin and its receptors UNC5 and DCC (deleted in colorectal cancer) guide axonal growth cones in navigating to their targets. Netrin also plays important roles in the regulation of cell migration, tissue morphogenesis and tumor growth. Here, we show that netrin induces UNC5 tyrosine phosphorylation and that this effect of netrin is dependent on its co-receptor DCC. UNC5 tyrosine phosphorylation is known to be important for netrin to induce cell migration and axonal repulsion. Src tyrosine kinase activity is required for netrin to stimulate UNC5 tyrosine phosphorylation in neurons and transfected cells. The SH2 domain of Src kinase directly interacts with the cytosolic domain of UNC5 in a tyrosine-phosphorylation-dependent manner. Furthermore, the tyrosine kinase focal adhesion kinase (FAK) is also involved in netrin-induced UNC5 tyrosine phosphorylation. Both Src and FAK can phosphorylate UNC5. Our data suggest a model in which netrin stimulates UNC5 tyrosine phosphorylation and signaling in a manner dependent on the co-receptor DCC, through the recruitment of Src and FAK kinases.

    Funded by: NCI NIH HHS: R01 CA107193, R01 CA107193-03

    Journal of cell science 2006;119;Pt 1;47-55

  • Altered exon usage in the juxtamembrane domain of mouse and human RON regulates receptor activity and signaling specificity.

    Wei X, Hao L, Ni S, Liu Q, Xu J and Correll PH

    Department of Veterinary Science, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.

    Alternative splicing of signaling proteins can contribute to the complexity of signaling networks. We find that expression of mouse RON, but not human RON, results in constitutive receptor autophosphorylation, ligand-independent activation of the mitogen-activated protein kinase pathway, and association of the receptor with c-Src. Using chimeric receptors, we mapped the region for this difference in signaling capacity of mouse and human RON to the juxtamembrane domain. Expression of these receptors in primary erythroid progenitor cells also demonstrated a functional difference in the ability of mouse and human RON to support erythropoietin-independent colony formation that mapped to the juxtamembrane domain. Splicing of the mouse RON receptor tyrosine kinase transcript results in the constitutive deletion of an exon used by all other known RON orthologs that encodes part of the juxtamembrane domain of the receptor. Mutational analysis indicated that the two tyrosines present in this region in human RON, one of which has been previously shown to be a c-Cbl binding site, are not responsible for this difference. However, deletion of this region in the context of human RON enhanced receptor phosphorylation, activation of mitogen-activated protein kinase, and association of c-Src at levels comparable with those observed with mouse RON. These data provide direct evidence that the divergence of exon usage among different species can generate a protein with novel activity and subsequently add to the complexity of cellular signaling regulation.

    Funded by: NHLBI NIH HHS: R01 HL66471

    The Journal of biological chemistry 2005;280;48;40241-51

  • Tyrosine 740 phosphorylation of discoidin domain receptor 2 by Src stimulates intramolecular autophosphorylation and Shc signaling complex formation.

    Yang K, Kim JH, Kim HJ, Park IS, Kim IY and Yang BS

    Biomedical Research Center, Korea Institute of Science and Technology, 39-1, Hawolgok-Dong, Sungbuk-Ku, Seoul 136-791, Korea.

    DDR2 is a receptor tyrosine kinase whose activating ligands are various collagens. DDR2-mediated cellular signaling has been shown to require Src activity. However, the precise mechanism underlying the Src dependence of DDR2 signaling is unknown. Here, using baculoviral co-expression of the DDR2 cytosolic domain and Src, we show that Src targets three tyrosine residues (Tyr-736, Tyr-740, and Tyr-741) in the activation loop of DDR2 for phosphorylation. This phosphorylation by Src stimulates DDR2 cis-autophosphorylation of additional tyrosine residues. In vitro Shc binding assays demonstrate that phosphotyrosines resulting from DDR2 autophosphorylation are involved in Shc binding to the DDR2 cytosolic domain. Mutating tyrosine 740 of DDR2 to phenylalanine stimulates autophosphorylation of DDR2 to an extent similar to that resulting from Src phosphorylation of DDR2. In addition, the DDR2 Y740F mutant protein displays collagen-independent, constitutively activated signaling. These findings suggest that tyrosine 740 inhibits DDR2 autophosphorylation. Collectively, our findings are consistent with the following mechanism for Src-dependent DDR2 activation and signaling: 1) ligand binding promotes phosphorylation of Tyr-740 in the DDR2 activation loop by Src; 2) Tyr-740 phosphorylation stimulates intramolecular autophosphorylation of DDR2; 3) DDR2 autophosphorylation generates cytosolic domain phosphotyrosines that promote the formation of DDR2 cytosolic domain-Shc signaling complexes.

    The Journal of biological chemistry 2005;280;47;39058-66

  • Bone morphogenetic protein receptor type II C-terminus interacts with c-Src: implication for a role in pulmonary arterial hypertension.

    Wong WK, Knowles JA and Morse JH

    Department of Medicine, Columbia University College of Physicians and Surgeons, and the New York State Psychiatric Institute, New York, New York 10032, USA. wpw2001@columbia.edu

    Mutations of bone morphogenetic protein receptor type II (BMPR-II) have been associated with familial and idiopathic pulmonary arterial hypertension (PAH). BMPR-II is a member of the transforming growth factor-beta receptor superfamily. It consists of extracellular, transmembrane, and kinase domains, and a unique C-terminus with mostly unknown function. However, a number of PAH-causing mutations are predicted to truncate the C-terminus, suggesting that this domain plays an important role in the homeostasis of pulmonary vessels. In this study, we sought to elucidate the functional role of this C-terminus by seeking its interacting partners. Using yeast two-hybrid screening, we identified c-Src tyrosine kinase as a binding partner of this C-terminus. In vitro co-immunoprecipitation confirmed their interaction. Mutations truncating the C-terminus disrupted their interaction, while missense mutation within kinase domain reduced their interaction. In addition, BMPR-II and c-Src tyrosine kinase colocalized within intracellular aggregates when overexpressed in HEK293 cells. Moreover, mutations truncating the C-terminus disrupted their colocalization, whereas missense mutation within kinase domain had no effect on their colocalization. Furthermore, BMP ligand stimulation decreased c-Src-activating phosphorylation at Tyrosine 418 in pulmonary smooth muscle cells in both time- and concentration-dependent manners. Mutations that truncated the C-terminus abolished this response. Taken together, these results suggest a model in which proliferative effect of c-Src by vasoactive molecules is balanced by opposing effect of BMP signaling in basal state, and the loss of this balance due to BMPR2 mutations leads to increased c-Src activity and subsequently cell growth.

    Funded by: NHLBI NIH HHS: HL60056

    American journal of respiratory cell and molecular biology 2005;33;5;438-46

  • Dual function of focal adhesion kinase in regulating integrin-induced MMP-2 and MMP-9 release by human T lymphoid cells.

    Segarra M, Vilardell C, Matsumoto K, Esparza J, Lozano E, Serra-Pages C, Urbano-Márquez A, Yamada KM and Cid MC

    Department of Internal Medicine, Hospital Clínic, University of Barcelona, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2005;19;13;1875-7

  • Crystal structures of active SRC kinase domain complexes.

    Breitenlechner CB, Kairies NA, Honold K, Scheiblich S, Koll H, Greiter E, Koch S, Schäfer W, Huber R and Engh RA

    Abteilung Strukturforschung, Max-Planck-Institut für Biochemie, 82152 Martinsried, Germany.

    c-Src was the first proto-oncoprotein to be identified, and has become the focus of many drug discovery programs. Src structures of a major inactive form have shown how the protein kinase is rigidified by several interdomain interactions; active configurations of Src are generated by release from this "assembled" or "bundled" form. Despite the importance of Src as a drug target, there is relatively little structural information available regarding the presumably more flexible active forms. Here we report three crystal structures of a dimeric active c-Src kinase domain, in an apo and two ligand complexed forms, with resolutions ranging from 2.9A to 1.95A. The structures show how the kinase domain, in the absence of the rigidifying interdomain interactions of the inactivation state, adopts a more open and flexible conformation. The ATP site inhibitor CGP77675 binds to the protein kinase with canonical hinge hydrogen bonds and also to the c-Src specific threonine 340. In contrast to purvalanol B binding in CDK2, purvalanol A binds in c-Src with a conformational change in a more open ATP pocket.

    Journal of molecular biology 2005;353;2;222-31

  • Uncoupling ligand-dependent and -independent mechanisms for mitogen-activated protein kinase activation by the murine Ron receptor tyrosine kinase.

    Wei X, Ni S and Correll PH

    Graduate Program in Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802-3500, USA.

    Receptor tyrosine kinases (RTKs) activate downstream signaling through cognate growth factor receptor-induced dimerization and autophosphorylation. Overexpression of RTKs can lead to constitutive activation due to increased dimerization in the absence of ligand, and downstream signals are presumed to be the same as the ligand-induced signals. We have shown that the murine Ron (mRon) receptor tyrosine kinase exhibits constitutive activation of the MAP kinase pathway that is independent of the two docking site tyrosines, whereas activation of this pathway in response to ligand (macrophage-stimulating protein) is abolished in the absence of these tyrosines. Furthermore, we identified three tyrosines (Tyr-1175, Tyr-1265, and Tyr-1294) within the kinase domain that play critical but overlapping roles in controlling constitutive Erk activation by mRon. Phenylalanine mutations at these three tyrosines results in a receptor that fails to constitutively activate the Erk pathway but retains the ability to induce Erk phosphorylation in response to ligand stimulation. The ability of mRon to activate the MAP kinase pathway is dependent on c-Src activity, and we have shown that c-Src co-immunoprecipitates with mRon. c-Src fails to interact with mRon when the three tyrosines required for MAP kinase activation are mutated, whereas the presence of any one of these tyrosines alone restores Erk phosphorylation and recruitment of c-Src. Thus, the ligand-dependent and -independent activity of mRon can be uncoupled through the alteration of selective sets of tyrosines.

    Funded by: NHLBI NIH HHS: R01-HL66471

    The Journal of biological chemistry 2005;280;42;35098-107

  • Towards a proteome-scale map of the human protein-protein interaction network.

    Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N, Berriz GF, Gibbons FD, Dreze M, Ayivi-Guedehoussou N, Klitgord N, Simon C, Boxem M, Milstein S, Rosenberg J, Goldberg DS, Zhang LV, Wong SL, Franklin G, Li S, Albala JS, Lim J, Fraughton C, Llamosas E, Cevik S, Bex C, Lamesch P, Sikorski RS, Vandenhaute J, Zoghbi HY, Smolyar A, Bosak S, Sequerra R, Doucette-Stamm L, Cusick ME, Hill DE, Roth FP and Vidal M

    Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA.

    Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.

    Funded by: NCI NIH HHS: R33 CA132073; NHGRI NIH HHS: P50 HG004233, R01 HG001715, RC4 HG006066, U01 HG001715; NHLBI NIH HHS: U01 HL098166

    Nature 2005;437;7062;1173-8

  • c-Src regulates constitutive and EGF-mediated VEGF expression in pancreatic tumor cells through activation of phosphatidyl inositol-3 kinase and p38 MAPK.

    Summy JM, Trevino JG, Baker CH and Gallick GE

    University of Texas MD Anderson Cancer Center, Department of Cancer Biology, Houston, TX 77054, USA.

    Objectives: Multiple signaling proteins may be aberrantly activated and/or overexpressed in pancreatic tumors, including the nonreceptor protein tyrosine kinase Src. The goal of this study was to determine the role of Src in regulating VEGF expression and angiogenic potential in pancreatic cancer cell lines.

    Methods: Src activity was inhibited using the Src family kinase selective inhibitor PP2, and c-Src expression was down-regulated via siRNA. The activities of downstream signaling molecules phosphatidyl inositol 3'-kinase (PI3K) and p38 mitogen-activated protein kinase (MAPK) were disrupted via selective inhibitors. In vivo angiogenesis was assessed through the use of a gel-foam assay.

    Results: Inhibition of Src activity or expression decreases both constitutive and EGF-induced VEGF production. Both the PI3K/Akt and p38 MAPK pathways are activated in a Src family kinase-dependent fashion on EGF-R activation and are important for EGF-mediated VEGF production in pancreatic cancer cells. Additionally, media from Src-inhibited L3.6pl cells fail to promote angiogenesis into gel foams implanted subcutaneously into mice, whereas media from control cells promote a robust angiogenic response.

    Conclusions: Src activity contributes to constitutive and EGF-induced VEGF expression and angiogenic potential in pancreatic cancer cells. Therefore, Src may be a viable target for antiangiogenesis therapy in pancreatic cancer.

    Funded by: NCI NIH HHS: T32 CA 09599, U54CA 090810

    Pancreas 2005;31;3;263-74

  • A human protein-protein interaction network: a resource for annotating the proteome.

    Stelzl U, Worm U, Lalowski M, Haenig C, Brembeck FH, Goehler H, Stroedicke M, Zenkner M, Schoenherr A, Koeppen S, Timm J, Mintzlaff S, Abraham C, Bock N, Kietzmann S, Goedde A, Toksöz E, Droege A, Krobitsch S, Korn B, Birchmeier W, Lehrach H and Wanker EE

    Max Delbrueck Center for Molecular Medicine, 13092 Berlin-Buch, Germany.

    Protein-protein interaction maps provide a valuable framework for a better understanding of the functional organization of the proteome. To detect interacting pairs of human proteins systematically, a protein matrix of 4456 baits and 5632 preys was screened by automated yeast two-hybrid (Y2H) interaction mating. We identified 3186 mostly novel interactions among 1705 proteins, resulting in a large, highly connected network. Independent pull-down and co-immunoprecipitation assays validated the overall quality of the Y2H interactions. Using topological and GO criteria, a scoring system was developed to define 911 high-confidence interactions among 401 proteins. Furthermore, the network was searched for interactions linking uncharacterized gene products and human disease proteins to regulatory cellular pathways. Two novel Axin-1 interactions were validated experimentally, characterizing ANP32A and CRMP1 as modulators of Wnt signaling. Systematic human protein interaction screens can lead to a more comprehensive understanding of protein function and cellular processes.

    Cell 2005;122;6;957-68

  • Physical and functional association of c-Src and adhesion and degranulation promoting adaptor protein (ADAP) in osteoclastogenesis in vitro.

    Koga S, Yogo K, Yoshikawa K, Samori H, Goto M, Uchida T, Ishida N and Takeya T

    Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan.

    c-Src plays a crucial role in osteoclastogenesis. In this study, we searched for c-Src-binding proteins using a combination of pull-down assays and mass spectrometric analysis, and identified the association of adhesion and degranulation promoting adaptor protein (ADAP) with c-Src in RAW264 cells and osteoclast precursors prepared from bone marrow cells. The kinase activity and the SH2 domain of c-Src were required for this association and Tyr807 in the extreme carboxyl terminus of ADAP was identified as a major recognition site. ADAP was found to be expressed in cells at the prefusion stage and localized mainly in the leading edge of lamellipodia and in pseudopodia. Tyrosine phosphorylation of ADAP was induced in an integrin-dependent manner, and the level was Src kinase-dependent. ADAP-knockdown RAW264 cells showed retarded migration and formed few multinucleated cells. Cas, known to be phosphorylated by c-Src, was identified as a major tyrosine-phosphorylated protein in differentiating RAW264 cells and the phosphorylation appeared to be decreased in ADAP-knockdown cells. ADAP thus may play an important role as a partner of c-Src for cell migration and progression to the multinucleated cell stage in osteoclastogenesis in vitro.

    The Journal of biological chemistry 2005;280;36;31564-71

  • Tyrosine phosphorylation of VE-cadherin prevents binding of p120- and beta-catenin and maintains the cellular mesenchymal state.

    Potter MD, Barbero S and Cheresh DA

    Moores Cancer Center, University of California at San Diego, La Jolla, California 92093, USA.

    In several pathological conditions, epithelial cells demonstrate a breakdown of barrier function and acquire an invasive phenotype. Endothelial cells in particular are maintained in a mesenchymal state during the cell invasion phase of angiogenesis. We show here that tyrosine phosphorylation of the adherens junction protein VE-cadherin at two critical tyrosines, Tyr-658 and Tyr-731, via tyrosine kinase activation or phosphatase inactivation was sufficient to prevent the binding of p120- and beta-catenin, respectively, to the cytoplasmic tail of VE-cadherin. In fact, phosphorylation at either site led to the inhibition of cell barrier function. Cells expressing wild-type VE-cadherin showed decreased cell migration compared with cells lacking VE-cadherin, whereas expression of VE-cadherin with a simple phosphomimetic tyrosine-to-glutamic acid mutation of Y658E or Y731E was sufficient to restore the migratory response. These findings demonstrate that a single phosphorylation event within the VE-cadherin cytoplasmic tail is sufficient to maintain cells in a mesenchymal state.

    Funded by: NCI NIH HHS: 5F32CA097885, CA45726, CA50286, CA78045, CA95262; NEI NIH HHS: EY14174; NHLBI NIH HHS: HL57900

    The Journal of biological chemistry 2005;280;36;31906-12

  • alpha(v)beta3-integrin-dependent activation of focal adhesion kinase mediates NF-kappaB activation and motogenic activity by HIV-1 Tat in endothelial cells.

    Urbinati C, Bugatti A, Giacca M, Schlaepfer D, Presta M and Rusnati M

    General Pathology and Immunology, Department of Biomedical Sciences and Biotechnology, School of Medicine, University of Brescia, viale Europe 11, 25123 Brescia, Italy.

    Once in the extracellular environment, the transactivator protein HIV-1 Tat exerts several pleiotropic effects by interacting with different cellular receptors, including integrin alpha(v)beta3. Real-time surface plasmon resonance analysis reveals that Tat/alpha(V)beta3 interaction occurs with rapid kinetics (association and dissociation rates equal to 1.16 x 10(7) M(-1) s(-1) and 3.78 x 10(-1) s(-1), respectively) and high affinity (dissociation constant = 32 nM). Through this interaction, substratum-immobilized Tat promotes adhesion and motogenic activity in endothelial cells. Also, alpha(v)beta(3)/Tat interaction triggers the activation of focal adhesion kinase, RhoA and pp60src. Overexpression of the dominant negative form of focal adhesion kinase, but not of an inactive Leu1034Ser substitution mutant isoform, impairs the activation of focal adhesion kinase and RhoA, but not that of pp60src, without affecting endothelial cell adhesion and spreading. alpha(v)beta3/Tat interaction triggers the activation of NF-kappaB in endothelial cells in a focal adhesion kinase-, RhoA- and pp60src-dependent manner, as shown in dominant negative focal adhesion kinase transfectants or using specific pharmacological inhibitors. Finally, the activation of focal adhesion kinase, RhoA, NF-kappaB and pp60src are required to mediate the motogenic activity of Tat in endothelial cells. Since Tat accumulates in an immobilized form in the extracellular matrix, these results provide new biochemical and biological insights about alpha(v)beta3/Tat interaction exploitable for the design of anti-Tat strategies.

    Journal of cell science 2005;118;Pt 17;3949-58

  • Integrins and Src: dynamic duo of adhesion signaling.

    Shattil SJ

    Hematology-Oncology Division, Department of Medicine, University of California-San Diego, La Jolla, CA 92093, USA. sshattil@ucsd.edu

    Src family protein tyrosine kinases (SFKs) play important roles downstream of integrin adhesion receptors, and they are necessary for the generation of "outside-in signals" that regulate cytoskeletal organization, cell motility and gene expression in response to cell adhesion. One relatively under-explored facet of this relationship is the possible physical interaction of integrins with SFKs. Recently, it has been established that beta3 integrins and c-Src can interact directly, and this pool of c-Src is activated by cell adhesion to initiate outside-in signaling in platelets, osteoclasts and cells of the vasculature. Here, the biochemical basis for and biological significance of this integrin-SFK interaction is summarized, and I propose a general mechanism for initiation of outside-in integrin signaling.

    Trends in cell biology 2005;15;8;399-403

  • Semaphorin 4D/plexin-B1 induces endothelial cell migration through the activation of PYK2, Src, and the phosphatidylinositol 3-kinase-Akt pathway.

    Basile JR, Afkhami T and Gutkind JS

    Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA. sg39v@nih.gov

    Semaphorins are cell surface and secreted proteins that provide axonal guidance in neuronal tissues and regulate cell motility in many cell types. They act by binding a family of transmembrane receptors known as plexins, which belong to the c-Met family of scatter factor receptors but lack an intrinsic tyrosine kinase domain. Interestingly, we have recently shown that Plexin-B1 is highly expressed in endothelial cells and that its activation by Semaphorin 4D elicits a potent proangiogenic response (J. R. Basile, A. Barac, T. Zhu, K. L. Guan, and J. S. Gutkind, Cancer Res. 64:5212-5224, 2004). In searches for the underlying molecular mechanism, we observed that Semaphorin 4D-stimulated endothelial cell migration requires the activation of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway. Surprisingly, we found that Plexin-B1 stimulates PI3K-Akt through the activation of an intracellular tyrosine kinase cascade that involves the sequential activation of PYK2 and Src. This results in the tyrosine phosphorylation of Plexin-B1, the rapid recruitment of a multimeric signaling complex that includes PYK2, Src, and PI3K to Plexin-B1 and the activation of Akt. These findings suggest that Plexin-B1 may achieve its numerous physiological functions through the direct activation of intracellular tyrosine kinase cascades.

    Molecular and cellular biology 2005;25;16;6889-98

  • Calcium-induced human keratinocyte differentiation requires src- and fyn-mediated phosphatidylinositol 3-kinase-dependent activation of phospholipase C-gamma1.

    Xie Z, Singleton PA, Bourguignon LY and Bikle DD

    Endocrine Unit, Veterans Affairs Medical Center, Northern California Institute for Research and Education and University of California-San Francisco, San Francisco, CA 94121, USA. zjxie@itsa.ucsf.edu

    We have previously demonstrated that phospholipase C (PLC)-gamma1 is required for calcium-induced human keratinocyte differentiation. In the present study, we investigated whether the activation of PLC-gamma1 by nonreceptor kinases such as src and fyn plays a role in mediating this process. Our results showed that the combination of dominant negative src and fyn blocked calcium-stimulated PLC-gamma1 activity and human keratinocyte differentiation, whereas each separately has little effect. However, unlike the activation of PLC-gamma1 by epidermal growth factor, calcium-induced activation of PLC-gamma1 was not a result of direct tyrosine phosphorylation. Therefore, we examined an alternative mechanism, in particular phosphatidylinositol 3,4,5-triphosphate (PIP3) formed as a product of phosphatidylinositol 3-kinase (PI3K) activity. PIP3 binds to and activates PLC-gamma1. The combination of dominant negative src and fyn blocked calcium-induced tyrosine phosphorylation of the regulatory subunit of PI3K, p85alpha, and the activity of the catalytic subunit of PI3K. PI3K inhibitors blocked calcium activation of PLC-gamma1 as well as the induction of keratinocyte differentiation markers involucrin and transglutaminase. These data indicate that calcium activates PLC-gamma1 via increased PIP3 formation mediated by c-src- and fyn-activated PI3K. This activation is required for calcium-induced human keratinocyte differentiation.

    Molecular biology of the cell 2005;16;7;3236-46

  • Dynamin forms a Src kinase-sensitive complex with Cbl and regulates podosomes and osteoclast activity.

    Bruzzaniti A, Neff L, Sanjay A, Horne WC, De Camilli P and Baron R

    Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510, USA. angela.bruzzaniti@yale.edu

    Podosomes are highly dynamic actin-containing adhesion structures found in osteoclasts, macrophages, and Rous sarcoma virus (RSV)-transformed fibroblasts. After integrin engagement, Pyk2 recruits Src and the adaptor protein Cbl, forming a molecular signaling complex that is critical for cell migration, and deletion of any molecule in this complex disrupts podosome ring formation and/or decreases osteoclast migration. Dynamin, a GTPase essential for endocytosis, is also involved in actin cytoskeleton remodeling and is localized to podosomes where it has a role in actin turnover. We found that dynamin colocalizes with Cbl in the actin-rich podosome belt of osteoclasts and that dynamin forms a complex with Cbl in osteoclasts and when overexpressed in 293VnR or SYF cells. The association of dynamin with Cbl in osteoclasts was decreased by Src tyrosine kinase activity and we found that destabilization of the dynamin-Cbl complex involves the recruitment of Src through the proline-rich domain of Cbl. Overexpression of dynamin increased osteoclast bone resorbing activity and migration, whereas overexpression of dynK44A decreased osteoclast resorption and migration. These studies suggest that dynamin, Cbl, and Src coordinately participate in signaling complexes that are important in the assembly and remodeling of the actin cytoskeleton, leading to changes in osteoclast adhesion, migration, and resorption.

    Funded by: NIAMS NIH HHS: AR42927, R01 AR042927

    Molecular biology of the cell 2005;16;7;3301-13

  • Kinetics of Src homology 3 domain association with the proline-rich domain of dynamins: specificity, occlusion, and the effects of phosphorylation.

    Solomaha E, Szeto FL, Yousef MA and Palfrey HC

    Department of Neurobiology, Pharamacology, and Physiology, University of Chicago, IL 60637, USA.

    Dynamin function is mediated in part through association of its proline-rich domain (PRD) with the Src homology 3 (SH3) domains of several putative binding proteins. To assess the specificity and kinetics of this process, we undertook surface plasmon resonance studies of the interaction between isolated PRDs of dynamin-1 and -2 and several purified SH3 domains. Glutathione S-transferase-linked SH3 domains bound with high affinity (K(D) approximately 10 nm to 1 microm) to both dynamin-1 and -2. The simplest interaction appeared to take place with the amphiphysin-SH3 domain; this bound to a single high affinity site (K(D) approximately 10 nm) in the C terminus of dynamin-1 PRD, as predicted by previous studies. Binding to the dynamin-2 PRD was also monophasic but with a slightly lower affinity (K(D) approximately 25 nm). Endophilin-SH3 binding to both dynamin-1 and -2 PRDs was biphasic, with one high affinity site (K(D) approximately 14 nm) in the N terminus of the PRD and another lower affinity site (K(D) approximately 60 nm) in the C terminus of dynamin-1. The N-terminal site in dynamin-2 PRD had a 10-fold lower affinity for endophilin-SH3. Preloading of dynamin-1 PRD with the amphiphysin-SH3 domain partially occluded binding of the endophilin-SH3 domain, indicating overlap between the binding sites in the C terminus, but endophilin was still able to interact with the high affinity N-terminal site. This shows that more than one SH3 domain can simultaneously bind to the PRD and suggests that competition probably occurs in vivo between different SH3-containing proteins for the limited number of PXXP motifs. Endophilin-SH3 binding to the high affinity site was disrupted when dynamin-1 PRD was phosphorylated with Cdk5, indicating that this site overlaps the phosphorylation sites, but amphiphysin-SH3 binding was unaffected. Other SH3 domains showed similarly complex binding characteristics, and substantial differences were noted between the PRDs from dynamin-1 and -2. For example, SH3 domains from c-Src, Grb2, and intersectin bound only to the C-terminal half of dynamin-2 PRD but to both the N- and C-terminal portions of dynamin-1 PRD. Thus, differential binding of SH3 domain-containing proteins to dynamin-1 and -2 may contribute to the distinct functions performed by these isoforms.

    The Journal of biological chemistry 2005;280;24;23147-56

  • The crystal structure of a c-Src complex in an active conformation suggests possible steps in c-Src activation.

    Cowan-Jacob SW, Fendrich G, Manley PW, Jahnke W, Fabbro D, Liebetanz J and Meyer T

    Discovery Technologies, Novartis Institutes for Biomedical Research, CH-4056 Basel, Switzerland. sandra.jacob@novartis.com

    The regulation of the activity of Abl and Src family tyrosine kinases is mediated by intramolecular interactions between the SH3, SH2, and kinase (SH1) domains. We have determined the crystal structure of an unphosphorylated form of c-Src in which the SH2 domain is not bound to the C-terminal tail. This results in an open structure where the kinase domain adopts an active conformation and the C terminus binds within a hydrophobic pocket in the C-terminal lobe. NMR binding studies support the hypothesis that an N-terminal myristate could bind in this pocket, as observed for Abl, suggesting that c-Src may also be regulated by myristate binding. In addition, the structure contains a des-methyl analog of the antileukemia drug imatinib (STI571; Gleevec). This structure reveals why the drug shows a low affinity for active kinase conformations, contributing to its excellent kinase selectivity profile.

    Structure (London, England : 1993) 2005;13;6;861-71

  • Src kinase regulation by phosphorylation and dephosphorylation.

    Roskoski R

    Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, 1100 Florida Avenue, New Orleans, LA 70119, USA. biocrr@lsuhsc.edu <biocrr@lsuhsc.edu&gt;

    Src and Src-family protein-tyrosine kinases are regulatory proteins that play key roles in cell differentiation, motility, proliferation, and survival. The initially described phosphorylation sites of Src include an activating phosphotyrosine 416 that results from autophosphorylation, and an inhibiting phosphotyrosine 527 that results from phosphorylation by C-terminal Src kinase (Csk) and Csk homologous kinase. Dephosphorylation of phosphotyrosine 527 increases Src kinase activity. Candidate phosphotyrosine 527 phosphatases include cytoplasmic PTP1B, Shp1 and Shp2, and transmembrane enzymes include CD45, PTPalpha, PTPepsilon, and PTPlambda. Dephosphorylation of phosphotyrosine 416 decreases Src kinase activity. Thus far PTP-BL, the mouse homologue of human PTP-BAS, has been shown to dephosphorylate phosphotyrosine 416 in a regulatory fashion. The platelet-derived growth factor receptor protein-tyrosine kinase mediates the phosphorylation of Src Tyr138; this phosphorylation has no direct effect on Src kinase activity. The platelet-derived growth factor receptor and the ErbB2/HER2 growth factor receptor protein-tyrosine kinases mediate the phosphorylation of Src Tyr213 and activation of Src kinase activity. Src kinase is also a substrate for protein-serine/threonine kinases including protein kinase C (Ser12), protein kinase A (Ser17), and CDK1/cdc2 (Thr34, Thr46, and Ser72). Of the three protein-serine/threonine kinases, only phosphorylation by CDK1/cdc2 has been demonstrated to increase Src kinase activity. Although considerable information on the phosphoprotein phosphatases that catalyze the hydrolysis of Src phosphotyrosine 527 is at hand, the nature of the phosphatases that mediate the hydrolysis of phosphotyrosine 138 and 213, and phosphoserine and phosphothreonine residues has not been determined.

    Biochemical and biophysical research communications 2005;331;1;1-14

  • Src-mediated tyrosine phosphorylation of p47phox in hyperoxia-induced activation of NADPH oxidase and generation of reactive oxygen species in lung endothelial cells.

    Chowdhury AK, Watkins T, Parinandi NL, Saatian B, Kleinberg ME, Usatyuk PV and Natarajan V

    Department of Medicine, Division of Pulmonary and Critical Care Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21224, USA.

    Superoxide (O(2)(-)) production by nonphagocytes, similar to phagocytes, is by activation of the NADPH oxidase multicomponent system. Although activation of neutrophil NADPH oxidase involves extensive serine phosphorylation of p47(phox), the role of tyrosine phosphorylation of p47(phox) in NADPH oxidase-dependent O(2)(-) production is unclear. We have shown recently that hyperoxia-induced NADPH oxidase activation in human pulmonary artery endothelial cells (HPAECs) is regulated by mitogen-activated protein kinase signal transduction. Here we provided evidence on the role of nonreceptor tyrosine kinase, Src, in hyperoxia-induced tyrosine phosphorylation of p47(phox) and NADPH oxidase activation in HPAECs. Exposure of HPAECs to hyperoxia for 1 h resulted in increased O(2)(-) and reactive oxygen species (ROS) production and enhanced tyrosine phosphorylation of Src as determined by Western blotting with phospho-Src antibodies. Pretreatment of HPAECs with the Src kinase inhibitor PP2 (1 mum) or transient expression of a dominant-negative mutant of Src attenuated hyperoxia-induced tyrosine phosphorylation of Src and ROS production. Furthermore, exposure of cells to hyperoxia enhanced tyrosine phosphorylation of p47(phox) and its translocation to cell peripheries that were attenuated by PP2. In vitro, Src phosphorylated recombinant p47(phox) in a time-dependent manner. Src immunoprecipitates of cell lysates from control cells revealed the presence of immunodetectable p47(phox) and p67(phox), suggesting the association of oxidase components with Src under basal conditions. Moreover, exposure of HPAECs to hyperoxia for 1 h enhanced the association of p47(phox), but not p67(phox), with Src. These results indicated that Src-dependent tyrosine phosphorylation of p47(phox) regulates hyperoxia-induced NADPH oxidase activation and ROS production in HPAECs.

    Funded by: NHLBI NIH HHS: P01 HL 58064, R01 HL 69909

    The Journal of biological chemistry 2005;280;21;20700-11

  • Connexin32 as a tumor suppressor gene in a metastatic renal cell carcinoma cell line.

    Fujimoto E, Sato H, Shirai S, Nagashima Y, Fukumoto K, Hagiwara H, Negishi E, Ueno K, Omori Y, Yamasaki H, Hagiwara K and Yano T

    Department of Food Science Research for Health, National Institute of Health and Nutrition, 1-23-1 Toyama, Shinjuku, 162-8636 Tokyo, Japan.

    Connexin genes expressing gap junction proteins have tumor-suppressive effects on primary cancers with certain cell specificity, but the suppressive effects on metastatic cancers are still conflicting. In this study, we show that connexin32 (Cx32) has a strong tumor-suppressive effect on a human metastatic renal cell carcinoma cell line (Caki-1 cell). Cx32 expression in Caki-1 cells reduced in vitro malignant phenotypes of the cells such as anchorage independency and invasion capacity. Furthermore, the Cx32 expression drastically reduced the development of Caki-1 cells in nude mice. We also determined that Cx32 reduced the malignant phenotypes in Caki-1 cells mainly through the inactivation of Src signaling. Especially, Cx32-dependent inactivation of Src decreased the production of vascular epithelial growth factor (VEGF) via the suppression of signal transducers and activators of transcription 3 (Stat3) activation, and we confirmed this result using short interfering RNA. In nude mice, Cx32-transfected Caki-1 cells showed lower serum level of VEGF comparing mock transfectant, and the development of the cells in nude mice positively related to the VEGF level. These data suggest that Cx32 acts as a tumor suppressor gene in Caki-1 cells and that the tumor-suppressive effect partly depends on the inhibition of Src-Stat3-VEGF signal pathway.

    Oncogene 2005;24;22;3684-90

  • HIF-1alpha, STAT3, CBP/p300 and Ref-1/APE are components of a transcriptional complex that regulates Src-dependent hypoxia-induced expression of VEGF in pancreatic and prostate carcinomas.

    Gray MJ, Zhang J, Ellis LM, Semenza GL, Evans DB, Watowich SS and Gallick GE

    Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, 1515 Holcomb Boulevard, Houston, TX 77030, USA.

    Hypoxia stimulates a number of pathways critical to cancer cell survival, including the activation of vascular endothelial growth factor (VEGF) transcription. In normal fibroblasts, hypoxia-induced activation of the protein tyrosine kinase, Src, is required for VEGF expression. We show here in both pancreatic and prostate carcinoma cell lines cobalt chloride (used to mimic hypoxia) -induced VEGF expression requires Src activation and leads to increased steady-state levels of HIF-1alpha and increased phosphorylation of signal and transducer of transcription 3 (STAT3). STAT3 and hypoxia-inducible factor (HIF)-1alpha bind simultaneously to the VEGF promoter, where they form a molecular complex with the transcription coactivators CBP/p300 and Ref-1/APE. Expression of activated Src from an inducible promoter is sufficient to increase VEGF expression and form these STAT3/HIF-1alpha-containing promoter complexes. Inhibition of DNA binding by expression of either STAT3 or HIF-1alpha dominant negative mutants significantly reduces VEGF expression. These data suggest that the binding of both STAT3 and HIF-1alpha to the VEGF promoter is required for maximum transcription of VEGF mRNA following hypoxia.

    Funded by: NCI NIH HHS: 2RO-1 CA65527, U54 CA 090810-01

    Oncogene 2005;24;19;3110-20

  • Phosphorylation of the human Fhit tumor suppressor on tyrosine 114 in Escherichia coli and unexpected steady state kinetics of the phosphorylated forms.

    Garrison PN, Robinson AK, Pekarsky Y, Croce CM and Barnes LD

    Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229-3900, USA.

    The human tumor suppressor Fhit is a homodimeric histidine triad (HIT) protein of 147 amino acids which has Ap(3)A hydrolase activity. We have recently discovered that Fhit is phosphorylated in vivo and is phosphorylated in vitro by Src kinase [Pekarsky, Y., Garrison, P. N., Palamarchuk, A., Zanesi, N., Aqeilan, R. I., Huebner, K., Barnes, L. D., and Croce, C. M. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 3775-3779]. Now we have coexpressed Fhit with the elk tyrosine kinase in Escherichia coli to generate phosphorylated forms of Fhit. Unphosphorylated Fhit, Fhit phosphorylated on one subunit, and Fhit phosphorylated on both subunits were purified to apparent homogeneity by column chromatography on anion-exchange and gel filtration resins. MALDI-TOF and HPLC-ESI tandem mass spectrometry of intact Fhit and proteolytic peptides of Fhit demonstrated that Fhit is phosphorylated on Y(114) on either one or both subunits. Monophosphorylated Fhit exhibited monophasic kinetics with K(m) and k(cat) values approximately 2- and approximately 7-fold lower, respectively, than the corresponding values for unphosphorylated Fhit. Diphosphorylated Fhit exhibited biphasic kinetics. One site had K(m) and k(cat) values approximately 2- and approximately 140-fold lower, respectively, than the corresponding values for unphosphorylated Fhit. The second site had a K(m) approximately 60-fold higher and a k(cat) approximately 6-fold lower than the corresponding values for unphosphorylated Fhit. The unexpected kinetic patterns for the phosphorylated forms suggest the system may be enzymologically novel. The decreases in the values of K(m) and k(cat) for the phosphorylated forms in comparison to those of unphosphorylated Fhit favor the formation and lifetime of the Fhit-Ap(3)A complex, which may enhance the tumor suppressor activity of Fhit.

