G2Cdb::Gene report

Gene id
G00001439
Gene symbol
YES1 (HGNC)
Species
Homo sapiens
Description
v-yes-1 Yamaguchi sarcoma viral oncogene homolog 1
Orthologue
G00000190 (Mus musculus)

Databases (9)

Curated Gene
OTTHUMG00000068164 (Vega human gene)
Gene
ENSG00000176105 (Ensembl human gene)
7525 (Entrez Gene)
350 (G2Cdb plasticity & disease)
YES1 (GeneCards)
Literature
164880 (OMIM)
Marker Symbol
HGNC:12841 (HGNC)
Protein Expression
4370 (human protein atlas)
Protein Sequence
P07947 (UniProt)

Synonyms (3)

  • HsT441
  • Yes
  • c-yes

Literature (73)

Pubmed - other

  • Coexpression network based on natural variation in human gene expression reveals gene interactions and functions.

    Nayak RR, Kearns M, Spielman RS and Cheung VG

    Medical Scientist Training Program, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

    Genes interact in networks to orchestrate cellular processes. Analysis of these networks provides insights into gene interactions and functions. Here, we took advantage of normal variation in human gene expression to infer gene networks, which we constructed using correlations in expression levels of more than 8.5 million gene pairs in immortalized B cells from three independent samples. The resulting networks allowed us to identify biological processes and gene functions. Among the biological pathways, we found processes such as translation and glycolysis that co-occur in the same subnetworks. We predicted the functions of poorly characterized genes, including CHCHD2 and TMEM111, and provided experimental evidence that TMEM111 is part of the endoplasmic reticulum-associated secretory pathway. We also found that IFIH1, a susceptibility gene of type 1 diabetes, interacts with YES1, which plays a role in glucose transport. Furthermore, genes that predispose to the same diseases are clustered nonrandomly in the coexpression network, suggesting that networks can provide candidate genes that influence disease susceptibility. Therefore, our analysis of gene coexpression networks offers information on the role of human genes in normal and disease processes.

    Funded by: Howard Hughes Medical Institute; NIEHS NIH HHS: R01 ES015733-01; NIGMS NIH HHS: GM081930, R01 GM081930-08, T32 GM008216

    Genome research 2009;19;11;1953-62

  • 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

  • Resequencing analysis of the human tyrosine kinase gene family in pancreatic cancer.

    Kubo T, Kuroda Y, Kokubu A, Hosoda F, Arai Y, Hiraoka N, Hirohashi S and Shibata T

    National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan.

    Objectives: Pancreatic cancer is one of the most intractable of cancers. However, the comprehensive view of somatic mutations in this tumor is far from clear. The tyrosine kinase (TK) gene family, which encodes important regulators of various signal transduction pathways, is one of the most frequently altered gene families in human cancer.

    Methods: To clarify the somatic mutation profile of TKs in pancreatic cancer, we performed a systematic screening of mutations in the kinase domains of all human TK genes (636 exons of 90 genes in total) in 11 pancreatic cancer cell lines and 29 microdissected primary tumors.

    Results: We identified 15 nonsynonymous alterations that included 9 DNA alterations in cell lines and 6 somatic mutations in primary tumors. In particular, we identified the previously reported pathogenic mutation of NTRK3 in a KRAS/BRAF wild-type tumor and 2 somatic mutations in the Src family of kinases (YES1 and LYN) that would be expected to cause structural changes.

    Conclusions: Our genome-wide resequencing approach revealed novel oncogenic pathways in pancreatic cancers.

    Pancreas 2009;38;7;e200-6

  • Coeliac disease-associated risk variants in TNFAIP3 and REL implicate altered NF-kappaB signalling.

    Trynka G, Zhernakova A, Romanos J, Franke L, Hunt KA, Turner G, Bruinenberg M, Heap GA, Platteel M, Ryan AW, de Kovel C, Holmes GK, Howdle PD, Walters JR, Sanders DS, Mulder CJ, Mearin ML, Verbeek WH, Trimble V, Stevens FM, Kelleher D, Barisani D, Bardella MT, McManus R, van Heel DA and Wijmenga C

    Genetics Department, University Medical Centre, University of Groningen, Groningen, The Netherlands.

    Objective: Our previous coeliac disease genome-wide association study (GWAS) implicated risk variants in the human leucocyte antigen (HLA) region and eight novel risk regions. To identify more coeliac disease loci, we selected 458 single nucleotide polymorphisms (SNPs) that showed more modest association in the GWAS for genotyping and analysis in four independent cohorts.

    Design: 458 SNPs were assayed in 1682 cases and 3258 controls from three populations (UK, Irish and Dutch). We combined the results with the original GWAS cohort (767 UK cases and 1422 controls); six SNPs showed association with p<1 x 10(-04) and were then genotyped in an independent Italian coeliac cohort (538 cases and 593 controls).

    Results: We identified two novel coeliac disease risk regions: 6q23.3 (OLIG3-TNFAIP3) and 2p16.1 (REL), both of which reached genome-wide significance in the combined analysis of all 2987 cases and 5273 controls (rs2327832 p = 1.3 x 10(-08), and rs842647 p = 5.2 x 10(-07)). We investigated the expression of these genes in the RNA isolated from biopsies and from whole blood RNA. We did not observe any changes in gene expression, nor in the correlation of genotype with gene expression.

    Conclusions: Both TNFAIP3 (A20, at the protein level) and REL are key mediators in the nuclear factor kappa B (NF-kappaB) inflammatory signalling pathway. For the first time, a role for primary heritable variation in this important biological pathway predisposing to coeliac disease has been identified. Currently, the HLA risk factors and the 10 established non-HLA risk factors explain approximately 40% of the heritability of coeliac disease.

    Funded by: British Heart Foundation: G0000934; Medical Research Council: G0000934; Wellcome Trust: 068545/Z/02, GR068094MA

    Gut 2009;58;8;1078-83

  • 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

  • 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

  • Differential mitotic activation of endogenous c-Src, c-Yes, and Lyn in HeLa cells.

    Kuga T, Nakayama Y, Hoshino M, Higashiyama Y, Obata Y, Matsuda D, Kasahara K, 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.

    Src-family tyrosine kinases (SFKs) play an important role in mitosis. Despite overlapping expression of multiple SFK members, little is known about how individual SFK members are activated in M phase. Here, we examined mitotic activation of endogenous c-Src, c-Yes, and Lyn, which are co-expressed in HeLa cells. c-Src, c-Yes, and Lyn were activated at different levels in M phase, and the activation was inhibited by Cdc2 inactivation. Mitotic c-Src and c-Yes exhibited normal- and retarded-electrophoretic-mobility forms on SDS-polyacrylamide gels, whereas Lyn did not show mobility retardation. Like c-Src, the retardation of electrophoretic mobility of c-Yes was caused by Cdc2-mediated phosphorylation. The normal- and retarded-mobility forms of c-Src were comparably activated, but activation of the retarded-mobility form of c-Yes was higher than that of the normal-mobility form of c-Yes. Thus, these results suggest that endogenous c-Src, c-Yes, and Lyn are differentially activated through Cdc2 activation during M phase.

    Archives of biochemistry and biophysics 2007;466;1;116-24

  • Increases in c-Yes expression level and activity promote motility but not proliferation of human colorectal carcinoma cells.

    Barraclough J, Hodgkinson C, Hogg A, Dive C and Welman A

    Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK.

    Increases in the levels and/or activity of nonreceptor tyrosine kinases c-Src and c-Yes are often associated with colorectal carcinogenesis. The physiological consequences of increased c-Yes activity during the early and late stages of tumorigenesis, in addition to the degree of redundancy between c-Yes and c-Src in colorectal cancer cells, remain elusive. To study the consequences of increases in c-Yes levels and activity in later stages of colorectal carcinogenesis, we developed human colorectal cancer cell lines in which c-Yes levels and activity can be inducibly increased by a tightly controlled expression of wild-type c-Yes or by constitutively active mutants of c-Yes, c-YesY537F, and c-Yes Delta t6aa. c-Yes induction resulted in increased cell motility but did not promote proliferation either in vitro or in vivo. These results suggest that in later stages of colorectal carcinogenesis, elevations in c-Yes levels/activity may promote cancer spread and metastasis rather than tumor growth.

    Neoplasia (New York, N.Y.) 2007;9;9;745-54

  • Crystallographic structure of the SH3 domain of the human c-Yes tyrosine kinase: loop flexibility and amyloid aggregation.

    Martín-García JM, Luque I, Mateo PL, Ruiz-Sanz J and Cámara-Artigas A

    Department of Physical Chemistry and Institute of Biotechnology, Faculty of Sciences, University of Granada, 18071 Granada, Spain.

    SH3 domains from the Src family of tyrosine kinases represent an interesting example of the delicate balance between promiscuity and specificity characteristic of proline-rich ligand recognition by SH3 domains. The development of inhibitors of therapeutic potential requires a good understanding of the molecular determinants of binding affinity and specificity and relies on the availability of high quality structural information. Here, we present the first high-resolution crystal structure of the SH3 domain of the c-Yes oncogen. Comparison with other SH3 domains from the Src family revealed significant deviations in the loop regions. In particular, the n-Src loop, highly flexible and partially disordered, is stabilized in an unusual conformation by the establishment of several intramolecular hydrogen bonds. Additionally, we present here the first report of amyloid aggregation by an SH3 domain from the Src family.

    FEBS letters 2007;581;9;1701-6

  • 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

  • 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

  • Identification of c-Yes expression in the nuclei of hepatocellular carcinoma cells: involvement in the early stages of hepatocarcinogenesis.

    Nonomura T, Masaki T, Morishita A, Jian G, Uchida N, Himoto T, Izuishi K, Iwama H, Yoshiji H, Watanabe S, Kurokohchi K and Kuriyama S

    Department of Gastroenterology and Neurology, Kagawa University School of Medicine, Miki-cho, Kita-gun, Kagawa 761-0793, Japan.

    It is thought that the subcellular distribution of Src-family tyrosine kinases, including c-Yes binding to the cellular membrane, is membranous and/or cytoplasmic. c-Yes protein tyrosine kinase is known to be related to malignant transformation. However, the expression patterns of c-Yes in hepatocellular carcinoma (HCC) remains unknown. In the present study, we report that c-Yes is expressed not only in the membrane and cytoplasm, but also in the nuclei of cancer cells in some human HCC tissues and in a human HCC cell line. We examined the expression and localization of c-Yes in human HCC cell lines (HLE, HLF, PLC/PRF/5 and Hep 3B) by Western blotting and immunohistochemical analyses; we also examined the expression of c-Yes by immunohistochemistry and Western blotting in the tissues of various liver diseases, including 39 samples from HCC patients. We used an antibody array to detect proteins that bind to nuclear c-Yes in PLC/PRF/5 cell line. c-Yes was found to be expressed in the membranes and cytoplasm of HLE, HLF and Hep 3B HCC cells; it was also detected in the nuclei in addition to the membranes and cytoplasm of PLC/PRF/5 HCC cells. HCC with nuclear c-Yes was detected in 5 of 39 cases (13.0%), and nuclear c-Yes expression was not detected in normal, chronic hepatitis or cirrhotic livers. All HCCs with nuclear c-Yes expression were well-differentiated, small tumors at the early stages. In the PLC/PRF/5 cell line, the nuclear localization of c-Yes with cyclin-dependent kinase 1 was confirmed by a protein antibody array. In conclusion, nuclear c-Yes expression was found in cancer cells at the early stages of hepatocarcinogenesis, suggesting that nucleus-located c-Yes may be a useful marker to detect early-stage HCC.

    International journal of oncology 2007;30;1;105-11

  • Comparison of gene expression profiles between primary tumor and metastatic lesions in gastric cancer patients using laser microdissection and cDNA microarray.

    Wang L, Zhu JS, Song MQ, Chen GQ and Chen JL

    Department of Gastroenterology, Sixth People's Hospital Affiliated to Shanghai Jiaotong University, Shanghai 200233, China.

    Aim: To study the differential gene expression profiles of target cells in primary gastric cancer and its metastatic lymph nodes using laser microdissection (LMD) in combination with cDNA microarray.

    Methods: Normal gastric tissue samples from 30 healthy individuals, 36 cancer tissue samples from primary gastric carcinoma and lymph node metastasis tissue samples from 58 patients during gastric cancer resection were obtained using LMD in combination with cDNA microarray independently. After P27-based amplification, aRNA from 36 of 58 patients (group 1) with lymph node metastasis and metastatic tissue specimens from the remaining 22 patients (group 2) were applied to cDNA microarray. Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemical assay verified the results of microarray in group 2 and further identified genes differentially expressed in the progression of gastric cancer.

    Results: The expression of 10 genes was up-regulated while the expression of 15 genes was down-regulated in 22 gastric carcinoma samples compared with that of genes in the normal controls. The results were confirmed at the level of mRNA and protein, and suggested that four genes (OPCML, RNASE1, YES1 and ACK1) could play a key role in the tumorigenesis and metastasis of gastric cancer. The expression pattern of 3 genes (OPCML, RNASE1 and YES1) was similar to tumor suppressor genes. For example, the expression level of these genes was the highest in normal gastric epithelium, which was decreased in primary carcinoma, and further decreased in metastatic lymph nodes. On the contrary, the expression pattern of gene ACK1 was similar to that of oncogene. Four genes were further identified as differentially expressed genes in the majority of the cases in the progression of gastric cancer.