    Funded by: NCI NIH HHS: CA56036, CA77738, P30CA54174

    Biochemistry 2005;44;16;6286-92

  • The C2 domain of PKCdelta is a phosphotyrosine binding domain.

    Benes CH, Wu N, Elia AE, Dharia T, Cantley LC and Soltoff SP

    Department of Medicine, Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA.

    In eukaryotic cells, the SH2 and PTB domains mediate protein-protein interactions by recognizing phosphotyrosine residues on target proteins. Here we make the unexpected finding that the C2 domain of PKCdelta directly binds to phosphotyrosine peptides in a sequence-specific manner. We provide evidence that this domain mediates PKCdelta interaction with a Src binding glycoprotein, CDCP1. The crystal structure of the PKCdelta C2 domain in complex with an optimal phosphopeptide reveals a new mode of phosphotyrosine binding in which the phosphotyrosine moiety forms a ring-stacking interaction with a histidine residue of the C2 domain. This is also the first example of a protein Ser/Thr kinase containing a domain that binds phosphotyrosine.

    Funded by: NIDCR NIH HHS: DE10877, DE14721; NIDDK NIH HHS: P30DK34854; NIGMS NIH HHS: GM56203, R01 GM056203

    Cell 2005;121;2;271-80

  • c-src activating mutation analysis in Chinese patients with colorectal cancer.

    Tan YX, Wang HT, Zhang P, Yan ZH, Dai GL, Wu MC and Wang HY

    International Co-operational Laboratory on Signal Transduction, Eastern Hepatobiliary Institute, Shanghai 200438, China.

    Aim: To investigate the occurrence of cellular src (c-src) activating mutation at codon 531 in colorectal cancer patients from Chinese mainland.

    Methods: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay followed by sequencing and single-strand conformation polymor-phism analysis were carried out to screen 110 samples of primary colorectal cancer and 20 colorectal liver metastases.

    Results: Only one sample showed PCR-RFLP-positive results and carried somatic codon 531 mutations. No additional mutation of c-src exon 12 was found.

    Conclusion: c-src codon 531 mutation in colorectal cancer is not the cause of c-src activation.

    World journal of gastroenterology : WJG 2005;11;15;2351-3

  • The transcriptional co-activator protein p100 recruits histone acetyltransferase activity to STAT6 and mediates interaction between the CREB-binding protein and STAT6.

    Välineva T, Yang J, Palovuori R and Silvennoinen O

    Institute of Medical Technology, University of Tampere, FIN-33014 Tampere, Finland.

    STAT6 is a critical regulator of transcription for interleukin-4 (IL-4)-induced genes. Activation of gene expression involves recruitment of coactivator proteins that function as bridging factors connecting sequence-specific transcription factors to the basal transcription machinery, and as chromatin-modifying enzymes. Coactivator proteins CBP/p300 have been implicated in regulation of transcription in all STATs. CBP is also required for STAT6-mediated gene activation, but the underlying molecular mechanisms are still elusive. In this study we investigated the mechanisms by which STAT6 recruits CBP and chromatin-modifying activities to the promoter. Our results indicate that while STAT1-interacted directly with CBP, the interaction between STAT6 and CBP was found to be mediated through p100 protein, a coactivator protein that has previously been shown to stimulate the transcription of IL-4-induced genes. The staphylococcal nuclease-like (SN)-domains of p100 directly interacted with amino acids 1099-1758 of CBP, while p100 did not associate with SRC-1, another coactivator of STAT6. p100 was found to recruit histone acetyltransferase (HAT) activity to STAT6 in vivo. Chromatin immunoprecipitation studies demonstrated that p100 increases the STAT6-p100-CBP ternary complex formation in the human Igepsilon promoter. p100 also increased the amount of acetylated histone H4 at the Igepsilon promoter, and siRNAs directed against p100 effectively inhibited Igepsilon reporter gene expression. Our results suggest that p100 has an important role in the assembly of STAT6 transcriptosome, and that p100 stimulates IL-4-dependent transcription by mediating interaction between STAT6 and CBP and recruiting chromatin modifying activities to STAT6-responsive promoters.

    The Journal of biological chemistry 2005;280;15;14989-96

  • Delayed activation of insulin-like growth factor-1 receptor/Src/MAPK/Egr-1 signaling regulates clusterin expression, a pro-survival factor.

    Criswell T, Beman M, Araki S, Leskov K, Cataldo E, Mayo LD and Boothman DA

    Department of Radiation Oncology and Program in Molecular and Cellular Basis of Disease, Laboratory of Molecular Stress Responses, Case Comprehensive Cancer Center, Cleveland, Ohio 44106-7285, USA.

    Secretory clusterin protein (sCLU) is a general genotoxic stress-induced, pro-survival gene product implicated in aging, obesity, heart disease, and cancer. However, the regulatory signal transduction processes that control sCLU expression remain undefined. Here, we report that induction of sCLU is delayed, peaking 72 h after low doses of ionizing radiation, and is dependent on the up-regulation of insulin-like growth factor-1 as well as phosphorylation-dependent activation of its receptor (IGF-1 and IGF-1R, respectively). Activated IGF-1R then stimulates the downstream Src-Mek-Erk signal transduction cascade to ultimately transactivate the early growth response-1 (Egr-1) transcription factor, required for sCLU expression. Thus, ionizing radiation exposure causes stress-induced activation of IGF-1R-Src-Mek-Erk-Egr-1 signaling that regulates the sCLU pro-survival cascade pathway, important for radiation resistance in cancer therapy.

    Funded by: NCI NIH HHS: P30 CA142543, P30 CA43703, R01 CA078530

    The Journal of biological chemistry 2005;280;14;14212-21

  • Involvement of c-Src and protein kinase C delta in the inhibition of Cl(-)/OH- exchange activity in Caco-2 cells by serotonin.

    Saksena S, Gill RK, Tyagi S, Alrefai WA, Sarwar Z, Ramaswamy K and Dudeja PK

    Section of Digestive Diseases and Nutrition, Department of Medicine, University of Illinois and Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois 60612, USA.

    Serotonin (5-hydroxytryptamine (5-HT)) is an important neurotransmitter and intercellular messenger regulating various gastrointestinal functions, including electrolyte transport. To date, however, no information is available with respect to its effects on the human intestinal apical anion exchanger Cl(-)/OH- (HCO3-). The present studies were therefore undertaken to examine the direct effects of serotonin on OH- gradient-driven 4,4'-diisothiocyanato-stilbene-2, 2'-disulfonic acid-sensitive 36Cl- uptake utilizing the post-confluent transformed human intestinal epithelial cell line Caco-2. Our results demonstrate that serotonin inhibits Cl(-)/OH- exchange activity in Caco-2 cells via both tyrosine kinase and Ca(2+)-independent protein kinase C delta-mediated pathways involving either 5-HT3 or 5-HT4 receptor subtype. The data consistent with our inference are as follows. (i) The short term treatment of cells with 5-HT (0.1 microM) for 15-60 min significantly decreased Cl(-)/OH- exchange (50-70%, p < 0.05). (ii) The specific agonists for 5-HT3, m-chlorophenylbiguanide, and 5-HT4, 3-(4-allylpiperazin-1-yl)-2-quinoxaline chloronitrile, mimicked the effects of serotonin. (iii) Tropisetron dual inhibitor for both the 5-HT3/4 receptor subtypes significantly blocked the inhibition, whereas specific 5-HT3 (Y-25130) or 5-HT4 receptor (RS39604) antagonist failed to block the inhibitory effects of 5-HT. (iv) The Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl ester) had no effect on the serotonin-induced inhibition. (v) The specific protein kinase C (PKC) inhibitors chelerythrine chloride or calphostin C completely blocked the inhibition by 5-HT. (vi) The specific inhibitor for PKC delta, rottlerin, significantly blocked the inhibition by 5-HT. (vii) The specific tyrosine kinase inhibitor, herbimycin, or Src family kinase inhibitor, PP1, abolished the 5-HT-mediated inhibition of Cl(-)/OH- exchange activity. (viii) 5-HT stimulated tyrosine phosphorylation of c-Src kinase and PKC delta.

    Funded by: NIDDK NIH HHS: DK 33349, DK 67990, DK 68324, DK54016, DK62221

    The Journal of biological chemistry 2005;280;12;11859-68

  • TGF-beta1-mediated activations of c-Src and Rac1 modulate levels of cyclins and p27(Kip1) CDK inhibitor in hepatoma cells replated on fibronectin.

    Kim HP, Kim TY, Lee MS, Jong HS, Kim TY, Lee JW and Bang YJ

    National Research Laboratory for Cancer Epigenetics, Cancer Research Institute, Department of Tumor Biology, College of Medicine, Seoul National University, Seoul 110-799, South Korea.

    Integrin-mediated cell adhesion transduces signals to regulate actin cytoskeleton and cell proliferation. While understanding how integrin signals cross-talk with the TGF-beta1 pathways, we observed lamellipodia formation and cyclin regulation in Hep3B cells, following TGF-beta1 treatment. To answer if integrin signaling via actin organization might regulate cell cycle progression after TGF-beta1 treatment, we analyzed cross-talk between the two receptor-mediated pathways in hepatoma cells on specific ECMs. We found that basal and TGF-beta1-mediated activation of c-Src and Rac1, expression of cyclins E and A, and suppression of p27Kip1 were significant in cells replated on fibronectin, but not in cells on collagen I, indicating a different integrin-mediated cellular response to TGF-beta1 treatment. Levels of tyrosine phosphorylation and actin-enriched lamellipodia on fibronectin were also more prominent than in cells on collagen I. Studies using pharmacological inhibitors or transient transfections revealed that the preferential TGF-beta1 effects in cells on fibronectin required c-Src family kinase activity. These observations suggest that a specific cross-talk between TGF-beta1 and fibronectin-binding integrin signal pathways leads to the activation of c-Src/Rac1/actin-organization, leading to changes in cell cycle regulator levels in hepatoma cells. Therefore, this study represents another mechanism to regulate cell cycle regulators when integrin signaling is collaborative with TGF-beta1 pathways.

    Biochimica et biophysica acta 2005;1743;1-2;151-61

  • Src phosphorylates ezrin at tyrosine 477 and induces a phosphospecific association between ezrin and a kelch-repeat protein family member.

    Heiska L and Carpén O

    Department of Pathology, Neuroscience Program, Biomedicum Helsinki, University of Helsinki and Helsinki University Hospital, FIN-00014 Helsinki, Finland.

    Ezrin, a linker between plasma membrane and actin cytoskeleton possesses morphogenic properties and can promote dissemination of tumor cells. Ezrin is phosphorylated on tyrosine, but a detailed picture of the signaling pathways involved in this modification is lacking. The transforming tyrosine kinase Src has various cytoskeletal substrates and is involved in regulation of cellular adhesion. We studied the role of Src in tyrosine phosphorylation of ezrin in adherent cells. We show that ezrin is phosphorylated in human embryonic kidney 293 cells in a Src family-dependent way. In SYF cells lacking Src, Yes, and Fyn, ezrin was not tyrosine-phosphorylated but reintroduction of wild-type Src followed by Src activation or introduction of active Src restored phosphorylation. Mapping of the Src-catalyzed tyrosine in vitro and in vivo by site-directed mutagenesis demonstrated Tyr(477) as the primary target residue. We generated a pTyr(477)-phosphospecific antibody, which confirmed that Tyr(477) becomes phosphorylated in cells in a Src-dependent manner. Tyr(477) phosphorylation did not affect ezrin head-to-tail association or phosphorylation of ezrin on threonine 566, indicating that the function of Tyr(477) phosphorylation is not related to the intramolecular regulation of ezrin. A modified yeast two-hybrid screen in which ezrin bait was phosphorylated by Src identified a novel interaction with a kelch-repeat protein family member, KBTBD2 (Kelch-repeat and BTB/POZ domain containing 2). The Src dependence of the interaction was further verified by affinity precipitation assays. Identification of a functional interplay with Src opens novel avenues for further characterization of the biological activities of ezrin.

    The Journal of biological chemistry 2005;280;11;10244-52

  • Associations and interactions between Ets-1 and Ets-2 and coregulatory proteins, SRC-1, AIB1, and NCoR in breast cancer.

    Myers E, Hill AD, Kelly G, McDermott EW, O'Higgins NJ, Buggy Y and Young LS

    Department of Surgery, Saint Vincent's University Hospital University College Dublin, Dublin 4, Ireland.

    Purpose: Associations between p160 coactivator proteins and the development of resistance to endocrine treatment have been described. We hypothesized that nuclear receptor coregulatory proteins may interact with nonsteroid receptors. We investigated the mitogen-activated protein kinase-activated transcription factors, Ets, as possible interaction proteins for the coactivators SRC-1 and AIB1 and the corepressor NCoR in human breast cancer.

    Expression and coexpression of Ets and the coregulatory proteins was investigated using immunohistochemistry and immunofluorescence in a cohort of breast tumor patients (N = 134). Protein expression, protein-DNA interactions and protein-protein interactions were assessed using Western blot, electromobility shift, and coimmunoprecipitation analysis, respectively.

    Results: Ets-1 and Ets-2 associated with reduced disease-free survival (P < 0.0292, P < 0.0001, respectively), whereas NCoR was a positive prognostic indicator (P < 0.0297). Up-regulation of Ets-1 protein expression in cell cultures derived from patient tumors in the presence of growth factors associated with tumor grade (P < 0.0013; n = 28). In primary breast tumor cell cultures and in the SKBR3 breast cell line, growth factors induced interaction between Ets and their DNA response element, induced recruitment of coactivators to the transcription factor-DNA complex, and up-regulated protein expression of HER2. Ets-1 and Ets-2 interacted with the coregulators under basal conditions, and growth factors up-regulated Ets-2 interaction with SRC-1 and AIB1. Coexpression of Ets-2 and SRC-1 significantly associated with the rate of recurrence and HER expression, compared with patients who expressed Ets-2 but not SRC-1 (P < 0.0001 and P < 0.0001, respectively).

    Conclusions: These data describe associations and interactions between nonsteroid transcription factors and coregulatory proteins in human breast cancer.

    Clinical cancer research : an official journal of the American Association for Cancer Research 2005;11;6;2111-22

  • The steroid receptor co-activator-1 (SRC-1) potentiates TGF-beta/Smad signaling: role of p300/CBP.

    Dennler S, Pendaries V, Tacheau C, Costas MA, Mauviel A and Verrecchia F

    INSERM U697, Institut de recherche sur la peau, Hôpital Saint-Louis, Paris, France.

    The three related 160-kDa proteins, SRC-1, TIF-2 and RAC-3, were initially identified as factors interacting with nuclear receptors. They have also been reported to potentiate the activity of other transcription factors such as AP-1 or NF-kappaB. The aim of this work was to identify whether SRC-1 interferes with the TGF-beta/Smad signaling pathway, and if so, to identify its underlying mechanisms of action. Using transient cell transfection experiments performed in human dermal fibroblasts with the Smad3/4-specific (SBE)4-lux reporter construct, as well as the human PAI-1 promoter, we determined that SRC-1 enhances TGF-beta-induced, Smad-mediated, transcription. Likewise, SRC-1 overexpression potentiated TGF-beta-induced upregulation of PAI-1 steady-state mRNA levels. Using a mammalian two-hybrid system, we demonstrated that SRC-1 interacts with the transcriptional co-activators p300/CBP, but not with Smad3. Overexpression of the adenovirus E1A oncoprotein, an inhibitor of CBP/p300 activity, prevented the enhancing effect of SRC-1 on Smad3/4-mediated transcription, indicating that p300/CBP may be required for SRC-1 effect. Such hypothesis was validated, as expression of a mutant form of SRC-1 lacking the CBP/p300-binding site failed to upregulate Smad3/4-dependent transcription, while full-length SRC-1 potentiated p300.Smad3 interactions. These results identify SRC-1 as a novel Smad3/4 transcriptional partner, facilitating the functional link between Smad3 and p300/CBP.

    Oncogene 2005;24;11;1936-45

  • Phorbol 12-myristate 13-acetate induces epidermal growth factor receptor transactivation via protein kinase Cdelta/c-Src pathways in glioblastoma cells.

    Amos S, Martin PM, Polar GA, Parsons SJ and Hussaini IM

    Department of Pathology, University of Virginia Health System, Charlottesville 22908, USA. sa7h@virginia.edu

    Both the epidermal growth factor receptor (EGFR) and protein kinase C (PKC) play important roles in glioblastoma invasive growth; however, the interaction between the EGFR and PKC is not well characterized in glioblastomas. Treatment with EGF stimulated global phosphorylation of the EGFR at Tyr(845), Tyr(992), Tyr(1068), and Tyr(1045) in glioblastoma cell lines (U-1242 MG and U-87 MG). Interestingly, phorbol 12-myristate 13-acetate (PMA) stimulated phosphorylation of the EGFR only at Tyr(1068) in the two glioblastoma cell lines. Phosphorylation of the EGFR at Tyr(1068) was not detected in normal human astrocytes treated with the phorbol ester. PMA-induced phosphorylation of the EGFR at Tyr(1068) was blocked by bisindolylmaleimide (BIM), a PKC inhibitor, and rottlerin, a PKCdelta-specific inhibitor. In contrast, Go 6976, an inhibitor of classical PKC isozymes, had no effect on PMA-induced EGFR phosphorylation. Furthermore, gene silencing with PKCdelta small interfering RNA (siRNA), siRNA against c-Src, and mutant c-Src(S12C/S48A) and treatment with a c-Src inhibitor (4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine) abrogated PMA-induced EGFR phosphorylation at Tyr(1068). PMA induced serine/threonine phosphorylation of Src, which was blocked by both BIM and rottlerin. Inhibition of the EGFR with AG 1478 did not significantly alter PMA-induced EGFR Tyr(1068) phosphorylation, but completely blocked EGF-induced phosphorylation of the EGFR. The effects of PMA on MAPK phosphorylation and glioblastoma cell proliferation were reduced by BIM, rottlerin, the MEK inhibitor U0126, and PKCdelta and c-Src siRNAs. Taken together, our data demonstrate that PMA transactivates the EGFR and increases cell proliferation by activating the PKCdelta/c-Src pathway in glioblastomas.

    Funded by: NCI NIH HHS: CA90851, R01 CA090851-01A2, R01 CA090851-02, R01 CA090851-03; NINDS NIH HHS: NS35122

    The Journal of biological chemistry 2005;280;9;7729-38

  • Phosphoryl transfer step in the C-terminal Src kinase controls Src recognition.

    Lieser SA, Shindler C, Aubol BE, Lee S, Sun G and Adams JA

    Department of Pharmacology, University of California at San Diego, La Jolla, California 92093-0728, USA.

    All members of the Src family of nonreceptor protein tyrosine kinases are phosphorylated and subsequently down-regulated by the C-terminal Src kinase, Csk. Although the recognition of Src protein substrates is essential for a diverse set of signaling events linked to cellular growth and differentiation, the factors controlling this critical protein-protein interaction are not well known. To understand how Csk recognizes Src, the chemical/physical events that modulate apparent substrate affinity and turnover were investigated. Src is phosphorylated in a biphasic manner in rapid quench flow experiments, suggesting that the phosphoryl transfer step is fast and highly favorable and does not limit overall turnover. As opposed to other kinase-substrate pairs, turnover is not limited by the physical release of ADP based on stopped-flow fluorescence and catalytic trapping experiments, suggesting that other steps control net phosphorylation. The K(d) for Src is considerably larger than the K(m) based on single turnover kinetic and equilibrium sedimentation experiments. Taken together, the data are consistent with a mechanism whereby Csk achieves a low K(m) for the substrate Src, not by stabilizing protein-protein interactions but rather by facilitating a fast phosphoryl transfer step. In this manner, the phosphoryl transfer step functions as a chemical clamp facilitating substrate recognition.

    Funded by: NCRR NIH HHS: 1P20 RR16457; NIGMS NIH HHS: GM 07752, GM 68168

    The Journal of biological chemistry 2005;280;9;7769-76

  • The Src kinase pathway promotes tamoxifen agonist action in Ishikawa endometrial cells through phosphorylation-dependent stabilization of estrogen receptor (alpha) promoter interaction and elevated steroid receptor coactivator 1 activity.

    Shah YM and Rowan BG

    Department of Biochemistry and Molecular Biology, Medical College of Ohio, 3035 Arlington Ave., Toledo, Ohio 43614-5804, USA.

    Tamoxifen is the most widely used selective estrogen receptor modulator for breast cancer in clinical use today. However, tamoxifen agonist action in endometrium remains a major hurdle for tamoxifen therapy. Activation of the nonreceptor tyrosine kinase src promotes tamoxifen agonist action, although the mechanisms remain unclear. To examine these mechanisms, the effect of src kinase on estrogen and tamoxifen signaling in tamoxifen-resistant Ishikawa endometrial adenocarcinoma cells was assessed. A novel connection was identified between src kinase and serine 167 phosphorylation in estrogen receptor (ER)-alpha via activation of AKT kinase. Serine 167 phosphorylation stabilized ER interaction with endogenous ER-dependent promoters. Src kinase exhibited the additional function of potentiating the transcriptional activity of Gal-steroid receptor coactivator 1 (SRC-1) and Gal-cAMP response element binding protein-binding protein in endometrial cancer cells while having no effect on Gal-p300-associated factor and Gal fusions of the other p160 coactivators glucocorticoid-interacting protein 1 (transcriptional intermediary factor 2/nuclear coactivator-2/SRC-2) and amplified in breast cancer 1 (receptor-associated coactivator 3/activator of transcription of nuclear receptor/SRC-3). Src effects on ER phosphorylation and SRC-1 activity both contributed to tamoxifen agonist action on ER-dependent gene expression in Ishikawa cells. Taken together, these data demonstrate that src kinase potentiates tamoxifen agonist action through serine 167-dependent stabilization of ER promoter interaction and through elevation of SRC-1 and cAMP response element binding protein-binding protein coactivation of ER.

    Funded by: NIDDK NIH HHS: R01 DK06832

    Molecular endocrinology (Baltimore, Md.) 2005;19;3;732-48

  • Selective regulation of tumor necrosis factor-induced Erk signaling by Src family kinases and the T cell protein tyrosine phosphatase.

    van Vliet C, Bukczynska PE, Puryer MA, Sadek CM, Shields BJ, Tremblay ML and Tiganis T

    Department of Biochemistry and Molecular Biology, Monash University, Victoria 3800, Australia.

    The proinflammatory cytokine tumor necrosis factor (TNF) modulates cellular responses through the mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-kappaB) signaling pathways, but the molecular mechanisms underlying MAPK activation are unknown. T cell protein tyrosine phosphatase (TCPTP) is essential for hematopoietic development and negatively regulates inflammatory responses. Using TCPTP-deficient fibroblasts, we show here that TCPTP regulates TNF-induced MAPK but not NF-kappaB signaling. TCPTP interacted with the adaptor protein TRAF2, and dephosphorylated and inactivated Src tyrosine kinases to suppress downstream signaling through extracellular signal-regulated kinases and production of interleukin 6. These results link TCPTP and Src tyrosine kinases to the selective regulation of TNF-induced MAPK signaling and identify a previously unknown mechanism for modulating inflammatory responses mediated by TNF.

    Nature immunology 2005;6;3;253-60

  • Src-dependent ezrin phosphorylation in adhesion-mediated signaling.

    Srivastava J, Elliott BE, Louvard D and Arpin M

    Morphogenèse et Signalisation Cellulaires, UMR144 CNRS-Institut Curie, 75248 Paris Cedex 05, France.

    In addition to providing a regulated linkage between the membrane and the actin cytoskeleton, ezrin participates in signal transduction pathways. Here we describe that expression of the ezrin Y145F mutant delays epithelial cell spreading on fibronectin by inhibiting events leading to FAK activation. The defect in spreading was rescued by the overexpression of catalytically functional Src. We demonstrate that ezrin Y145 is phosphorylated in A431 cells stimulated with epidermal growth factor (EGF) and in v-Src-transformed cells. Moreover in cells devoid of Src, SYF-/- fibroblasts, ezrin Y145 phosphorylation could only be detected upon the introduction of an active form of Src. The phosphorylation of ezrin at Y145 required prior binding of the Src SH2 domain to ezrin. Our results further show that Src activity influences its binding to ezrin and a positive feedback mechanism for Src-mediated Y145 phosphorylation is implied. Interestingly, cells expressing ezrin Y145F did not proliferate when cultured in a 3D collagen gel. Collectively, our results demonstrate a key signaling input of Src-dependent ezrin phosphorylation in adhesion-mediated events in epithelial cells.

    Molecular biology of the cell 2005;16;3;1481-90

  • Src is necessary and sufficient for human airway smooth muscle cell proliferation and migration.

    Krymskaya VP, Goncharova EA, Ammit AJ, Lim PN, Goncharov DA, Eszterhas A and Panettieri RA

    Pulmonary, Allergy and Critical Care Division, Department of Medicine, University of Pennsylvania, 421 Curie Blvd., BRB II/III, Philadelphia, PA 19104-6160, USA. krymskay@mail.med.upenn.edu

    Airway smooth muscle (ASM) hypertrophy and hyperplasia, important pathological features in chronic severe asthma, likely contribute to irreversible airflow obstruction. Despite considerable research effort, the precise cellular mechanisms that modulate ASM growth remain unknown. Src, a nonreceptor tyrosine kinase proto-oncogene, reportedly modulates cell proliferative responses to growth factors, contractile agonists, and inflammatory mediators. Here, we show that Src activation is required for human ASM mitogenesis and motility. Platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and thrombin induce rapid activation of Src, and inhibition of Src induces a concentration-dependent abrogation of PDGF-, EGF-, and thrombin-induced ASM cell proliferation. Src immunoprecipitates had associated phosphatidylinositol 3-kinase, or PI3K, activation in response to PDGF and thrombin but not EGF. Further, Src activation is both necessary and sufficient for the stimulation of DNA synthesis as demonstrated by dominant negative Src inhibition of PDGF-, EGF-, and thrombin-induced DNA synthesis. Human ASM cell migration was also attenuated by transfection of cells with dominant negative Src. Further, expression of constitutively active Src promoted cell migration. Collectively, these data demonstrate that Src modulates human ASM cell proliferation and migration, suggesting that Src may play an important role in promoting ASM cell growth and migration that occur in airway remodeling found in asthma and chronic obstructive pulmonary disease, or COPD.

    Funded by: NHLBI NIH HHS: HL071106, HL55301, HL64063, HL67663

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2005;19;3;428-30

  • Functional interaction of protein kinase Calpha with the tyrosine kinases Syk and Src in human platelets.

    Pula G, Crosby D, Baker J and Poole AW

    Department of Pharmacology, School of Medical Sciences, University Walk, Bristol BS8 1TD, United Kingdom.

    There is a high degree of cross-talk between tyrosine phosphorylation and the serine/threonine phosphorylation signaling pathways. Here we show a physical and functional interaction between the classical protein kinase C isoform (cPKC), PKCalpha, and two major nonreceptor tyrosine kinases in platelets, Syk and Src. In the presence of the cPKC-selective inhibitor Go6976, platelet 5-hydroxytryptamine release was abolished in response to co-activation of glycoproteins VI and Ib-IX-V by the snake venom alboaggregin A, whereas platelet aggregation was substantially inhibited. Of the two platelet cPKCs, PKCalpha but not PKCbeta was activated, occurring in an Syk- and phospholipase C-dependent manner. Syk and PKCalpha associate in a stimulation-dependent manner, requiring Syk but not PKC activity. PKCalpha and Syk also co-translocate from the cytosol to the plasma membrane upon platelet activation, in a manner dependent upon the activities of both kinases. Although PKCalpha is phosphorylated on tyrosine downstream of Syk, we provide evidence against phosphorylation of Syk by PKCalpha, consistent with a lack of effect of PKCalpha inhibition on Syk activity. PKCalpha also associates with Src; although in contrast to interaction with Syk, PKCalpha activity is required for the association of these kinases but not the stimulation-induced translocation of Src to the cell membrane. Finally, the activity of Src is negatively regulated by PKC, as shown by potentiation of Src activity in the presence of the PKC inhibitors GF109203X or Go6976. Therefore, there is a complex interplay between PKCalpha, Syk, and Src involving physical interaction, phosphorylation, translocation within the cell, and functional activity regulation.

    The Journal of biological chemistry 2005;280;8;7194-205

  • A novel role for Gab1 and SHP2 in epidermal growth factor-induced Ras activation.

    Montagner A, Yart A, Dance M, Perret B, Salles JP and Raynal P

    Département Lipoprotéines et Médiateurs Lipidiques, INSERM U563, Hôpital Purpan, 31300 Toulouse, France.

    SHP2 was recently found to down-regulate PI3K activation by dephosphorylating Gab1 but the mechanisms explaining the positive role of the Gab1/SHP2 pathway in EGF-induced Ras activation remain ill defined. Substrate trapping experiments now suggest that SHP2 dephosphorylates other Gab1 phosphotyrosines located within a central region displaying four YXXP motifs. Because these sites are potential docking motifs for Ras-GAP, we tested whether SHP2 dephosphorylates them to facilitate Ras activation. We observed that a Gab1 construct preventing SHP2 recruitment promoted membrane relocation of RasGAP. Moreover, a RasGAP-inactive mutant restored the activation of Ras in cells transfected with SHP2-inactivating Gab1 mutant or in SHP2-deficient fibroblasts, supporting the hypothesis that RasGAP is a downstream target of SHP2. To determine whether Gab1 is a RasGAP-binding partner, a Gab1 mutant deleted of four YXXP motifs was produced. The deletion suppressed RasGAP redistribution and restored the defective Ras activation caused by SHP2-inactivating mutations. Moreover, Gab1 was found to interact with RasGAP SH2 domains, only under conditions where SHP2 is not activated. To identify Ras-GAP-binding sites, Tyr to Phe mutants of Gab1 YXXP motifs were produced. Gab1 constructs mutated on Tyr(317) were severely affected in RasGAP binding and were the most active in compensating for Ras-defective activation and blocking RasGAP redistribution induced by SHP2 inactivation. We have thus localized on Gab1 a Ras-negative regulatory tyrosine phosphorylation site involved in RasGAP binding and showed that an important SHP2 function is to down-regulate its phosphorylation to disengage RasGAP and sustain Ras activation.

    The Journal of biological chemistry 2005;280;7;5350-60

  • Src phosphorylation of Alix/AIP1 modulates its interaction with binding partners and antagonizes its activities.

    Schmidt MHH, Dikic I and Bögler O

    William and Karen Davidson Laboratory of Brain Tumor Biology, Hermelin Brain Tumor Center, Department of Neurosurgery, Henry Ford Hospital, Detroit, Michigan 48202, USA.

    Alix/AIP1 is an adaptor protein involved in regulating the function of receptor and cytoskeleton-associated tyrosine kinases. Here, we investigated its interaction with and regulation by Src. Tyr319 of Alix bound the isolated Src homology-2 (SH2) domain and was necessary for interaction with intact Src. A proline-rich region in the C terminus of Alix bound the Src SH3 domain, but this interaction was dependent on the release of the Src SH2 domain from its Src internal ligand either by interaction with Alix Tyr319 or by mutation of Src Tyr527. Src phosphorylated Alix at a C-terminal region rich in tyrosines, an activity that was stimulated by the presence of the Alix binding partner SETA/CIN85. Phosphorylation of Alix by Src caused it to translocate from the membrane and cytoskeleton to the cytoplasm and reduced its interaction with binding partners SETA/CIN85, epidermal growth factor receptor, and Pyk2. As a consequence of this, Src antagonized the negative regulation of receptor tyrosine kinase internalization and cell adhesion by Alix. We propose a model whereby Src antagonizes the effects of Alix by phosphorylation of its C terminus, leading to the disruption of interactions with target proteins.

    Funded by: NCI NIH HHS: CA-R01-108500, CA-R01-84109, R01 CA084109, R01 CA108500

    The Journal of biological chemistry 2005;280;5;3414-25

  • c-Src is involved in regulating signal transmission from PDGFbeta receptor-GPCR(s) complexes in mammalian cells.

    Waters CM, Connell MC, Pyne S and Pyne NJ

    Department of Physiology and Pharmacology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, 27 Taylor St., Glasgow G4 ONR, UK.

    We have reported that the platelet-derived growth factor receptor-beta (PDGFbeta) forms a novel signaling complex with G protein-coupled receptors (GPCR) (e.g. S1P(1) receptor) that enables more efficient activation of p42/p44 mitogen-activated protein kinase (MAPK) in response to PDGF and sphingosine 1-phosphate (S1P). We now demonstrate that c-Src participates in regulating the endocytosis of PDGFbeta receptor-GPCR complexes in response to PDGF. This leads to association of cytoplasmic p42/p44 MAPK with the receptor complex in endocytic vesicles. c-Src is regulated by G protein betagamma subunits and can interact with beta-arrestin. Indeed, the PDGF-dependent activation of p42/p44 MAPK was reduced by over-expression of the C-terminal domain of GRK2 (sequesters Gbetagamma subunits), the clathrin-binding domain of beta-arrestin and by inhibitors of c-Src and clathrin-mediated endocytosis. Moreover, PDGF and S1P induce the recruitment of c-Src to the PDGFbeta receptor-S1P(1) receptor complex. This leads to a G protein/c-Src-dependent tyrosine phosphorylation of Gab1 and accumulation of dynamin II at the plasma membrane, a step required for endocytosis of the PDGFbeta receptor-GPCR complex. These findings provide important information concerning the molecular organisation of novel receptor tyrosine kinase (RTK)-GPCR signal relays in mammalian cells.

    Cellular signalling 2005;17;2;263-77

  • c-Src regulates clathrin adapter protein 2 interaction with beta-arrestin and the angiotensin II type 1 receptor during clathrin- mediated internalization.

    Fessart D, Simaan M and Laporte SA

    Hormones and Cancer Research Unit, Department of Medicine, McGill University, Royal Victoria Hospital, 687 Pine Avenue West, Montréal, Québec, Canada H3A 1A1.

    Beta-arrestins are multifunctional adapters involved in the internalization and signaling of G protein-coupled receptors (GPCRs). They target receptors to clathrin-coated pits (CCPs) through binding with clathrin and clathrin adapter 2 (AP-2) complex. They also act as transducers of signaling by recruiting c-Src kinase to certain GPCRs. Here we sought to determine whether c-Src regulates the recruitment of AP-2 to beta-arrestin and the angiotensin II (Ang II) type 1 receptor (AT1R) during internalization. We show that the agonist stimulation of native AT1R in vascular smooth muscle cells (VSMCs) induces the formation of an endogenous complex containing c-Src, beta-arrestins and AP-2. In vitro studies using coimmunoprecipitation experiments and a yeast three-hybrid assay reveal that c-Src stabilizes the agonist-independent association between beta-arrestin2 and the beta-subunit of AP-2 independently of the kinase activity of c-Src. However, although c-Src expression promoted the rapid dissociation of AP-2 from both beta-arrestin and AT1R after receptor stimulation, a kinase-inactive mutant of c-Src failed to induce the dissociation of AP-2 from the agonist-occupied receptor. Thus, the consequence of c-Src in regulating the dissociation of AP-2 from the receptor was also examined on the internalization of AT1R by depleting c-Src in human embryonic kidney (HEK) 293 cells using a small interfering RNA strategy. Experiments in c-Src depleted cells reveal that AT1R remained mostly colocalized with AP-2 at the plasma membrane after Ang II stimulation, consistent with the observed delay in receptor internalization. Moreover, coimmunoprecipitation experiments in c-Src depleted HEK 293 cells and VSMCs showed an increased association of AP-2 to the agonist-occupied AT1R and beta-arrestin, respectively. Together, our results support a role for c-Src in regulating the dissociation of AP-2 from agonist-occupied AT1R and beta-arrestin during the clathrin-mediated internalization of receptors and suggest a novel function for c-Src kinase in the internalization of AT1R.

    Molecular endocrinology (Baltimore, Md.) 2005;19;2;491-503

  • Functional role of c-Src in IL-1-induced NF-kappa B activation: c-Src is a component of the IKK complex.

    Funakoshi-Tago M, Tago K, Andoh K, Sonoda Y, Tominaga S and Kasahara T

    Department of Biochemistry and Immunology, Kyoritsu University of Pharmacy, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512.

    Interleukin-1 (IL-1) mediates numerous host responses through the rapid activation of nuclear factor-kappa B (NF-kappa B), but the signal pathways leading to NF-kappa B activation are regulated at multiple stages. Here, we propose a novel regulatory system for IL-1-induced NF-kappa B activation by a tyrosine kinase, c-Src. The kinase activity of c-Src increases in an IL-1-dependent manner and the ectopic expression of c-Src augments IL-1-induced NF-kappa B activation, suggesting the involvement of c-Src in IL-1 signaling. However, a Src family inhibitor, PP2 failed to inhibit IL-1-induced NF-kappa B activation, and the expression of a c-Src mutant lacking kinase activity (c-Src KD) augmented IL-1-induced NF-kappa B activation as well as wild type c-Src, indicating that the tyrosine kinase activity is not required for IL-1-induced NF-kappa B activation. Furthermore, a physiological interaction between c-Src and I kappa B kinase gamma (IKK gamma) was observed, implying the involvement of c-Src in the IKK-complex. While c-Src augmented IL-1-induced IKK activation independent of its kinase activity, the region comprising amino acids 361-440 in the c-Src kinase domain are required for NF-kappa B activation. The same region of c-Src is also required for IL-1-induced IKK activation and the association with IKK gamma. Taken together, our results suggest that c-Src plays a critical role in IL-1-induced NF-kappa B activation through the IKK complex.

    Journal of biochemistry 2005;137;2;189-97

  • Mapping DNA-protein interactions in large genomes by sequence tag analysis of genomic enrichment.

    Kim J, Bhinge AA, Morgan XC and Iyer VR

    Center for Systems and Synthetic Biology, Institute for Cellular and Molecular Biology & Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, Texas 78712-0159, USA.

    Identifying the chromosomal targets of transcription factors is important for reconstructing the transcriptional regulatory networks underlying global gene expression programs. We have developed an unbiased genomic method called sequence tag analysis of genomic enrichment (STAGE) to identify the direct binding targets of transcription factors in vivo. STAGE is based on high-throughput sequencing of concatemerized tags derived from target DNA enriched by chromatin immunoprecipitation. We first used STAGE in yeast to confirm that RNA polymerase III genes are the most prominent targets of the TATA-box binding protein. We optimized the STAGE protocol and developed analysis methods to allow the identification of transcription factor targets in human cells. We used STAGE to identify several previously unknown binding targets of human transcription factor E2F4 that we independently validated by promoter-specific PCR and microarray hybridization. STAGE provides a means of identifying the chromosomal targets of DNA-associated proteins in any sequenced genome.

    Nature methods 2005;2;1;47-53

  • Nef: "necessary and enforcing factor" in HIV infection.

    Joseph AM, Kumar M and Mitra D

    National Centre for Cell Science, Ganeshkhind, Pune-411007, India.

    The Human Immunodeficiency Virus -1 (HIV-1) Nef protein that was originally identified as a viral negative factor is a 27kDa myristoylated protein. However, this so called dispensable viral protein has emerged as one of the most important proteins for viral life cycle. Nef not only establishes the host cell environment suitable for viral replication and pathogenesis but also facilitates the progression of the infection into disease. Previous efforts have been focussed to explain how Nef down modulates host cell receptors like CD4 and MHC-1 molecules, thereby helping the virus to evade host defense and to increase viral infectivity. Nef also ably modulates specific processes like apoptosis in favour of viral life cycle other than being the stimulus for cell activation and signal transduction pathways. After much maligning over its reported positive or negative functions on the HIV-1 Long Terminal Repeat (LTR) promoter, the Nef protein is now perceived to enhance viral replication and infection through a combination of different effector functions. Recent reports emphasize a role for Nef in viral gene expression and place it in a prime position to oversee and optimize viral replication. Nef may do so by enhancing Tat mediated gene expression from the LTR by activating signalling pathways that result in a concomitant increase in the activation of general transcription factors, and also by mediating translocation of repression factors from the nucleus. Thus, Nef not only enhances infection but also plays an important role in viral replication and pathogenesis.

    Current HIV research 2005;3;1;87-94

  • Substrate-trapping techniques in the identification of cellular PTP targets.

    Blanchetot C, Chagnon M, Dubé N, Hallé M and Tremblay ML

    McGill Cancer Centre, McGill University, 3655 Sir William-Osler, Room 715, Montreal, Quebec, H3G1Y6 Canada. christophe.blanchetot@mcgill.ca

    Tyrosine phosphorylation is negatively regulated by the protein-tyrosine phosphatases (PTPs). In order to find the physiological substrates of these enzymes, diverse PTP mutants that do not possess any catalytic activities but appear to bind tightly to their tyrosine phosphorylated substrates have been designed. Hence, they can be used as tools to pull out their respective substrates from heterogeneous extracts. Named PTP "substrate-trapping" mutants by the Tonks laboratory, they represent a diverse variety of defective PTPs that are epitomized by the Cys to Ser mutant (C/S) where the active cysteine residue of the signature motif is mutated to a serine residue. In addition, new mutants have been developed which are expected to help characterize novel and less abundant substrates. In this article, we review and describe all the different substrate-trapping mutants that have successfully been used or that hold interesting promises. We present their methodology to identify substrates in vivo (co-immunoprecipitation) and in vitro (GST pulldown), and provide a current list of substrates that have been identified using these technologies.

    Methods (San Diego, Calif.) 2005;35;1;44-53

  • Novel insights into c-Src.

    Alper O and Bowden ET

    National Institute of Health, National Institute of Neurological Disorders, Surgical Neurology, Bldg. 10, Rm. 5D37, Bethesda, MD 20892-1414, USA. alpero@mail.nih.gov

    Since identifying a transmissible agent responsible for tumorigenesis in chickens, the v-Src oncogene, significant progress has been made in determining the functions of its cellular homologue. c-Src is the product of the SRC gene and has been found both over-expressed and highly activated in a number of human cancers. In fact the relationship between c-Src activation and cancer progression is significant. Furthermore c-Src may play a role in the acquisition of the invasive and metastatic phenotype. In this review we will summarize some of the latest evidence for the role of c-Src in tumorigenesis and particularly in human tumor progression. In this review, specifically, we will address growth signals, adhesion, migration, invasion, angiogenesis and functional genomics.

    Current pharmaceutical design 2005;11;9;1119-30

  • Src, Fyn and Yes play differential roles in VEGF-mediated endothelial cell events.

    Werdich XQ and Penn JS

    Department of Ophthalmology and Visual Sciences, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.

    Widely coexpressed Src family kinase (SFK) members Src, Fyn and Yes are involved in various cellular events, often acting downstream of receptor tyrosine kinases, such as vascular endothelial growth factor (VEGF) receptors. They are well known for their functional redundancy; any unique features remain largely undefined. Utilizing RNA interference, we have selectively knocked down Src, Fyn and Yes in human retinal microvascular endothelial cells (HRMECs). Cells with single SFK knockdown showed that all three kinases were required for VEGF mitogenic signaling. VEGF-induced cell migration was significantly increased in Fyn-deficient cells and decreased in Yes-deficient cells. Selective interference of Fyn, but not Src or Yes, impaired VEGF-induced tube formation in HRMECs. Cells in which all three SFKs were targeted showed significant inhibition of all three cellular events. In addition, interference of Src, Fyn and Yes did not affect the anti-apoptotic effect of VEGF in HRMECs, as determined by DNA fragmentation analysis. These results provide direct evidence that Src, Fyn and Yes maintain distinct properties in the regulation of VEGF-mediated endothelial cell events.

    Funded by: NEI NIH HHS: EY07533, EY08126

    Angiogenesis 2005;8;4;315-26

  • SRC tyrosine kinase inhibitor, m475271, suppresses subcutaneous growth and production of lung metastasis via inhibition of proliferation, invasion, and vascularization of human lung adenocarcinoma cells.

    Zheng R, Yano S, Matsumori Y, Nakataki E, Muguruma H, Yoshizumi M and Sone S

    Department of Internal Medicine and Molecular Therapeutics, University of Tokushima Graduate School, Tokushima, Japan.

    Src, a proto-oncogene, has been strongly implicated in the growth, progression and metastasis of a number of human cancers. Its role in lung cancer is, however, still unknown. In the present study, we assessed the expression of Src in three different human lung adenocarcinoma cell lines (PC-9, PC14PE6, A549), and explored the effect of a novel Src kinase inhibitor, M475271, on the behavior of the cell lines. The three cell lines expressed various levels of auto-phosphorylated Src. While M475271 reduced Src-phosphorylation and invasiveness of all three cell lines, it inhibited the proliferation of PC-9 and A549 cells with highly phosphorylated Src, but not PC14PE6 cells. We further examined the effect of M475271 on subcutaneous tumors and lung metastasis caused by PC-9 and/or A549 cells in NK-cell depleted SCID mice. Daily oral treatment with M475271 inhibited the growth of subcutaneous tumors with PC-9 and A549 cells via inhibition of tumor cells proliferation, VEGF production and/or vascularization in the mice in a dose-dependent manner. In the metastasis model with A549 cells, the lung weight in the M475271 (50 mg/kg)-treated group was less than that of the control group, despite no difference in the number of metastatic nodules. Our results suggest that inhibition of tyrosine kinase Src by M475271 could reduce the growth, invasion and VEGF-mediated neovascularization of lung adenocarcinoma cells, resulting in inhibition of growth of subcutaneous tumors and lung metastasis. Therefore, a novel Src tyrosine kinase inhibitor, M475271, might be helpful for controlling the progression of human lung adenocarcinoma.

    Clinical & experimental metastasis 2005;22;3;195-204

  • Substance P receptor in U373 MG human astrocytoma cells activates mitogen-activated protein kinases ERK1/2 through Src.

    Yamaguchi K, Kugimiya T and Miyazaki T

    Department of Anesthesiology and Pain Medicine, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. keisuke@med.juntendo.ac.jp

    Substance P (SP) acting through substance P receptor (SPR) increases the proliferation of glioblastoma cells. At the molecular level, stimulation of SPR in human U373 MG glioblastoma cells results in phosphorylation of mitogen-activated protein kinases ERK1/2. Examination of the underlying mechanism reveals that SPR mediates ERK1/2 phosphorylation in a calcium-dependent manner. Surprisingly, blockade of epidermal growth factor receptor (EGFR), which is transactivated by SPR, has a minimal effect on SPR-mediated ERK1/2 phosphorylation. However, SPR-mediated ERK1/2 phosphorylation is significantly reduced by the Src kinase inhibitor PP2. Interestingly, ERK1/2 in U373 MG cells is also activated by several other mitogenic G protein-coupled receptors (GPCRs) including alpha(1B)-adrenergic, M(3)-muscarinic, and H(1)-histaminergic in an Src-dependent manner. We conclude that c-Src is a mediator of SP-stimulated ERK1/2 phosphorylation in human U373 MG glioblastoma cells.

    Brain tumor pathology 2005;22;1;1-8

  • G16-mediated activation of nuclear factor kappaB by the adenosine A1 receptor involves c-Src, protein kinase C, and ERK signaling.

    Liu AM and Wong YH

    Department of Biochemistry, Molecular Neuroscience Center, and Biotechnology Research Institute, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.

    The G(i)-linked adenosine A1 receptor has been shown to mediate anti-inflammatory actions, possibly via modulation of the transcription factor nuclear factor-kappaB (NFkappaB). Here we demonstrate that an adenosine A1 agonist, N(6)-cyclohexyladenosine (CHA), activated IKKalpha/beta phosphorylation through PTX-insensitive G proteins in human lymphoblastoma Reh cells. To delineate the mechanism of action, different PTX-insensitive G proteins were expressed in human embryonic kidney 293 cells. Only Galpha(16) supported the CHA-induced IKK phosphorylation and NFkappaB-driven luciferase activity in time-dependent, dose-dependent, and PTX-insensitive manners. Gbetagamma subunits also modulated IKK/NFkappaB, as indicated by the stimulatory actions of Gbeta(1)gamma(2) and the abrogation of CHA-induced response by transducin. The participation of phospholipase Cbeta, protein kinase C, and calmodulin-dependent kinase II in CHA-induced IKK/NFkappaB activation were demonstrated by employing specific inhibitors and dominant-negative mutants. Inhibition of c-Src and numerous intermediates along the extracellular signal-regulated (ERK) kinase cascade including Ras, Raf-1 kinase, and MEK1/2 abolished the CHA-induced IKK/NFkappaB activation. Although c-Jun N-terminal kinase and p38 MAPK were also activated by CHA, they were not required for the IKK/NFkappaB regulation. Similar results were obtained using Reh cells. These data suggest that the G(16)-mediated activation of IKK/NFkappaB by CHA required a complex signaling network composed of multiple intermediates.

    The Journal of biological chemistry 2004;279;51;53196-204

  • Type I collagen synergistically enhances PDGF-induced smooth muscle cell proliferation through pp60src-dependent crosstalk between the alpha2beta1 integrin and PDGFbeta receptor.

    Hollenbeck ST, Itoh H, Louie O, Faries PL, Liu B and Kent KC

    Columbia Weill Cornell Division of Vascular Surgery, Weill Medical College of Cornell University, USA. sth9005@med.cornell.edu

    Smooth muscle cells (SMCs) are exposed to both platelet-derived growth factor (PDGF) and type I collagen (CNI) at the time of arterial injury. In these studies we explore the individual and combined effects of these agonists on human saphenous vein SMC proliferation. PDGF-BB produced a 5.5-fold increase in SMC DNA synthesis whereas CNI stimulated DNA synthesis to a much lesser extent (1.6-fold increase). Alternatively, we observed an 8.3-fold increase in DNA synthesis when SMCs were co-incubated with CNI and PDGF-BB. Furthermore, stimulation of SMCs with PDGF-BB produced a significant increase in ERK-2 activity whereas CNI alone had no effect. Co-incubation of SMCs with PDGF-BB and CNI resulted in ERK-2 activity that was markedly greater than that produced by PDGF-BB alone. In a similar fashion, PDGF-BB induced phosphorylation of the PDGF receptor beta (PDGFRbeta) and CNI did not, whereas concurrent agonist stimulation produced a synergistic increase in receptor activity. Blocking antibodies to the alpha2 and beta1 subunits eliminated this synergistic interaction, implicating the alpha2beta1 integrin as the mediator of this effect. Immunoprecipitation of the alpha2beta1 integrin in unstimulated SMCs followed by immunoblotting for the PDGFRbeta as well as Src family members, pp60(src), Fyn, Lyn, and Yes demonstrated coassociation of alpha2beta1 and the PDGFRbeta as well as pp60(src). Incubation of cells with CNI and/or PDGF-BB did not change the degree of association. Finally, inhibition of Src activity with SU6656 eliminated the synergistic effect of CNI on PDGF-induced PDGFRbeta phosphorylation suggesting an important role for pp60(src) in the observed receptor crosstalk. Together, these data demonstrate that CNI synergistically enhances PDGF-induced SMC proliferation through Src-dependent crosstalk between the alpha2beta1 integrin and the PDGFRbeta.

    Funded by: NCI NIH HHS: T32 CA68971; NHLBI NIH HHS: HL55465, HL68673

    Biochemical and biophysical research communications 2004;325;1;328-37

  • Tyrosine phosphatase epsilonM stimulates migration and survival of porcine aortic endothelial cells by activating c-Src.

    Nakagawa Y, Yamada N, Shimizu H, Shiota M, Tamura M, Kim-Mitsuyama S and Miyazaki H

    Gene Research Center, University of Tsukuba, Ibaraki 305-8572, Japan.

    The cell growth, survival, and migration of vascular endothelial cells (ECs) are positively regulated by several protein tyrosine kinase receptors. Therefore, protein tyrosine phosphatases (PTPs) must also be important for these processes. The present study found that transmembranal PTPepsilonM, but not cytoplasmic PTPepsilonC, is expressed in porcine ECs and in rat smooth muscle cells, both of which were prepared from the aorta. The overexpression of wild-type PTPepsilonM promoted cell survival and migration in porcine aortic ECs even in medium without and with 1% serum, respectively. A catalytically inactive, substrate-trapping mutant of PTPepsilonM, respectively, did not affect and conversely suppressed cell survival and migration. Interestingly, the forced expression of wild-type PTPepsilonC reduced cell viability in contrast to PTPepsilonM in ECs lacking endogenous PTPepsilonC, indicating the biological significance of selective expression of PTPepsilon isoforms in the vasculature. PTPepsilonM activated c-Src kinase probably by directly dephosphorylating phospho-Tyr527, a negative regulatory site of c-Src. The increases in cell survival and migration induced by overexpressed PTPepsilonM were suppressed by the c-Src inhibitor SU6656. Considering the behaviors of vascular ECs in the pathogenesis of atherosclerosis, these data suggest that PTPepsilonM negatively regulates the development of this disease by activating c-Src.

    Biochemical and biophysical research communications 2004;325;1;314-9

  • ARAP3 is transiently tyrosine phosphorylated in cells attaching to fibronectin and inhibits cell spreading in a RhoGAP-dependent manner.

    I ST, Nie Z, Stewart A, Najdovska M, Hall NE, He H, Randazzo PA and Lock P

    Department of Surgery, University of Melbourne, Level 5 Clinical Sciences Building, Royal Melbourne Hospital, VIC 3050, Australia.

    ARAP3 is a GTPase activating protein (GAP) for Rho and Arf GTPases that is implicated in phosphoinositide 3-kinase (PI 3-kinase) signalling pathways controlling lamellipodia formation and actin stress fibre assembly. We have identified ARAP3 as a phosphorylated target of protein tyrosine kinases. In cells, ARAP3 was tyrosine phosphorylated when co-expressed with Src-family kinases (SFKs), upon stimulation with growth factors and during adhesion to the extracellular matrix (ECM) substrate fibronectin. Adhesion-induced phosphorylation of ARAP3 was suppressed by selective inhibitors of Src-family kinases and PI 3-kinase and by a Src dominant interfering mutant. Inducible expression of ARAP3 in HEK293 epithelial cells resulted in increased cell rounding, membrane process formation and cell clustering on ECM substrates. In contrast, ARAP3 dramatically slowed the kinetics of cell spreading on fibronectin but had no effect on cell adhesion. These effects of ARAP3 required a functional Rho GAP domain and were associated with reduced cellular levels of active RhoA and Rac1 but did not require the sterile alpha motif (SAM) or Arf GAP domains. Mutation of two phosphorylation sites, Y1399 and Y1404, enhanced some ARAP3 activities, suggesting that ARAP3 may be negatively regulated by phosphorylation on these tyrosine residues. These results implicate ARAP3 in integrin-mediated tyrosine kinase signalling pathways controlling Rho GTPases and cell spreading.

    Journal of cell science 2004;117;Pt 25;6071-84

  • FRS2-dependent SRC activation is required for fibroblast growth factor receptor-induced phosphorylation of Sprouty and suppression of ERK activity.

    Li X, Brunton VG, Burgar HR, Wheldon LM and Heath JK

    CR-UK Growth Factor Group, School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.

    Activation of signalling by fibroblast growth factor receptor leads to phosphorylation of the signalling attenuator human Sprouty 2 (hSpry2) on residue Y55. This event requires the presence of the signalling adaptor fibroblast growth factor receptor substrate 2 (FRS2). The phosphorylation of hSpry2 is therefore mediated by an intermediate kinase. Using a SRC family kinase-specific inhibitor and mutant cells, we show that hSpry2 is a direct substrate for SRC family kinases, including SRC itself. Activation of SRC via fibroblast growth factor signalling is dependent upon FRS2 and fibroblast growth factor receptor kinase activity. SRC forms a complex with hSpry2 and this interaction is enhanced by hSpry2 phosphorylation. Phosphorylation of hSpry2 is required for hSpry2 to inhibit activation of the extracellular signal-regulated kinase pathway. These results show that recruitment of SRC to FRS2 leads to activation of signal attenuation pathways.

    Journal of cell science 2004;117;Pt 25;6007-17

  • PKA phosphorylation of Src mediates Rap1 activation in NGF and cAMP signaling in PC12 cells.

    Obara Y, Labudda K, Dillon TJ and Stork PJ

    The Vollum Institute, L474, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA.

    Recent studies suggest that the tyrosine kinase Src plays an important role in the hormonal regulation of extracellular signal-regulated kinases (ERKs) via cyclic AMP (cAMP). Src has also been proposed to mediate signals downstream of nerve growth factor (NGF). Here, we report that the cAMP-dependent protein kinase A (PKA) induced the phosphorylation of Src at residue serine17 (S17) in multiple cell types including PC12, Hek293, AtT-20 and CHO cells. In PC12 cells, Src phosphorylation on S17 participates in the activation of the small G protein Rap1 by both cAMP and NGF. In these cells, Rap1 is required for cAMP/PKA signaling to ERKs and also for the sustained activation of ERKs by NGF. The activation of Rap1 by both cAMP and NGF was blocked by PP2, an inhibitor of Src family kinases, and by a Src mutant incapable of being phosphorylated by PKA (SrcS17A), consistent with the requirement of PKA phosphorylation of Src at S17 in these actions. PP2 and SrcS17A also inhibited the Rap1-dependent activation of ERKs by both agents. These results strongly indicate that PKA phosphorylation of Src at S17 is essential for cAMP and NGF signaling in PC12 cells and identify PKA as an important downstream target of NGF. PKA phosphorylation of Src may therefore be required for Rap1 activation in PC12 cells.

    Journal of cell science 2004;117;Pt 25;6085-94

  • Induction of cyclooxygenase-2 overexpression in human gastric epithelial cells by Helicobacter pylori involves TLR2/TLR9 and c-Src-dependent nuclear factor-kappaB activation.

    Chang YJ, Wu MS, Lin JT, Sheu BS, Muta T, Inoue H and Chen CC

    Department of Pharmacology, College of Medicine, National Taiwan University and National Taiwan University Hospital, Taipei, Taiwan.

    Gastric epithelial cells were incubated with a panel of clinical isolates of Helicobacter pylori, including nonulcer dyspepsia with gastritis (HS, n = 20), gastric ulcer (HU, n = 20), duodenal ulcer (HD, n = 21), and gastric cancer (HC, n = 20). HC strains induced a higher cyclooxygenase-2 (COX-2) expression than those from HS, HD, and HU. The bacterial virulence factors and the host cellular pathways were investigated. Virulence genes of iceA, vacA, babA2, cagA 3' repeat region, and hrgA failed to show any association with the disease status and COX-2 expression. Methylation-specific polymerase chain reaction revealed HC strains not affecting the methylation status of COX-2 promoter. Nuclear factor (NF)-kappaB, NF-interleukin 6, and cAMP response element were found to be involved in COX-2 induction. We explored a novel NF-kappaB activation pathway. The mutants of TLR2 and TLR9, but not TLR4, inhibited H. pylori-induced COX-2 promoter activity, and neutralizing antibodies for TLR2 and TLR9 abolished H. pylori-induced COX-2 expression. Phosphatidylinositol-specific phospholipase C (PI-PLC), protein kinase C (PKC), and Src inhibitors inhibited COX-2 induction. The dominant-negative mutants of NIK and various IkappaB kinase complexes, including IKKbeta (Y188F), IKKbeta (Y199F), and IKKbeta (FF), inhibited the COX-2 promoter activity. Phosphorylation of GST-IKKbeta (132-206) at Tyr188 and Tyr199 by c-Src was found after H. pylori infection. In summary, H. pylori induces COX-2 expression via activations of NF-kappaB, NF-interleukin 6, the cAMP response element. In NF-kappaB activation, H. pylori acts through TLR2/TLR9 to activate both the cascade of PI-PLCgamma/PKCalpha/c-Src/IKKalpha/beta and the cascade of NIK/IKKalpha/beta, resulting in the IkappaBalpha degradation and the expression of COX-2 gene. The COX-2 overexpression may contribute to the carcinogenesis in patients colonized with these strains.

    Molecular pharmacology 2004;66;6;1465-77

  • Fibronectin and type IV collagen activate ERalpha AF-1 by c-Src pathway: effect on breast cancer cell motility.

    Sisci D, Aquila S, Middea E, Gentile M, Maggiolini M, Mastroianni F, Montanaro D and Andò S

    Dipartimento Farmaco-Biologico, Università della Calabria, Arcavacata di Rende, Italy.

    The expression of estrogen receptor alpha (ERalpha) is generally associated with a less invasive and aggressive phenotype in breast carcinoma. In an attempt to understand the role of ERalpha in regulating breast cancer cells invasiveness, we have demonstrated that cell adhesion on fibronectin (Fn) and type IV Collagen (Col) induces ERalpha-mediated transcription and reduces cell migration in MCF-7 and in MDA-MB-231 cell lines expressing ERalpha. Analysis of deleted mutants of ERalpha indicates that the transcriptional activation function (AF)-1 is required for ERalpha-mediated transcription as well as for the inhibition of cell migration induced by cell adhesion on extracellular matrix (ECM) proteins. In addition, the nuclear localization signal region and some serine residues in the AF-1 of the ERalpha are both required for the regulation of cell invasiveness as we have observed in HeLa cells. It is worth noting that c-Src activation is coincident with adhesion of cells to ECM proteins and that the inhibition of c-Src activity by PP2 or the expression of a dominant-negative c-Src abolishes ERalpha-mediated transcription and partially reverts the inhibition of cell invasiveness in ERalpha-positive cancer cells. These findings address the integrated role of ECM proteins and ERalpha in influencing breast cancer cell motility through a mechanism that involves c-Src and seems not to be related to a specific cell type.

    Oncogene 2004;23;55;8920-30

  • Regulation of cell motility by tyrosine phosphorylated villin.

    Tomar A, Wang Y, Kumar N, George S, Ceacareanu B, Hassid A, Chapman KE, Aryal AM, Waters CM and Khurana S

    Department of Physiology, University of Tennessee Health Science Center, Memphis, TN 38163, USA.

    Temporal and spatial regulation of the actin cytoskeleton is vital for cell migration. Here, we show that an epithelial cell actin-binding protein, villin, plays a crucial role in this process. Overexpression of villin in doxycyline-regulated HeLa cells enhanced cell migration. Villin-induced cell migration was modestly augmented by growth factors. In contrast, tyrosine phosphorylation of villin and villin-induced cell migration was significantly inhibited by the src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) as well as by overexpression of a dominant negative mutant of c-src. These data suggest that phosphorylation of villin by c-src is involved in the actin cytoskeleton remodeling necessary for cell migration. We have previously shown that villin is tyrosine phosphorylated at four major sites. To further investigate the role of tyrosine phosphorylated villin in cell migration, we used phosphorylation site mutants (tyrosine to phenylalanine or tyrosine to glutamic acid) in HeLa cells. We determined that tyrosine phosphorylation at residues 60, 81, and 256 of human villin played an essential role in cell migration as well as in the reorganization of the actin cytoskeleton. Collectively, these studies define how biophysical events such as cell migration are actuated by biochemical signaling pathways involving tyrosine phosphorylation of actin binding proteins, in this case villin.

    Funded by: NIDDK NIH HHS: DK-54755, DK-65006

    Molecular biology of the cell 2004;15;11;4807-17

  • Src tyrosine kinase regulates insulin-induced activation of protein kinase C (PKC) delta in skeletal muscle.

    Rosenzweig T, Aga-Mizrachi S, Bak A and Sampson SR

    Gonda-Goldschmeid Center, Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel.

    Insulin stimulation of skeletal muscle results in rapid activation of protein kinase Cdelta (PKCdelta), which is associated with its tyrosine phosphorylation and physical association with insulin receptor (IR). The mechanisms underlying tyrosine phosphorylation of PKCdelta have not been determined. In this study, we investigated the possibility that the Src family of nonreceptor tyrosine kinases may be involved upstream insulin signaling. Studies were done on differentiated rat skeletal myotubes in primary culture. Insulin caused an immediate stimulation of Src and induced its physical association with both IR and PKCdelta. Inhibition of Src by treatment with the Src family inhibitor PP2 reduced insulin-stimulated Src-PKCdelta association, PKCdelta tyrosine phosphorylation and PKCdelta activation. PP2 inhibition of Src also decreased insulin-induced IR tyrosine phosphorylation, IR-PKCdelta association and association of Src with both PKCdelta and IR. Finally, inhibition of Src decreased insulin-induced glucose uptake. We conclude that insulin activates Src tyrosine kinase, which regulates PKCdelta activity. Thus, Src tyrosine kinase may play an important role in insulin-induced tyrosine phosphorylation of both IR and PKCdelta. Moreover, both Src and PKCdelta appear to be involved in IR activation and subsequent downstream signaling.

    Cellular signalling 2004;16;11;1299-308

  • Dual phosphorylations underlie modulation of unitary KCNQ K(+) channels by Src tyrosine kinase.

    Li Y, Langlais P, Gamper N, Liu F and Shapiro MS

    Department of Physiology, University of Texas Health Science Center, San Antonio, Texas 78229, USA.

    Src tyrosine kinase suppresses KCNQ (M-type) K(+) channels in a subunit-specific manner representing a mode of modulation distinct from that involving G protein-coupled receptors. We probed the molecular and biophysical mechanisms of this modulation using mutagenesis, biochemistry, and both whole-cell and single channel modes of patch clamp recording. Immunoprecipitation assays showed that Src associates with KCNQ2-5 subunits but phosphorylates only KCNQ3-5. Using KCNQ3 as a background, we found that mutation of a tyrosine in the amino terminus (Tyr-67) or one in the carboxyl terminus (Tyr-349) abolished Src-dependent modulation of heterologously expressed KCNQ2/3 heteromultimers. The tyrosine phosphorylation was much weaker for either the KCNQ3-Y67F or KCNQ3-Y349F mutants and wholly absent in the KCNQ3-Y67F/Y349F double mutant. Biotinylation assays showed that Src activity does not alter the membrane abundance of channels in the plasma membrane. In recordings from cell-attached patches containing a single KCNQ2/3 channel, we found that Src inhibits the open probability of the channels. Kinetic analysis was consistent with the channels having two discrete open times and three closed times. Src activity reduced the durations of the longest open time and lengthened the longest closed time of the channels. The implications for the mechanisms of channel regulation by the dual phosphorylations on both channel termini are discussed.

    Funded by: NIDDK NIH HHS: R01 DK52933; NINDS NIH HHS: R01 NS43394

    The Journal of biological chemistry 2004;279;44;45399-407

  • A juxtamembrane tyrosine in the colony stimulating factor-1 receptor regulates ligand-induced Src association, receptor kinase function, and down-regulation.

    Rohde CM, Schrum J and Lee AW

    Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109, USA.

    Recent literature implicates a regulatory function of the juxtamembrane domain (JMD) in receptor tyrosine kinases. Mutations in the JMD of c-Kit and Flt3 are associated with gastrointestinal stromal tumors and acute myeloid leukemias, respectively. Additionally, autophosphorylated Tyr559 in the JMD of the colony stimulating factor-1 (CSF-1) receptor (CSF-1R) binds to Src family kinases (SFKs). To investigate SFK function in CSF-1 signaling we established stable 32D myeloid cell lines expressing CSF-1Rs with mutated SFK binding sites (Tyr559-TFI). Whereas binding to I562S was not significantly perturbed, Y559F and Y559D exhibited markedly decreased CSF-1-dependent SFK association. All JMD mutants retained intrinsic kinase activity, but Y559F, and less so Y559D, showed dramatically reduced CSF-1-induced autophosphorylation. CSF-1-mediated wild-type (WT)-CSF-1R phosphorylation was not markedly affected by SFK inhibition, indicating that lack of SFK binding is not responsible for diminished Y559F phosphorylation. Unexpectedly, cells expressing Y559F were hyperproliferative in response to CSF-1. Hyperproliferation correlated with prolonged activation of Akt, ERK, and Stat5 in the Y559F mutant. Consistent with a defect in receptor negative regulation, c-Cbl tyrosine phosphorylation and CSF-1R/c-Cbl co-association were almost undetectable in the Y559F mutant. Furthermore, Y559F underwent reduced multiubiquitination and delayed receptor internalization and degradation. In conclusion, we propose that Tyr559 is a switch residue that functions in kinase regulation, signal transduction and, indirectly, receptor down-regulation. These findings may have implications for the oncogenic conversion of c-Kit and Flt3 with JMD mutations.

    Funded by: NCI NIH HHS: R01-CA85368

    The Journal of biological chemistry 2004;279;42;43448-61

  • Changes in androgen receptor nongenotropic signaling correlate with transition of LNCaP cells to androgen independence.

    Unni E, Sun S, Nan B, McPhaul MJ, Cheskis B, Mancini MA and Marcelli M

    Department of Medicine, Baylor College of Medicine and VA Medical Center, Houston, Texas, USA.

    A cure for prostate cancer (CaP) will be possible only after a complete understanding of the mechanisms causing this disease to progress from androgen dependence to androgen independence. To carry on a careful characterization of the phenotypes of CaP cell lines before and after acquisition of androgen independence, we used two human CaP LNCaP sublines: LNCaP(nan), which is androgen dependent (AD), and LNCaP-HP, which is androgen independent (AI). In AD LNCaP(nan) cells, dihydrotestosterone (DHT) stimulated in an androgen receptor (AR)-dependent way a phosphorylation signaling pathway involving steroid receptor coactivator (Src)-mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)-1/2-ERK-1/2-cAMP-response element binding-protein (CREB). Activation of this pathway was associated with increased [(3)H]thymidine incorporation and resistance to apoptosis. Use of dominant-negative forms of MEK-1/2 and CREB demonstrated in LNCaP(nan) cells that DHT induced [(3)H]thymidiine incorporation through a thus far unidentified molecule activated downstream of MEK-1/2, and antiapoptosis through phosphorylation of the transcription factor CREB. In contrast, in AI LNCaP-HP cells, the Src-MEK-1/2-ERK-1/2-CREB pathway was constitutively active. Because it was not further stimulated by addition of DHT, no increase of [(3)H]thymidine incorporation or apoptosis resistance was demonstrated in LNCaP-HP cells. Additional experiments showed that Src and the scaffold protein MNAR coimmunoprecipitated with AR, indicating a role for Src as an apical molecule in the Src-MEK-1/2-ERK-1/2-CREB pathway. Interestingly, differences between the two cell lines were that in LNCaP-HP cells presence of an AI phenotype and lack of response to DHT were associated with constitutive activation of the protein kinase Src and interaction among Src, AR, and MNAR. In contrast, in LNCaP(nan) cells, presence of an AD phenotype and ability to respond to DHT were associated with DHT-dependent activation of Src kinase activity and interaction among Src, AR, and MNAR. Intriguingly, in LNCaP(nan) cells, we found that transcription through the prototypical CREB-responsive promoter c-fos could be induced in a DHT-dependent way, and this action was inhibited by the AR antagonist Casodex and MEK-1 inhibitor PD98059. In contrast, transcription through the PSA P/E promoter, a prototypical AR-dependent promoter directly activated by agonist, was obliterated only by Casodex. Additional experiments with genital skin fibroblasts derived from patients with a variety of AR abnormalities indicated that nongenotropic AR signaling does not depend on an intact DNA-binding domain or on the ability of AR to translocate to the nucleus. The results suggest the following: (1) Constitutive activation of the Src-MEK-1/2-ERK-1/2-CREB pathway is associated with the AI phenotype observed in LNCaP-HP cells. (2) Activation of the Src-MEK-1/2-ERK-1/2-CREB pathway is DHT dependent in AD LNCaP(nan) cells. (3) DHT activation of this pathway is associated with induction of [(3)H]thymidine incorporation by a molecule activated downstream of MEK-1/2 and of antiapoptosis through activation of the transcription factor CREB in AD LNCaP(nan) cells. (4) AR regulates transcription either directly upon ligand binding and nuclear translocation or indirectly through kinase pathways leading to activation of downstream transcription factors. (5) Nuclear translocation and ability of the DNA-binding domain of AR to interact with DNA are not prerequisites for nongenotropic AR activity.

    Funded by: NIDDK NIH HHS: DK 55622, DK03892

    Cancer research 2004;64;19;7156-68

  • SHEP1 function in cell migration is impaired by a single amino acid mutation that disrupts association with the scaffolding protein cas but not with Ras GTPases.

    Dail M, Kalo MS, Seddon JA, Côté JF, Vuori K and Pasquale EB

    The Burnham Institute, La Jolla, California 92037, USA.

    SHEP1 is a signaling protein that contains a guanine nucleotide exchange factor-like domain, which binds Ras family GTPases and also forms a stable complex with the scaffolding protein Crk-associated substrate (Cas). SHEP1 and Cas have several common functions, such as increasing c-Jun N-terminal kinase activity, promoting T cell activation, and regulating the actin cytoskeleton. However, it is unclear whether a physical association between SHEP1 and Cas is required for these activities. We reported previously that SHEP1 is tyrosine-phosphorylated downstream of the EphB2 receptor; in this study, we further demonstrate that activated EphB2 inhibits SHEP1 association with Cas. To investigate whether phosphorylation negatively regulates the SHEP1-Cas complex, we have identified by mass spectrometry several SHEP1 tyrosine phosphorylation sites downstream of EphB2; of particular interest among them is tyrosine 635 in the Cas association/exchange factor domain. Mutation of this tyrosine to glutamic acid, but not to phenylalanine, disrupts Cas binding to SHEP1 without inhibiting Ras GTPase binding. The glutamic acid mutation also makes SHEP1 unable to promote Cas-Crk association, membrane ruffling, and cell migration toward epidermal growth factor (EGF), implying that these activities of SHEP1 depend upon a physical interaction with Cas. Association with Cas also seems to be necessary for EGF-induced SHEP1 tyrosine phosphorylation, which is mediated by a Src family kinase. It is noteworthy that EGF stimulation does not cause dissociation of SHEP1 from Cas. These data show that SHEP1 regulates membrane ruffling and cell migration and that binding to Cas is probably critical for these functions. Furthermore, the SHEP1-Cas complex may have different roles downstream of EphB2 and the EGF receptor.

    The Journal of biological chemistry 2004;279;40;41892-902

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • In silico activation of Src tyrosine kinase reveals the molecular basis for intramolecular autophosphorylation.

    Mendieta J and Gago F

    Departamento de Farmacología, Universidad de Alcalá, E-28871 Alcalá de Henares, Madrid, Spain.

    Structural data suggest that important hinge-bending motions of the two lobes that shape the catalytic domain of Src tyrosine kinase, together with reorganization of an alpha helix (helix C), are needed for the activation loop to adopt the catalytically competent conformation. The phosphorylation of a Tyr residue (Tyr-416) in this loop also seems to be essential for enzyme activation. However, no information is available about the dynamics of this activation process. By comparing the inactive and active forms of the catalytic domains of Src and Lck, another member of the Src family, we first identified a short stretch that can act as a hinge for the interlobe motion. The opening of the lobes was then simulated using a targeted molecular dynamics approach. The results obtained suggested that pulling the two lobes apart is not enough to induce the required conformational change in the activation loop. Rather unexpectedly, however, swinging of the lobes situated Tyr-416 in a suitable position for intramolecular autophosphorylation, and further simulation of Tyr-416-phosphorylated Src in the presence of ADP did then result in a conformational change that placed the activation loop in a position similar to that found in the active open conformation of Lck. Taken together, our results establish a physical link between intramolecular autophosphorylation and loop activation.

    Journal of molecular graphics & modelling 2004;23;2;189-98

  • Obligatory role of Src kinase in the signaling mechanism for TRPC3 cation channels.