    Conclusion: LMD in combination with cDNA microarray provides a unique support foe the identification of early expression profiles of differential genes and the expression pattern of 3 genes (OPCML, RNASE1 and YES1) associated with the progression of gastric cancer. Further study is needed to reveal the molecular mechanism of lymph node metastasis in patients with gastric cancer.

    World journal of gastroenterology 2006;12;43;6949-54

  • Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.

    Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P and Mann M

    Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark.

    Cell signaling mechanisms often transmit information via posttranslational protein modifications, most importantly reversible protein phosphorylation. Here we develop and apply a general mass spectrometric technology for identification and quantitation of phosphorylation sites as a function of stimulus, time, and subcellular location. We have detected 6,600 phosphorylation sites on 2,244 proteins and have determined their temporal dynamics after stimulating HeLa cells with epidermal growth factor (EGF) and recorded them in the Phosida database. Fourteen percent of phosphorylation sites are modulated at least 2-fold by EGF, and these were classified by their temporal profiles. Surprisingly, a majority of proteins contain multiple phosphorylation sites showing different kinetics, suggesting that they serve as platforms for integrating signals. In addition to protein kinase cascades, the targets of reversible phosphorylation include ubiquitin ligases, guanine nucleotide exchange factors, and at least 46 different transcriptional regulators. The dynamic phosphoproteome provides a missing link in a global, integrative view of cellular regulation.

    Cell 2006;127;3;635-48

  • Activation of c-Yes in hepatocellular carcinoma: a preliminary study.

    Feng H, Masaki T, Nonomura T, Morishita A, Jian G, Nakai S, Deguchi A, Uchida N, Himoto T, Iwama H, Usuki H, Wakabayashi H, Izuishi K, Yoshiji H, Kurokohchi K and Kuriyama S

    World journal of gastroenterology : WJG 2006;12;35;5743-5

  • Wnt-5a/Ca2+-induced NFAT activity is counteracted by Wnt-5a/Yes-Cdc42-casein kinase 1alpha signaling in human mammary epithelial cells.

    Dejmek J, Säfholm A, Kamp Nielsen C, Andersson T and Leandersson K

    Experimental Pathology, Department of Laboratory Medicine, Lund University, U-MAS, Entrance 78, SE-205 02 Malmö, Sweden.

    Wnt-5a has been shown to influence the metastatic behavior of human breast cancer cells, and the loss of Wnt-5a expression is associated with metastatic disease. We show here that NFAT1, a transcription factor connected with breast cancer metastasis, is activated by Wnt-5a through a Ca2+ signaling pathway in human breast epithelial cells. This activation was simultaneously counteracted by a Wnt-5a-induced Yes/Cdc42 signaling pathway. The observation that inhibition of the Wnt-5a/Yes/Cdc42 signal prolonged the duration of ionomycin-induced NFAT1 activation revealed the general importance of this pathway. The Wnt-5a-induced inhibition of NFAT1 did not require glycogen synthase kinase 3beta, JNK, or Pak1 activity or modulation of the cytoskeleton. Instead, we observed that Wnt-5a induced a complex formation of NFAT1/casein kinase 1alpha, even upon treatment with ionomycin, which was blocked upon inhibition of the Wnt-5a/Yes/Cdc42 signaling pathway. Our results explain why Wnt-5a/Ca2+-induced NFAT activity is hard to detect and suggest a novel mechanism by which Wnt-5a can suppress tumor-specific, agonist-induced NFAT activity and thus the metastatic behavior of breast cancer cells.

    Molecular and cellular biology 2006;26;16;6024-36

  • Docking-based substrate recognition by the catalytic domain of a protein tyrosine kinase, C-terminal Src kinase (Csk).

    Lee S, Ayrapetov MK, Kemble DJ, Parang K and Sun G

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

    Protein tyrosine kinases are key enzymes of mammalian signal transduction. Substrate specificity is a fundamental property that determines the specificity and fidelity of signaling by protein tyrosine kinases. However, how protein tyrosine kinases recognize the protein substrates is not well understood. C-terminal Src kinase (Csk) specifically phosphorylates Src family kinases on a C-terminal Tyr residue, which down-regulates their activities. We have previously determined that Csk recognizes Src using a substrate-docking site away from the active site. In the current study, we identified the docking determinants in Src recognized by the Csk substrate-docking site and demonstrated an interaction between the docking determinants of Src and the Csk substrate-docking site for this recognition. A similar mechanism was confirmed for Csk recognition of another Src family kinase, Yes. Although both Csk and MAP kinases used docking sites for substrate recognition, their docking sites consisted of different substructures in the catalytic domain. These results helped establish a docking-based substrate recognition mechanism for Csk. This model may provide a framework for understanding substrate recognition and specificity of other protein tyrosine kinases.

    Funded by: NCI NIH HHS: 1R01CA111687; NCRR NIH HHS: 1 P20 RR16457

    The Journal of biological chemistry 2006;281;12;8183-9

  • 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

  • Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes.

    Kimura K, Wakamatsu A, Suzuki Y, Ota T, Nishikawa T, Yamashita R, Yamamoto J, Sekine M, Tsuritani K, Wakaguri H, Ishii S, Sugiyama T, Saito K, Isono Y, Irie R, Kushida N, Yoneyama T, Otsuka R, Kanda K, Yokoi T, Kondo H, Wagatsuma M, Murakawa K, Ishida S, Ishibashi T, Takahashi-Fujii A, Tanase T, Nagai K, Kikuchi H, Nakai K, Isogai T and Sugano S

    Life Science Research Laboratory, Central Research Laboratory, Hitachi, Ltd., Kokubunji, Tokyo, 185-8601, Japan.

    By analyzing 1,780,295 5'-end sequences of human full-length cDNAs derived from 164 kinds of oligo-cap cDNA libraries, we identified 269,774 independent positions of transcriptional start sites (TSSs) for 14,628 human RefSeq genes. These TSSs were clustered into 30,964 clusters that were separated from each other by more than 500 bp and thus are very likely to constitute mutually distinct alternative promoters. To our surprise, at least 7674 (52%) human RefSeq genes were subject to regulation by putative alternative promoters (PAPs). On average, there were 3.1 PAPs per gene, with the composition of one CpG-island-containing promoter per 2.6 CpG-less promoters. In 17% of the PAP-containing loci, tissue-specific use of the PAPs was observed. The richest tissue sources of the tissue-specific PAPs were testis and brain. It was also intriguing that the PAP-containing promoters were enriched in the genes encoding signal transduction-related proteins and were rarer in the genes encoding extracellular proteins, possibly reflecting the varied functional requirement for and the restricted expression of those categories of genes, respectively. The patterns of the first exons were highly diverse as well. On average, there were 7.7 different splicing types of first exons per locus partly produced by the PAPs, suggesting that a wide variety of transcripts can be achieved by this mechanism. Our findings suggest that use of alternate promoters and consequent alternative use of first exons should play a pivotal role in generating the complexity required for the highly elaborated molecular systems in humans.