    Vazquez G, Wedel BJ, Kawasaki BT, Bird GS and Putney JW

    Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709, USA. vazquez@niehs.nih.gov

    Members of the canonical transient receptor potential (TRPC) subfamily of cation channels are candidates for capacitative and non-capacitative Ca2+ entry channels. When ectopically expressed in cell lines, TRPC3 can be activated by phospholipase C-mediated generation of diacylglycerol or by addition of synthetic diacylglycerols, independently of Ca2+ store depletion. Apart from this mode of regulation, little is known about other receptor-dependent signaling events that modulate TRPC3 activity. In the present study the role of tyrosine kinases in receptor- and diacylglycerol-dependent activation of TRPC3 was investigated. In HEK293 cells stably expressing TRPC3, pharmacological inhibition of tyrosine kinases, and specifically of Src kinases, abolished activation of TRPC3 by muscarinic receptor stimulation and by diacylglycerol. Channel regulation was lost following expression of a dominant-negative mutant of Src, or when TRPC3 was expressed in an Src-deficient cell line. In both instances, wild-type Src restored TRPC3 regulation. We conclude that Src plays an obligatory role in the mechanism for receptor and diacylglycerol activation of TRPC3.

    The Journal of biological chemistry 2004;279;39;40521-8

  • Prolactin induces c-Myc expression and cell survival through activation of Src/Akt pathway in lymphoid cells.

    Domínguez-Cáceres MA, García-Martínez JM, Calcabrini A, González L, Porque PG, León J and Martín-Pérez J

    Instituto de Investigaciones Biomédicas, CSIC, Calle Arturo Duperier, 4, Madrid 28029, Spain.

    Stimulation of resting W53 cells (lymphoid murine cells expressing prolactin (PRL) receptor) by PRL induced expression of growth-related immediate-early genes (IEG), and proliferation through activation of the Src kinases. Since IEG are essential for cell cycle progression, we have studied how PRL controls expression of c-Myc mRNA and c-Fos. Stimulation of W53 cell proliferation by PRL required activation of MAPK, as the Mek1/2 inhibitor PD184352 eliminated Erk1/2 stimulation, cell proliferation, and expression of c-Fos mRNA. In contrast, PD184352 did not alter PRL activation of c-Myc mRNA expression or stimulation of p70S6K, Akt, and the Jak2/Stat5 pathway. Activation of the PI3K by PRL was necessary for the expression of c-MycmRNA and W53 cell proliferation, as the PI3K inhibitor LY294002 abolished them. However, it did not modify PRL stimulation of c-Fos mRNA expression or activation of Erk1/2 and Stat5. Furthermore, rapamycin, an inhibitor of mTOR and consequently of p70S6K, did not alter PRL stimulation of c-Myc and c-Fos mRNA expression and it had a very minor inhibitory effect on PRL stimulation of W53 cell proliferation. In addition, rapamycin did not affect PRL stimulation of Akt or Stat5. However, it reinforced PRL activation of Erk1/2. Overexpression of a constitutively activated Akt (myristoylated Akt) in W53 cells overcame the inhibitory effect of LY294002 on c-Myc expression, as well as cell death upon PRL deprivation. Consistently, inducible expression of Akt-CAAX Box in W53 cells caused inhibition of c-Myc expression. PRL stimulation of W53 cells resulted in Akt translocation to the nucleus, phosphorylation of FKHRL1 transcription factor, and its nuclear exclusion. In contrast, induced expression of Akt-CAAX Box caused inhibition of FKHRL1 phosphorylation. Furthermore, transient expression of nonphosphorylatable FKHRL1-A3 mutant impaired PRL-induced activation of the c-Myc promoter. Akt activation also resulted in phosphorylation and inhibition of glycogen synthetase kinase 3 (GSK3), which in turn promoted c-Myc stability. Consistently, treatment of W53 with selective inhibitors of GSK3 such as SB415286 and lithium salts resulted in increased levels of c-Myc. Also, overexpression of c-Myc in W53 cells overcame the decrease in cell proliferation induced by LY294002. These findings defined a PRL-signalling cascade in W53 cells, involving Src kinases/PI3K/Akt/FKHRL1-GSK3, that mediates stimulation of c-Myc expression.

    Oncogene 2004;23;44;7378-90

  • The phosphorylation of vinculin on tyrosine residues 100 and 1065, mediated by SRC kinases, affects cell spreading.

    Zhang Z, Izaguirre G, Lin SY, Lee HY, Schaefer E and Haimovich B

    Department of Surgery and the Cancer Institute of New Jersey, Robert Wood Johnson Medical School-University of Medicine and Dentistry of New Jersey, New Brunswick, NJ 08903, USA.

    Vinculin is a conserved actin binding protein localized in focal adhesions and cell-cell junctions. Here, we report that vinculin is tyrosine phosphorylated in platelets spread on fibrinogen and that the phosphorylation is Src kinases dependent. The phosphorylation of vinculin on tyrosine was reconstituted in vanadate treated COS-7 cells coexpressing c-Src. The tyrosine phosphorylation sites in vinculin were mapped to residues 100 and 1065. A phosphorylation-specific antibody directed against tyrosine residue 1065 reacted with phosphorylated platelet vinculin but failed to react with vinculin from unstimulated platelet lysates. Tyrosine residue 1065 located in the vinculin tail domain was phosphorylated by c-Src in vitro. When phosphorylated, the vinculin tail exhibited significantly less binding to the vinculin head domain than the unphosphorylated tail. In contrast, the phosphorylation did not affect the binding of vinculin to actin in vitro. A double vinculin mutant protein Y100F/Y1065F localized to focal adhesion plaques. Wild-type vinculin and single tyrosine phosphorylation mutant proteins Y100F and Y1065F were significantly more effective at rescuing the spreading defect of vinculin null cells than the double mutant Y100F/Y1065F. The phosphorylation of vinculin by Src kinases may be one mechanism by which these kinases regulate actin filament assembly and cell spreading.

    Funded by: NHLBI NIH HHS: HL54104, R01 HL054104, R29 HL054104

    Molecular biology of the cell 2004;15;9;4234-47

  • Identification of a novel family of ankyrin repeats containing cofactors for p160 nuclear receptor coactivators.

    Zhang A, Yeung PL, Li CW, Tsai SC, Dinh GK, Wu X, Li H and Chen JD

    Department of Pharmacology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854-5635, USA.

    Members of the p160 nuclear receptor coactivators interact with liganded nuclear receptors to enhance transcription of target genes. Here we identify a novel family of ankyrin repeats containing cofactors (ANCOs) that interact with the p160 coactivators. ANCO-1 binds to the conserved Per-Arnt-Sim (PAS) region of the p160 coactivators. It encodes a large nuclear protein with five ankyrin repeats, and parts of its sequences have been reported as nasopharyngeal carcinoma susceptibility protein and medulloblastoma antigen. Immunofluorescence staining reveals discrete nuclear foci of ANCO-1 that are distinct from known nuclear structures. Intriguingly, ANCO-1 also colocalizes and interacts with histone deacetylases. Transient reporter gene assay shows that ANCO-1 expression inhibits ligand-dependent transactivation by both steroid and nonsteroid nuclear receptors. Taken together, we have identified a novel family of ankyrin repeats containing cofactors that may recruit histone deacetylases to the p160 coactivators/nuclear receptor complex to inhibit ligand-dependent transactivation.

    Funded by: NIDDK NIH HHS: DK52542, DK52888, R01 DK052542-02, R01 DK052542-03, R01 DK052542-04, R01 DK052542-05, R01 DK052542-06, R01 DK052888-04, R01 DK052888-05

    The Journal of biological chemistry 2004;279;32;33799-805

  • Regulation of alternative SRC promoter usage in HepG2 hepatocellular carcinoma cells.

    Dehm SM and Bonham K

    Cancer Research Unit, Health Research Division, Saskatchewan Cancer Agency, Saskatoon, Canada. dehm.scott@mayo.edu

    Alternative promoters allow for increased spatial and temporal diversity in expression patterns for a single gene. The human SRC gene, encoding the non-receptor c-Src tyrosine kinase, is regulated by two alternative promoters separated by approximately 1 kb. The distal SRC1alpha promoter is tissue-restricted, while expression of the proximal SRC1A promoter appears to be ubiquitous. A barrier to elucidating the mechanisms of SRC transcriptional regulation has been the finding that the individual strengths of the SRC promoters in isolation do not match their relative strength of use seen in vivo. For example, in HepG2 hepatocellular carcinoma cells, SRC1A is significantly stronger in isolation than SRC1alpha, despite SRC1alpha being the predominant promoter used in this cell line. Previously, we have shown that HepG2 cells, as well as various colon cancer cell lines, display activated SRC transcription, which is linked to the elevated c-Src expression and activity necessary for growth and survival of these cells. These findings thus highlight the importance of understanding the mechanisms of SRC transcriptional regulation in human cancer. We hypothesize the discrepancy between individual SRC promoter strength and relative usage in vivo stems from a lack of linked promoter context. Therefore, we have developed and validated a novel dual SRC promoter reporter strategy to allow the simultaneous mechanistic study of both SRC promoters in their natural linked context. This approach has yielded evidence that SRC activation proceeds through genomic element(s) outside the promoter region in HepG2 cells. Therefore, we performed a preliminary study of DNaseI hypersensitive (DH) site composition within the SRC locus. This approach identified a HepG2-specific DH site that displayed activating potential towards the SRC1alpha promoter. These results thus provide important insight to the mechanism of SRC transcriptional activation in liver cancer cells.

    Gene 2004;337;141-50

  • The intranuclear localization and function of YT521-B is regulated by tyrosine phosphorylation.

    Rafalska I, Zhang Z, Benderska N, Wolff H, Hartmann AM, Brack-Werner R and Stamm S

    University of Erlangen, Institute for Biochemistry, Germany.

    YT521-B is a ubiquitously expressed nuclear protein that changes alternative splice site usage in a concentration dependent manner. YT521-B is located in a dynamic nuclear compartment, the YT body. We show that YT521-B is tyrosine phosphorylated by c-Abl in the nucleus. The protein shuttles between nucleus and cytosol, where it can be phosphorylated by c-Src or p59(fyn). Tyrosine phosphorylation causes dispersion of YT521-B from YT bodies to the nucleoplasm. Whereas YT bodies are soluble in non-denaturing buffers, the phosphorylated, dispersed form is non-soluble. Non-phosphorylated YT521-B changes alternative splice site selection of the IL-4 receptor, CD44 and SRp20, but phosphorylation of c-Abl minimizes this concentration dependent effect. We propose that tyrosine phosphorylation causes sequestration of YT521-B in an insoluble nuclear form, which abolishes the ability of YT521-B to change alternative splice sites.

    Human molecular genetics 2004;13;15;1535-49

  • RACK1 regulates G1/S progression by suppressing Src kinase activity.

    Mamidipudi V, Zhang J, Lee KC and Cartwright CA

    Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5187, USA.

    Cancer genes exert their greatest influence on the cell cycle by targeting regulators of a critical checkpoint in late G(1). Once cells pass this checkpoint, they are fated to replicate DNA and divide. Cancer cells subvert controls at work at this restriction point and remain in cycle. Previously, we showed that RACK1 inhibits the oncogenic Src tyrosine kinase and NIH 3T3 cell growth. RACK1 inhibits cell growth, in part, by prolonging G(0)/G(1). Here we show that RACK1 overexpression induces a partial G(1) arrest by suppressing Src activity at the G(1) checkpoint. RACK1 works through Src to inhibit Vav2, Rho GTPases, Stat3, and Myc. Consequently, cyclin D1 and cyclin-dependent kinases 4 and 2 (CDK4 and CDK2, respectively) are suppressed, CDK inhibitor p27 and retinoblastoma protein are activated, E2F1 is sequestered, and G(1)/S progression is delayed. Conversely, downregulation of RACK1 by short interference RNA activates Src-mediated signaling, induces Myc and cyclin D1, and accelerates G(1)/S progression. RACK1 suppresses Src- but not mitogen-activated protein kinase-dependent platelet-derived growth factor signaling. We also show that Stat3 is required for Rac1 induction of Myc. Our results reveal a novel mechanism of cell cycle control in late G(1) that works via an endogenous inhibitor of the Src kinase.

    Funded by: NIDDK NIH HHS: DK 43743, R01 DK043743

    Molecular and cellular biology 2004;24;15;6788-98

  • N-terminal kinase, and c-Src are activated in human aortic smooth muscle cells by pressure stress.

    Morita N, Iizuka K, Murakami T and Kawaguchi H

    Department of Pathophysiology and Laboratory Medicine, Hokkaido University Graduate School of Medicine, N-15 W-7, Kita-Ku Sapporo, Japan.

    Mechanical forces related to pressure and flow are important for cell hypertrophy and proliferation. There are still a few studies that examine responses of human vascular smooth muscle cells to pure pressure stress (transmural pressure). It is unclear as to which mechanisms are involved in cellular responses to pressure elevation. On the other hand, although the involvement of the local renin-angiotensin system (RAS) in pressure-induced responses was reported, the results were contradictory. It still remains to be determined whether RAS in human vascular smooth muscle cells is activated by pure pressure stress. We studied the upstream signal transduction events of extracellular signal kinase (ERK) in response to atmospheric pressure stress and involvement of angiotensin II in pressure-induced cell proliferation in human aortic smooth muscle cells (HASMC). A pressure-loading apparatus was set up to examine the effects of atmospheric pressure on human aortic smooth muscle cells. Pressure application of 160 mmHg for 3 h produced cell proliferation and activated ERK and c-JUN N-terminal kinase (JNK). ACE inhibitor suppressed all of them. ERK kinase (MEK) inhibitor also suppressed cell proliferation stimulated by pure pressure. The phosphorylated c-Src was increased by pure pressure stress. The treatment with c-Src kinase inhibitor suppressed pressure-induced proliferative response. In summary, our study found that ERK activation mediated pure pressure-induced proliferative response of HASMC. This activation was partly mediated by c-Src.

    Molecular and cellular biochemistry 2004;262;1-2;71-8

  • Activation of diacylglycerol kinase alpha is required for VEGF-induced angiogenic signaling in vitro.

    Baldanzi G, Mitola S, Cutrupi S, Filigheddu N, van Blitterswijk WJ, Sinigaglia F, Bussolino F and Graziani A

    Department of Medical Sciences, University Amedeo Avogadro of Piemonte Orientale, v. Solaroli 17, 28100, Novara, Italy.

    Vascular endothelial growth factor-A (VEGF-A) promotes angiogenesis by stimulating migration, proliferation and organization of endothelium, through the activation of signaling pathways involving Src tyrosine kinase. As we had previously shown that Src-mediated activation of diacylglycerol kinase-alpha (Dgk-alpha) is required for hepatocytes growth factor-stimulated cell migration, we asked whether Dgk-alpha is involved in the transduction of angiogenic signaling. In PAE-KDR cells, an endothelial-derived cell line expressing VEGFR-2, VEGF-A165, stimulates the enzymatic activity of Dgk-alpha: activation is inhibited by R59949, an isoform-specific Dgk inhibitor, and is dependent on Src tyrosine kinase, with which Dgk-alpha forms a complex. Conversely in HUVEC, VEGF-A165-induced activation of Dgk is only partially sensitive to R59949, suggesting that also other isoforms may be activated, albeit still dependent on Src tyrosine kinase. Specific inhibition of Dgk-alpha, obtained in both cells by R59949 and in PAE-KDR by expression of Dgk-alpha dominant-negative mutant, impairs VEGF-A165-dependent chemotaxis, proliferation and in vitro angiogenesis. In addition, in HUVEC, specific downregulation of Dgk-alpha by siRNA impairs in vitro angiogenesis on matrigel, further suggesting the requirement for Dgk-alpha in angiogenic signaling in HUVEC. Thus, we propose that activation of Dgk-alpha generates a signal essential for both proliferative and migratory response to VEGF-A165, suggesting that it may constitute a novel pharmacological target for angiogenesis control.

    Oncogene 2004;23;28;4828-38

  • Transcriptional regulation by the repressor of estrogen receptor activity via recruitment of histone deacetylases.

    Kurtev V, Margueron R, Kroboth K, Ogris E, Cavailles V and Seiser C

    Institute of Medical Biochemistry, Max F. Perutz Laboratories, Medical University of Vienna, Vienna Biocenter, Dr. Bohr-Gasse 9/2, A-1030 Vienna, Austria.

    Histone acetyltransferases and deacetylases are recruited by transcription factors and adapter proteins to regulate specific subsets of target genes. We were interested in identifying interaction partners of histone deacetylase 1 (HDAC1) that might be involved in conferring target or substrate specificity. Using the yeast two-hybrid system, we isolated the repressor of estrogen receptor activity (REA) as a novel HDAC1-associated protein. We demonstrated the in vivo interaction of REA with HDAC1 and characterized the respective domains required for their interaction in vitro. In addition, we found that REA also associates with the class II histone deacetylase HDAC5. In luciferase reporter assays, REA decreased transcription, and this repression was sensitive to the deacetylase inhibitor trichostatin A. Finally, we showed that REA specifically interacts with the chicken ovalbumin upstream binding transcription factors and II. The nuclear receptor chicken ovalbumin upstream binding transcription factor I was found to cooperate with REA and histone deacetylases in the repression of target genes. We, therefore, propose a novel function for REA as a mediator of transcriptional repression by nuclear hormone receptors via recruitment of histone deacetylases.

    The Journal of biological chemistry 2004;279;23;24834-43

  • Mitochondrial AKAP121 binds and targets protein tyrosine phosphatase D1, a novel positive regulator of src signaling.

    Cardone L, Carlucci A, Affaitati A, Livigni A, DeCristofaro T, Garbi C, Varrone S, Ullrich A, Gottesman ME, Avvedimento EV and Feliciello A

    Dipartimento di Biologia e Patologia Molecolare e Cellulare, via S. Pansini, 5, 80131 Naples, Italy. feliciel@unina.it

    A-kinase anchor protein 121 (AKAP121) and its spliced isoform AKAP84 anchor protein kinase A (PKA) to the outer membrane of mitochondria, focusing and enhancing cyclic AMP signal transduction to the organelle. We find that AKAP121/84 also binds PTPD1, a src-associated protein tyrosine phosphatase. A signaling complex containing AKAP121, PKA, PTPD1, and src is assembled in vivo. PTPD1 activates src tyrosine kinase and increases the magnitude and duration of epidermal growth factor (EGF) signaling. EGF receptor phosphorylation and downstream activation of ERK 1/2 and Elk1-dependent gene transcription are enhanced by PTPD1. Expression of a PTPD1 mutant lacking catalytic activity inhibits src and downregulates ERK 1/2 but does not affect the activity of c-Jun N-terminal kinase 1/2 and p38alpha mitogen-activated protein kinase. AKAP121 binds to and redistributes PTPD1 from the cytoplasm to mitochondria and inhibits EGF signaling. Our findings indicate that PTPD1 is a novel positive regulator of src signaling and a key component of the EGF transduction pathway. By binding and/or targeting the phosphatase on mitochondria, AKAP121 modulates the amplitude and persistence of src-dependent EGF transduction pathway. This represents the first example of physical and functional interaction between AKAPs and a protein tyrosine phosphatase.

    Molecular and cellular biology 2004;24;11;4613-26

  • Src SH3/2 domain-mediated peripheral accumulation of Src and phospho-myosin is linked to deregulation of E-cadherin and the epithelial-mesenchymal transition.

    Avizienyte E, Fincham VJ, Brunton VG and Frame MC

    Beatson Institute for Cancer Research, Cancer Research UK Beatson Laboratories, Bearsden, Glasgow G61 1BD, United Kingdom. e.avizienyte@beatson.gla.co.uk

    Elevated Src kinase in epithelial cancer cells induces adhesion changes that are associated with a mesenchymal-like state. We recently showed that Src induces dynamic integrin adhesions in KM12C colon cancer cells, whereas E-cadherin-dependent cell-cell contacts become disorganized. This promotes a fibroblastic-like morphology and expression of the mesenchymal marker vimentin. Furthermore, Src-induced deregulation of E-cadherin, and the associated mesenchymal transition, is dependent on integrin signaling (Avizienyte et al., Nat. Cell Biol. 2002, 4, 632-638), although the nature of downstream signals that mediate these Src- and integrin-dependent effects are unknown. Here we show that the SH2 and SH3 domains of Src mediate peripheral accumulation of phospho-myosin, leading to integrin adhesion complex assembly, whereas loss of SH2 or SH3 function restores normal regulation of E-cadherin and inhibits vimentin expression. Inhibitors of MEK, ROCK, or MLCK also suppress peripheral accumulation of phospho-myosin and Src-induced formation of integrin-dependent adhesions, whereas at the same time restoring E-cadherin redistribution to regions of cell-cell contact. Our data therefore implicate peripheral phospho-myosin activity as a point of convergence for upstream signals that regulate integrin- and E-cadherin-mediated adhesions. This further implicates spatially regulated contractile force as a determinant of epithelial cell plasticity, particularly in cancer cells that can switch between epithelial and mesenchymal-like states.

    Molecular biology of the cell 2004;15;6;2794-803

  • Transcriptional cofactors exhibit differential preference toward peroxisome proliferator-activated receptors alpha and delta in uterine cells.

    Lim HJ, Moon I and Han K

    Department of Obstetrics & Gynecology, Washington University School of Medicine, Campus Box 8064, 4566 Scott Avenue, St. Louis, Missouri 63110, USA. limj@wustl.edu

    We previously showed that peroxisome proliferator-activated receptor delta (PPARdelta) is crucial for embryo implantation as a receptor for cyclooxygenase-2-derived prostacyclin in mice. PPARs belong to the nuclear receptor superfamily. They form heterodimer with a retinoid X receptor, recruit transcriptional cofactors, and bind to a specific recognition element for regulation of target genes. Although cofactors are generally shared by various nuclear receptors, some are involved in cell-specific events. The objective of this investigation was to examine interactions of transcriptional cofactors with PPARdelta in uterine cells for its effectiveness in regulating gene expression. We chose two uterine cellular systems: periimplantation mouse uterus and AN(3)CA human uterine cell line. As examined by in situ hybridization, steroid receptor coactivator (SRC)-2, SRC-3, PPAR-interacting protein, receptor-interacting protein 140 (RIP140), nuclear receptor corepressor (N-CoR), and silencing mediator for retinoid and thyroid hormone receptor (SMRT) exhibit overlapping expression with that of PPARdelta in the periimplantation mouse uterus. Glutathione-S-transferase (GST) pull-down assays show that PPARdelta physically interacts with SRC 1-3, RIP140, PPAR-binding protein, N-CoR, and SMRT in the absence of ligands, suggesting their potent interactions with PPARdelta. Transient transfection assays in AN(3)CA cells show that among members of the SRC family, only SRC-2 serves as a true coactivator for PPARdelta, whereas all SRC members could enhance PPARalpha-induced transcriptional activation. Interestingly, N-CoR and SMRT potently repress PPARdelta-induced transcriptional activation but fail to repress PPARalpha activity. RIP140 is effective in repressing basal and PPAR-induced transcriptional activation. Collectively, the results suggest that gene regulation by PPARdelta in the uterine cells uniquely responds to SRC-2, N-CoR, SMRT, or RIP140, and these interactions may be operative during implantation when these cofactors are abundantly expressed.

    Funded by: NICHD NIH HHS: HD40810

    Endocrinology 2004;145;6;2886-95

  • SH3P2 in complex with Cbl and Src.

    Szymkiewicz I, Destaing O, Jurdic P and Dikic I

    Institute of Biochemistry II, Goethe University Medical School, Frankfurt, Germany.

    In this report, we describe SH3P2, an SH3-domain containing protein, as a novel Cbl-interacting molecule that is a substrate of tyrosine kinase Src. We identified a specific polyproline motif of Cbl responsible for binding of SH3P2 and Src, and observed mutual sequestration of Src and SH3P2 from monomer Cbl molecules. In adherent cells, SH3P2 associated with Cbl and fibrilar actin and was localized at focal contacts in fibroblasts as well as at the apical part of podosome rings in differentiated osteoclasts. Our data implicate that SH3P2, a novel component of adhesion sites, is involved in Cbl and Src-mediated pathways.

    FEBS letters 2004;565;1-3;33-8

  • Regulation of TRPC6 channel activity by tyrosine phosphorylation.

    Hisatsune C, Kuroda Y, Nakamura K, Inoue T, Nakamura T, Michikawa T, Mizutani A and Mikoshiba K

    Laboratory for Developmental Neurobiology, RIKEN Brain Science Institute (BSI), 2-1 Hirosawa, Wako City, Saitama 351-0198, Japan. chihiro@brain.riken.go.jp

    Various hormonal stimuli and growth factors activate the mammalian canonical transient receptor potential (TRPC) channel through phospholipase C (PLC) activation. However, the precise mechanism of the regulation of TRPC channel activity remains unknown. Here, we provide the first evidence that direct tyrosine phosphorylation by Src family protein-tyrosine kinases (PTKs) is a novel mechanism for modulating TRPC6 channel activity. We found that TRPC6 is tyrosine-phosphorylated in COS-7 cells when coexpressed with Fyn, a member of the Src family PTKs. We also found that Fyn interacts with TRPC6 and that the interaction is mediated by the SH2 domain of Fyn and the N-terminal region of TRPC6 in a phosphorylation-independent manner. In addition, we demonstrated the physical association of TRPC6 with Fyn in the mammalian brain. Moreover, we showed that stimulation of the epidermal growth factor receptor induced rapid tyrosine phosphorylation of TRPC6 in COS-7 cells. This epidermal growth factor-induced tyrosine phosphorylation of TRPC6 was significantly blocked by PP2, a specific inhibitor of Src family PTKs, and by a dominant negative form of Fyn, suggesting that the direct phosphorylation of TRPC6 by Src family PTKs could be caused by physiological stimulation. Furthermore, using single channel recording, we showed that Fyn modulates TRPC6 channel activity via tyrosine phosphorylation. Thus, our findings demonstrated that tyrosine phosphorylation by Src family PTKs is a novel regulatory mechanism of TRPC6 channel activity.

    The Journal of biological chemistry 2004;279;18;18887-94

  • Tyrosine 981, a novel ret autophosphorylation site, binds c-Src to mediate neuronal survival.

    Encinas M, Crowder RJ, Milbrandt J and Johnson EM

    Department of Neurology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

    The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) are neurotrophic factors that influence several aspects of the developing and injured nervous system. GFLs signal through a common receptor tyrosine kinase (Ret) and one of the four ligand-binding co-receptors (GFRalpha1 to 4). Ligand-induced translocation of Ret to lipid rafts, where it interacts with the nonreceptor tyrosine kinase Src, is a prerequisite for full biological activity of these neurotrophic factors. This interaction and subsequent activation of Src are required for GFL-mediated neuronal survival, neurite outgrowth, or cell proliferation. Here we show by multiple approaches that Ret tyrosine 981 constitutes the major binding site of the Src homology 2 domain of Src and therefore the primary residue responsible for Src activation upon Ret engagement. Other tyrosines such as 1015 and 1029 may contribute to the overall interaction between Ret and Src, as judged by overexpression experiments. By generating a phosphospecific antibody, we demonstrate that tyrosine 981 is a novel autophosphorylation site in Ret. Importantly, we also show that this tyrosine becomes phosphorylated in dissociated sympathetic neurons after ligand stimulation. Mutation of tyrosine 981 to phenylalanine reduces GDNF-mediated survival in a transfected cerebellar granule neuron paradigm.

    Funded by: NIA NIH HHS: AG13729, AG13730

    The Journal of biological chemistry 2004;279;18;18262-9

  • Autocrine CSF-1R activation promotes Src-dependent disruption of mammary epithelial architecture.

    Wrobel CN, Debnath J, Lin E, Beausoleil S, Roussel MF and Brugge JS

    Dept. of Cell Biology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115, USA.

    Elevated coexpression of colony-stimulating factor receptor (CSF-1R) and its ligand, CSF-1, correlates with invasiveness and poor prognosis of a variety of epithelial tumors (Kacinski, B.M. 1995. Ann. Med. 27:79-85). Apart from recruitment of macrophages to the tumor site, the mechanisms by which CSF-1 may potentiate invasion are poorly understood. We show that autocrine CSF-1R activation induces hyperproliferation and a profound, progressive disruption of junctional integrity in acinar structures formed by human mammary epithelial cells in three-dimensional culture. Acini coexpressing receptor and ligand exhibit a dramatic relocalization of E-cadherin from the plasma membrane to punctate intracellular vesicles, accompanied by its loss from the Triton-insoluble fraction. Interfering with Src kinase activity, either by pharmacological inhibition or mutation of the Y561 docking site on CSF-1R, prevents E-cadherin translocation, suggesting that CSF-1R disrupts cell adhesion by uncoupling adherens junction complexes from the cytoskeleton and promoting cadherin internalization through a Src-dependent mechanism. These findings provide a mechanistic basis whereby CSF-1R could contribute to invasive progression in epithelial cancers.

    The Journal of cell biology 2004;165;2;263-73

  • Pyk2 amplifies epidermal growth factor and c-Src-induced Stat3 activation.

    Shi CS and Kehrl JH

    B Cell Molecular Immunology Section, Laboratory of Immunoregulation, NIAID, National Institutes of Health, Bethesda, Maryland 20892-1876, USA.

    Signal transducers and activators of transcription factors (STATs) mediate many of the cellular responses that occur following cytokine, growth factor, and hormone signaling. STATs are activated by tyrosine and serine phosphorylation, which normally occurs as a tightly regulated process. Dysregulated STAT activity may facilitate oncogenesis, as constitutively activated STATs have been found in many human tumors as well as in v-abl- and v-src-transformed cell lines. Pyk2 is a member of the focal adhesion kinase family and can be activated by c-Src, epidermal growth factor receptor (EGFR), Janus kinase 1, tyrosine kinases, and G-protein-coupled receptor signaling. Although Pyk2 has been implicated in Janus kinase-dependent activation of MAPK and Stat1, no role for Pyk2 in the activation of other STAT proteins has been ascribed. Here, we provide evidence that Pyk2, along with c-Src, facilitates EGFR-mediated Stat3 activation. Pyk2 expression in HeLa cells induces Stat3 reporter gene activation and Stat3 phosphorylation on amino acid residues Tyr-705 and Ser-727. Together Pyk2 and c-Src potently activate Stat3, and Pyk2 enhances Stat3-induced cell proliferation. Moreover, the expression of a dominant negative version of Pyk2 impairs c-Src-induced Stat3 activation and cell proliferation. The treatment of A431 cells with EGF results in the recruitment of c-Src, Pyk2, and Stat3 to the EGFR and the phosphorylation of c-Src, Pyk2, and Stat3. Expression of constructs for dominant negative forms of either Pyk2 or c-Src impair EGF-induced Stat3 phosphorylation. These results indicate that Pyk2 facilitates EGFR- and c-Src-mediated Stat3 activation, thereby implicating Pyk2 activation as a potential co-mediator in triggering Stat3-induced oncogenesis.

    The Journal of biological chemistry 2004;279;17;17224-31

  • Stimulation of hTAFII68 (NTD)-mediated transactivation by v-Src.

    Lee HJ, Kim S, Pelletier J and Kim J

    Laboratory of Molecular and Cellular Biology, Department of Life Science, Sogang University, Seoul 121-743, South Korea.

    The three genes hTAF(II)68, EWS, and TLS (called the TET family) encode related RNA binding proteins containing an RNA recognition motif and three glycine-, arginine-, and proline-rich regions in the C-terminus and a degenerated repeat containing the consensus sequence Ser-Tyr-Gly-Gln-Ser in the N-terminus. In many human cancers, the N-terminal portion of hTAF(II)68, EWS, or TLS is fused to the DNA binding domain of one of several transcription factors including Fli-1, ERG, ETV1, E1AF, WT1, ATF-1, CHOP, or TEC. We have recognized the presence of several potential tyrosine phosphorylation sites within the amino-terminal domain of hTAF(II)68 and have investigated the potential effects of cytoplasmic signaling on hTAF(II)68 function. Herein, we find that hTAF(II)68 is phosphorylated on tyrosine residue(s) by ectopic expression of v-Src protein tyrosine kinase in vitro and in vivo. The hTAF(II)68 protein can associated with the SH3 domains of several cell signaling proteins, including v-Src protein tyrosine kinase. We also document that full-length v-Src can stimulate hTAF(II)68-mediated transcriptional activation, whereas deletion mutants of v-Src are unable to exert this effect. In addition, cellular Src activity appears important for hTAF(II)68 function since hTAF(II)68-mediated transactivation is reduced in a dose-dependent fashion by ectopic overexpression of a dominant-negative mutant of Src. Taken together, our results suggest that the biological activities of hTAF(II)68 are linked to the cytoplasmic Src signal transduction pathway.

    FEBS letters 2004;564;1-2;188-98

  • Unique domain anchoring of Src to synaptic NMDA receptors via the mitochondrial protein NADH dehydrogenase subunit 2.

    Gingrich JR, Pelkey KA, Fam SR, Huang Y, Petralia RS, Wenthold RJ and Salter MW

    Brain and Behaviour Program, Hospital for Sick Children, Department of Physiology, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada M5G 1X8.

    Src is the prototypic protein tyrosine kinase and is critical for controlling diverse cellular functions. Regions in Src define structural and functional domains conserved in many cell signaling proteins. Src also contains a region of low sequence conservation termed the unique domain, the function of which has until now remained enigmatic. Here, we show that the unique domain of Src is a protein-protein interaction region and we identify NADH dehydrogenase subunit 2 (ND2) as a Src unique domain-interacting protein. ND2 is a subunit of complex I in mitochondria, but we find that ND2 interacts with Src outside this organelle at excitatory synapses in the brain. ND2 acts as an adapter protein anchoring Src to the N-methyl-d-aspartate (NMDA) receptor complex, and is crucial for Src regulation of synaptic NMDA receptor activity. By showing an extramitochondrial action for a protein encoded in the mitochondrial genome, we identify a previously unsuspected means by which mitochondria regulate cellular function, suggesting a new paradigm that may be of general relevance for control of Src signaling.

    Proceedings of the National Academy of Sciences of the United States of America 2004;101;16;6237-42

  • Adhesion or plasmin regulates tyrosine phosphorylation of a novel membrane glycoprotein p80/gp140/CUB domain-containing protein 1 in epithelia.

    Brown TA, Yang TM, Zaitsevskaia T, Xia Y, Dunn CA, Sigle RO, Knudsen B and Carter WG

    Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA. wcarter@fhcrc.org

    Suspension of cultured human foreskin keratinocytes (HKs) with trypsin phosphorylates tyrosine residues on an 80-kDa membrane glycoprotein, p80 (Xia, Y., Gil, S. G., and Carter, W. G. (1996) J. Cell Biol. 132, 727-740). Readhesion dephosphorylates p80. Sequencing of a p80 cDNA established identity to CUB domain-containing protein 1 (CDCP1), a gene elevated in carcinomas. CDCP1/p80 cDNA encodes three extracellular CUB domains, a transmembrane domain, and two putative cytoplasmic Tyr phosphorylation sites. Treatment of adherent HKs with suramin, a heparin analogue, or inhibitors of phosphotyrosine phosphatases (PTPs; vanadate or calpeptin) increases phosphorylation of p80 and a novel 140-kDa membrane glycoprotein, gp140. Phosphorylated gp140 was identified as a trypsin-sensitive precursor to p80. Identity was confirmed by digestion and phosphorylation studies with recombinant gp140-GFP. Plasmin, a serum protease, also converts gp140 to p80, providing biological significance to the cleavage in wounds. Phosphorylation of gp140 and p80 are mediated by Src family kinases at multiple Tyr residues including Tyr(734). Dephosphorylation is mediated by PTP(s). Conversion of gp140 to p80 prolongs phosphorylation of p80 in response to suramin and changes in adhesion. This distinguishes gp140 and p80 and explains the relative abundance of phosphorylated p80 in trypsinized HKs. We conclude that phosphorylation of gp140 is dynamic and balanced by Src family kinase and PTPs yielding low equilibrium phosphorylation. We suggest that the balance is altered by conversion of gp140 to p80 and by adhesion, providing a novel transmembrane phosphorylation signal in epithelial wounds.

    Funded by: NCI NIH HHS: CA49259; NIAMS NIH HHS: AR047963

    The Journal of biological chemistry 2004;279;15;14772-83

  • Mechanical pressure-induced phosphorylation of p38 mitogen-activated protein kinase in epithelial cells via Src and protein kinase C.

    Hofmann M, Zaper J, Bernd A, Bereiter-Hahn J, Kaufmann R and Kippenberger S

    Department of Dermatology and Venerology, University Hospital, Johann Wolfgang Goethe-University, D-60590 Frankfurt/Main, Germany.

    Mechanical stimulation is known to modulate cell physiology in a variety of different tissues. Particularly, epithelial cells are permanently exposed to mechanical stimulation generated by externally applied forces. The present in vitro study demonstrated mechanical pressure as a trigger-factor of the p38 mitogen-activated protein kinase (MAPK) pathway in epithelial cells. Mechanical pressure applied by teflon weights (1.02g/cm(2)) led to a rapid phosphorylation of p38 peaking between 5 and 10min. Furthermore, phosphorylation of the small heat shock protein 27 (HSP27) was shown in response to mechanical pressure. Suppression of p38 function by using specific inhibitors blocked the pressure-mediated phosphorylation of HSP27. In order to identify upstream regulators of p38, a contribution of Src and protein kinase C (PKC) in pressure-signaling was investigated. We could demonstrate that inhibition of Src or PKC suppressed the pressure-induced phosphorylation of p38. These findings suggest mechanical pressure as a new type of effector stimulus for the p38 pathway with implications to (patho-) physiological conditions.

    Biochemical and biophysical research communications 2004;316;3;673-9

  • Activation of a Dab1/CrkL/C3G/Rap1 pathway in Reelin-stimulated neurons.

    Ballif BA, Arnaud L, Arthur WT, Guris D, Imamoto A and Cooper JA

    Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109, USA. jcooper@fhcrc.org

    During brain development, many neurons migrate long distances before settling and differentiating. These migrations are coordinated to ensure normal development. The secreted protein Reelin controls the locations of many types of neurons, and its absence causes the classic "Reeler" phenotype. Reelin action requires tyrosine phosphorylation of the intracellular protein Dab1 by Src-family kinases. However, little is known about signaling pathways downstream of Dab1. Here, we identify several proteins in embryonic brain extract that bind to tyrosine-phosphorylated, but not non-phosphorylated, Dab1. Of these, the Crk-family proteins (CrkL, CrkI, and CrkII ), bind significant quantities of Dab1 when embryonic cortical neurons are exposed to Reelin. CrkL binding to Dab1 involves two tyrosine phosphorylation sites, Y220 and 232, that are critical for proper positioning of migrating cortical plate neurons. CrkL also binds C3G, an exchange factor (GEF) for the small GTPase Rap1 that is activated in other systems by tyrosine phosphorylation. We report that Reelin stimulates tyrosine phosphorylation of C3G and activates Rap1. C3G and Rap1 regulate adhesion of fibroblasts and other cell types. Regulation of Crk/CrkL, C3G, and Rap1 by Reelin may be involved in coordinating neuron migrations during brain development.

    Funded by: NCI NIH HHS: CA 41072, T32 CA 09657; NINDS NIH HHS: F32 NS046492

    Current biology : CB 2004;14;7;606-10

  • HIV/SIV escape from immune surveillance: focus on Nef.

    Tolstrup M, Ostergaard L, Laursen AL, Pedersen SF and Duch M

    Department of Infectious Disease Q, Skejby Hospital, Denmark.

    During a progressive HIV-1 infection, the gradual decrease in functional CD4+ T(helper) cells leads to immunodeficiency and eventually death in the untreated patient. The virulence role of the lentiviral accessory gene nef was first reported from deletion studies in the macaque model, and research during the past decade has revealed a pluripotent protein capable of multiple points of interference with cellular mechanisms. Importantly, Nef has the capacity to modify the plasma membrane signalling by regulation of receptor/ligand endocytosis as well as to modulate cellular regulation such as apoptosis and lymphocyte activation. This effective defence against an apparent vigorous and specific immune response is crucial for the ability of HIV-1 to persist in the host. Here we review the multitude of functions exerted by Nef and discuss the functional domains of the protein in terms of cellular interaction partners and the effect of nef mutations in the course of AIDS disease progression.

    Current HIV research 2004;2;2;141-51

  • SRC gene expression in human cancer: the role of transcriptional activation.

    Dehm SM and Bonham K

    Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada. dehm.scott@mayo.edu

    Human pp60c-Src (or c-Src) is a 60 kDa nonreceptor tyrosine kinase encoded by the SRC gene and is the cellular homologue to the potent transforming v-Src viral oncogene. c-Src functions at the hub of a vast array of signal transduction cascades that influence cellular proliferation, differentiation, motility, and survival. c-Src activation has been documented in upwards of 50% of tumors derived from the colon, liver, lung, breast, and pancreas. Therefore, a major focus has been to understand the mechanisms of c-Src activation in human cancer. Early studies concentrated on post-translational mechanisms that lead to increased c-Src kinase activity, which often correlated with overexpression of c-Src protein. More recently, the discovery of an activating SRC mutation in a small subset of advanced colon tumors has been reported. In addition, elevated SRC transcription has been identified as yet another mechanism contributing significantly to c-Src activation in a subset of human colon cancer cell lines. Interestingly, histone deacetylase (HDAC) inhibitors, agents with well-documented anti-cancer activity, repress SRC transcription in a wide variety of human cancer cell lines. Analysis of the mechanisms behind HDAC inhibitor mediated repression could be utilized in the future to specifically inhibit SRC gene expression in human cancer.

    Biochemistry and cell biology = Biochimie et biologie cellulaire 2004;82;2;263-74

  • Functional association between Wwox tumor suppressor protein and p73, a p53 homolog.

    Aqeilan RI, Pekarsky Y, Herrero JJ, Palamarchuk A, Letofsky J, Druck T, Trapasso F, Han SY, Melino G, Huebner K and Croce CM

    Kimmel Cancer Institute, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107, USA.

    The WWOX gene is a recently cloned tumor suppressor gene that spans the FRA16D fragile region. Wwox protein contains two WW domains that are generally known to mediate protein-protein interaction. Here we show that Wwox physically interacts via its first WW domain with the p53 homolog, p73. The tyrosine kinase, Src, phosphorylates Wwox at tyrosine 33 in the first WW domain and enhances its binding to p73. Our results further demonstrate that Wwox expression triggers redistribution of nuclear p73 to the cytoplasm and, hence, suppresses its transcriptional activity. In addition, we show that cytoplasmic p73 contributes to the proapoptotic activity of Wwox. Our findings reveal a functional cross-talk between p73 and Wwox tumor suppressor protein.

    Proceedings of the National Academy of Sciences of the United States of America 2004;101;13;4401-6

  • Gonadotropin-releasing hormone-induced activation of diacylglycerol kinase-zeta and its association with active c-src.

    Davidson L, Pawson AJ, López de Maturana R, Freestone SH, Barran P, Millar RP and Maudsley S

    MRC Human Reproductive Sciences Unit, Edinburgh Royal Infirmary, The University of Edinburgh Chancellor's Building, 49 Little France Crescent, Edinburgh EH16 4SB, Scotland, United Kingdom.

    Gonadotropin-releasing hormone (GnRH)-induced receptor activation has been demonstrated to entrain a wide variety of signaling modalities. Most signaling pathways are concerned with the control of serine, threonine, or tyrosine-protein kinases, however, in the current article we demonstrate that in both a model cell line and in gonadotropes, GnRH additionally mediates the activation of lipid-directed kinases. We have shown that there is a functional connection between protein-tyrosine kinase modulation and lipid kinase activation. In HEK293 cells stably expressing the Type I mammalian GnRH receptor, we employed a proteomic approach to identify novel protein binding partners for GnRH-activated c-Src. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry we identified a GnRH-induced association between c-Src and the lipid kinase, diacylglycerol kinase-zeta (DGK-zeta). Using reciprocal co-immunoprecipitation we show that there is a significant elevation of the association between catalytically active c-Src with DGK-zeta in both HEK293 cells and murine gonadotrope LbetaT2 cells. Employing lipid kinase assays we have shown that the catalytic activity of DGK-zeta is significantly heightened in both HEK293 and LbetaT2 cells by GnRH. In addition, we demonstrate that the activation of DGK-zeta exerts a functional role in the murine gonadotrope LbetaT2 cell line. Elevated expression of DGK-zeta resulted in a shortening of the time scale of ERK activation in these cells suggesting a potential role of endogenous DGK-zeta in controlling the induction of LHbeta transcription by ERK1/2.

    Funded by: Medical Research Council: MC_U127685842, U.1276.00.005.00002.01 (85842)

    The Journal of biological chemistry 2004;279;12;11906-16

  • TRIP6 enhances lysophosphatidic acid-induced cell migration by interacting with the lysophosphatidic acid 2 receptor.

    Xu J, Lai YJ, Lin WC and Lin FT

    Department of Cell Biology, University of Alabama at Birmingham, Alabama 35294-0005, USA.

    Lysophosphatidic acid (LPA) induces actin rearrangement, focal adhesion assembly, and cell migration through the activation of small G protein Rho and its downstream effectors. These diverse cellular responses are mediated by its associated G protein-coupled receptors. However, the mechanisms and specificity by which these LPA receptors mediate LPA actions are still poorly understood. Here we show that LPA stimulation promotes the interaction of the LPA(2) receptor with a focal adhesion molecule, TRIP6 (thyroid receptor interacting protein 6)/ZRP-1 (zyxin-related protein 1). TRIP6 directly binds to the carboxyl-terminal tail of the LPA(2) receptor through its LIM domains. LPA-dependent recruitment of TRIP6 to the plasma membrane promotes its targeting to focal adhesions and co-localization with actin stress fibers. In addition, TRIP6 associates with the components of focal complexes including paxillin, focal adhesion kinase, c-Src, and p130(cas) in an agonist-dependent manner. Overexpression of TRIP6 augments LPA-induced cell migration; in contrast, suppression of endogenous TRIP6 expression by a TRIP6-specific small interfering RNA reduces it in SKOV3 ovarian cancer cells. Strikingly, the association with TRIP6 is specific to the LPA(2) receptor but not LPA(1) or LPA(3) receptor, indicating a specific role for TRIP6 in regulating LPA(2) receptor-mediated signaling. Taken together, our results suggest that TRIP6 functions at a point of convergence between the activated LPA(2) receptor and downstream signals involved in cell adhesion and migration.

    Funded by: NCI NIH HHS: CA100848, R01 CA100848, R01 CA100857

    The Journal of biological chemistry 2004;279;11;10459-68

  • Cbl-c suppresses v-Src-induced transformation through ubiquitin-dependent protein degradation.

    Kim M, Tezuka T, Tanaka K and Yamamoto T

    Division of Oncology, Department of Cancer Biology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

    The Cbl family proteins Cbl, Cbl-b, and Cbl-c/Cbl-3 are thought to regulate signaling through protein-tyrosine kinases, positively as scaffold proteins and negatively as ubiquitin ligases. However, the precise signaling pathways and target proteins for each Cbl family member are not well understood. Here we show that Src is a preferential target of Cbl-c for degradation. Although exogenous expression of all Cbl family proteins suppressed the anchorage-independent growth of v-Src-transformed NIH3T3 cells, only Cbl-c caused reversion of the refractile morphology. The level of v-Src protein was reduced by Cbl-c, possibly through a lysosome-dependent pathway. The TKB domain and RING finger of Cbl-c were important for its antioncogenic activity. Wild-type Cbl-c promoted ubiquitination of Src in 293T cells, whereas a RING finger mutant did not. Cbl-c bound specifically to Src phosphorylated at Tyr419. Furthermore, Cbl-c together with UbcH5 induced ubiquitination of Src in vitro. Importantly, the Tyr419 nonphosphorylated form of Src was not ubiquitinated by Cbl-c. Therefore, activated Src may be a direct target of Cbl-c in vivo. Our results suggest that Cbl and Cbl-b suppress v-Src-induced transformation through mechanisms distinct from that of Cbl-c.

    Oncogene 2004;23;9;1645-55

  • Mechanisms of androgen receptor signalling via steroid receptor coactivator-1 in prostate.

    Powell SM, Christiaens V, Voulgaraki D, Waxman J, Claessens F and Bevan CL

    Prostate Cancer Research Group, Department of Cancer Medicine, Imperial College London, Hammersmith Hospital, London W12 0NN, UK.

    The androgen receptor (AR) is a member of the nuclear receptor superfamily. These ligand-activated transcription factors usually contain two activation functions, a ligand-independent activation function 1(AF1) in the divergent N-terminal domain and a ligand-dependent AF2 in the more conserved C-terminal ligand-binding domain. To promote transcription from target promoters, DNA-bound nuclear receptors recruit coactivator proteins that promote transcription by modifying histones within nucleosomes, resulting in altered topology of chromatin to allow access of the basal transcriptional machinery, or stabilising the pre-initiation complex. It is well known that most coactivators interact with AF2 of many nuclear receptors via conserved, helical LxxLL motifs (where L is leucine and x is any amino acid). The AF2 of the AR is very weak, but we were able to demonstrate that its intrinsic ligand-dependent activity is potentiated by steroid receptor coactivator-1 (SRC1) and that this region interacts with coactivators via LxxLL motifs. However, a mutant SRC1 coactivator with no functional LxxLL motifs was still able to potentiate AR activity. We found that SRC1 can also be recruited to (and increase activity of) AF1 of the AR via a conserved, glutamine-rich region. Point mutations within this region abolish SRC1 interaction with AF1 and also abolish or severely impair its ability to potentiate AR activity on all promoters tested. Thus the AR interacts with SRC1 via two different regions and the AF1 interaction is functionally the more important, although the contribution of the two interactions varies in a promoter-dependent fashion. SRC1 then potentiates receptor activity via recruitment of CBP/p300, a histone acetyltranferase. This is important in the context of prostate cancer as SRC1 and other coactivators including CBP are coexpressed with AR in the luminal epithelial cells of the prostate, where over 90% of prostate tumours arise. There is a need for effective second-line prostate cancer therapy aimed at blocking the AR pathway when anti-androgen therapy has failed. Since there is growing evidence that nuclear receptor cofactors may be implicated in the progression of hormone-dependent tumours to hormone-independent states, novel targets could include the interaction of AR with coactivator proteins. We suggest that the N-terminal interaction would be a more specific and effective target in the case of prostate cancer than the LxxLL/AF2 interaction.

    Endocrine-related cancer 2004;11;1;117-30

  • Selective down-regulation of angiotensin II receptor type 1A signaling by protein tyrosine phosphatase SHP-2 in vascular smooth muscle cells.

    Doan T, Farmer P, Cooney T and Ali MS

    Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA.

    The heptahelical AT(1) G-protein-coupled receptor lacks inherent tyrosine kinase activity. Angiotensin II binding to AT(1) nevertheless activates several tyrosine kinases and stimulates both tyrosine phosphorylation and phosphatase activity of the SHP-2 tyrosine phosphatase in vascular smooth muscle cells. Since a balance between tyrosine kinase and tyrosine phosphatase activities is essential in angiotensin II signaling, we investigated the role of SHP-2 in modulating tyrosine kinase signaling pathways by stably transfecting vascular smooth muscle cells with expression vectors encoding wild-type SHP-2 protein or a catalytically inactive SHP-2 mutant. Our data indicate that SHP-2 is an efficient negative regulator of angiotensin II signaling. SHP-2 inhibited c-Src catalytic activity by dephosphorylating a positive regulatory tyrosine 418 within the Src kinase domain. Importantly, SHP-2 expression also abrogated angiotensin II-induced activation of ERK, whereas expression of catalytically inactive SHP-2 caused sustained ERK activation. Thus, SHP-2 likely regulates angiotensin II-induced MAP kinase signaling by inactivating c-Src. These SHP-2 effects were specific for a subset of angiotensin II signaling pathways, since SHP-2 overexpression failed to influence Jak2 tyrosine phosphorylation or Fyn catalytic activity. These data show SHP-2 represents a critical negative regulator of angiotensin II signaling, and further demonstrate a new function for this phosphatase in vascular smooth muscle cells.

    Funded by: NHLBI NIH HHS: HL 61710

    Cellular signalling 2004;16;3;301-11

  • Src homology 3 binding sites in the P2Y2 nucleotide receptor interact with Src and regulate activities of Src, proline-rich tyrosine kinase 2, and growth factor receptors.

    Liu J, Liao Z, Camden J, Griffin KD, Garrad RC, Santiago-Pérez LI, González FA, Seye CI, Weisman GA and Erb L

    Department of Biochemistry, University of Missouri-Columbia, Columbia, Missouri 65212, USA.

    Many G protein-coupled receptors activate growth factor receptors, although the mechanisms controlling this transactivation are unclear. We have identified two proline-rich, SH3 binding sites (PXXP) in the carboxyl-terminal tail of the human P2Y(2) nucleotide receptor that directly associate with the tyrosine kinase Src in protein binding assays. Furthermore, Src co-precipitated with the P2Y(2) receptor in 1321N1 astrocytoma cells stimulated with the P2Y(2) receptor agonist UTP. A mutant P2Y(2) receptor lacking the PXXP motifs was found to stimulate calcium mobilization and serine/threonine phosphorylation of the Erk1/2 mitogen-activated protein kinases, like the wild-type receptor, but was defective in its ability to stimulate tyrosine phosphorylation of Src and Src-dependent tyrosine phosphorylation of the proline-rich tyrosine kinase 2, epidermal growth factor receptor (EGFR), and platelet-derived growth factor receptor. Dual immunofluorescence labeling of the P2Y(2) receptor and the EGFR indicated that UTP caused an increase in the co-localization of these receptors in the plasma membrane that was prevented by the Src inhibitor PP2. Together, these data suggest that agonist-induced binding of Src to the SH3 binding sites in the P2Y(2) receptor facilitates Src activation, which recruits the EGFR into a protein complex with the P2Y(2) receptor and allows Src to efficiently phosphorylate the EGFR.

    Funded by: NCRR NIH HHS: RR15565; NIA NIH HHS: AG18357

    The Journal of biological chemistry 2004;279;9;8212-8

  • Vascular endothelial growth factor-mediated activation of p38 is dependent upon Src and RAFTK/Pyk2.

    McMullen M, Keller R, Sussman M and Pumiglia K

    Center for Cell Biology and Cancer Research, Albany Medical College, Albany NY, USA.

    Vascular endothelial growth factor (VEGF) induces activation of p38 mitogen-activated protein kinase (MAPK) in primary endothelial cells and may be critical for VEGF-induced angiogenesis. We investigated the molecular basis for p38 activation in response to VEGF. The expression of a C-terminal splice variant of FAK, FRNK, had no affect on VEGF-induced activation of p38; however, expression of a dominant-negative RAFTK/Pyk2 mutant led to a decrease in the activation of p38, but had no affect on extracellular signal-regulated kinase (ERK). Since calcium regulates RAFTK/Pyk2, we investigated its role in p38 activity. Preincubation with EGTA suppressed p38 activation, while calcium ionophore induced p38 activity. Inhibition of phospholipase C (PLC) resulted in complete inhibition of ERK, while having no affect on p38 activity. These data suggested a bifurcation in the regulation of MAPKs that occurs at the level of PLC and RAFTK/Pyk2 activation. Src family kinases interact with RAFTK/Pyk2. Inhibition of Src by either pharmacological or genetic means decreased p38 activity. Finally, we found that both Src and RAFTK/Pyk2 were essential for endothelial cell migration. These data identified a novel regulatory network involving extracellular calcium, RAFTK/Pyk2, Src and p38. This signaling network appears to be critical for VEGF-induced endothelial cell migration.

    Funded by: NCI NIH HHS: R01 CA081419, R01-CA-81419; NHLBI NIH HHS: T32-HL-07194

    Oncogene 2004;23;6;1275-82

  • Activation of platelet-activating factor receptor-coupled G alpha q leads to stimulation of Src and focal adhesion kinase via two separate pathways in human umbilical vein endothelial cells.

    Deo DD, Bazan NG and Hunt JD

    Department of Biochemistry and Molecular Biology, Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA.

    Platelet-activating factor (PAF), a phospholipid second messenger, has diverse physiological functions, including responses in differentiated endothelial cells to external stimuli. We used human umbilical vein endothelial cells (HUVECs) as a model system. We show that PAF activated pertussis toxin-insensitive G alpha(q) protein upon binding to its seven transmembrane receptor. Elevated cAMP levels were observed via activation of adenylate cyclase, which activated protein kinase A (PKA) and was attenuated by a PAF receptor antagonist, blocking downstream activity. Phosphorylation of Src by PAF required G alpha(q) protein and adenylate cyclase activation; there was an absolute requirement of PKA for PAF-induced Src phosphorylation. Immediate (1 min) PAF-induced STAT-3 phosphorylation required the activation of G alpha(q) protein, adenylate cyclase, and PKA, and was independent of these intermediates at delayed (30 min) and prolonged (60 min) PAF exposure. PAF activated PLC beta 3 through its G alpha(q) protein-coupled receptor, whereas activation of phospholipase C gamma 1 (PLC gamma 1) by PAF was independent of G proteins but required the involvement of Src at prolonged PAF exposure (60 min). We demonstrate for the first time in vascular endothelial cells: (i) the involvement of signaling intermediates in the PAF-PAF receptor system in the induction of TIMP2 and MT1-MMP expression, resulting in the coordinated proteolytic activation of MMP2, and (ii) a receptor-mediated signal transduction cascade for the tyrosine phosphorylation of FAK by PAF. PAF exposure induced binding of p130(Cas), Src, SHC, and paxillin to FAK. Clearly, PAF-mediated signaling in differentiated endothelial cells is critical to endothelial cell functions, including cell migration and proteolytic activation of MMP2.

    Funded by: NIEHS NIH HHS: ES00358-03

    The Journal of biological chemistry 2004;279;5;3497-508

  • Activation of beta-catenin-TCF-mediated transcription by non-receptor tyrosine kinase v-Src.

    Haraguchi K, Nishida A, Ishidate T and Akiyama T

    Laboratory of Molecular and Genetic Information, Institute for Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.

    Activation of Wnt signaling is an early event in colorectal tumorigenesis, while aberrant activation of non-receptor tyrosine kinase c-Src occurs during tumor progression. Here, we show that v-Src and receptor tyrosine kinase ErbB2 activate beta-catenin-TCF-mediated transcription. The effect of v-Src was abrogated by a dominant-negative mutant of TCF and the tumor suppressor APC. Furthermore, the effect of v-Src was partially abrogated by a dominant-negative mutant of MAP kinase, suggesting that v-Src exerts its effect at least in part via the MAP kinase pathway. Our finding raises the possibility that aberrantly activated c-Src may enhance Wnt signaling and this may contribute to tumor progression.

    Biochemical and biophysical research communications 2004;313;4;841-4

  • Complete sequencing and characterization of 21,243 full-length human cDNAs.

    Ota T, Suzuki Y, Nishikawa T, Otsuki T, Sugiyama T, Irie R, Wakamatsu A, Hayashi K, Sato H, Nagai K, Kimura K, Makita H, Sekine M, Obayashi M, Nishi T, Shibahara T, Tanaka T, Ishii S, Yamamoto J, Saito K, Kawai Y, Isono Y, Nakamura Y, Nagahari K, Murakami K, Yasuda T, Iwayanagi T, Wagatsuma M, Shiratori A, Sudo H, Hosoiri T, Kaku Y, Kodaira H, Kondo H, Sugawara M, Takahashi M, Kanda K, Yokoi T, Furuya T, Kikkawa E, Omura Y, Abe K, Kamihara K, Katsuta N, Sato K, Tanikawa M, Yamazaki M, Ninomiya K, Ishibashi T, Yamashita H, Murakawa K, Fujimori K, Tanai H, Kimata M, Watanabe M, Hiraoka S, Chiba Y, Ishida S, Ono Y, Takiguchi S, Watanabe S, Yosida M, Hotuta T, Kusano J, Kanehori K, Takahashi-Fujii A, Hara H, Tanase TO, Nomura Y, Togiya S, Komai F, Hara R, Takeuchi K, Arita M, Imose N, Musashino K, Yuuki H, Oshima A, Sasaki N, Aotsuka S, Yoshikawa Y, Matsunawa H, Ichihara T, Shiohata N, Sano S, Moriya S, Momiyama H, Satoh N, Takami S, Terashima Y, Suzuki O, Nakagawa S, Senoh A, Mizoguchi H, Goto Y, Shimizu F, Wakebe H, Hishigaki H, Watanabe T, Sugiyama A, Takemoto M, Kawakami B, Yamazaki M, Watanabe K, Kumagai A, Itakura S, Fukuzumi Y, Fujimori Y, Komiyama M, Tashiro H, Tanigami A, Fujiwara T, Ono T, Yamada K, Fujii Y, Ozaki K, Hirao M, Ohmori Y, Kawabata A, Hikiji T, Kobatake N, Inagaki H, Ikema Y, Okamoto S, Okitani R, Kawakami T, Noguchi S, Itoh T, Shigeta K, Senba T, Matsumura K, Nakajima Y, Mizuno T, Morinaga M, Sasaki M, Togashi T, Oyama M, Hata H, Watanabe M, Komatsu T, Mizushima-Sugano J, Satoh T, Shirai Y, Takahashi Y, Nakagawa K, Okumura K, Nagase T, Nomura N, Kikuchi H, Masuho Y, Yamashita R, Nakai K, Yada T, Nakamura Y, Ohara O, Isogai T and Sugano S

    Helix Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan.

    As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

    Nature genetics 2004;36;1;40-5

  • Agonist-antagonist induced coactivator and corepressor interplay on the human androgen receptor.

    Dotzlaw H, Papaioannou M, Moehren U, Claessens F and Baniahmad A

    Genetic Institute, Justus-Liebig-University, Heinrich-Buff-Ring 58-62, D-35392 Giessen, Germany.

    The human androgen receptor (AR) is a member of the nuclear hormone receptor superfamily. However, in contrast to other members of this family the amino-(N)-terminus of AR harbors the major transactivation function. Previously we have shown that hormone antagonists that bind to the carboxy-terminal ligand-binding domain repress AR through recruitment of corepressors that are recruited to the receptor N-terminus. Here we show by a modified mammalian two-hybrid system that both the AR interacting domains of the coactivator SRC1 and of the corepressor SMRT compete for interaction with the AR N-terminus. In contrast to other members of the nuclear receptor superfamily the LXXLL motifs of SRC1e are not required for this interaction, instead a stretch of 135 amino acids of the glutamine rich region (Qr) of SRC1e is essential to bind to the AR N-terminus. We show that the Qr-region of SRC1 is able to inhibit the interaction of SMRT with AR. Also, we demonstrate that the corepressor mediated repression decreases the antagonist-induced transactivation while, surprisingly, it increases the agonist-induced transactivation. This may indicate that coactivators and corepressors act in concert to dictate the overall receptor-mediated action dependent on the type of ligand.

    Molecular and cellular endocrinology 2003;213;1;79-85

  • Constitutively active Galpha16 stimulates STAT3 via a c-Src/JAK- and ERK-dependent mechanism.

    Lo RK, Cheung H and Wong YH

    Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.

    The hematopoietic-specific Galpha16 protein has recently been shown to mediate receptor-induced activation of the signal transducer and activator of transcription 3 (STAT3). In the present study, we have delineated the mechanism by which Galpha16 stimulates STAT3 in human embryonic kidney 293 cells. A constitutively active Galpha16 mutant, Galpha16QL, stimulated STAT3-dependent luciferase activity as well as the phosphorylation of STAT3 at both Tyr705 and Ser727. Galpha16QL-induced STAT3 activation was enhanced by overexpression of extracellular signal-regulated kinase 1 (ERK1), but was inhibited by U0126, a Raf-1 inhibitor, and coexpression of the dominant negative mutants of Ras and Rac1. Inhibition of phospholipase Cbeta, protein kinase C, and calmodulin-dependent kinase II by their respective inhibitors also suppressed Galpha16QL-induced STAT3 activation. The involvement of tyrosine kinases such as c-Src and Janus kinase 2 and 3 (JAK2 and JAK3) in Galpha16QL-induced activation of STAT3 was illustrated by the combined use of selective inhibitors and dominant negative mutants. In contrast, c-Jun N-terminal kinase, p38 MAPK, RhoA, Cdc42, phosphatidylinositol 3-kinase, and the epidermal growth factor receptor did not appear to be required. Similar observations were obtained with human erythroleukemia cells, where STAT3 phosphorylation was stimulated by C5a in a PTX-insensitive manner. Collectively, these results highlight the important regulatory roles of the Ras/Raf/MEK/ERK and c-Src/JAK pathways on the stimulation of STAT3 by activated Galpha16. Demonstration of the involvement of different kinases in Galpha16QL-induced STAT3 activation supports the involvement of multiple signaling pathways in the regulation of transcription by G proteins.

    The Journal of biological chemistry 2003;278;52;52154-65

  • GIT1 mediates Src-dependent activation of phospholipase Cgamma by angiotensin II and epidermal growth factor.

    Haendeler J, Yin G, Hojo Y, Saito Y, Melaragno M, Yan C, Sharma VK, Heller M, Aebersold R and Berk BC

    Center for Cardiovascular Research and Department of Medicine, University of Rochester, Rochester, New York 14642, USA.

    Critical events for vasoconstrictor and growth factor signal transduction include stimulation of phospholipase Cgamma (PLCgamma) and elevation of intracellular calcium. c-Src has been proposed as a common mediator for these signals activated by both G protein-coupled receptors (GPCRs) and tyrosine kinase-coupled receptors (TKRs). Here we show that the GPCR kinase-interacting protein-1 (GIT1) is a substrate for c-Src that undergoes tyrosine phosphorylation in response to angiotensin II (AngII) and EGF in vascular smooth muscle and 293 cells. GIT1 associates with PLCgamma via the PLCgamma Src homology 2 and 3 domains constitutively, and the interaction is unaltered by AngII and EGF. GIT1 interaction with PLCgamma is required for PLCgamma activation based on inhibition of tyrosine phosphorylation and calcium mobilization after GIT1 knockdown with antisense GIT1 oligonucleotides. GIT1 interacts with PLCgamma via a novel Spa homology domain (SHD) and a coiled-coil domain. Deletion mutation analysis showed that GIT1(SHD) is required for AngII- and EGF-mediated PLCgamma activation (measured by phosphorylation of Tyr783 and inositol 1,4,5-trisphosphate formation). We propose that GIT1 is a novel regulator of PLCgamma function that mediates PLCgamma activation by c-Src and integrates signal transduction by GPCRs and TKRs.

    Funded by: NHLBI NIH HHS: R01 HL49192, R01 HL59975

    The Journal of biological chemistry 2003;278;50;49936-44

  • Activation of SRC tyrosine kinases in response to ICAM-1 ligation in pulmonary microvascular endothelial cells.

    Wang Q, Pfeiffer GR and Gaarde WA

    Division of Integrative Biology, Department of Pediatrics, Rainbow Babies and Children's Hospital and Case Western Reserve University, Cleveland, Ohio 44106, USA. qxw9@po.cwru.edu

    Previous studies demonstrated that ICAM-1 ligation on human pulmonary microvascular endothelial cells (ECs) sequentially induces activation of xanthine oxidase and p38 MAPK. Inhibition of these signaling events reduces neutrophil migration to the EC borders. This study examined the role of SRC tyrosine kinases in ICAM-1-initiated signaling within these ECs. Cross-linking ICAM-1 on tumor necrosis factor-alpha-pretreated ECs induced an increase in the activity of SRC tyrosine kinases. This increase was inhibited by allopurinol (a xanthine oxidase inhibitor), Me2SO (a hydroxyl radical scavenger), or deferoxamine (an iron chelator). Phenylarsine oxide, a tyrosine phosphatase inhibitor, reduced the base-line activity of SRC as well as the increase in SRC activity induced by ICAM-1 cross-linking. Specific inhibition of the protein expression of the SRC homology 2-containing protein-tyrosine phosphatase-2 (SHP-2) by an antisense oligonucleotide prevented the induced SRC activation but had no effect on the basal SRC activity. Activation of SRC tyrosine kinases was accompanied by tyrosine phosphorylation of ezrin at Tyr-146, which was inhibited by PP2, an SRC tyrosine kinase inhibitor. Moreover, PP2 completely inhibited p38 activation, suggesting a role for SRC tyrosine kinases in p38 activation. These data demonstrate that ICAM-1 ligation activates SRC tyrosine kinases and that this activation requires SHP-2 as well as production of reactive oxygen species generated from xanthine oxidase. Activation of SRC tyrosine kinases in turn leads to tyrosine phosphorylation of ezrin, as well as activation of p38, a kinase previously identified to be required for cytoskeletal changes induced by ICAM-1 ligation and for neutrophil migration along the EC surface.

    Funded by: NHLBI NIH HHS: HL07009, HL48160

    The Journal of biological chemistry 2003;278;48;47731-43

  • Src phosphorylates Cas on tyrosine 253 to promote migration of transformed cells.

    Goldberg GS, Alexander DB, Pellicena P, Zhang ZY, Tsuda H and Miller WT

    Department of Physiology and Biophysics, School of Medicine, Basic Science Tower T6, Health Science Complex, State University of New York at Stony Brook, Stony Brook, NY 11794-8661, USA. gary.goldberg@stonybrook.edu

    Cas is a member of the focal adhesion complex. Phosphorylation of Cas by Src is an important event leading to cell transformation. Using mass spectrometry, we have mapped 11 sites in Cas that are phosphorylated by Src. These sites are all located between residues 132 and 414 of Cas, in a region that is required for binding to a number of other proteins including Crk. We tested synthetic peptides modeled on Cas phosphorylation sites, and found that the sequence containing tyrosine 253 was phosphorylated by Src most efficiently. Using cells derived from Cas-deficient mice, we confirmed that Cas greatly enhanced the ability of Src to transform cells. Phosphorylation of Cas on tyrosine 253 was not required for Src to increase growth rate, suppress contact inhibition, or suppress anchorage dependence. Yet, in contrast to these growth characteristics, phosphorylation of Cas on tyrosine 253 was required for Src to promote cell migration. Thus, a single phosphorylation site on this focal adhesion adaptor protein can effectively separate cell migration from other transformed growth characteristics.

    Funded by: NCI NIH HHS: CA 58530, CA 88805-01A2, R01 CA058530, R01 CA058530-10A1, R01 CA088805

    The Journal of biological chemistry 2003;278;47;46533-40

  • Src regulates Golgi structure and KDEL receptor-dependent retrograde transport to the endoplasmic reticulum.

    Bard F, Mazelin L, Péchoux-Longin C, Malhotra V and Jurdic P

    UCSD Biological Sciences Division, Cell and Developmental Biology Department, University of California-San Diego, La Jolla, CA 92093-0347, USA. fabard@biomail.ucsd.edu

    The tyrosine kinase Src is present on the Golgi membranes. Its role, however, in the overall function and organization of the Golgi apparatus is unclear. We have found that in a cell line called SYF, which lacks the three ubiquitous Src-like kinases (Src, Yes, and Fyn), the organization of the Golgi apparatus is perturbed. The Golgi apparatus is composed of collapsed stacks and bloated cisternae in these cells. Expression of an activated form of Src relocated the KDEL receptor (KDEL-R) from the Golgi apparatus to the endoplasmic reticulum. Other Golgi-specific marker proteins were not affected under these conditions. Because of the specific effect of Src on the location of KDEL-R, we tested whether protein transport between ER and the Golgi apparatus involves Src. Transport of Pseudomonas exotoxin, which is transported to the ER by binding to the KDEL-R is accelerated by inhibition or genetic ablation of Src. Protein transport from ER to the Golgi apparatus however, is unaffected by Src deletion or inhibition. We propose that Src has an appreciable role in the organization of the Golgi apparatus, which may be linked to its involvement in protein transport from the Golgi apparatus to the endoplasmic reticulum.

    The Journal of biological chemistry 2003;278;47;46601-6

  • Src mediates prolactin-dependent proliferation of T47D and MCF7 cells via the activation of focal adhesion kinase/Erk1/2 and phosphatidylinositol 3-kinase pathways.

    Acosta JJ, Muñoz RM, González L, Subtil-Rodríguez A, Dominguez-Caceres MA, García-Martínez JM, Calcabrini A, Lazaro-Trueba I and Martín-Pérez J

    Instituto de Investigaciones Biomédicas A Sols, Consejo Superior de Investigaciones Cientificas, Madrid, Spain.

    Prolactin (PRL) stimulates breast cancer cell proliferation; however, the involvement of PRL-activated signaling molecules in cell proliferation is not fully established. Here we studied the role of c-Src on PRL-stimulated proliferation of T47D and MCF7 breast cancer cells. We initially observed that PRL-dependent activation of focal adhesion kinase (Fak), Erk1/2, and cell proliferation was mediated by c-Src in T47D cells, because expression of a dominant-negative form of c-Src (SrcDM, K295A/Y527F) blocked the PRL-dependent effects. The Src inhibitor PP1 abrogated PRL-dependent in vivo activation of Fak, Erk1/2, p70S6K, and Akt and the proliferation of T47D and MCF7 cells; Janus kinase 2 (Jak2) activation was not affected. However, in vitro, Fak and Jak2 kinases were not directly inhibited by PP1, demonstrating the effect of PP1 on c-Src kinase as an upstream activator of Fak. Expression of Fak mutant Y397F abrogated PRL-dependent activation of Fak, Erk1/2, and thymidine incorporation, but had no effect on p70S6K and Akt kinases. MAPK kinase 1/2 (Mek1/2) inhibitor PD184352 blocked PRL-induced stimulation of Erk1/2 and cell proliferation; however, p70S6K and Akt activation were unaffected. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 abolished cell proliferation and activation of p70S6K and Akt; however, PRL-dependent activation of Erk1/2 was not modified. Moreover, we show that both c-Src/PI3K and c-Src/Fak/Erk1/2 pathways are involved in the up-regulation of c-myc and cyclin d1 expression mediated by PRL. The previous findings suggest the existence of two PRL-dependent signaling cascades, initiated by the c-Src-mediated activation of Fak/Erk1/2 and PI3K pathways that, subsequently, control the expression of c-Myc and cyclin D1 and the proliferation of T47D and MCF7 breast cancer cells.

    Molecular endocrinology (Baltimore, Md.) 2003;17;11;2268-82

  • Activation of phosphatidylinositol 3-kinase/Akt pathway by androgen through interaction of p85alpha, androgen receptor, and Src.

    Sun M, Yang L, Feldman RI, Sun XM, Bhalla KN, Jove R, Nicosia SV and Cheng JQ

    Departments of Pathology and Interdisciplinary Oncology, University of South Florida College of Medicine and H. Lee Moffitt Cancer Center, Tampa, Florida 33612, USA.

    Recent studies have demonstrated that the cell growth and antiapoptotic actions of androgen could be dissociated from the transcriptional activity of the receptor and were, instead, mediated by activation of a mitogen-activated protein kinase pathway. This finding suggests an important cellular function of androgen receptor (AR) outside the nucleus. In this report, we demonstrate that androgen activates phosphatidylinositol 3-kinase (PI3K) and Akt, including AKT1 and AKT2, in AR-positive cells. Androgen-induced cell growth and survival were inhibited by PI3K inhibitor and dominant-negative Akt. AR interacts with the p85alpha regulatory subunit of PI3K, and its binding affinity is increased after androgen stimulation. The sites of interaction on the two proteins were mapped to the C-terminal Src-homology 2 domain of p85alpha and N terminus of AR. Activation of PI3K/Akt by androgen was inhibited by dominant-negative Src. Neither N-terminal truncated nor proline-rich region-deleted AR mutants, which are unable to bind to p85alpha and Src, respectively, was able to mediate androgen-induced PI3K/Akt activation. AR with deletion of C-terminal region including ligand binding domain, however, retains the ability to activate PI3K/Akt upon androgen stimulation, which supports the notion that nongenomic function of androgen is mediated by its interaction with membrane receptors (1, 3, 4). These findings indicate that a triple complex between AR, p85alpha, and Src is required for androgen-stimulated PI3K/Akt activation, and that the PI3K/Akt pathway, in addition to mitogen-activated protein kinase, mediates androgen-induced cell growth and cell survival.