    Genome research 2006;16;1;55-65

  • The c-Yes 3'-UTR contains adenine/uridine-rich elements that bind AUF1 and HuR involved in mRNA decay in breast cancer cells.

    Sommer S, Cui Y, Brewer G and Fuqua SA

    Breast Center, Baylor College of Medicine and The Methodist Hospital, Houston, TX 77030, USA.

    c-Yes is a member of the c-Src family of tyrosine kinases and has been implicated in intracellular signaling, cell morphology, and adhesion. Changes in its expression have also been associated with the aggressiveness of human breast and colon cancer cells. In MDA-MB-231 human breast cancer cells, overexpression of the small heat shock protein 27 (hsp27) results in a downregulation of c-Yes levels, concomitant with increased in vitro invasiveness and in vivo metastatic behavior. Very little is known, however, about the mechanisms regulating c-Yes expression. Here, we demonstrate that hsp27-induced c-Yes downregulation is not due to a reduction in transcriptional activity. However, the 3'-untranslated region (3'-UTR) of the c-Yes gene may be involved in its own regulation, since this region affects heterologous reporter gene activity in transactivation assays. This down-regulatory effect maps to three adenine/uridine-rich elements (AREs) that bind to cellular HuR and AUF1 (hnRNP D), two ARE-binding proteins (ARE-BPs) implicated in accelerated mRNA degradation. Our results suggest that the c-Yes 3'-UTR contains at least three newly identified AREs which are bound specifically by ARE-BPs, and provide a structural basis for post-transcriptional regulation of c-Yes expression.

    Funded by: NCI NIH HHS: CA58183, P50 CA58183

    The Journal of steroid biochemistry and molecular biology 2005;97;3;219-29

  • Time-resolved mass spectrometry of tyrosine phosphorylation sites in the epidermal growth factor receptor signaling network reveals dynamic modules.

    Zhang Y, Wolf-Yadlin A, Ross PL, Pappin DJ, Rush J, Lauffenburger DA and White FM

    Biological Engineering Division, Massachusetts Institute of Technnology, Cambridge, Massachusetts 02139, USA.

    Ligand binding to cell surface receptors initiates a cascade of signaling events regulated by dynamic phosphorylation events on a multitude of pathway proteins. Quantitative features, including intensity, timing, and duration of phosphorylation of particular residues, may play a role in determining cellular response, but experimental data required for analysis of these features have not previously been available. To understand the dynamic operation of signaling cascades, we have developed a method enabling the simultaneous quantification of tyrosine phosphorylation of specific residues on dozens of key proteins in a time-resolved manner, downstream of epidermal growth factor receptor (EGFR) activation. Tryptic peptides from four different EGFR stimulation time points were labeled with four isoforms of the iTRAQ reagent to enable downstream quantification. After mixing of the labeled samples, tyrosine-phosphorylated peptides were immunoprecipitated with an anti-phosphotyrosine antibody and further enriched by IMAC before LC/MS/MS analysis. Database searching and manual confirmation of peptide phosphorylation site assignments led to the identification of 78 tyrosine phosphorylation sites on 58 proteins from a single analysis. Replicate analyses of a separate biological sample provided both validation of this first data set and identification of 26 additional tyrosine phosphorylation sites and 18 additional proteins. iTRAQ fragment ion ratios provided time course phosphorylation profiles for each site. The data set of quantitative temporal phosphorylation profiles was further characterized by self-organizing maps, which resulted in identification of several cohorts of tyrosine residues exhibiting self-similar temporal phosphorylation profiles, operationally defining dynamic modules in the EGFR signaling network consistent with particular cellular processes. The presence of novel proteins and associated tyrosine phosphorylation sites within these modules indicates additional components of this network and potentially localizes the topological action of these proteins. Additional analysis and modeling of the data generated in this study are likely to yield more sophisticated models of receptor tyrosine kinase-initiated signal transduction, trafficking, and regulation.

    Funded by: NCI NIH HHS: CA96504; NIDDK NIH HHS: DK070172, DK42816; NIGMS NIH HHS: GM68762

    Molecular & cellular proteomics : MCP 2005;4;9;1240-50

  • Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry.

    Tao WA, Wollscheid B, O'Brien R, Eng JK, Li XJ, Bodenmiller B, Watts JD, Hood L and Aebersold R

    The Bindley Bioscience Center and Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907, USA.

    We present a robust and general method for the identification and relative quantification of phosphorylation sites in complex protein mixtures. It is based on a new chemical derivatization strategy using a dendrimer as a soluble polymer support and tandem mass spectrometry (MS/MS). In a single step, phosphorylated peptides are covalently conjugated to a dendrimer in a reaction catalyzed by carbodiimide and imidazole. Modified phosphopeptides are released from the dendrimer via acid hydrolysis and analyzed by MS/MS. When coupled with an initial antiphosphotyrosine protein immunoprecipitation step and stable-isotope labeling, in a single experiment, we identified all known tyrosine phosphorylation sites within the immunoreceptor tyrosine-based activation motifs (ITAM) of the T-cell receptor (TCR) CD3 chains, and previously unknown phosphorylation sites on total 97 tyrosine phosphoproteins and their interacting partners in human T cells. The dynamic changes in phosphorylation were quantified in these proteins.

    Funded by: NHLBI NIH HHS: N01-HV-28179

    Nature methods 2005;2;8;591-8

  • 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

  • Immunoaffinity profiling of tyrosine phosphorylation in cancer cells.

    Rush J, Moritz A, Lee KA, Guo A, Goss VL, Spek EJ, Zhang H, Zha XM, Polakiewicz RD and Comb MJ

    Cell Signaling Technology Inc., 166B Cummings Center, Beverly, Massachusetts 01915, USA.

    Tyrosine kinases play a prominent role in human cancer, yet the oncogenic signaling pathways driving cell proliferation and survival have been difficult to identify, in part because of the complexity of the pathways and in part because of low cellular levels of tyrosine phosphorylation. In general, global phosphoproteomic approaches reveal small numbers of peptides containing phosphotyrosine. We have developed a strategy that emphasizes the phosphotyrosine component of the phosphoproteome and identifies large numbers of tyrosine phosphorylation sites. Peptides containing phosphotyrosine are isolated directly from protease-digested cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. Applying this approach to several cell systems, including cancer cell lines, shows it can be used to identify activated protein kinases and their phosphorylated substrates without prior knowledge of the signaling networks that are activated, a first step in profiling normal and oncogenic signaling networks.

    Funded by: NCI NIH HHS: 1R43CA101106

    Nature biotechnology 2005;23;1;94-101

  • 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

  • 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

  • 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

  • The Src family of tyrosine kinases is important for embryonic stem cell self-renewal.

    Annerén C, Cowan CA and Melton DA

    Howard Hughes Medical Institute and Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.

    cYes, a member of the Src family of non-receptor tyrosine kinases, is highly expressed in mouse and human embryonic stem (ES) cells. We demonstrate that cYes kinase activity is regulated by leukemia inhibitory factor (LIF) and serum and is down-regulated when cells differentiate. Moreover, selective chemical inhibition of Src family kinases decreases growth and expression of stem cell genes that mark the undifferentiated state, including Oct3/4, alkaline phosphatase, fibroblast growth factor 4, and Nanog. A synergistic effect on differentiation is observed when ES cells are cultured with an Src family inhibitor and low levels of retinoic acid. Src family kinase inhibition does not interfere with LIF-induced JAK/STAT3 (Janus-associated tyrosine kinases/signal transducer and activator of transcription 3) or p42/p44 MAPK (mitogen-activated protein kinase) phosphorylation. Together the results suggest that the activation of the Src family is important for maintaining mouse and human ES in an undifferentiated state and may represent a third, independent pathway, downstream of LIF in mouse ES cells.

    The Journal of biological chemistry 2004;279;30;31590-8

  • The Src family kinase Yes triggers hyperosmotic activation of the epidermal growth factor receptor and CD95.

    Reinehr R, Becker S, Höngen A and Haüssinger D

    Clinic for Gastroenterology, Hepatology and Infectiology, Heinrich-Heine-University, Moorenstrasse 5, Düsseldorf D-40225, Germany.

    Hyperosmotic exposure of rat hepatocytes triggers epidermal growth factor receptor (EGFR) activation, which results in an activation of the CD95 system and sensitizes the cells toward apoptosis (Reinehr, R., Schliess, F., and Haüssinger, D. (2003) FASEB J. 17, 731-733). The mechanisms underlying the hyperosmotic EGFR activation were studied. Hyperosmotic exposure (405 mosm) resulted in a rapid activation of the Src kinase family members Yes, Fyn, and Lck. Hyperosmotic Yes, but not Fyn activation, was antioxidant-sensitive and was followed by a rapid Yes/EGFR association. PP-2 abolished the hyperosmotic activation of Fyn and Lck but not activation of Yes and EGFR and their association. However, these latter processes were prevented in the presence of SU6656. SU6656 and antioxidants, but not PP-2 and AG1478, also inhibited the hyperosmotic JNK activation. Cyclic AMP had no effect on hyperosmotic Yes and JNK activation but prevented EGFR/Yes association and EGFR activation in an H89-sensitive way. When the hyperosmolarity-induced Yes-EGFR protein complex started to disappear after 30 min, an association between EGFR and CD95 became apparent, which was followed by CD95 tyrosine phosphorylation and activation. SU6656 but not PP-2 also inhibited EGFR/CD95 association, CD95 tyrosine phosphorylation, CD95 membrane trafficking, and death-inducing signaling complex (DISC) formation. EGFR knockdown had no effect on hyperosmotic Yes activation but prevented CD95 tyrosine phosphorylation, membrane targeting, and DISC formation. Hyperosmotic EGFR and CD95 activation was also largely blunted following Yes knockdown. The data suggest that hyperosmotic signaling triggers an oxidative stress-dependent Yes activation, which is followed by JNK and EGFR activation and subsequent activation of the CD95 system. However, the functional relevance of hyperosmolarity-induced Fyn and Lck activation remains to be elucidated.

    The Journal of biological chemistry 2004;279;23;23977-87

  • Protein tyrosine phosphatase epsilon activates Yes and Fyn in Neu-induced mammary tumor cells.

    Granot-Attas S and Elson A

    Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot 76100, Israel.

    The receptor-type form of protein tyrosine phosphatase epsilon (RPTP) is among the few tyrosine phosphatases that can support the transformed phenotype of tumor cells. Accordingly, cells from mammary epithelial tumors induced by activated Neu in mice genetically lacking RPTP appear morphologically less transformed and exhibit reduced proliferation. The effect of RPTP in these cells is mediated at least in part by its ability to activate Src, the prototypic member of a family of related kinases. We show here that RPTP is a physiological activator of two additional Src family kinases, Yes and Fyn. Activities of both kinases are inhibited in mammary tumor cells lacking RPTP, and phosphorylation at their C-terminal inhibitory tyrosines is increased. In agreement, opposite effects on activities and phosphorylation of Yes and Fyn are observed following increased expression of PTP. RPTP also forms stable complexes with either kinase, providing physical opportunity for their activation by RPTP. Surprisingly, expression of Yes or of Fyn does not rescue the morphological phenotype of RPTP-deficient tumor cells in contrast with the strong ability of Src to do so. We conclude that RPTP activates Src, Yes, and Fyn, but that these related kinases play distinct roles in Neu-induced mammary tumor cells.

    Experimental cell research 2004;294;1;236-43

  • 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

  • Fyn binds to and phosphorylates the kidney slit diaphragm component Nephrin.

    Verma R, Wharram B, Kovari I, Kunkel R, Nihalani D, Wary KK, Wiggins RC, Killen P and Holzman LB

    Department of Veterans Affairs, Ann Arbor, Michigan 48105, USA.

    Recent investigations have focused on characterizing the molecular components of the podocyte intercellular junction, because several of these components, including Nephrin, are functionally necessary for development of normal podocyte structure and filter integrity. Accumulating evidence suggests that the Nephrin-associated protein complex is a signaling nexus. As such, Nephrin-dependent signaling might be mediated in part through Nephrin phosphorylation. Described are biochemical and mouse genetics experiments demonstrating that membrane-associated Nephrin is tyrosine-phosphorylated by the Src family kinase Fyn. Nephrin fractionated in detergent-resistant glomerular membrane fractions with Fyn and Yes. Fyn directly bound Nephrin via its SH3 domain, and Fyn directly phosphorylated Nephrin. Glomeruli in which Fyn, Yes, or Fyn and Yes were genetically deleted in mice were characterized to explore the relationship between these kinases and Nephrin. Fyn deletion resulted in coarsening of podocyte foot processes and marked attenuation of Nephrin phosphorylation in isolated glomerular detergent-resistant membrane fractions. Yes deletion had no identifiable effect on podocyte morphology but dramatically increased Nephrin phosphorylating activity. Similar to Fyn deletion, simultaneous deletion of Fyn and Yes reduced Nephrin phosphorylating activity. These results demonstrate that endogenous Fyn catalyzes Nephrin phosphorylation in podocyte detergent-resistant membrane fractions. Although Yes appears to effect the regulation of Nephrin phosphorylation, the mechanism by which this occurs requires investigation.