    Funded by: NCI NIH HHS: CA77935, CA89242

    The Journal of biological chemistry 2003;278;44;42992-3000

  • Endothelin-1 decreases gap junctional intercellular communication by inducing phosphorylation of connexin 43 in human ovarian carcinoma cells.

    Spinella F, Rosanò L, Di Castro V, Nicotra MR, Natali PG and Bagnato A

    Laboratories of Molecular Pathology and Ultrastructure and Immunology, Regina Elena Cancer Institute, Rome 00158, Italy.

    Endothelin-1 (ET-1) is overexpressed in ovarian carcinoma and acts as an autocrine factor selectively through the ETA receptor (ETAR) to promote tumor cell proliferation, survival, neovascularization, and invasiveness. Loss of gap junctional intercellular communication (GJIC) is critical for tumor progression by allowing the cells to escape growth control. Exposure of HEY and OVCA 433 ovarian carcinoma cell lines to ET-1 led to a 50-75% inhibition in intercellular communication and to a decrease in the connexin 43 (Cx43)-based gap junction plaques. To investigate the phosphorylation state of Cx43, ovarian carcinoma cell lysates were immunoprecipitated and transient tyrosine phosphorylation of Cx43 was detected in ET-1-treated cells. BQ 123, a selective ETAR antagonist, blocked the ET-1-induced Cx43 phosphorylation and cellular uncoupling. Gap junction closure was prevented by tyrphostin 25 and by the selective c-Src inhibitor, PP2. Furthermore, the increased Cx43 tyrosine phosphorylation was correlated with ET-1-induced increase of c-Src activity, and PP2 suppressed the ET-1-induced Cx43 tyrosine phosphorylation, indicating that inhibition of Cx43-based GJIC is mainly mediated by the Src tyrosine kinase pathway. In vivo, the inhibition of human ovarian tumor growth in nude mice induced by the potent ETAR antagonist, ABT-627, was associated with a reduction of Cx43 phosphorylation. These findings indicate that the signaling mechanisms involved in GJIC disruption on ovarian carcinoma cells depend on ETAR activation, which leads to the Cx43 tyrosine phosphorylation mediated by c-Src, suggesting that ETAR blockade may contribute to the control of ovarian carcinoma growth and progression also by preventing the loss of GJIC.

    The Journal of biological chemistry 2003;278;42;41294-301

  • v-SRC specifically regulates the nucleo-cytoplasmic delocalization of the major isoform of TEL (ETV6).

    Lopez RG, Carron C and Ghysdael J

    CNRS UMR146-Institut Curie, Centre Universitaire, Bat 110, 91405 Orsay, France.

    TEL is a frequent target of chromosomal translocations in human cancer and an alleged tumor suppressor gene. TEL encodes two isoforms: a major TEL-M1 isoform as well as TEL-M43, which lacks the first 42 amino acid residues of TEL-M1. Both isoforms are potent transcriptional repressors that can inhibit RAS-induced transformation. Here we show that the v-SRC protein-tyrosine kinase relieves the repressive activity of TEL-M1, an activity that is associated with the v-SRC-induced delocalization of TEL-M1 from the nucleus to the cytoplasm. TEL-M1 delocalization requires the kinase activity of v-SRC and is not induced by oncogenic RAS or AKT. Cytoplasmic delocalization of TEL-M1 in response to v-SRC critically depends upon its unique amino-terminal domain (SRCD domain) because (i). v-SRC did not inhibit the repressive properties of TEL-M43, nor affected TEL-M43 nuclear localization; (ii). fusion of the first 52 amino acid residues of TEL-M1 to FLI-1, an ETS protein insensitive to v-SRC-induced delocalization, is sufficient to confer v-SRC-induced delocalization to this TEL/FLI-1 chimeric protein. The v-SRC-induced nucleo-cytoplasmic delocalization of TEL-M1 does not involve phosphorylation of the SRCD and does not require TEL self-association and repressive domains. Finally, enforced expression of the v-SRC-insensitive TEL-M43, but not of TEL-M1, inhibits v-SRC-induced transformation of NIH3T3 fibroblasts. These results identify a regulatory domain in TEL that specifically impinges on the subcellular localization of its major TEL-M1 isoform. They, furthermore, indicate that inhibition of TEL-M1 nuclear function is required for v-SRC to induce cellular transformation.

    The Journal of biological chemistry 2003;278;42;41316-25

  • An increase in the expression and total activity of endogenous p60(c-Src) in several factor-independent mutants of a human GM-CSF-dependent leukemia cell line (TF-1).

    Horn S, Meyer J, Stocking C, Ostertag W and Jücker M

    Zentrum für Experimentelle Medizin, Institut für Biochemie und Molekularbiologie I, Zelluläre Signaltransduktion, Universitätsklinikum Hamburg-Eppendorf, Martinistr 52, D-20246 Hamburg, Germany.

    Growth factor independence of hematopoietic cells can be induced by ectopic expression of a variety of oncogenes encoding receptor or cytoplasmic tyrosine kinases. To examine whether the activation of tyrosine kinases occurs in factor-independent mutants in vivo, the tyrosine-phosphorylated proteins from 14 factor-independent mutants of a GM-CSF-dependent cell line (TF-1) were analysed. These mutants did not secrete any growth-stimulating activity for TF-1 cells, suggesting that activation of intracellular signaling rather than an autocrine stimulation by secreted growth factors is responsible for their factor-independent growth. In 11 out of 14 GM-CSF-independent mutants analysed, a constitutively tyrosine-phosphorylated protein of 60 kDa was detected, which was subsequently identified as p60(c-Src). The kinase activity of p60(c-Src) was increased up to 12-fold in these mutants, which was at least in part due to overexpression of the c-src gene on the RNA and protein level. The Src substrate Sam68 showed an increased phosphorylation in mutants with high Src activity, suggesting that p60(c-Src) triggers downstream signaling in these cells. Treatment of the factor-independent mutants with the Src kinase inhibitor PP2 resulted in a reduced proliferation, demonstrating that Src kinases are essential for these cells for maximal proliferation. Further analysis of factor-independent mutants with low or undetectable Src activity revealed a constitutive phosphorylation of the common beta chain of the GM-CSF receptor and STAT5. Our data indicate an increase in the expression and total activity of endogenous p60(c-Src) in several GM-CSF-independent TF-1 mutants, further underlining the role of Src in the process of autonomous growth of hematopoietic cells.

    Oncogene 2003;22;46;7170-80

  • Coexpression of IGF-1R and c-Src proteins in human pancreatic ductal adenocarcinoma.

    Hakam A, Fang Q, Karl R and Coppola D

    Department of Interdisciplinary Oncology , Moffitt Cancer Center and Research Institute, University of South Florida, 12902 Magnolia Drive, Tampa, Florida 33612-9497, USA.

    Aberrant c-Src protein kinase activation has been identified as one of the molecular alterations involved in human pancreatic carcinogenesis. It has been postulated that c-Src may induce transformation by causing the overexpression of the insulinlike growth factor-1 receptor (IGF-1R) in pancreatic tumor cell lines. To further study the interaction between c-Src and IGF-1R proteins in human pancreatic cancer, we examined their coexpression in 47 human pancreatic ductal adenocarcinomas (PDA). Formalin-fixed, paraffin-embedded sections from 47 cases of PDA were stained using the immunohistochemical avidin-biotin-peroxidase method. We used an anti-human IGF-1R mouse monoclonal antibody (dilution 1:100 with antigen retrieval), and an anti-c-Src mouse monoclonal antibody (dilution 1:100 with antigen retrieval). The stains were semiquantitatively evaluated using the Allred score system, assessing intensity of stain and percentage of positive tumor cells. High cytoplasmic c-Src expression (Allred score 7-8) was seen in 33/47 (70%) tumors. In only 4 cases was c-Src either negative or low (Allred score 3). Strong and diffuse membranous IGF-1R stain (Allred score 7-8) was identified in 30/47 (64%) tumors. IGF-1R staining was low (Alled score 2-4) in 2 cases and negative in 1. Interestingly, in 40/47 (85%) cases c-Src and IGF-1R stains had similar scores. An inverse staining pattern was detected in only 6/47 (13%) tumors. Normal pancreatic ducts as well as areas of chronic pancreatitis were negative for IGF-1R. In conclusion, our data support the role of IGF-1R and c-Src in human pancreatic carcinogenesis; the coexpression of both these molecules may play an important role in transformation of pancreatic ductal cells.

    Digestive diseases and sciences 2003;48;10;1972-8

  • Tyrosine phosphorylation of plakoglobin causes contrary effects on its association with desmosomes and adherens junction components and modulates beta-catenin-mediated transcription.

    Miravet S, Piedra J, Castaño J, Raurell I, Francí C, Duñach M and García de Herreros A

    Unitat de Biofísica, Departament de Bioquímica i Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain.

    Plakoglobin is a protein closely related to beta-catenin that links desmosomal cadherins to intermediate filaments. Plakoglobin can also substitute for beta-catenin in adherens junctions, providing a connection between E-cadherin and alpha-catenin. Association of beta-catenin with E-cadherin and alpha-catenin is regulated by phosphorylation of specific tyrosine residues; modification of beta-catenin Tyr654 and Tyr142 decreases binding to E-cadherin and alpha-catenin, respectively. We show here that plakoglobin can also be phosphorylated on tyrosine residues, but unlike beta-catenin, this modification is not always associated with disrupted association with junctional components. Protein tyrosine kinases present distinct specificities on beta-catenin and plakoglobin, and phosphorylation of beta-catenin-equivalent Tyr residues of plakoglobin affects its interaction with components of desmosomes or adherens junctions differently. For instance, Src, which mainly phosphorylates Tyr86 in beta-catenin, modifies Tyr643 in plakoglobin, decreasing the interaction with E-cadherin and alpha-catenin and increasing the interaction with the alpha-catenin-equivalent protein in desmosomes, desmoplakin. The tyrosine kinase Fer, which modifies beta-catenin Tyr142, lessening its association with alpha-catenin, phosphorylates plakoglobin Tyr549 and exerts the contrary effect: it raises the binding of plakoglobin to alpha-catenin. These results suggest that tyrosine kinases like Src or Fer modulate desmosomes and adherens junctions differently. Our results also indicate that phosphorylation of Tyr549 and the increased binding of plakoglobin to components of adherens junctions can contribute to the upregulation of the transcriptional activity of the beta-catenin-Tcf-4 complex observed in many epithelial tumor cells.

    Molecular and cellular biology 2003;23;20;7391-402

  • Plasma membrane phospholipid scramblase 1 promotes EGF-dependent activation of c-Src through the epidermal growth factor receptor.

    Nanjundan M, Sun J, Zhao J, Zhou Q, Sims PJ and Wiedmer T

    Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.

    Phospholipid scramblase (PLSCR1) is a multiply palmitoylated, calcium-binding endofacial membrane protein proposed to mediate transbilayer movement of plasma membrane phospholipids. PLSCR1 is a component of membrane lipid rafts and has been shown to both physically and functionally interact with activated epidermal growth factor (EGF) receptors and other raft-associated cell surface receptors. Cell stimulation by EGF results in Tyr phosphorylation of PLSCR1, its association with both Shc and EGF receptors, and rapid cycling of PLSCR1 between plasma membrane and endosomal compartments. We now report evidence that upon EGF stimulation, PLSCR1 is phosphorylated by c-Src, within the tandem repeat sequence 68VYNQPVYNQP77. The in vivo interaction between PLSCR1 and Shc requires the Src-mediated phosphorylation on tyrosines 69 and 74. In in vitro pull down studies, phosphorylated PLSCR1 was found to bind directly to Shc through the phosphotyrosine binding domain. Consistent with the potential role of PLSCR1 in growth factor signaling pathways, granulocyte precursors derived from mice deficient in PLSCR1 show impaired proliferation and maturation under cytokine stimulation. Using PLSCR1-/- embryonic fibroblasts and kidney epithelial cells, we now demonstrate that deletion of PLSCR1 from the plasma membrane reduces the activation of c-Src by EGF, implying that PLSCR1 normally facilitates receptor-dependent activation of this kinase. We propose that PLSCR1, through its interaction with Shc, promotes Src kinase activation through the EGF receptor.

    Funded by: NHLBI NIH HHS: HL36946, HL61200, HL63819

    The Journal of biological chemistry 2003;278;39;37413-8

  • Lithium reduced N-methyl-D-aspartate receptor subunit 2A tyrosine phosphorylation and its interactions with Src and Fyn mediated by PSD-95 in rat hippocampus following cerebral ischemia.

    Ma J and Zhang GY

    Research Center for Biochemistry and Molecular Biology, Xuzhou Medical College, 84 West Huai-hai Road, Xuzhou, 221002 Jiangsu, PR China.

    Recently, the neuroprotective effects of lithium against excitotoxicity mediated by N-methyl-D-aspartate (NMDA) receptors have been demonstrated. Since brain ischemia results in NMDA receptor over-excitation and Src family protein tyrosine kinase-mediated tyrosine phosphorylation of NMDA receptor subunit 2A (NR2A) enhances NMDA receptor activity, we examined the effects of lithium on tyrosine phosphorylation of NR2A and its interactions with Src and Fyn (two members of the Src family of protein tyrosine kinases) mediated by PSD-95 (postsynaptic density protein 95 kDa) after 6 h of reperfusion following 15 min of ischemia (I/R), which was induced by occlusion of the four vessels in Sprague-Dawley rats. After abdominal injection of LiCl (2 mg/kg) for 7 days, the data showed that together with the significant decrease in I/R-induced tyrosine phosphorylation of NR2A, the interactions of NR2A with Src and Fyn mediated by PSD-95 were also decreased significantly. However, lithium pretreatment did not alter the total protein levels of NR2A, Src, Fyn and PSD-95. These results suggest that the inhibition of NR2A tyrosine phosphorylation and its interactions with Src and Fyn mediated by PSD-95 may contribute to the lithium-induced downregulation of NMDA receptor function and provide neuroprotection against excitotoxicity.

    Neuroscience letters 2003;348;3;185-9

  • GC-GAP, a Rho family GTPase-activating protein that interacts with signaling adapters Gab1 and Gab2.

    Zhao C, Ma H, Bossy-Wetzel E, Lipton SA, Zhang Z and Feng GS

    Burnham Institute, La Jolla, California 92037, USA.

    Gab1 and Gab2 are scaffolding proteins acting downstream of cell surface receptors and interact with a variety of cytoplasmic signaling proteins such as Grb2, Shp-2, phosphatidylinositol 3-kinase, Shc, and Crk. To identify new binding partners for GAB proteins and better understand their functions, we performed a yeast two-hybrid screening with hGab2-(120-587) as bait. This work led to identification of a novel GTPase-activating protein (GAP) for Rho family GTPases. The GAP domain shows high similarity to the recently cloned CdGAP and displays activity toward RhoA, Rac1, and Cdc42 in vitro. The protein was named GC-GAP for its ability to interact with GAB proteins and its activity toward Rac and Cdc42. GC-GAP is predominantly expressed in the brain with low levels detected in other tissues. Antibodies directed against GC-GAP recognized a protein of approximately 200 kDa. Expression of GC-GAP in 293T cells led to a reduction in active Rac1 and Cdc42 levels but not RhoA. Suppression of GC-GAP expression by siRNA inhibited proliferation of C6 astroglioma cells. In addition, GC-GAP contains several classic proline-rich motifs, and it interacts with the first SH3 domain of Crk and full-length Nck in vitro. We propose that Gab1 and Gab2 in cooperation with other adapter molecules might regulate the cellular localization of GC-GAP under specific stimuli, acting to regulate precisely Rac and Cdc42 activities. Given that GC-GAP is specifically expressed in the nervous system and that it is localized to the dendritic processes of cultured neurons, GC-GAP may play a role in dendritic morphogenesis and also possibly in neural/glial cell proliferation.

    Funded by: NHLBI NIH HHS: R01HL66208; NIGMS NIH HHS: R01GM53660

    The Journal of biological chemistry 2003;278;36;34641-53

  • Rac1 function is required for Src-induced transformation. Evidence of a role for Tiam1 and Vav2 in Rac activation by Src.

    Servitja JM, Marinissen MJ, Sodhi A, Bustelo XR and Gutkind JS

    Oral and Pharyngeal Cancer Branch, National Institutes of Dental and Craniofacial Research, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892, USA.

    The proto-oncogene c-Src has been implicated in the development and progression of a number of human cancers including those of colon and breast. Accumulating evidence indicates that activated alleles of Src may induce cell transformation through Ras-ERK-dependent and -independent pathways. Here we show that Rac1 activity is strongly elevated in Src-transformed cells and that this small G protein is a critical component of the pathway connecting oncogenic Src with cell transformation. We further show that Vav2 and the ubiquitously expressed Rac1 guanine nucleotide exchange factor Tiam1 are phosphorylated in tyrosine residues in cells transfected with active and oncogenic Src. Moreover, phosphorylation of Tiam1 in cells treated with pervanadate, a potent inhibitor of tyrosine phosphatases, was partially inhibited by the Src inhibitor SU6656. Using truncated mutants of Tiam1, we demonstrate that multiple sites can be tyrosine-phosphorylated by Src. Furthermore, Tiam1 cooperated with Src to induce activation of Rac1 in vivo and the formation of membrane ruffles. Similarly, activation of JNK and the c-jun promoter by Src were also potently increased by Tiam1. Together, these results suggest that Vav2 and Tiam1 may act as downstream effectors of Src, thereby regulating Rac1-dependent pathways that participate in Src-induced cell transformation.

    The Journal of biological chemistry 2003;278;36;34339-46

  • Src kinase regulates the activation of a novel FGD-1-related Cdc42 guanine nucleotide exchange factor in the signaling pathway from the endothelin A receptor to JNK.

    Miyamoto Y, Yamauchi J and Itoh H

    Department of Cell Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan.

    Small GTPases act as binary switches by cycling between an inactive (GDP-bound) and an active (GTP-bound) state. Upon stimulation with extracellular signals, guanine-nucleotide exchange factors (GEFs) stimulate the exchange of GDP to GTP to shift toward the active forms of small GTPases, recognizing the downstream targets. Here we show that KIAA0793, containing substantial sequence homology with the catalytic Dbl homology domain of the faciogenital dysplasia gene product (FGD1), is a specific GEF for Cdc42. We, therefore, tentatively named it FRG (FGD1-related Cdc42-GEF). Src kinase directly phosphorylates and activates FRG, as Vav family GEFs. Additionally, FRG is involved in the signaling pathway from the endothelin A receptor to c-Jun N-terminal kinase, resulting in the inhibition of cell motility. These results suggest that FRG is a member of Cdc42-GEF and plays an important role in the signaling pathway downstream of G protein-coupled receptors.

    The Journal of biological chemistry 2003;278;32;29890-900

  • Identification of Tyr900 in the kinase domain of c-Kit as a Src-dependent phosphorylation site mediating interaction with c-Crk.

    Lennartsson J, Wernstedt C, Engström U, Hellman U and Rönnstrand L

    Ludwig Institute for Cancer Research, Biomedical Centre, P.O. Box 595, SE-751 24 Uppsala, Sweden.

    We have previously demonstrated that ligand-stimulation of c-Kit induces phosphorylation of Tyr568 and Tyr570 in the juxtamembrane region of the receptor, leading to recruitment, phosphorylation and activation of members of the Src family of tyrosine kinases. In this paper, we demonstrate that members of the Src family of tyrosine kinases are able to phosphorylate c-Kit selectively on one particular tyrosine residue, Tyr900, located in the second part of the tyrosine kinase domain. In order to identify potential docking partners of Tyr900, a synthetic phosphopeptide corresponding to the amino acid sequence surrounding Tyr900 was used as an affinity matrix. By use of MALDI-TOF mass spectrometry, CrkII was identified as a protein that specifically bound to Tyr900 in a phosphorylation dependent manner, possibly via the p85 subunit of PI3-kinase. Expression of a mutant receptor where Tyr900 had been replaced with a phenylalanine residue (Y900F) resulted in a receptor with reduced ability to phosphorylate CrkII. Together these data support a model where c-Src phosphorylates the receptor, thereby creating docking sites for SH2 domain containing proteins, leading to recruitment of Crk to the receptor.

    Experimental cell research 2003;288;1;110-8

  • Clustering-induced tyrosine phosphorylation of nephrin by Src family kinases.

    Lahdenperä J, Kilpeläinen P, Liu XL, Pikkarainen T, Reponen P, Ruotsalainen V and Tryggvason K

    Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.

    Background: Nephrin is a recently discovered protein of the immunoglobulin (Ig) superfamily. In the kidney, it is located at the slit diaphragm, which forms the decisive size-selective filter of glomerular ultrafiltration barrier and locates between the interdigitating foot processes of podocytes. Nephrin is mutated in congenital nephrosis of the Finnish type (NPHS1) and has been demonstrated to be an essential component of the slit diaphragm. Based on its domain structure, nephrin is likely to be a cell-cell or cell-matrix adhesion protein that may have a signaling function. In this study, we hypothesized that the clustering of nephrin with antibodies on cell surface mimics the situation where the interaction between nephrin and its extracellular ligand(s) is altered.

    Methods: Nephrin was clustered on the surface of stably transfected HEK293 cells by a monoclonal antinephrin antibody and polyclonal secondary antibody. Clusters were visualized by immunofluorescence microscopy. Changes in protein phosphorylation were studied employing immunoprecipitations and Western blot analysis. A specific inhibitor and cotransfection experiments were used to investigate role of Src family kinases in nephrin phosphorylation.

    Results: Clustering of nephrin induced its own tyrosine phosphorylation. This phosphorylation was inhibited by PP2, an inhibitor of Src family kinases. Several members of Src family kinases were able to induce nephrin phosphorylation when cotransfected to HEK293 cells with nephrin. Moreover, the Src family kinase Fyn was consistently found to be coimmunoprecipitated with nephrin. Interestingly, clustering of nephrin induced also tyrosine phosphorylation of a 46 kD protein that was as well found to be coimmunoprecipitated with nephrin.

    Conclusion: Nephrin is a signaling protein phosphorylated by Src family kinases.

    Funded by: NIDDK NIH HHS: DK54724

    Kidney international 2003;64;2;404-13

  • Inhibition of tyrosine kinase Src suppresses pancreatic cancer invasiveness.

    Ito H, Gardner-Thorpe J, Zinner MJ, Ashley SW and Whang EE

    Department of Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.

    Background: Src is a 60-kDa tyrosine kinase that plays a critical role in signal transduction associated with cell-extracellular matrix interactions. We tested the hypothesis that Src inhibition might suppress pancreatic cancer cellular invasiveness.

    Methods: We tested the effects of pyrazolopyrimidine (a Src kinase-specific inhibitor) on 3 human pancreatic cancer cell lines: BXPC-3, MIAPaCa-2, and PANC-1. Src expression was assayed with Western blotting. Pyrazolopyrimidine-mediated inhibition of Src phosphorylation was confirmed by immunoprecipitation. Matrix metalloproteinase (MMP) activities and cellular invasive potential were assessed by use of zymography and Boyden chamber assays, respectively. Cell growth was assessed with the MTT assay.

    Results: Src was expressed in all 3 pancreatic cancer cell lines tested. Pyrazolopyrimidine completely suppressed Src phosphorylation, inhibited MMP2 (72kDa) and MMP9 (92kDa) activities by 40% to 34% (P <.05), and suppressed cellular invasiveness by more than 90% (P <.05) in all 3 cell lines. Pyrazolopyrimidine had variable effects on cell growth: 50% reduction (P <.05) in BXPC-3, 7% reduction (P >.05) in MIAPaCa-2, and 22% reduction (P <.05) in PANC-1.

    Conclusions: Inhibition of Src signaling results in a marked reduction of pancreatic cancer cellular invasiveness. Src may represent a novel therapeutic target for this deadly cancer.

    Surgery 2003;134;2;221-6

  • Simultaneous inhibition of focal adhesion kinase and SRC enhances detachment and apoptosis in colon cancer cell lines.

    Golubovskaya VM, Gross S, Kaur AS, Wilson RI, Xu LH, Yang XH and Cance WG

    Department of Surgery, University of Florida, Gainesville, FL 32610-0286, USA.

    Focal adhesion kinase (FAK) and Src have been shown to be overexpressed in colon cancer. We have studied the role of these two kinases in resistance to apoptosis. Adenovirus-containing FAK-CD (Ad-FAK-CD), a dominant-negative, COOH-terminal portion of FAK, was used to inhibit FAK and cause apoptosis. Colon cancer cell lines were more resistant to Ad-FAK-CD-induced detachment and apoptosis than the breast cancer cell line, BT474. Colon cancer cell lines overexpressed highly active Src and FAK. Ad-FAK-CD-induced apoptosis was significantly increased by PP2, an inhibitor of Src family kinases. Activation of caspase-3, down-regulation of FAK, and Src and AKT activities were demonstrated in Ad-FAK-CD + PP2-treated colon cancer cells undergoing apoptosis. The results suggest that FAK and Src are both important survival factors, playing a role in protecting colon cancer cell lines from Ad-FAK-CD-induced apoptosis. Dual inhibition of these kinases may be important for therapies designed to enhance the apoptosis in colon cancers.

    Molecular cancer research : MCR 2003;1;10;755-64

  • Mutation of Leu-536 in human estrogen receptor-alpha alters the coupling between ligand binding, transcription activation, and receptor conformation.

    Zhao C, Koide A, Abrams J, Deighton-Collins S, Martinez A, Schwartz JA, Koide S and Skafar DF

    Department of Physiology, Wayne State University School of Medicine, Barbara Ann Karmanos Cancer Institute, Detroit, Michigan 48201, USA.

    The estrogen receptor (ER), of which there are two forms, ERalpha and ERbeta, is a ligand-modulated transcription factor important in both normal biology and as a target for agents to prevent and treat breast cancer. Crystallographic studies of the ERalpha ligand-binding domain suggest that Leu-536 may be involved in hydrophobic interactions at the start of a helix, "helix 12," that is crucial in the agonist-stimulated activity of ERalpha, as well as in the ability of antagonists to block the activity of ERalpha. We found that certain mutations of Leu-536 increased the ligand-independent activity of ERalpha although greatly reducing or eliminating the agonist activity of 17beta-estradiol (E2) and 4-hydroxytamoxifen (4OHT), on an estrogen response element-driven and an AP-1-driven reporter. The mutations impaired the interaction of the ER ligand-binding domain with the SRC1 receptor-interacting domain in a mammalian two-hybrid system. When tested in the yeast two-hybrid system, mutation of Leu-536 increased the basal reactivity of ERalpha to probes that recognize the agonist-bound conformation but did not significantly alter its reactivity to these probes in the presence of E2. Most interestingly, mutation of Leu-536 reduced the interaction of the 4OHT-bound ERalpha and increased the reactivity of the raloxifene- or ICI 182,780-bound ERalpha, with probes that recognize the 4OHT-bound ERalpha conformation in a yeast two-hybrid system. These results show that Leu-536 is critical in coupling the binding of ligand to the modulation of the conformation and activity of ERalpha.

    Funded by: NCI NIH HHS: P30-CA14599; NIDDK NIH HHS: 1R01-DK56934-01A1, R01-DK62316; NIGMS NIH HHS: R29-GM55042

    The Journal of biological chemistry 2003;278;29;27278-86

  • Two new substrates in insulin signaling, IRS5/DOK4 and IRS6/DOK5.

    Cai D, Dhe-Paganon S, Melendez PA, Lee J and Shoelson SE

    Joslin Diabetes Center and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215, USA.

    We have identified two new human genes that encode proteins with tandem pleckstrin homology-phosphotyrosine binding (PH-PTB) domains at their amino termini. Because the other known PH-PTB proteins (insulin receptor substrates: IRS-1, IRS-2, IRS-3, and IRS-4, and the downstream of kinases: DOK-1, DOK-2, and DOK-3) are substrates of insulin and insulin-like growth factor (IGF)-1 receptors, we asked whether these new proteins, termed IRS5/DOK4 and IRS6/DOK5, might also have roles in insulin and IGF-1 signaling. Northern analyses indicate that IRS5/DOK4 is ubiquitously expressed but most abundant in kidney and liver. IRS6/DOK5 expression is highest in skeletal muscle. Both proteins are tyrosine-phosphorylated in response to insulin and IGF-1 in transfected cells, although the kinetics differ. Insulin receptor-phosphorylated IRS5/DOK4 associates with RasGAP, Crk, Src, and Fyn, but not phosphatidylinositol 3-kinase p85, Grb2, SHP-2, Nck, or phospholipase Cgamma Src homology 2 domains, and activates MAPK in cells. IRS6/DOK5 neither associates with these Src homology 2 domains nor activates MAPK. IRS5/DOK4 and IRS6/DOK5 represent two new signaling proteins with potential roles in insulin and IGF-1 action.

    Funded by: NIDDK NIH HHS: DK36836, F32 DK61187, R01 DK43123

    The Journal of biological chemistry 2003;278;28;25323-30

  • Interaction of guanylyl cyclase C with SH3 domain of Src tyrosine kinase. Yet another mechanism for desensitization.

    Singh R

    Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, India. ghrika_s@yahoo.com

    Protein-protein interactions mediated by the Src homology 3 (SH3) domain have been implicated in the regulation of receptor functions for subcellular localization of proteins and the reorganization of cytoskeleton. The experiments described in this article begin to identify the interaction of the SH3 domain of Src tyrosine kinase with the guanylyl cyclase C receptor after activation with Escherichia coli heat-stable enterotoxin (ST). Only one of two post-translationally modified forms of guanylyl cyclase C from T84 colonic carcinoma cells bind to GST-SH3 fusion protein of Src and Hck tyrosine kinases. Interestingly, the GST-Src-SH3 fusion protein showed 2-fold more affinity to native guanylyl cyclase C in solution than the GST-Hck-SH3 fusion protein. The affinity of the GST-Src-SH3 fusion protein to guanylyl cyclase C increased on desensitization of receptor in vivo. An in vitro cyclase assay in the presence of GST-Src-SH3 fusion protein indicated inhibition of the catalytic activity of guanylyl cyclase C. The catalytic domain recombinant protein (GST-GCD) of guanylyl cyclase C could pull-down a 60-kDa protein that reacted with Src tyrosine antibody and also showed autophosphorylation. These data suggest that SH3 domain-mediated protein-protein interaction with the catalytic domain of guanylyl cyclase C inhibited the cyclase activity and that such an interaction, possibly mediated by Src tyrosine kinase or additional proteins, might be pivotal for the desensitization phenomenon of the guanylyl cyclase C receptor.

    The Journal of biological chemistry 2003;278;27;24342-9

  • Activation and inhibition of cellular calcium and tyrosine kinase signaling pathways identify targets of the HBx protein involved in hepatitis B virus replication.

    Bouchard MJ, Puro RJ, Wang L and Schneider RJ

    Department of Microbiology, New York University School of Medicine, New York, NY 10016, USA.

    Human hepatitis B virus (HBV) HBx protein is a multifunctional protein that activates cellular signaling pathways and is thought to be essential for viral infection. Woodchuck HBV mutants that lack HBx are unable to replicate in vivo or are severely impaired. HBV replication in HepG2 cells, a human hepatoblastoma cell line, is stimulated 5- to 10-fold by HBx protein. We have utilized the HepG2, HBx-dependent HBV replication system to study the effects of activators and inhibitors of cytosolic calcium and tyrosine kinase signaling pathways on viral replication. By transfecting either a wild-type HBV genome or an HBV genome that does not express HBx and then treating transfected cells with activators or inhibitors of signaling pathways, we identified compounds that either impair wild-type HBV replication or rescue HBx-deficient HBV replication. Geldanamycin or herbimycin A, tyrosine kinase inhibitors, blocked HBV replication. Derivatives of cyclosporine, i.e., cyclosporine A, cyclosporine H, and SDZ NIM811, which block cytosolic calcium signaling and specifically the mitochondrial permeability transition pore (SDZ NIM811), also impaired HBV replication. Treatment of cells with compounds that increase cytosolic calcium levels by a variety of mechanisms rescued replication of an HBx-deficient HBV mutant. Transcription of viral RNA and production of viral capsids were only minimally affected by these treatments. These results define a functional signaling circuit for HBV replication that includes calcium signaling and activation of cytosolic signaling pathways involving Src kinases, and they suggest that these pathways are stimulated by HBx acting on the mitochondrial transition pore.

    Funded by: NCI NIH HHS: CA 56533, F32 CA 4476; NIAID NIH HHS: T32 AI 07180

    Journal of virology 2003;77;14;7713-9

  • Hydrogen peroxide triggers nuclear export of telomerase reverse transcriptase via Src kinase family-dependent phosphorylation of tyrosine 707.

    Haendeler J, Hoffmann J, Brandes RP, Zeiher AM and Dimmeler S

    Molecular Cardiology, Department of Internal Medicine IV. Department of Physiology, University of Frankfurt, Frankfurt, Germany.

    The regulation of telomerase reverse transcriptase (TERT) plays an important role in the proliferative capacity and survival of cells. Here, we report that exogenously as well as endogenously induced oxidative stress leads to translocation of endogenous as well as overexpressed human TERT from the nucleus into the cytosol. TERT is transported through the nuclear pores in a leptomycin-sensitive and Ran GTPase-dependent process. H(2)O(2)-induced nuclear export of TERT is preceded by TERT tyrosine phosphorylation at position 707 and prevented by the Src kinase family inhibitor PP1. Oxidative stress-induced nuclear export of TERT depends on association with the Ran GTPase. In contrast, mutation of tyrosine 707 inhibits phosphorylation induced by oxidative stress and prevents association with Ran and nuclear export of TERT. Moreover, inhibition of tyrosine phosphorylation at 707 increases the antiapoptotic capacity of TERT. Taken together, depletion of nuclear TERT by tyrosine phosphorylation-dependent nuclear export of TERT is a novel mechanism for regulation of TERT localization, which reduces the antiapoptotic activity of TERT.

    Molecular and cellular biology 2003;23;13;4598-610

  • H-Ras modulates N-methyl-D-aspartate receptor function via inhibition of Src tyrosine kinase activity.

    Thornton C, Yaka R, Dinh S and Ron D

    Ernest Gallo Clinic and Research Center, University of California San Francisco, San Francisco, California 94110-3518, USA.

    Tyrosine phosphorylation of the NR2A and NR2B subunits of the N-methyl-d-aspartate (NMDA) receptor by Src protein-tyrosine kinases modulates receptor channel activity and is necessary for the induction of long term potentiation (LTP). Deletion of H-Ras increases both NR2 tyrosine phosphorylation and NMDA receptor-mediated hippocampal LTP. Here we investigated whether H-Ras regulates phosphorylation and function of the NMDA receptor via Src family protein-tyrosine kinases. We identified Src as a novel H-Ras binding partner. H-Ras bound to Src but not Fyn both in vitro and in brain via the Src kinase domain. Cotransfection of H-Ras and Src inhibited Src activity and decreased NR2A tyrosine phosphorylation. Treatment of rat brain slices with Tat-H-Ras depleted NR2A from the synaptic membrane, decreased endogenous Src activity and NR2A phosphorylation, and decreased the magnitude of hippocampal LTP. No change was observed for NR2B. We suggest that H-Ras negatively regulates Src phosphorylation of NR2A and retention of NR2A into the synaptic membrane leading to inhibition of NMDA receptor function. This mechanism is specific for Src and NR2A and has implications for studies in which regulation of NMDA receptor-mediated LTP is important, such as synaptic plasticity, learning, and memory and addiction.

    Funded by: NIAAA NIH HHS: R01 AA013438, R01AA/MH13438-01A1

    The Journal of biological chemistry 2003;278;26;23823-9

  • Localization of phospho-beta-dystroglycan (pY892) to an intracellular vesicular compartment in cultured cells and skeletal muscle fibers in vivo.

    Sotgia F, Bonuccelli G, Bedford M, Brancaccio A, Mayer U, Wilson MT, Campos-Gonzalez R, Brooks JW, Sudol M and Lisanti MP

    Department of Molecular Pharmacology and The Albert Einstein Cancer Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.

    beta-Dystroglycan is a ubiquitously expressed integral membrane protein that undergoes tyrosine phosphorylation in an adhesion-dependent manner. Tyrosine 892 is now thought to be the principal site for recognition by the c-Src tyrosine kinase; however, little is known about the regulation of this phosphorylation event in vivo. Here, we generated a novel monoclonal antibody probe that recognizes only tyrosine 892 phosphorylated beta-dystroglycan (pY892). We show that upon tyrosine phosphorylation, beta-dystroglycan undergoes a profound change in its sub-cellular localization (e.g., from the plasma membrane to an internal membrane compartment). One possibility is that the net negative charge at position 892 causes the redistribution of beta-dystroglycan to this intracellular vesicular location. In support of this notion, mutation of tyrosine 892 to glutamate (Y892E) is sufficient to drive this intracellular localization, while other point mutants (Y892F and Y892A) remain at the plasma membrane. Interestingly, our colocalization studies with endosomal markers (EEA1, transferrin, and transferrin receptor) suggest that these phospho-beta-dystroglycan containing internal vesicles represent a subset of recycling endosomes. At the level of these internal vesicular structures, we find that tyrosine phosphorylated beta-dystroglycan is colocalized with c-Src. In addition, we demonstrate that known ligands for alpha-dystroglycan, namely, agrin and laminin, are able to induce the tyrosine phosphorylation of beta-dystroglycan. Finally, we show that tyrosine phosphorylated beta-dystroglycan is also detectable in skeletal muscle tissue lysates and is localized to an internal vesicular membrane compartment in skeletal muscle fibers in vivo. The generation of a phospho-specific beta-dystroglycan (pY892) mAb probe provides a new powerful tool for dissecting the role of dystroglycan phosphorylation in normal cellular functioning and in the pathogenesis of muscular dystrophies.