    The Journal of biological chemistry 2003;278;23;20716-23

  • 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

  • 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

  • Specificity in signaling by c-Yes.

    Summy JM, Sudol M, Eck MJ, Monteiro AN, Gatesman A and Flynn DC

    Dept. of Microbiology and Immunology and Mary Babb Randolph Cancer Center, West Virginia University School of Medicine, Morgantown WV 26506-9300, USA.

    c-Yes and c-Src are the two most closely related members of the Src family of nonreceptor tyrosine kinases. Although there is much evidence to support redundancy in signaling between these two kinases, there is also a growing body of evidence to indicate specificity in signaling. In this review, we summarize c-Yes, its potential functions and its ability to modulate signals that are distinct from c-Src.

    Funded by: NCI NIH HHS: CA60731, CA92309; NCRR NIH HHS: RR16640; NIEHS NIH HHS: T32 ES10953

    Frontiers in bioscience : a journal and virtual library 2003;8;s185-205

  • 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

  • Src kinase becomes preferentially associated with the VEGFR, KDR/Flk-1, following VEGF stimulation of vascular endothelial cells.

    Chou MT, Wang J and Fujita DJ

    Department of Biochemistry and Molecular Biology, University of Calgary Health Sciences Center, 3330 Hospital Dr, N,W, Calgary, AB, Canada T2N 4N1. mthchou@ucalgary.ca

    Background: The cytoplasmic tyrosine kinase, Src, has been found to play a crucial role in VEGF (vascular endothelial growth factor) - dependent vascular permeability involved in angiogenesis. The two main VEGFRs present on vascular endothelial cells are KDR/Flk-1 (kinase insert domain-containing receptor/fetal liver kinase-1) and Flt-1 (Fms-like tyrosine kinase-1). However, to date, it has not been determined which VEGF receptor (VEGFR) is involved in binding to and activating Src kinase following VEGF stimulation of the receptors.

    Results: In this report, we demonstrate that Src preferentially associates with KDR/Flk-1 rather than Flt-1 in human umbilical vein endothelial cells (HUVECs), and that VEGF stimulation resulted in an increase of Src activity associated with activated KDR/Flk-1. These findings were determined through immunoprecipitation-kinase experiments and coimmunoprecipitation studies, and were further confirmed by GST-pull-down assays and Far Western studies. However, Fyn and Yes, unlike Src, were found to associate preferentially with Flt-1.

    Conclusions: Thus, Src preferentially associates with KDR/Flk-1, rather than with Flt-1, upon VEGF stimulation in endothelial cells. Our findings further highlight the potential significance of upregulated KDR/Flk-1-associated Src activity in the process of angiogenesis, and help to elucidate more clearly the specific roles and mechanisms involving Src family tyrosine kinase in VEGF-stimulated signal transduction events.

    BMC biochemistry 2002;3;32

  • QM, a putative tumor suppressor, regulates proto-oncogene c-yes.

    Oh HS, Kwon H, Sun SK and Yang CH

    Division of Chemistry and Molecular Engineering, Seoul National University, Seoul 151-742, Korea.

    The QM gene encodes a 24.5 kDa ribosomal protein L10 known to be highly homologous to a Jun-binding protein (Jif-1), which inhibits the formation of Jun-Jun dimers. Here we have carried out screening with the c-Yes protein and found that a QM homologous protein showed interactions with c-Yes and other Src family members. We have found that two different regions of QM protein were associated with the SH3 domain of c-Yes. The QM protein does not contain canonical SH3 binding motifs or previously reported amino acid fragments showing interaction with SH3 domains. Several c-Yes kinase activity assays indicated that the QM protein reduced c-Yes kinase activity by 70% and that this suppression is related not only to the two SH3 binding regions but also to the C-terminal region of QM. Moreover, our autophosphorylation assays clarified that this regulation resulted from the inhibition of c-Yes autophosphorylation. Immunofluorescence studies showed that the QM proteins and c-Yes are able to interact in various tumor cell lines in vivo. The increases of the c-Yes protein and mRNA levels were detected when the QM was transfected. These results suggest that the QM protein might be a regulator for various signal transduction pathways involving SH3 domain-containing membrane proteins.

    The Journal of biological chemistry 2002;277;39;36489-98

  • Regulation of the human sperm tyrosine kinase c-yes. Activation by cyclic adenosine 3',5'-monophosphate and inhibition by Ca(2+).

    Leclerc P and Goupil S

    Endocrinologie de la Reproduction, Department of OB/GYN, Centre de Recherche du CHUQ, Université Laval, Pavillon Saint-François d'Assise 10, de l'Espinay, Quebec, PQ, Canada G1L 3L5. pierre.leclerc@crsfa.ulaval.ca

    During the process of capacitation, spermatozoa go through a whole set of signaling cascade events in order to become fully competent at fertilizing the egg. An increase in sperm protein tyrosine phosphorylation has been described during this final maturational event in different animal species as well as in humans. Although the phosphotyrosine content of sperm protein is modulated by cAMP, Ca(2+), BSA, oxygen derivatives, and cholesterol, no protein tyrosine kinase (PTK) nor the phosphotyrosine protein phosphatase (PTPase) directly involved in the control of the phosphotyrosine content of sperm protein has been identified. Therefore, the goal of the present study was to identify the tyrosine kinases putatively responsible for the increases in sperm protein phosphotyrosine content. In the present study, we show that the src-related tyrosine kinase c-yes is present in the head of human spermatozoa in both membranes and Triton X-100-insoluble extracts. Our hypothesis was that c-yes is a tyrosine kinase responsible for at least some of the capacitation-induced increase in protein tyrosine phosphorylation. When spermatozoa were previously incubated in the presence of 3-isobutyl-1-methylxanthine or 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, treatments known to increase the phosphotyrosine content of human sperm proteins, an increase in the kinase activity of immunoprecipitated yes was measured using enolase as a substrate. These results suggest that cAMP activates while Ca(2+) inhibits human sperm c-yes kinase activity.

    Biology of reproduction 2002;67;1;301-7

  • Nonreceptor tyrosine kinase c-Yes interacts with occludin during tight junction formation in canine kidney epithelial cells.

    Chen YH, Lu Q, Goodenough DA and Jeansonne B

    Department of Anatomy and Cell Biology, East Carolina University School of Medicine, Greenville, NC 27858, USA. cheny@mail.ecu.edu

    Occludin is an integral membrane protein that is tyrosine phosphorylated when localized at tight junctions. When Ca(2+) was depleted from the culture medium, occludin tyrosine phosphorylation was diminished from Madin-Darby canine kidney epithelial cells in 2 min. This dephosphorylation was correlated with a significant reduction in transepithelial electrical resistance (TER), indicating a global loss of the tight junction barrier function. Reconstitution of Ca(2+) resulted in a robust tyrosine rephosphorylation of occludin that was temporally associated with an increase in TER. Moreover, we demonstrate in this study that occludin was colocalized with the nonreceptor tyrosine kinase c-Yes at cell junction areas and formed an immunoprecipitable complex with c-Yes in vivo. This complex dissociated when the cells were incubated in medium without Ca(2+) or treated with a c-Yes inhibitor, CGP77675. In the presence of CGP77675 after Ca(2+) repletion, occludin tyrosine phosphorylation was completely abolished and both tight junction formation and the increase of the TER were inhibited. Our study thus provides strong evidence that occludin tyrosine phosphorylation is tightly linked to tight junction formation in epithelial cells, and that the nonreceptor tyrosine kinase c-Yes is involved in the regulation of this process.

    Funded by: NIDDK NIH HHS: DK-34854, P30 DK034854; NIGMS NIH HHS: GM-18974, GM-37751, R01 GM018974, R01 GM037751

    Molecular biology of the cell 2002;13;4;1227-37

  • CD46 is phosphorylated at tyrosine 354 upon infection of epithelial cells by Neisseria gonorrhoeae.

    Lee SW, Bonnah RA, Higashi DL, Atkinson JP, Milgram SL and So M

    Department of Molecular Microbiology and Immunology, L220, Oregon Health and Science University, Portland, OR 97201.

    The Neisseria type IV pilus promotes bacterial adhesion to host cells. The pilus binds CD46, a complement-regulatory glycoprotein present on nucleated human cells (Källström et al., 1997). CD46 mutants with truncated cytoplasmic tails fail to support bacterial adhesion (Källström et al., 2001), suggesting that this region of the molecule also plays an important role in infection. Here, we report that infection of human epithelial cells by piliated Neisseria gonorrhoeae (GC) leads to rapid tyrosine phosphorylation of CD46. Studies with wild-type and mutant tail fusion constructs demonstrate that Src kinase phosphorylates tyrosine 354 in the Cyt2 isoform of the CD46 cytoplasmic tail. Consistent with these findings, infection studies show that PP2, a specific Src family kinase inhibitor, but not PP3, an inactive variant of this drug, reduces the ability of epithelial cells to support bacterial adhesion. Several lines of evidence point to the role of c-Yes, a member of the Src family of nonreceptor tyrosine kinases, in CD46 phosphorylation. GC infection causes c-Yes to aggregate in the host cell cortex beneath adherent bacteria, increases binding of c-Yes to CD46, and stimulates c-Yes kinase activity. Finally, c-Yes immunoprecipitated from epithelial cells is able to phosphorylate the wild-type Cyt2 tail but not the mutant derivative in which tyrosine 354 has been substituted with alanine. We conclude that GC infection leads to rapid tyrosine phosphorylation of the CD46 Cyt2 tail and that the Src kinase c-Yes is involved in this reaction. Together, the findings reported here and elsewhere strongly suggest that pilus binding to CD46 is not a simple static process. Rather, they support a model in which pilus interaction with CD46 promotes signaling cascades important for Neisseria infectivity.

    Funded by: NHLBI NIH HHS: HL63755, R01 HL063755; NIAID NIH HHS: AI32493, AI4997301, R01 AI032493

    The Journal of cell biology 2002;156;6;951-7

  • beta -Arrestin-mediated recruitment of the Src family kinase Yes mediates endothelin-1-stimulated glucose transport.

    Imamura T, Huang J, Dalle S, Ugi S, Usui I, Luttrell LM, Miller WE, Lefkowitz RJ and Olefsky JM

    Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, California 92093-0673, USA.

    The insulin and the endothelin type A (ETA) receptor both can couple into the heterotrimeric G protein alpha(q/11) (Galpha(q/11)), leading to Galpha(q/11) tyrosine phosphorylation, phosphatidylinositol 3-kinase activation, and subsequent stimulation of glucose transport. In this study, we assessed the potential role of Src kinase in ET-1 signaling to glucose transport in 3T3-L1 adipocytes. Src kinase inhibitor PP2 blocked ET-1-induced Src kinase activity, Galpha(q/11) tyrosine phosphorylation, and glucose transport stimulation. To determine which Src family kinase member was involved, we microinjected anti-c-Src, -c-Fyn, or -c-Yes antibody into these cells and found that only anti-c-Yes antibody blocked GLUT4 translocation (70% decreased). Overexpression or microinjection of a dominant negative mutant (K298M) of Src kinase also inhibited ET-1-induced Galpha(q/11) tyrosine phosphorylation and GLUT4 translocation. In co-immunoprecipitation experiments, we found that beta-arrestin 1 associated with the ETA receptor in an agonist-dependent manner and that beta-arrestin 1 recruited Src kinase to a molecular complex that included the ETA receptor. Microinjection of beta-arrestin 1 antibody inhibited ET-1- but not insulin-stimulated GLUT4 translocation. In conclusion, 1) the Src kinase Yes can induce tyrosine phosphorylation of Galpha(q/11) in response to ET-1 stimulation, and 2) beta-arrestin 1 and Src kinase form a molecular complex with the ETA receptor to mediate ET-1 signaling to Galpha(q/11) with subsequent glucose transport stimulation.

    Funded by: NIDDK NIH HHS: DK-33651

    The Journal of biological chemistry 2001;276;47;43663-7

  • EGF-R signaling through Fyn kinase disrupts the function of integrin alpha6beta4 at hemidesmosomes: role in epithelial cell migration and carcinoma invasion.

    Mariotti A, Kedeshian PA, Dans M, Curatola AM, Gagnoux-Palacios L and Giancotti FG

    Cellular Biochemistry and Biophysics Program, Department of Surgery, Sloan-Kettering Institute for Cancer Research, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.