    Biochemistry 2003;42;23;7110-23

  • Involvement of G(i) proteins and Src tyrosine kinase in TNFalpha production induced by lipopolysaccharide, group B Streptococci and Staphylococcus aureus.

    Fan H, Teti G, Ashton S, Guyton K, Tempel GE, Halushka PV and Cook JA

    Department of Physiology and Neuroscience, Medical University of South Carolina, 173 Ashley Avenue, BSB Room 403, Charleston, SC 29425, USA.

    Previous studies have suggested that heterotrimeric G(i) proteins, Src tyrosine kinase and phosphatidylinositol-3 kinase (PI3 Kinase) are involved in signaling events induced by lipopolysaccharide (LPS) leading to pro-inflammatory cytokines gene expression. To investigate the involvement of these mediators in Gram-positive bacteria induced pro-inflammatory cytokine expression, LPS (10 ng/ml), heat killed group B Streptococci (GBS 1 microg/ml) and Staphylococcus aureus (SA 10 microg/ml) were used to induce TNFalpha production in the murine J774A.1 macrophage (MØ) cell line and human promonocytic THP-1 cell line. Pertussis toxin (PTx, 1 microg/ml), an inhibitor of G(i) protein; pyrazolopyrimidine-2 (PP2, 1 or 25 microM), a Src tyrosine kinase inhibitor; and LY294002 (100 nM), an inhibitor of PI3 Kinase were used to examine the involvement of G(i), Src tyrosine kinase and PI3 Kinase, respectively, in TNFalpha production. In J774A.1 cells, pretreatment with PTx and PP2 attenuated TNFalpha production induced by LPS (60+/-9% and 81+/-11% inhibition, n=3, p<0.05, respectively), GBS (95+/-1% and 80+/-6% inhibition, n=3, p<0.05, respectively) and SA (51+/-18% and 68+/-16% inhibition, n=4, p<0.05, respectively). However, pretreatment with LY 294002 inhibited LPS induced TNFalpha production (82+/-13% inhibition, n=3, p<0.05), but did not inhibit GBS or SA induced TNFalpha production. In THP-1 cells, pretreatment with PTx, PP2 and LY 294002 inhibited TNFalpha production induced by LPS (84+/-3%, 59+/-12% and 84+/-4% inhibition, n=3, p<0.05, respectively) and SA (56+/-7%, 87+/-1% and 35+/-6% inhibition, n=3, p<0.05, respectively). These data support our hypothesis that G(i)-coupled and Src tyrosine kinase-coupled signaling pathways are involved in both Gram-negative and Gram-positive bacteria induced pro-inflammatory cytokine expression. However, unlike LPS, involvement of PI3 Kinase in Gram-positive bacteria induced signaling pathways are species dependent.

    Funded by: NIGMS NIH HHS: GM27673

    Cytokine 2003;22;5;126-33

  • v-Src induces Shc binding to tyrosine 63 in the cytoplasmic domain of the LDL receptor-related protein 1.

    Barnes H, Ackermann EJ and van der Geer P

    Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0601, USA.

    We recently observed that the LDL receptor-related protein 1 (LRP-1) is tyrosine phosphorylated in v-Src-transformed cells. Using a GST-fusion protein containing the cytoplasmic domain of LRP-1, we show that LRP-1 is a direct substrate for v-Src in vitro. To study LRP-1 phosphorylation in vivo, we constructed an LRP-1 minireceptor composed of the beta chain linked at the amino-terminus to a Myc epitope (Myc-LRPbeta). When expressed together with v-Src, Myc-LRPbeta becomes phosphorylated on tyrosine. Of the four tyrosine residues present in the cytoplasmic domain of LRP-1, only Tyr 63 is phosphorylated by v-Src in vivo or in vitro. Using fibroblasts deficient in Src, Yes and Fyn, we were able to show that there are multiple kinases present in the cell that can phosphorylate LRP-1. Tyrosine-phosphorylated LRP-1 associates with Shc, a PTB and SH2 domain containing signaling protein that is involved in the activation of Ras. Binding of the purified Shc PTB domain to Tyr 63 containing peptides shows that the interaction between LRP-1 and Shc is direct. We found that DAB, a PTB domain containing signaling protein that is involved in signaling by LDL receptor-related proteins in the nervous system, did not bind to full-length LRP-1. Our observations suggest that LRP-1 may be involved in normal and malignant signal transduction through a direct interaction with Shc adaptor proteins.

    Funded by: NCI NIH HHS: R29 CA78629; NIDDK NIH HHS: DK 07233

    Oncogene 2003;22;23;3589-97

  • c-Src induces phosphorylation and translocation of p47phox: role in superoxide generation by angiotensin II in human vascular smooth muscle cells.

    Touyz RM, Yao G and Schiffrin EL

    Multidisciplinary Research Group on Hypertension, Clinical Research Institute of Montreal, University of Montreal, Montreal, Canada. touyzr@ircm.qc.ca

    Objective: The aim of this study was to determine molecular mechanisms whereby c-Src regulates angiotensin II (Ang II)-mediated NAD(P)H oxidase-derived *O2- in human vascular smooth muscle cells (VSMCs).

    VSMCs from human small arteries were studied. Ang II increased NAD(P)H oxidase-mediated generation of *O2- and H2O2 (P<0.01). PP2, c-Src inhibitor, attenuated these effects by 70% to 80%. Immunoprecipitation of p47phox, followed by immunoblotting with antiphosphoserine antibody, demonstrated a rapid increase (1.5- to 2-fold) in p47phox phosphorylation in Ang II-stimulated cells. This was associated with p47phox translocation from cytosol to membrane, as assessed by immunoblotting and immunofluorescence. PP2 abrogated these effects. Long-term Ang II stimulation (6 to 24 hours) increased NAD(P)H oxidase subunit expression. c-Src inhibition decreased abundance of gp91phox, p22phox, and p47phox. Confirmation of c-Src-dependent regulation of NAD(P)H oxidase was tested in VSMCs from c-Src-/- mice. Ang II-induced *O2- generation was lower in c-Src-/- than c-Src+/+ counterparts. This was associated with decreased p47phox phosphorylation, blunted Ang II-stimulated NAD(P)H oxidase activation, and failure of Ang II to increase subunit expression.

    Conclusions: c-Src regulates NAD(P)H oxidase-derived *O2- generation acutely by stimulating p47phox phosphorylation and translocation and chronically by increasing protein content of gp91phox, p22phox, and p47phox in Ang II-stimulated cells. These novel findings identify NAD(P)H oxidase subunits, particularly p47phox, as downstream targets of c-Src.

    Arteriosclerosis, thrombosis, and vascular biology 2003;23;6;981-7

  • Heregulin and HER2 signaling selectively activates c-Src phosphorylation at tyrosine 215.

    Vadlamudi RK, Sahin AA, Adam L, Wang RA and Kumar R

    Department of Molecular and Cellular Oncology, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA. rvadlamu@mdanderson.org

    To elucidate the molecular mechanisms by which human epidermal growth factor receptor/heregulin (HER2/HRG) influence the migratory potential of breast cancer cells, we have used phospho-specific antibodies against c-Src kinase and focal adhesion kinase (FAK). This study establishes that HER2/HRG signaling selectively upregulates Tyr phosphorylation of c-Src at Tyr-215 located within the SH2 domain, increases c-Src kinase activity and selectively upregulates Tyr phosphorylation of FAK at Tyr-861. HER2-overexpressing tumors showed increased levels of c-Src phosphorylation at Tyr-215. These findings suggest that HER2/HRG influence metastasis of breast cancer cells through a novel signaling pathway involving phosphorylation of FAK tyrosine 861 via activation of c-Src tyrosine 215.

    Funded by: NCI NIH HHS: CA90970

    FEBS letters 2003;543;1-3;76-80

  • The inhibitory gamma subunit of the type 6 retinal cGMP phosphodiesterase functions to link c-Src and G-protein-coupled receptor kinase 2 in a signaling unit that regulates p42/p44 mitogen-activated protein kinase by epidermal growth factor.

    Wan KF, Sambi BS, Tate R, Waters C and Pyne NJ

    Department of Physiology and Pharmacology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G4 ONR, Scotland, United Kingdom.

    The inhibitory gamma subunit of the retinal photoreceptor type 6 cGMP phosphodiesterase (PDEgamma) is phosphorylated by G-protein-coupled receptor kinase 2 on threonine 62 and regulates the epidermal growth factor- dependent stimulation of p42/p44 mitogen-activated protein kinase in human embryonic kidney 293 cells. We report here that PDEgamma is in a pre-formed complex with c-Src and that stimulation of cells with epidermal growth factor promotes the association of GRK2 with this complex. c-Src has a critical role in the stimulation of the p42/p44 mitogen-activated protein kinase cascade by epidermal growth factor, because c-Src inhibitors block the activation of this kinase by the growth factor. Mutation of Thr-62 (to Ala) in PDEgamma produced a GRK2 phosphorylation-resistant mutant that was less effective in associating with GRK2 in response to epidermal growth factor and did not potentiate the stimulation of p42/p44 mitogen-activated protein kinase by this growth factor. The transcript for a short splice variant version of PDEgamma lacking the Thr-62 phosphorylation site is also expressed in certain mammalian cells and, in common with the Thr-62 mutant, failed to potentiate the stimulatory effect of epidermal growth factor on p42/p44 mitogen-activated protein kinase. The mutation of Thr-22 (to Ala) in PDEgamma, which is a site for phosphorylation by p42/p44 mitogen-activated protein kinase, resulted in a prolonged activation of p42/p44 mitogen-activated protein kinase by epidermal growth factor, suggesting a role for this phosphorylation event in the negative feedback control of PDEgamma.

    The Journal of biological chemistry 2003;278;20;18658-63

  • Tyrosine phosphorylation of protein kinase D in the pleckstrin homology domain leads to activation.

    Storz P, Döppler H, Johannes FJ and Toker A

    Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.

    Protein kinase D (PKD) is a member of the AGC family of Ser/Thr kinases and is distantly related to protein kinase C (PKC). Formerly known as PKCmu, PKD contains protein domains not found in conventional PKC isoforms. A functional pleckstrin homology (PH) domain is critical for the regulation of PKD activity. Here we report that PKD is tyrosine-phosphorylated within the PH domain, leading to activation. This phosphorylation is mediated by a pathway that consists of the Src and Abl tyrosine kinases and occurs in response to stimulation with pervanadate and oxidative stress. Mutational analysis revealed three tyrosine phosphorylation sites (Tyr(432), Tyr(463), and Tyr(502)), which are regulated by the Src-Abl pathway, and phosphorylation of only one of these (Tyr(463)) leads to PKD activation. By using a phospho-specific antibody, we show that Abl directly phosphorylates PKD at Tyr(463) in vitro, and in cells phosphorylation of this site is sufficient to mediate full activation of PKD. Mutation of the other two sites, Tyr(432) and Tyr(502), had no significant influence on PKD activity. These data reveal a tyrosine phosphorylation-dependent activation mechanism for PKD and suggest that this event contributes to the release of the autoinhibitory PKD PH domain leading to kinase activation and downstream responses.

    Funded by: NCI NIH HHS: CA 75134

    The Journal of biological chemistry 2003;278;20;17969-76

  • Hypertonicity activates Na+/H+ exchange through Janus kinase 2 and calmodulin.

    Garnovskaya MN, Mukhin YV, Vlasova TM and Raymond JR

    Medical and Research Services, Ralph H. Johnson Veterans Affairs Medical Center, Charleston, South Carolina 29425, USA. garnovsk@musc.edu

    The type 1 sodium-hydrogen exchanger (NHE-1) is a ubiquitous electroneutral membrane transporter that is activated by hypertonicity in many cells. NHE-1 may be an important pathway for Na(+) entry during volume restoration, yet the molecular mechanisms underlying the osmotic regulation of NHE-1 are poorly understood. In the present study we conducted a screen for important signaling molecules that could be involved in hypertonicity-induced activation of NHE-1 in CHO-K1 cells. Hypertonicity rapidly activated NHE-1 in a concentration-dependent manner as assessed by proton microphysiometry and by measurements of intracellular pH on a FLIPR (fluorometric imaging plate reader). Inhibitors of Ca(2+)/calmodulin (CaM) and Janus kinase 2 (Jak2) attenuated this activation, whereas neither calcium chelation nor inhibitors of protein kinase C, the Ras-ERK1/2 pathway, Src kinase, and Ca(2+)/calmodulin-dependent enzymes had significant effects. Hypertonicity also resulted in the rapid tyrosine phosphorylation of Jak2 and STAT3 (the major substrate of Jak2) and CaM. Phosphorylation of Jak2 and CaM were blocked by AG490, an inhibitor of Jak2. Immunoprecipitation studies showed that hypertonicity stimulates the assembly of a signaling complex that includes CaM, Jak2, and NHE-1. Formation of the complex could be blocked by AG490. Thus, we propose that hypertonicity induces activation of NHE-1 in CHO-K1 cells in large part through the following pathway: hypertonicity --> Jak2 phosphorylation and activation --> tyrosine phosphorylation of CaM --> association of CaM with NHE-1 --> NHE-1 activation.

    Funded by: NIDDK NIH HHS: DK52448, KO1-DK02694

    The Journal of biological chemistry 2003;278;19;16908-15

  • Phosphorylation-dependent regulation of Kv2.1 Channel activity at tyrosine 124 by Src and by protein-tyrosine phosphatase epsilon.

    Tiran Z, Peretz A, Attali B and Elson A

    Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot 76100, Israel.

    Voltage-gated potassium (Kv) channels are a complex and heterogeneous family of proteins that play major roles in brain and cardiac excitability. Although Kv channels are activated by changes in cell membrane potential, tyrosine phosphorylation of channel subunits can modulate the extent of channel activation by depolarization. We have previously shown that dephosphorylation of Kv2.1 by the nonreceptor-type tyrosine phosphatase PTPepsilon (cyt-PTPepsilon) down-regulates channel activity and counters its phosphorylation and up-regulation by Src or Fyn. In the present study, we identify tyrosine 124 within the T1 cytosolic domain of Kv2.1 as a target site for the activities of Src and cyt-PTPepsilon. Tyr(124) is phosphorylated by Src in vitro; in whole cells, Y124F Kv2.1 is significantly less phosphorylated by Src and loses most of its ability to bind the D245A substrate-trapping mutant of cyt-PTPepsilon. Phosphorylation of Tyr(124) is critical for Src-mediated up-regulation of Kv2.1 channel activity, since Y124F Kv2.1-mediated K(+) currents are only marginally up-regulated by Src, in contrast with a 3-fold up-regulation of wild-type Kv2.1 channels by the kinase. Other properties of Kv2.1, such as expression levels, subcellular localization, and voltage dependence of channel activation, are unchanged in Y124F Kv2.1, indicating that the effects of the Y124F mutation are specific. Together, these results indicate that Tyr(124) is a significant site at which the mutually antagonistic activities of Src and cyt-PTPepsilon affect Kv2.1 phosphorylation and activity.

    The Journal of biological chemistry 2003;278;19;17509-14

  • Signal transduction pathways regulated by prolactin and Src result in different conformations of activated Stat5b.

    Kabotyanski EB and Rosen JM

    Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030-3498, USA.

    Stat5 is activated by a broad spectrum of cytokines, as well as non-receptor tyrosine kinases, such as Src. In this study, the DNA binding properties of the two closely related Stat5 proteins, Stat5a and Stat5b, induced either by prolactin (Prl) or by Src were analyzed by electrophoretic mobility shift assays using several different Stat5 binding sites. Src-induced Stat5b-DNA binding complexes consistently displayed a slightly faster mobility than those induced by Prl, as well as differences in their ability to be supershifted by anti-Stat5 antibodies. IP-Westerns performed using specific antibodies directed at the N and C termini of Stat5b suggested that depending on the activating stimulus, Stat5b exhibited different conformations, which influenced antibody accessibility at its C terminus. These conformational differences may in part be due to differential effects of Prl and Src on Stat5b tyrosine phosphorylation, since Src induced several additional sites of tyrosine phosphorylation of Stat5b at residues other than Tyr-699, including Tyr-724 and Tyr-679. The latter Tyr-679 is conserved in all mammalian Stat5bs, but is not present in Stat5a. A Stat 5bY679F mutant induced by Src kinase exhibited an altered pattern of nuclear localization as compared with wild-type Stat5b. Furthermore, this mutation inhibited v-Src-induced cyclin D1-luciferase reporter activity in transient transfection assays performed in Stat5a/b-deficient MEFs, suggesting that Tyr-679 phosphorylation may play a role in v-Src induced proliferation. Thus, depending on the signal transduction pathway responsible for activation, different conformations of activated Stat5 may result in selective biological responses.

    Funded by: NCI NIH HHS: CA16303

    The Journal of biological chemistry 2003;278;19;17218-27

  • Src-mediated RGS16 tyrosine phosphorylation promotes RGS16 stability.

    Derrien A, Zheng B, Osterhout JL, Ma YC, Milligan G, Farquhar MG and Druey KM

    Laboratory of Allergic Diseases, NIAID/National Institutes of Health, 12441 Parklawn Drive, Rockville, MD 20852, USA.

    The amplitude of signaling evoked by stimulation of G protein-coupled receptors may be controlled in part by the GTPase accelerating activity of the regulator of G protein signaling (RGS) proteins. In turn, subcellular targeting, protein-protein interactions, or post-translational modifications such as phosphorylation may shape RGS activity and specificity. We found previously that RGS16 undergoes tyrosine phosphorylation on conserved tyrosine residues in the RGS box. Phosphorylation on Tyr(168) was mediated by the epidermal growth factor receptor (EGFR). We show here that endogenous RGS16 is phosphorylated after epidermal growth factor stimulation of MCF-7 cells. In addition, p60-Src or Lyn kinase phosphorylated recombinant RGS16 in vitro, and RGS16 underwent phosphorylation in the presence of constitutively active Src (Y529F) in EGFR(-) CHO-K1 cells. Blockade of endogenous Src activity by selective inhibitors attenuated RGS16 phosphorylation induced by pervanadate or receptor stimulation. Furthermore, the rate of RGS16 degradation was reduced in cells expressing active Src or treated with pervanadate or a G protein-coupled receptor ligand (CXCL12). Induction of RGS16 tyrosine phosphorylation was associated with increased RGS16 protein levels and enhanced GAP activity in cell membranes. These results suggest that Src mediates RGS16 tyrosine phosphorylation, which may promote RGS16 stability.

    The Journal of biological chemistry 2003;278;18;16107-16

  • Interaction between von Willebrand factor and glycoprotein Ib activates Src kinase in human platelets: role of phosphoinositide 3-kinase.

    Wu Y, Asazuma N, Satoh K, Yatomi Y, Takafuta T, Berndt MC and Ozaki Y

    Department of Clinical and Laboratory Medicine, Faculty of Medicine, University of Yamanashi, Tamaho, Nakakoma, Yamanashi, Japan.

    The binding of von Willebrand factor (VWF) to glycoprotein (GP) Ib-IX-V stimulates transmembrane signaling events that lead to platelet adhesion and aggregation. Recent studies have implied that activation of Src family kinases is involved in GPIb-mediated platelet activation, although the related signal transduction pathway remains poorly defined. This study presents evidence for an important role of Src and GPIb association. In platelet lysates containing Complete, a broad-spectrum protease inhibitor mixture, Src and Lyn dynamically associated with GPIb on VWF-botrocetin stimulation. Cytochalasin D, which inhibits translocation of Src kinases to the cytoskeleton, further increased Src and GPIb association. Similar results were obtained with botrocetin and monomeric A1 domain, instead of intact VWF, with induction of both Src activation and association between GPIb and Src. These findings suggest that ligand binding of GPIb, without receptor clustering, is sufficient to activate Src. Immunoprecipitation studies demonstrated that Src, phosphoinositide 3- kinase (PI 3-kinase), and GPIb form a complex in GPIb-stimulated platelets. When the p85 subunit of PI 3-kinase was immunodepleted, association of Src with GPIb was abrogated. However, wortmannin, a specific PI 3-kinase inhibitor, failed to block complex formation between Src and GPIb. The Src-SH3 domain as a glutathione S-transferase (GST)-fusion protein coprecipitated the p85 subunit of PI 3-kinase and GPIb. These findings taken together suggest that the p85 subunit of PI 3-kinase mediates GPIb-related activation signals and activates Src independently of the enzymatic activity of PI 3- kinase.

    Blood 2003;101;9;3469-76

  • Tyrosine phosphorylation of I-kappa B kinase alpha/beta by protein kinase C-dependent c-Src activation is involved in TNF-alpha-induced cyclooxygenase-2 expression.

    Huang WC, Chen JJ, Inoue H and Chen CC

    Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan.

    The signaling pathway involved in TNF-alpha-induced cyclooxygenase-2 (COX-2) expression was further studied in human NCI-H292 epithelial cells. A protein kinase C (PKC) inhibitor (staurosporine), tyrosine kinase inhibitors (genistein and herbimycin A), or a Src kinase inhibitor (PP2) attenuated TNF-alpha- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced COX-2 promoter activity. TNF-alpha- or TPA-induced I-kappaB kinase (IKK) activation was also blocked by these inhibitors, which reversed I-kappaBalpha degradation. Activation of c-Src and Lyn kinases, two Src family members, was inhibited by the PKC, tyrosine kinase, or Src kinase inhibitors. The dominant-negative c-Src (KM) mutant inhibited induction of COX-2 promoter activity by TNF-alpha or TPA. Overexpression of the constitutively active PKCalpha (PKCalpha A/E) or wild-type c-Src plasmids induced COX-2 promoter activity, and these effects were inhibited by the dominant-negative c-Src (KM), NF-kappaB-inducing kinase (NIK) (KA), or IKKbeta (KM) mutant. The dominant-negative PKCalpha (K/R) or c-Src (KM) mutant failed to block induction of COX-2 promoter activity caused by wild-type NIK overexpression. In coimmunoprecipitation experiments, IKKalpha/beta was found to be associated with c-Src and to be phosphorylated on its tyrosine residues after TNF-alpha or TPA treatment. Two tyrosine residues, Tyr(188) and Tyr(199), near the activation loop of IKKbeta, were identified to be crucial for NF-kappaB activation. Substitution of these residues with phenylalanines attenuated COX-2 promoter activity and c-Src-dependent phosphorylation of IKKbeta induced by TNF-alpha or TPA. These data suggest that, in addition to activating NIK, TNF-alpha also activates PKC-dependent c-Src. These two pathways cross-link between c-Src and NIK and converge at IKKalpha/beta, and go on to activate NF-kappaB, via serine phosphorylation and degradation of IkappaB-alpha, and, finally, to initiate COX-2 expression.

    Journal of immunology (Baltimore, Md. : 1950) 2003;170;9;4767-75

  • Transmodulation between phospholipase D and c-Src enhances cell proliferation.

    Ahn BH, Kim SY, Kim EH, Choi KS, Kwon TK, Lee YH, Chang JS, Kim MS, Jo YH and Min DS

    Department of Physiology, College of Medicine, The Catholic University of Korea, Seoul, Korea.

    Phospholipase D (PLD) has been implicated in the signal transduction pathways initiated by several mitogenic protein tyrosine kinases. We demonstrate for the first time that most notably PLD2 and to a lesser extent the PLD1 isoform are tyrosine phosphorylated by c-Src tyrosine kinase via direct association. Moreover, epidermal growth factor induced tyrosine phosphorylation of PLD2 and its interaction with c-Src in A431 cells. Interaction between these proteins is via the pleckstrin homology domain of PLD2 and the catalytic domain of c-Src. Coexpression of PLD1 or PLD2 with c-Src synergistically enhances cellular proliferation compared with expression of either molecule. While PLD activity as a lipid-hydrolyzing enzyme is not affected by c-Src, wild-type PLDs but not catalytically inactive PLD mutants significantly increase c-Src kinase activity, up-regulating c-Src-mediated paxillin phosphorylation and extracellular signal-regulated kinase activity. These results demonstrate the critical role of PLD catalytic activity in the stimulation of Src signaling. In conclusion, we provide the first evidence that c-Src acts as a kinase of PLD and PLD acts as an activator of c-Src. This transmodulation between c-Src and PLD may contribute to the promotion of cellular proliferation via amplification of mitogenic signaling pathways.

    Molecular and cellular biology 2003;23;9;3103-15

  • Felic (CIP4b), a novel binding partner with the Src kinase Lyn and Cdc42, localizes to the phagocytic cup.

    Dombrosky-Ferlan P, Grishin A, Botelho RJ, Sampson M, Wang L, Rudert WA, Grinstein S and Corey SJ

    Department of Pediatrics, University of Pittsburgh, PA, USA.

    Through its Src homology 3 (SH3) and SH2 domains, the Src kinase Lyn interacts with a small number of phosphoproteins, such as Shc, Cbl, and Vav, which regulate cell cycle and the cytoskeleton. Using Lyn's Unique, SH3, and SH2 domains as bait in a yeast 2-hybrid screen, we isolated a novel gene product with features of a scaffolding protein. We named it Felic because it contains a domain homologous to the tyrosine kinase Fes and the cytoskeletal protein ezrin and forms a Lyn interaction with the GTPase Cdc42 (Felic). Felic was expressed in both hematopoietic and nonhematopoietic tissues. Because it represents an alternative splice product related to the Cdc42-interacting protein 4, CIP4, we also refer to Felic as CIP4b. Felic contains an SH3 recognition site RXPXXP and multiple tyrosine residues. In insulin or serum-stimulated HEK293 cells, Felic became tyrosine phosphorylated. Like CIP4, Felic associated with Cdc42 in its activated form only. Unlike CIP4, Felic does not possess a C-terminal SH3 domain. Coprecipitation studies show that Felic bound to Lyn or activated forms of Cdc42. Overexpression of Felic or CIP4 inhibited NIH 3T3 cell invasiveness in a Matrigel assay. Because Lyn and Cdc42 are involved in phagocytosis, we examined the distribution of Felic in RAW macrophages during particle ingestion. Felic was recruited more efficiently than CIP4 to the phagocytic cups. Altogether, these data suggest that CIP4/Felic constitute a novel family of cytoskeletal scaffolding proteins, integrating Src and Cdc42 pathways. The absence of an SH3 domain in Felic provides a structural basis for functional differences.

    Blood 2003;101;7;2804-9

  • HIV-1 Nef control of cell signalling molecules: multiple strategies to promote virus replication.

    Greenway AL, Holloway G, McPhee DA, Ellis P, Cornall A and Lidman M

    Macfarlane Burnet Institute for Medical Research and Public Health, Cnr Commercial and Punt Roads, Melbourne, Victoria 3004, Australia. greenway@burnet.edu.au

    HIV-1 has at its disposal numerous proteins encoded by its genome which provide the required arsenal to establish and maintain infection in its host for a considerable number of years. One of the most important and enigmatic of these proteins is Nef. The Nef protein of HIV-1 plays a fundamental role in the virus life cycle. This small protein of approximately 27 kDa is required for maximal virus replication and disease progression. The mechanisms by which it is able to act as a positive factor during virus replication is an area of intense research and although some controversy surrounds Nef much has been gauged as to how it functions. Its ability to modulate the expression of key cellular receptors important for cell activation and control signal transduction elements and events by interacting with numerous cellular kinases and signalling molecules, including members of the Src family kinases, leading to an effect on host cell function is likely to explain at least in part its role during infection and represents a finely tuned mechanism where this protein assists HIV-1 to control its host.

    Journal of biosciences 2003;28;3;323-35

  • p120 Catenin-associated Fer and Fyn tyrosine kinases regulate beta-catenin Tyr-142 phosphorylation and beta-catenin-alpha-catenin Interaction.

    Piedra J, Miravet S, Castaño J, Pálmer HG, Heisterkamp N, García de Herreros A and Duñach M

    Unitat de Biofísica, Departament de Bioquímica i Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain.

    beta-Catenin has a key role in the formation of adherens junction through its interactions with E-cadherin and alpha-catenin. We show here that interaction of beta-catenin with alpha-catenin is regulated by the phosphorylation of beta-catenin Tyr-142. This residue can be phosphorylated in vitro by Fer or Fyn tyrosine kinases. Transfection of these kinases to epithelial cells disrupted the association between both catenins. We have also examined whether these kinases are involved in the regulation of this interaction by K-ras. Stable transfectants of the K-ras oncogene in intestinal epithelial IEC18 cells were generated which show little alpha-catenin-beta-catenin association with respect to control clones; this effect is accompanied by increased Tyr-142 phosphorylation and activation of Fer and Fyn kinases. As reported for Fer, Fyn kinase is constitutively bound to p120 catenin; expression of K-ras induces the phosphorylation of p120 catenin on tyrosine residues increasing its affinity for E-cadherin and, consequently, promotes the association of Fyn with the adherens junction complex. Yes tyrosine kinase also binds to p120 catenin but only upon activation, and stimulates Fer and Fyn tyrosine kinases. These results indicate that p120 catenin acts as a docking protein facilitating the activation of Fer/Fyn tyrosine kinases by Yes and demonstrate the role of these p120 catenin-associated kinases in the regulation of beta-catenin-alpha-catenin interaction.

    Molecular and cellular biology 2003;23;7;2287-97

  • Protein tyrosine phosphatase PTP20 induces actin cytoskeleton reorganization by dephosphorylating p190 RhoGAP in rat ovarian granulosa cells stimulated with follicle-stimulating hormone.

    Shiota M, Tanihiro T, Nakagawa Y, Aoki N, Ishida N, Miyazaki K, Ullrich A and Miyazaki H

    Gene Research Center, University of Tsukuba, Ibaraki 305-8572, Japan.

    We identified 25 protein tyrosine phosphatases (PTPs) expressed in rat ovarian granulosa cells. Of these PTPs, the expression levels of at least PTP20, PTP-MEG1, PTPepsilonM, and PTPepsilonC significantly changed during the estrous cycle. We examined the cellular functions of PTP20 in granulosa cells by expressing the wild type, a catalytically inactive CS mutant in which Cys229 of PTP20 was changed to Ser, or a substrate-trapping DA mutant in which Asp197 was mutated to Ala, using an adenovirus vector. Overexpression of the wild type, but not of the CS mutant, induced retraction of the cell body with the extension of long, dendritic-like processes after stimulation with FSH, a critical factor for the survival and differentiation of these cells. In addition, cell adhesion to the substratum decreased in an FSH-dependent manner. Inhibiting Rho GTPase activity with C3 botulinum toxin caused similar morphological changes. The FSH-enhanced phosphotyrosine (p-Tyr) level of p190 RhoGAP was selectively reduced by the overexpressed wild type, but not by mutated PTP20. Although p190 RhoGAP is tyrosine phosphorylated by c-Src via the tyrosine kinase Pyk2, wild-type PTP20 had little effect on p-Tyr418 of c-Src and no effect on p-Tyr402 of Pyk2, which are required for full c-Src activity and for interacting between Pyk2 and c-Src, respectively. The CS and DA mutants as well as the wild type reduced the formation of p190 RhoGAP-p120 RasGAP complexes. Confocal microscopy analysis revealed that PTP20 intracellularly colocalizes with p190 RhoGAP. These results demonstrate that PTP20 regulates the functions of granulosa cells in an FSH-dependent manner by dephosphorylating p190 RhoGAP and subsequently inducing reorganization of the actin cytoskeleton. Moreover, our data suggest that PTPs play significant roles in controlling the dynamics of ovarian functions.

    Molecular endocrinology (Baltimore, Md.) 2003;17;4;534-49

  • Regulation of a transient receptor potential (TRP) channel by tyrosine phosphorylation. SRC family kinase-dependent tyrosine phosphorylation of TRPV4 on TYR-253 mediates its response to hypotonic stress.

    Xu H, Zhao H, Tian W, Yoshida K, Roullet JB and Cohen DM

    Division of Nephrology, Department of Medicine, Oregon Health & Science University and the Portland Veterans Affairs Medical Center, Portland, Oregon 97201, USA.

    The recently identified transient receptor potential (TRP) channel family member, TRPV4 (formerly known as OTRPC4, VR-OAC, TRP12, and VRL-2) is activated by hypotonicity. It is highly expressed in the kidney as well as blood-brain barrier-deficient hypothalamic nuclei responsible for systemic osmosensing. Apart from its gating by hypotonicity, little is known about TRPV4 regulation. We observed that hypotonic stress resulted in rapid tyrosine phosphorylation of TRPV4 in a heterologous expression model and in native murine distal convoluted tubule cells in culture. This tyrosine phosphorylation was sensitive to the inhibitor of Src family tyrosine kinases, PP1, in a dose-dependent fashion. TRPV4 associated with Src family kinases by co-immunoprecipitation studies and confocal immunofluorescence microscopy, and this interaction required an intact Src family kinase SH2 domain. One of these kinases, Lyn, was activated by hypotonic stress and phosphorylated TRPV4 in an immune complex kinase assay and an in vitro kinase assay using recombinant Lyn and TRPV4. Transfection of wild-type Lyn dramatically potentiated hypotonicity-dependent TRPV4 tyrosine phosphorylation whereas dominant negative-acting Lyn modestly inhibited it. Through mutagenesis studies, the site of tonicity-dependent tyrosine phosphorylation was mapped to Tyr-253, which is conserved across all species from which TRPV4 has been cloned. Importantly, point mutation of Tyr-253 abolished hypotonicity-dependent channel activity. In aggregate, these data indicate that hypotonic stress results in Src family tyrosine kinase-dependent tyrosine phosphorylation of the tonicity sensor TRPV4 at residue Tyr-253 and that this residue is essential for channel function in this context. This is the first example of direct regulation of TRP channel function through tyrosine phosphorylation.

    Funded by: NIDDK NIH HHS: DK52494

    The Journal of biological chemistry 2003;278;13;11520-7

  • STAP-2/BKS, an adaptor/docking protein, modulates STAT3 activation in acute-phase response through its YXXQ motif.

    Minoguchi M, Minoguchi S, Aki D, Joo A, Yamamoto T, Yumioka T, Matsuda T and Yoshimura A

    Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashiku, Fukuoka 812-8582, Japan.

    As a c-fms-interacting protein, we cloned a novel adaptor molecule, signal-transducing adaptor protein-2 (STAP-2), which contains pleckstrin homology- and Src homology 2-like (PH and SRC) domains and a proline-rich region. STAP-2 is structurally related to STAP-1/BRDG1 (BCR downstream signaling-1), which we had cloned previously from hematopoietic stem cells. STAP-2 is a murine homologue of a recently identified adaptor molecule, BKS, a substrate of BRK tyrosine kinase. STAP-2 was tyrosine-phosphorylated and translocated to the plasma membrane in response to epidermal growth factor when overexpressed in fibroblastic cells. To define the function of STAP-2, we generated mice lacking the STAP-2 gene. STAP-2 mRNA was strongly induced in the liver in response to lipopolysaccharide and in isolated hepatocytes in response to interleukin-6. In the STAP-2(-/-) hepatocytes, the interleukin-6-induced expression of acute-phase (AP) genes and the tyrosine-phosphorylation level of STAT3 were reduced specifically at the late phase (6-24 h) of the response. These data indicate that STAP-2 plays a regulatory role in the AP response in systemic inflammation. STAP-2 contains a YXXQ motif in the C-terminal region that is a potential STAT3-binding site. Overexpression of wild-type STAP-2, but not of mutants lacking this motif, enhanced the AP response element reporter activity and an AP protein production. These data suggest that STAP-2 is a new class of adaptor molecule that modulates STAT3 activity through its YXXQ motif.

    The Journal of biological chemistry 2003;278;13;11182-9

  • Defective activation of c-Src in cystic fibrosis airway epithelial cells results in loss of tumor necrosis factor-alpha-induced gap junction regulation.

    Huang S, Dudez T, Scerri I, Thomas MA, Giepmans BN, Suter S and Chanson M

    Laboratory of Clinical Investigation III, Department of Pediatrics, Geneva University Hospitals, Switzerland.

    Tumor necrosis factor-alpha (TNF-alpha) signaling is central to the transmission of the innate immune response and subsequent activation of the adaptive immune system. The functioning of both systems is required for optimal clearance of pathogens from the airways. In cystic fibrosis (CF), dysfunction of the CF transmembrane conductance regulator (CFTR) is associated with recurrent pulmonary infections despite an intense inflammatory and immune response. We reported recently that TNF-alpha decreased gap junction connectivity in non-CF airway cells, a mechanism that was absent in CF cells expressing the DeltaPhe-508 mutant of CFTR. We have now identified the tyrosine kinase c-Src as a possible pathway between the mediators of inflammation and the gap junction protein connexin43 (Cx43). Indeed, TNF-alpha increased the proportion of activated c-Src in non-CF airway cells. Moreover, pharmacological antagonists and expression in non-CF cells of a dominant negative construct of c-Src prevented Cx43 channel closure by TNF-alpha. Finally, gap junction channel closure was prevented by expression of a Cx43 mutant lacking tyrosine phosphorylation sites for c-Src. Additional experiments showed that activation of c-Src was defective in CF airway cells but rescued in CFTR-corrected CF cells. These data suggest that CFTR dysfunction is associated with altered TNF-alpha signaling, resulting in the persistence of gap junction connectivity in CF airway cells. We propose that altered regulation of c-Src may contribute to the dysregulated inflammatory response that is characteristic of the CF phenotype.

    The Journal of biological chemistry 2003;278;10;8326-32

  • Cysteine residues in the C-terminal lobe of Src: their role in the suppression of the Src kinase.

    Oo ML, Senga T, Thant AA, Amin AR, Huang P, Mon NN and Hamaguchi M

    Laboratory of Molecular Pathogenesis, Nagoya University School of Medicine, Japan.

    To evaluate the function of cysteine residues of the Src kinase, we constructed a series of Src mutants in which some of cysteines were replaced to alanines. With these mutants, we studied the effect of SH-alkylating agents, N-[p-(2-benzimidazolyl)phenyl] maleimide (BIPM) and N-(9-acridinyl) maleimide (NAM), on their kinase activity. Of 10 cysteine residues scattered over v-Src, either a single mutation at Cys520 or multiple mutations at the four clustered cyteines, Cys483, Cys487, Cys496 and Cys498, yielded clear resistance to the treatment with 10 microM BIPM or 1 microM NAM. In contrast, other cysteines including those in the SH2 domain and those in the catalytic cleft of the kinase domain were dispensable for the inactivation by BIPM and NAM. Similarly, deletion of SH2 and SH3 did not confer the resistance to v-Src, suggesting the inactivation by the SH-alkylating agents is SH2/SH3-independent. Although Cys520-mutated v-Src was resistant to 1 microM NAM, it was inactivated by 5 microM NAM. However, combined mutation including all of Cys483, Cys487, Cys496, Cys498 and Cys520 yielded clear resistance to 5 microM NAM. Among these mutants, those with double mutations in the four clustered cysteines yielded a temperature sensitive phenotype in the transfected cells, whereas Cys520 did not, suggesting that Cys520 has, at least in part, a discrete function. In contrast to v-Src, c-Src, which lacks cysteine at position 520, was resistant to 1 microM NAM but sensitive to 5 microM NAM. While replacement of Phe520 of c-Src to cysteine made it sensitive to 1 microM NAM, double mutation in clustered cysteines again yielded resistance to 5 microM NAM. Taken together, our results strongly suggest that the multiple cysteine residues clustered at the end of the C-terminal lobe are critical for the inhibition by the SH-alkylating agents and, thereby, have an allosteric repressor effect on the catalytic activity of Src in a SH2-phosphoTyr527 independent manner.

    Oncogene 2003;22;9;1411-7

  • Src promotes destruction of c-Cbl: implications for oncogenic synergy between Src and growth factor receptors.

    Bao J, Gur G and Yarden Y

    Department of Biological Regulation, The Weizmann Institute of Science, Rehovot 76100, Israel.

    Cellular Src and epidermal growth factor receptor (EGFR) collaborate in the progression of certain human malignancies, and their cooverexpression characterizes relatively aggressive animal tumors. Our study addressed the mode of oncogenic cooperation and reports that overexpression of c-Src in model cellular systems results in the accumulation of EGFR at the cell surface. The underlying mechanism involves inhibition of the normal, c-Cbl-regulated process of ligand-induced receptor down-regulation. In response to activation of c-Src, c-Cbl proteins undergo tyrosine phosphorylation that promotes their ubiquitylation and proteasomal destruction. Consequently, ubiquitylation of EGFR by c-Cbl is restrained in Src-transformed cells, and receptor sorting to endocytosis is impaired. In conclusion, by promoting destruction of c-Cbl, c-Src enables EGFR to evade desensitization, which explains Src-EGFR collaboration in oncogenesis.

    Funded by: NCI NIH HHS: CA72981, R01 CA072981, R37 CA072981

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;5;2438-43

  • Differential regulation of cell motility and invasion by FAK.

    Hsia DA, Mitra SK, Hauck CR, Streblow DN, Nelson JA, Ilic D, Huang S, Li E, Nemerow GR, Leng J, Spencer KS, Cheresh DA and Schlaepfer DD

    The Scripps Research Institute, Dept. of Immunology, IMM26, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA.

    Cell migration and invasion are fundamental components of tumor cell metastasis. Increased focal adhesion kinase (FAK) expression and tyrosine phosphorylation are connected with elevated tumorigenesis. Null mutation of FAK results in embryonic lethality, and FAK-/- fibroblasts exhibit cell migration defects in culture. Here we show that viral Src (v-Src) transformation of FAK-/- cells promotes integrin-stimulated motility equal to stable FAK reexpression. However, FAK-/- v-Src cells were not invasive, and FAK reexpression, Tyr-397 phosphorylation, and FAK kinase activity were required for the generation of an invasive cell phenotype. Cell invasion was linked to transient FAK accumulation at lamellipodia, formation of a FAK-Src-p130Cas-Dock180 signaling complex, elevated Rac and c-Jun NH2-terminal kinase activation, and increased matrix metalloproteinase expression and activity. Our studies support a dual role for FAK in promoting cell motility and invasion through the activation of distinct signaling pathways.

    Funded by: NCI NIH HHS: CA45726, CA50286, CA75240, CA78045, CA87038, P01 CA078045, R01 CA045726, R01 CA050286, R01 CA075240, R01 CA087038, R29 CA075240, R37 CA050286; NHLBI NIH HHS: HL54352, HL65754, R01 HL054352, R01 HL065754

    The Journal of cell biology 2003;160;5;753-67

  • Electrostatic modulation in steroid receptor recruitment of LXXLL and FXXLF motifs.

    He B and Wilson EM

    Laboratories for Reproductive Biology, Department of Biochemistry and Biophysics, University of North Carolina, 374 Medical Sciences Research Building, Chapel Hill, NC 27599-7500, USA.

    Coactivator recruitment by activation function 2 (AF2) in the steroid receptor ligand binding domain takes place through binding of an LXXLL amphipathic alpha-helical motif at the AF2 hydrophobic surface. The androgen receptor (AR) and certain AR coregulators are distinguished by an FXXLF motif that interacts selectively with the AR AF2 site. Here we show that LXXLL and FXXLF motif interactions with steroid receptors are modulated by oppositely charged residues flanking the motifs and charge clusters bordering AF2 in the ligand binding domain. An increased number of charged residues flanking AF2 in the ligand binding domain complement the two previously characterized charge clamp residues in coactivator recruitment. The data suggest a model whereby coactivator recruitment to the receptor AF2 surface is initiated by complementary charge interactions that reflect a reversal of the acidic activation domain-coactivator interaction model.

    Funded by: NICHD NIH HHS: HD 16910, R01 HD016910, R37 HD016910, U54 HD 35041, U54 HD035041

    Molecular and cellular biology 2003;23;6;2135-50

  • TRAF6 and C-SRC induce synergistic AP-1 activation via PI3-kinase-AKT-JNK pathway.

    Funakoshi-Tago M, Tago K, Sonoda Y, Tominaga S and Kasahara T

    Department of Biochemistry, Kyoritsu College of Pharmacy, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan.

    Interleukin-1 (IL-1) induces multiple genes via activation of transcription factors that include NF-kappa B and activator protein-1 (AP-1). We found that IL-1-mediated c-Src activation was required for AP-1 activation, but not for NF-kappa B activation and also revealed that c-Src-induced AP-1 activation was enhanced synergistically by the coexpression of TNF receptor associated factor 6 (TRAF6). In addition, c-Src interacts with TRAF6 in response to IL-1 and this interaction is required for c-Src activity. However, neither dominant negative mutants of TRAF6 (TRAF6 DN) nor kinase-dead mutant of c-Src (c-Src KD) counteracted each-induced AP-1 activation, suggesting no hierarchy between these two molecules. During the TRAF6 and c-Src-induced AP-1 activation, phosphatidylinositol 3 (PI3)-kinase, its downstream signaling molecule, Akt and c-Jun N-terminal kinase (JNK) were significantly activated and inhibition of these kinase activities down-regulated AP-1 activation through the suppression of c-fos expression. Furthermore, TRAF6 and c-Src-induced JNK activation was significantly inhibited by PI3-kinase inhibitor or a dominant negative mutant of Akt (Akt DN). Taken together, our results demonstrate that c-Src and TRAF6 are key mediators of IL-1-induced AP-1 activation and provide evidence of cross talk between c-Src and TRAF6 molecules through PI3 kinase-Akt-JNK pathways.

    European journal of biochemistry / FEBS 2003;270;6;1257-68

  • Two domains of the progesterone receptor interact with the estrogen receptor and are required for progesterone activation of the c-Src/Erk pathway in mammalian cells.

    Ballaré C, Uhrig M, Bechtold T, Sancho E, Di Domenico M, Migliaccio A, Auricchio F and Beato M

    Institut für Molekularbiologie und Tumorforschung, Philipps-Universität, D-35033 Marburg, Germany.

    In breast cancer cells, estrogens activate the Src/Erk pathway through an interaction of the estrogen receptor alpha (ERalpha) with the SH2 domain of c-Src. Progestins have been reported to activate also this pathway either via an interaction of the progesterone receptor isoform B (PRB) with ERalpha, which itself activates c-Src, or by direct interaction of PRB with the SH3 domain of c-Src. Here we identify two domains of PRB, ERID-I and -II, mediating a direct interaction with the ligand-binding domain of ERalpha. ERID-I and ERID-II flank a proline cluster responsible for binding of PRB to c-Src. In mammalian cells, the interaction of PRB with ERalpha and the progestin activation of the Src/Erk cascade are abolished by deletion of either ERID-I or ERID-II. These regions are not required for transactivation of a progesterone-responsive reporter gene. Mutations in the proline cluster of PRB that prevent a direct interaction with c-Src do not affect the strong activation of c-Src by progestins in the presence of ERalpha. Thus, in cells with ERalpha, ERID-I and ERID-II are necessary and sufficient for progestin activation of the endogenous Src/Erk pathway.

    Molecular and cellular biology 2003;23;6;1994-2008

  • Characterization of a novel negative regulator (DOC-2/DAB2) of c-Src in normal prostatic epithelium and cancer.

    Zhou J, Scholes J and Hsieh JT

    Department of Urology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9110, USA.

    DOC-2/DAB2 is a potent tumor suppressor in many cancer types including prostate cancer. In prostate cancer, expression of DOC-2/DAB2 can inhibit its growth. Our recent studies demonstrate that DOC-2/DAB2 can suppress both protein kinase C and peptide growth factor-elicited signal pathways via the Ras-mitogen-activated protein kinase pathway. In this study, we further showed that the proline-rich domain of DOC-2/DAB2 could also interact with proteins containing the Src homology 3 domain, such as Src and Fgr. The binding of c-Src to DOC-2/DAB2 was enhanced in cells treated with growth factor, and this interaction resulted in c-Src inactivation. The c-Src inactivation was evidenced by the decreased tyrosine 416 phosphorylation of c-Src and reduced downstream effector activation. It appears that DOC-2/DAB2 can bind to Src homology 3 domain of c-Src and maintain it in an inactive conformation. Thus, this study provides a new mechanism for modulating c-Src in prostatic epithelium and cancer.

    Funded by: NIDDK NIH HHS: DK 47657

    The Journal of biological chemistry 2003;278;9;6936-41

  • Global profiling of the cell surface proteome of cancer cells uncovers an abundance of proteins with chaperone function.

    Shin BK, Wang H, Yim AM, Le Naour F, Brichory F, Jang JH, Zhao R, Puravs E, Tra J, Michael CW, Misek DE and Hanash SM

    Departments of Pediatrics and Pathology, University of Michigan, Ann Arbor, Michigan 48109-0656, USA.

    There is currently limited data available pertaining to the global characterization of the cell surface proteome. We have implemented a strategy for the comprehensive profiling and identification of surface membrane proteins. This strategy has been applied to cancer cells, including the SH-SY5Y neuroblastoma, the A549 lung adenocarcinoma, the LoVo colon adenocarcinoma, and the Sup-B15 acute lymphoblastic leukemia (B cell) cell lines and ovarian tumor cells. Surface membrane proteins of viable, intact cells were subjected to biotinylation then affinity-captured and purified on monomeric avidin columns. The biotinylated proteins were eluted from the monomeric avidin columns as intact proteins and were subsequently separated by two-dimensional PAGE, transferred to polyvinylidene difluoride membranes, and visualized by hybridization with streptavidin-horseradish peroxidase. Highly reproducible, but distinct, two-dimensional patterns consisting of several hundred biotinylated proteins were obtained for the different cell populations analyzed. Identification of a subset of biotinylated proteins among the different cell populations analyzed using matrix-assisted laser desorption ionization and tandem mass spectrometry uncovered proteins with a restricted expression pattern in some cell line(s), such as CD87 and the activin receptor type IIB. We also identified more widely expressed proteins, such as CD98, and a sushi repeat-containing protein, a member of the selectin family. Remarkably, a set of proteins identified as chaperone proteins were found to be highly abundant on the cell surface, including GRP78, GRP75, HSP70, HSP60, HSP54, HSP27, and protein disulfide isomerase. Comprehensive profiling of the cell surface proteome provides an effective approach for the identification of commonly occurring proteins as well as proteins with restricted expression patterns in this compartment.

    The Journal of biological chemistry 2003;278;9;7607-16

  • Characterization of a brain-specific Rho GTPase-activating protein, p200RhoGAP.

    Moon SY, Zang H and Zheng Y

    Division of Experimental Hematology, Children's Hospital Research Foundation, Cincinnati, Ohio 45229, USA.

    The Rho GTPase-activating proteins (RhoGAPs) are a family of multifunctional molecules that transduce diverse intracellular signals by regulating Rho GTPase activities. A novel RhoGAP family member, p200RhoGAP, is cloned in human and mouse. The murine p200RhoGAP shares 86% sequence identity with the human homolog. In addition to a conserved RhoGAP domain at the N terminus, multiple proline-rich motifs are found in the C-terminal region of the molecules. Northern blot analysis revealed a brain-specific expression pattern of p200RhoGAP. The RhoGAP domain of p200RhoGAP stimulated the GTPase activities of Rac1 and RhoA in vitro and in vivo, and the conserved catalytic arginine residue (Arg-58) contributed to the GAP activity. Expression of the RhoGAP domain of p200RhoGAP in Swiss 3T3 fibroblasts inhibited actin stress fiber formation stimulated by lysophosphatidic acid and platelet-derived growth factor-induced membrane ruffling but not Bradykinin-induced filopodia formation. Endogenous p200RhoGAP was localized to cortical actin in naive N1E-115 neuroblastoma cells and to the edges of extended neurites of differentiated N1E-115 cells. Transient expression of the RhoGAP domain and the full-length molecule, but not the catalytic arginine mutants, readily induced a differentiation phenotype in N1E-115 cells. Finally, p200RhoGAP was capable of binding to the Src homology 3 domains of Src, Crk, and phospholipase Cgamma in vitro and became tyrosine-phosphorylated upon association with activated Src in cells. These results suggest that p200RhoGAP is involved in the regulation of neurite outgrowth by exerting its RhoGAP activity and that its cellular activity may be regulated through interaction with Src-like tyrosine kinases.

    Funded by: NIGMS NIH HHS: GM60523

    The Journal of biological chemistry 2003;278;6;4151-9

  • Tissue microarray analysis of signal transducers and activators of transcription 3 (Stat3) and phospho-Stat3 (Tyr705) in node-negative breast cancer shows nuclear localization is associated with a better prognosis.

    Dolled-Filhart M, Camp RL, Kowalski DP, Smith BL and Rimm DL

    Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.

    Purpose: Although a high frequency of tumors contain constitutively activated signal transducers and activators of transcription 3 (Stat3), its relationship to breast cancer and patient survival has not been determined in a large retrospective study of node-negative tumors. To further elucidate the role of Stat3 in breast cancers, the expression patterns of Stat3 and Phospho-tyrosine residue 705 (Tyr705) Stat3 were correlated with survival outcome and clinicopathological parameters in a large cohort of node-negative breast cancer tumors.

    Immunohistochemical analysis of Stat3 and Phospho-Stat3 was performed on a breast cancer tissue microarray of 346 node-negative breast cancer specimens. These results were correlated with overall survival and other clinicopathological data.

    Results: Positive Stat3 cytoplasmic expression was seen in 69.2% of tumors, and positive Phospho-Stat3 (Tyr705) cytoplasmic expression was seen in 19.6% of tumors. Neither cytoplasmic expression showed significant association with survival or other clinical parameters. However, 23.1% of tumors had positive Stat3 nuclear expression, and those patients had a significantly improved short-term survival (P = 0.0332) at 5 years of follow-up. Upon analysis of positive Phospho-Stat3 (Tyr705) nuclear expression, seen in 43.5% of tumors, positive tumors had a significantly improved survival at both short-term 5-year survival (P = 0.0054) and long-term 20-year (P = 0.0376) survival analysis. Additionally, positive Phospho-Stat3 (Tyr705) nuclear expression is an independent prognostic marker of better overall survival node-negative breast cancer by multivariate analyses that included the variables of nuclear grade, Ki-67, estrogen receptor staining, progesterone receptor staining, Her2 staining, age, and tumor size.

    Conclusions: These findings support a role for Stat3 and Phospho-Stat3 (Tyr705) overexpression in node-negative breast cancer and provide initial evidence that Phospho-Stat3 (Tyr705) may be a marker for improved overall survival independent of other prognostic markers.

    Funded by: NIGMS NIH HHS: R0-1 GM57604

    Clinical cancer research : an official journal of the American Association for Cancer Research 2003;9;2;594-600

  • Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry.

    Salomon AR, Ficarro SB, Brill LM, Brinker A, Phung QT, Ericson C, Sauer K, Brock A, Horn DM, Schultz PG and Peters EC

    Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121, USA.

    The reversible phosphorylation of tyrosine residues is an important mechanism for modulating biological processes such as cellular signaling, differentiation, and growth, and if deregulated, can result in various types of cancer. Therefore, an understanding of these dynamic cellular processes at the molecular level requires the ability to assess changes in the sites of tyrosine phosphorylation across numerous proteins simultaneously as well as over time. Here we describe a sensitive approach based on multidimensional liquid chromatography/mass spectrometry that enables the rapid identification of numerous sites of tyrosine phosphorylation on a number of different proteins from human whole cell lysates. We used this methodology to follow changes in tyrosine phosphorylation patterns that occur over time during either the activation of human T cells or the inhibition of the oncogenic BCR-ABL fusion product in chronic myelogenous leukemia cells in response to treatment with STI571 (Gleevec). Together, these experiments rapidly identified 64 unique sites of tyrosine phosphorylation on 32 different proteins. Half of these sites have been documented in the literature, validating the merits of our approach, whereas motif analysis suggests that a number of the undocumented sites are also potentially involved in biological pathways. This methodology should enable the rapid generation of new insights into signaling pathways as they occur in states of health and disease.

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;2;443-8

  • STAT5b, a Mediator of Synergism between c-Src and the Epidermal Growth Factor Receptor.

    Kloth MT, Laughlin KK, Biscardi JS, Boerner JL, Parsons SJ and Silva CM

    Departments of Internal Medicine and Microbiology and the Cancer Center, University of Virginia Health System, Charlottesville, Virginia 22908.

    Overexpression of the epidermal growth factor receptor (EGFR) and its association with the tyrosine kinase, c-Src, is correlated with increased cellular proliferation and tumorigenesis. Previous studies have shown that EGFR and c-Src co-overexpression and association leads to the c-Src-mediated phosphorylation of tyrosine 845 of the EGFR and that mutation of Tyr(845) ablates epidermal growth factor (EGF)-induced DNA synthesis. Here, we investigate the contribution of the signal transducers and activators of transcription (STAT5b) in the signaling pathways regulated by EGFR and c-Src overexpression in human breast tumor cell lines as well as in a mouse fibroblast model (C3H10T1/2). We demonstrate that 1) activation of STAT5b by EGF requires overexpression of the EGFR, 2) co-overexpression of c-Src alone does not result in EGF-induced activation of STAT5b but enhances that seen in EGFR-overexpressing cells, and 3) EGF-induced tyrosine phosphorylation of STAT5b requires Tyr(845) of the EGFR. Furthermore, the stable overexpression of a kinase-defective c-Src in the context of EGFR overexpression results in a decrease in the tyrosine phosphorylation of STAT5b in response to EGF and a more dramatic decrease in EGF-induced transcriptional activation of STAT5b, suggesting an integral role for c-Src in the physiological actions of STAT5b. Using a dominant negative STAT5b, we provide evidence that one such physiological action is to mediate EGF-induced DNA-synthesis. Finally, the use of site-specific tyrosine mutants demonstrates that EGF-induced phosphorylation of STAT5b involves not only tyrosine 699 of STAT5b, which is required for its transcriptional activation, but also three previously identified tyrosines in the C terminus of STAT5b (Tyr(725)/Tyr(740)/Tyr(743)).

    Funded by: NCI NIH HHS: CA39438, CA71449; NICHD NIH HHS: P30-HD28934

    The Journal of biological chemistry 2003;278;3;1671-9

  • Tyrosine phosphorylation of I kappa B alpha activates NF kappa B through a redox-regulated and c-Src-dependent mechanism following hypoxia/reoxygenation.

    Fan C, Li Q, Ross D and Engelhardt JF

    Molecular Biology Graduate Program, the Department of Anatomy and Cell Biology, University of Iowa, Iowa City, Iowa 52242, USA.

    NF kappa B is a critical transcription factor involved in modulating cellular responses to environmental injuries. Tyrosine 42 phosphorylation of I kappa B alpha has been shown to mediate NF kappa B activation following hypoxia/reoxygenation (H/R) or pervanadate treatment. This pathway differs from the canonical proinflammatory pathways, which mediate NF kappa B activation through serine phosphorylation of I kappa B alpha by the IKK complex. In the present study, we investigated the involvement of c-Src in the redox activation of NFkappaB following H/R or pervanadate treatment. Our results demonstrate that pervanadate or H/R treatment leads to tyrosine phosphorylation of I kappa B alpha and NF kappa B transcriptional activation independent of the IKK pathway. In contrast, inhibition of c-Src by pp2 treatment or in c-Src (-/-) knockout cell lines, demonstrated a significant reduction in I kappa B alpha tyrosine phosphorylation and NF kappa B activation following pervanadate or H/R treatment. Overexpression of glutathione peroxidase-1 or catalase, but not Mn-SOD or Cu,Zn-SOD, significantly reduced both NF kappa B activation and tyrosine phosphorylation of I kappa B alpha. In vitro kinase assays further demonstrated that immunoprecipitated c-Src has the capacity to directly phosphorylate GST-I kappa B alpha and that this I kappa B alpha kinase activity is significantly reduced by Gpx-1 overexpression. These results suggest that c-Src-dependent tyrosine phosphorylation of I kappa B alpha and subsequent activation of NF kappa B is controlled by intracellular H(2)O(2) and defines an important redox-regulated pathway for NF kappa B activation following H/R injury that is independent of the IKK complex.

    Funded by: NHLBI NIH HHS: P50 HL60316; NIDDK NIH HHS: DK51315, P30 DK54759

    The Journal of biological chemistry 2003;278;3;2072-80

  • Site-specific phosphorylation of platelet focal adhesion kinase by low-density lipoprotein.

    Relou IA, Bax LA, van Rijn HJ and Akkerman JW

    Laboratory for Thrombosis and Haemostasis, Department of Haematology, University Medical Center Utrecht, P.O. Box 85500, 3508 GA Utrecht, The Netherlands. i.relou@azu.nl

    Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase implicated in signalling pathways mediated by integrins and G-protein-coupled receptors (GPCRs). Upon stimulation FAK is phosphorylated on six tyrosine residues. Here we report the site-specific phosphorylation by low-density lipoprotein (LDL), which is known to induce integrin-independent FAK phosphorylation, and compare this with the effect of thrombin, which phosphorylates FAK via integrin alphaIIbbeta3. Stimulation with LDL reveals (i) a major role for Tyr-925 phosphorylation which surpasses the phosphorylation of the other residues, including Tyr-397, in rate and extent, (ii) alphaIIbbeta3-independent phosphorylation of Tyr-925 and Tyr-397, and (iii) complex formation between FAK and the Src-kinase Fgr but not with c-Src. These patterns differ profoundly from those induced by thrombin. LDL-induced phosphorylation of Tyr-925 and Tyr-397 was inhibited by 60-75% by receptor-associated protein, an inhibitor of members of the LDL receptor family. Thus these findings reveal a novel mechanism of FAK phosphorylation by signalling cascades involving a member of the LDL receptor family.

    The Biochemical journal 2003;369;Pt 2;407-16

  • Functional implication of the interaction between EGF receptor and c-Src.

    Leu TH and Maa MC

    Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan 70101, ROC. tzengleu@mail.ncku.edu.tw

    EGF receptor (EGFR) and c-Src are the prototypes that individually represent the receptor and nonreceptor tyrosine kinases respectively. Accumulated evidence reveals the association between EGF-activated EGFR and c-Src that leads to activation of both kinases. Importantly, their mutual interaction is required for many EGFR-mediated cellular functions including proliferation, migration, survival and EGFR endocytosis. Interestingly, activation of c-Src and its association with transactivated EGFR is also observed in cells stimulated with non-EGF agonists. This review will not only discuss the structure, function and regulation of these two tyrosine kinases, but also will summarize our current knowledge of the molecular mechanisms depicting the cellular events that require their participation.

    Frontiers in bioscience : a journal and virtual library 2003;8;s28-38

  • Activating signal cointegrator 2 belongs to a novel steady-state complex that contains a subset of trithorax group proteins.

    Goo YH, Sohn YC, Kim DH, Kim SW, Kang MJ, Jung DJ, Kwak E, Barlev NA, Berger SL, Chow VT, Roeder RG, Azorsa DO, Meltzer PS, Suh PG, Song EJ, Lee KJ, Lee YC and Lee JW

    Department of Life Science, Pohang University of Science and Technology, Korea.

    Many transcription coactivators interact with nuclear receptors in a ligand- and C-terminal transactivation function (AF2)-dependent manner. These include activating signal cointegrator 2 (ASC-2), a recently isolated transcriptional coactivator molecule, which is amplified in human cancers and stimulates transactivation by nuclear receptors and numerous other transcription factors. In this report, we show that ASC-2 belongs to a steady-state complex of approximately 2 MDa (ASC-2 complex [ASCOM]) in HeLa nuclei. ASCOM contains retinoblastoma-binding protein RBQ-3, alpha/beta-tubulins, and trithorax group proteins ALR-1, ALR-2, HALR, and ASH2. In particular, ALR-1/2 and HALR contain a highly conserved 130- to 140-amino-acid motif termed the SET domain, which was recently implicated in histone H3 lysine-specific methylation activities. Indeed, recombinant ALR-1, HALR, and immunopurified ASCOM exhibit very weak but specific H3-lysine 4 methylation activities in vitro, and transactivation by retinoic acid receptor appears to involve ligand-dependent recruitment of ASCOM and subsequent transient H3-lysine 4 methylation of the promoter region in vivo. Thus, ASCOM may represent a distinct coactivator complex of nuclear receptors. Further characterization of ASCOM will lead to a better understanding of how nuclear receptors and other transcription factors mediate transcriptional activation.

    Molecular and cellular biology 2003;23;1;140-9

  • Plasma membrane Ca2+-ATPase isoform 4b is phosphorylated on tyrosine 1176 in activated human platelets.

    Wan TC, Zabe M and Dean WL

    Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, Louisville, KY 40292, USA.

    Plasma membrane Ca(2+) -ATPase isoform 4b (PMCA4b) is phosphorylated on a tyrosine residue during platelet activation resulting in inhibition of its ATPase activity. We now report that tyrosine 1176 (Y(1176)) in the carboxyl (C-) terminal domain of PMCA4b is the phosphorylated residue. Two tyrosine residues located in the C-terminus of PMCA4b, Y(1122) and Y(1176) can be removed by calpain-dependent cleavage. This truncation removes all of the tyrosine phosphates added to PMCA during platelet activation. Sequence analysis indicates that Y(1176) is a likely substrate for focal adhesion kinase (FAK), while Y(1122) is not located in a tyrosine phosphorylation motif. This is the same residue we reported earlier to be phosphorylated by Src kinase in vitro. Thus we conclude that Y(1176) is the only tyrosine phosphorylated during platelet activation. Results of co-immunoprecipitation, treatment with tyrosine kinase inhibitors and integrin inhibition experiments suggest that FAK is responsible for PMCA4b tyrosine phosphorylation during platelet activation.

    Thrombosis and haemostasis 2003;89;1;122-31

  • Specific interactions of neuronal focal adhesion kinase isoforms with Src kinases and amphiphysin.

    Messina S, Onofri F, Bongiorno-Borbone L, Giovedì S, Valtorta F, Girault JA and Benfenati F

    Department of Experimental Medicine, Section of Human Physiology, University of Genova, Italy.

    Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that activates Src family kinases via SH2- and SH3-mediated interactions. Specific FAK isoforms (FAK+), responsive to depolarization and neurotransmitters, are enriched in neurons. We analyzed the interactions of endogenous FAK+ and recombinant FAK+ isoforms containing amino acid insertions (boxes 6,7,28) with an array of SH3 domains and the c-Src SH2/SH3 domain tandem. Endogenous FAK+ bound specifically to the SH3 domains of c-Src (but not n-Src), Fyn, Yes, phosphtidylinositol-3 kinase, amphiphysin II, amphiphysin I, phospholipase Cgamma and NH2-terminal Grb2. The inclusion of boxes 6,7 was associated with a significant decrease in the binding of FAK+ to the c-Src and Fyn SH3 domains, and a significant increase in the binding to the Src SH2 domain, as a consequence of the higher phosphorylation of Tyr-397. The novel interaction with the amphiphysin SH3 domain, involving the COOH-terminal proline-rich region of FAK, was confirmed by coimmunoprecipitation of the two proteins and a closely similar response to stimuli affecting the actin cytoskeleton. Moreover, an impairment of endocytosis was observed in synaptosomes after internalization of a proline-rich peptide corresponding to the site of interaction. The data account for the different subcellular distribution of FAK and Src kinases and the specific regulation of the transduction pathways linked to FAK activation in the brain and implicate FAK in the regulation of membrane trafficking in nerve terminals.

    Funded by: Telethon: 1131

    Journal of neurochemistry 2003;84;2;253-65

  • Activation of c-Src is inversely correlated with biological aggressiveness of breast carcinoma.

    Ito Y, Kawakatsu H, Takeda T, Tani N, Kawaguchi N, Noguchi S, Sakai T and Matsuura N

    Department of Surgery, Osaka Seamen's Insurance Hospital, Suita, Osaka, Japan.

    In order to investigate whether c-Src is involved in carcinogenesis and progression of breast carcinoma, we examined the expression of activated c-Src in tissue sections from surgically resected human breast specimens. First, we confirmed the specificity of the antibody against activated c-Src (Clone 28) using six cell lines established from human breast carcinomas by western blotting. As expected, activated c-Src was detected as a 60 kDa band in all cell lines tested. Immunofluorescence analysis demonstrated that the activated c-Src was mainly observed in cytoplasms of these cells. Then, we designed an immunohistochemical study with 73 human breast carcinoma tissues. Glandular epithelial and myoepithelial cells in normal mammary glands adjacent to carcinoma nests and infiltrating stromal cells were negative for activated c-Src. In contrast, 37 of the 73 breast carcinoma tested (50.7%) were positive for activated c-Src, and this positive staining was inversely correlated with Ki-67 labeling index (p < 0.0001), TNM stage (p < 0.0001), tumor size (p < 0.0001), an d histological grade (p = 0.0002). These results strongly suggest that the activation of c-Src would be related to the progression of breast carcinomas with low aggressiveness.

    Breast cancer research and treatment 2002;76;3;261-7

  • Expression and subcellular distribution of the active form of c-Src tyrosine kinase in differentiating human endometrial stromal cells.

    Yamamoto Y, Maruyama T, Sakai N, Sakurai R, Shimizu A, Hamatani T, Masuda H, Uchida H, Sabe H and Yoshimura Y

    Department of Obstetrics and Gynecology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.

    Decidual growth factors and locally produced cytokines are thought to activate specific phosphorylation signalling pathway(s), thereby eliciting a variety of decidual functions. We have previously reported the activation of c-Src tyrosine kinase during ovarian steroid-induced decidualization of cultured human endometrial stromal cells. As chicken c-Src is known to be activated upon dephosphorylation of tyrosine 527 (Y527, corresponding to Y530 in human), we here employed a monoclonal antibody, clone 28, directed against the active form of human c-Src whose Y530 is dephosphorylated, and investigated whether c-Src became dephosphorylated at Y530 and thereby activated during decidualization. We found that the active form of c-Src was up-regulated and demonstrated increased kinase activity during in-vitro decidualization. Immunohistochemistry revealed that decidual cells in early pregnancy decidua were intensely stained with clone 28 when compared with the stromal cells in the non-pregnant endometrium. Moreover, the active form of c-Src translocated from a perinuclear region to the cytoplasm upon decidualization. Thus, the Y530 dephosphorylation, kinase activation, and subcellular translocation of c-Src may be intracellular signalling events associated with decidualization in vivo as well as in vitro.

    Molecular human reproduction 2002;8;12;1117-24

  • Progesterone receptor interacting coregulatory proteins and cross talk with cell signaling pathways.

    Edwards DP, Wardell SE and Boonyaratanakornkit V

    Program in Molecular Biology, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, B216 Denver, CO 80262, USA. dean.edwards@uchsc.edu

    Progesterone receptor (PR) is a member of the nuclear receptor family of ligand-dependent transcription activators and is expressed as two different sized proteins from a single gene; PR-A and PR-B. The two PR isoforms are identical in their DNA binding domains (DBD) and C-terminal ligand binding domains (LBD), differing only in the N-terminal domain that is truncated in PR-A. PR also contains two autonomous transcription activation domains (AD), ligand-dependent AF-2 in the C-terminus and constitutive AF-1 in the N-terminus. AF-2 is highly conserved and a family of p160 coactivators that interacts with and mediates the activity of AF-2 has been well characterized. By contrast the N-terminal domain and AF-1 are not conserved and little is known about AF-1 coactivators. The N-terminal domain is functionally important as it is required for full transcription activity of PR and is responsible for the distinct activities of the two PR isoforms, as well as cell and promoter specific functions of PR. This paper describes our efforts to identify PR N-terminal interacting coregulatory proteins. We summarize our work on the role of jun dimerization protein-2 (JDP-2) as an AF-1 coactivator of PR. JDP-2, initially defined as a repressor of jun and other bZIP transcription factors, also functions as a potent PR selective coactivator. JDP-2 lacks an intrinsic activation domain and through association with the DBD, we propose that JDP-2 potentiates AF-1 by recruiting other coactivators independent of AF-2 and p160 pathways. We also discovered that PR contains an SH3 domain interaction motif in the N-terminus that mediates interaction with Src tyrosine kinases and other signaling molecules. This interaction mediates rapid progesterone activation of Src/MAP K signaling pathways and defines a molecular mechanism for some of the rapid non-genomic actions of progesterone.

    Funded by: NIDDK NIH HHS: DK 49030

    The Journal of steroid biochemistry and molecular biology 2002;83;1-5;173-86

  • Activation of STAT3 by the Src family kinase Hck requires a functional SH3 domain.

    Schreiner SJ, Schiavone AP and Smithgall TE

    Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.

    STAT3 is a member of a family of transcription factors with Src homology 2 (SH2) domains that are activated by tyrosine phosphorylation in response to a wide variety of cytokines and growth factors. In this study, we investigated the mechanism of STAT3 activation by the Src family of nonreceptor tyrosine kinases, which have been linked to STAT activation in both normal and transformed cell types. Using Sf-9 insect cells, we demonstrate direct STAT3 tyrosine phosphorylation and stimulation of DNA binding activity by five members of the Src kinase family (Src, Hck, Lyn, Fyn, and Fgr). We also observed stable STAT3.Src family kinase complex formation in this system. Recombinant Src family kinase SH3 domains were sufficient for interaction with STAT3, suggesting a mechanistic basis for the Src kinase-STAT3 interaction. To test the contribution of Src family kinase SH3 domains to the recruitment and activation of STAT3 in vivo, we used Rat-2 fibroblasts expressing activated mutants of the myeloid Src family member Hck. Transformation of fibroblasts by an activated Hck mutant lacking the negative regulatory tail tyrosine residue (Hck-YF) induced strong DNA binding activity of endogenous STAT3. Inactivation of Hck SH3 function by Ala replacement of a conserved Trp residue (W93A mutant) completely abolished STAT3 activation by Hck-YF and reduced transforming activity by 50% without affecting Hck kinase activity. Finally, overexpression of STAT3 in Rat-2 cells transiently stimulated Hck and c-Src kinase activity in the absence of extracellular signals, an effect that was dependent upon a putative SH3 binding motif in STAT3. These results support a model in which Src family kinases recruit STAT3 through an SH3-dependent mechanism, resulting in transient kinase activation and STAT3 phosphorylation.

    Funded by: NCI NIH HHS: CA81398

    The Journal of biological chemistry 2002;277;47;45680-7

  • Estrogen receptor-interacting protein that modulates its nongenomic activity-crosstalk with Src/Erk phosphorylation cascade.

    Wong CW, McNally C, Nickbarg E, Komm BS and Cheskis BJ

    Department of Cell Biology, Women's Health Research Institute, Wyeth Pharmaceuticals, 500 Arcolla Road, Collegeville, PA 19426, USA.

    Numerous studies have demonstrated that estrogens induce rapid and transient activation of the Src/Erk phosphorylation cascade. Activation of this cascade triggers vital cellular functions including cell proliferation and differentiation. However, the details of the molecular mechanism of this process remain to be elucidated. We have identified a previously uncharacterized nuclear receptor-interacting protein designated as modulator of nongenomic activity of estrogen receptor (MNAR). Here we show that MNAR modulates estrogen-receptor (ER) interaction with members of the Src family of tyrosine kinases, which leads to a stimulation of Src enzymatic activity and activation of Erk1 and Erk2 kinases. We also show that MNAR, through activation of the Src/Erk phosphorylation cascade, affects ER transcriptional activity and ultimately ER-mediated gene expression. These data reveal that MNAR mediates the crosstalk between two important classes of signal transducing molecules and suggest that ER "genomic" and "nongenomic" activities are interrelated.