    We have examined the mechanism and functional significance of hemidesmosome disassembly during normal epithelial cell migration and squamous carcinoma invasion. Our findings indicate that a fraction of EGF receptor (EGF-R) combines with the hemidesmosomal integrin alpha6beta4 in both normal and neoplastic keratinocytes. Activation of the EGF-R causes tyrosine phosphorylation of the beta4 cytoplasmic domain and disruption of hemidesmosomes. The Src family kinase inhibitors PP1 and PP2 prevent tyrosine phosphorylation of beta4 and disassembly of hemidesmosomes without interfering with the activation of EGF-R. Coimmunoprecipitation experiments indicate that Fyn and, to a lesser extent, Yes combine with alpha6beta4. By contrast, Src and Lck do not associate with alpha6beta4 to a significant extent. A dominant negative form of Fyn, but not Src, prevents tyrosine phosphorylation of beta4 and disassembly of hemidesmosomes. These observations suggest that the EGF-R causes disassembly of hemidesmosomes by activating Fyn, which in turn phosphorylates the beta4 cytoplasmic domain. Neoplastic cells expressing dominant negative Fyn display increased hemidesmosomes and migrate poorly in vitro in response to EGF. Furthermore, dominant negative Fyn decreases the ability of squamous carcinoma cells to invade through Matrigel in vitro and to form lung metastases following intravenous injection in nude mice. These results suggest that disruption of hemidesmosomes mediated by Fyn is a prerequisite for normal keratinocyte migration and squamous carcinoma invasion.

    Funded by: NCI NIH HHS: P30 CA008748, P30 CA08748, R01 CA058976, R01 CA58976

    The Journal of cell biology 2001;155;3;447-58

  • Src, Fyn, and Yes are not required for neuromuscular synapse formation but are necessary for stabilization of agrin-induced clusters of acetylcholine receptors.

    Smith CL, Mittaud P, Prescott ED, Fuhrer C and Burden SJ

    Molecular Neurobiology Program, Skirball Institute for Biomolecular Medicine, New York University Medical School, New York, New York 10016, USA.

    Mice deficient in src and fyn or src and yes move and breathe poorly and die perinatally, consistent with defects in neuromuscular function. Src and Fyn are associated with acetylcholine receptors (AChRs) in muscle cells, and Src and Yes can act downstream of ErbB2, suggesting roles for Src family kinases in signaling pathways regulating neuromuscular synapse formation. We studied neuromuscular synapses in src(-/-); fyn(-/-) and src(-/-); yes(-/-) mutant mice and found that muscle development, motor axon pathfinding, clustering of postsynaptic proteins, and synapse-specific transcription are normal in these double mutants, showing that these pairs of kinases are not required for early steps in synapse formation. We generated muscle cell lines lacking src and fyn and found that neural agrin and laminin-1 induced normal clustering of AChRs and that agrin induced normal tyrosine phosphorylation of the AChR beta subunit in the absence of Src and Fyn. Another Src family member, most likely Yes, was associated with AChRs and phosphorylated by agrin in myotubes lacking Src and Fyn, indicating that Yes may compensate for the loss of Src and Fyn. Nevertheless, PP1 and PP2, inhibitors of Src-class kinases, did not inhibit agrin signaling, suggesting that Src class kinase activity is dispensable for agrin-induced clustering and tyrosine phosphorylation of AChRs. AChR clusters, however, were less stable in myotubes lacking Src and Fyn but not in PP1- or PP2-treated wild-type cells. These data show that the stabilization of agrin-induced AChR clusters requires Src and Fyn and suggest that the adaptor activities, rather than the kinase activities, of these kinases are essential for this stabilization.

    Funded by: NINDS NIH HHS: NS27963, NS36193, R01 NS036193, R01 NS036193-04

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2001;21;9;3151-60

  • Yes-associated protein and p53-binding protein-2 interact through their WW and SH3 domains.

    Espanel X and Sudol M

    Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York, New York 10029-6574, USA. xavier.espanel@serono.com

    To understand the role of the Yes-associated protein (YAP), binding partners of its WW1 domain were isolated by a yeast two-hybrid screen. One of the interacting proteins was identified as p53-binding protein-2 (p53BP-2). YAP and p53BP-2 interacted in vitro and in vivo using their WW1 and SH3 domains, respectively. The YAP WW1 domain bound to the YPPPPY motif of p53BP-2, whereas the p53BP-2 SH3 domain interacted with the VPMRLR sequence of YAP, which is different from other known SH3 domain-binding motifs. By mutagenesis, we showed that this unusual SH3 domain interaction was due to the presence of three consecutive tryptophans located within the betaC strand of the SH3 domain. A point mutation within this triplet, W976R, restored the binding selectivity to the general consensus sequence for SH3 domains, the PXXP motif. A constitutively active form of c-Yes was observed to decrease the binding affinity between YAP and p53BP-2 using chloramphenicol acetyltransferase/enzyme-linked immunosorbent assay, whereas the overexpression of c-Yes did not modify this interaction. Since overexpression of an activated form of c-Yes resulted in tyrosine phosphorylation of p53BP-2, we propose that the p53BP-2 phosphorylation, possibly in the WW1 domain-binding motif, might negatively regulate the YAP.p53BP-2 complex.

    Funded by: NCI NIH HHS: CA45757; NIAMS NIH HHS: AR45626

    The Journal of biological chemistry 2001;276;17;14514-23

  • Meltrin alpha cytoplasmic domain interacts with SH3 domains of Src and Grb2 and is phosphorylated by v-Src.

    Suzuki A, Kadota N, Hara T, Nakagami Y, Izumi T, Takenawa T, Sabe H and Endo T

    Department of Biology, Faculty of Science, Chiba University, Japan.

    Meltrin alpha/ADAM12 is a member of the ADAM/MDC family proteins characterized by the presence of metalloprotease and disintegrin domains. This protein also contains a single transmembrane domain and a relatively long cytoplasmic domain containing several proline-rich sequences. These sequences are compatible with the consensus sequences for binding the Src homology 3 (SH3) domains. To determine whether the proline-rich sequences interact with SH3 domains in several proteins, binding of recombinant SH3 domains to the meltrin alpha cytoplasmic domain was analysed by pull-down assays. The SH3 domains of Src and Yes bound strongly, but that of Abl or phosphatidylinositol 3-kinase p85 subunit did not. Full-length Grb2/Ash bound strongly, whereas its N-terminal SH3 domain alone did less strongly. Src and Grb2 in bovine brain extracts also bound to meltrin alpha cytoplasmic domain on affinity resin. Furthermore, immunoprecipitation with a monoclonal antibody to meltrin alpha resulted in coprecipitation of Src and Grb2 with meltrin alpha in cell extracts, suggesting that Src and Grb2 are associated in vivo with meltrin alpha cytoplasmic domain. This notion was also supported by the findings that exogenously expressed meltrin cytoplasmic domain coexisted with Src and Grb2 on the membrane ruffles. The C-terminal Tyr901 of meltrin alpha was phosphorylated both in vitro and in cultured cells by v-Src. These results may imply that meltrin alpha cytoplasmic domain is involved in a signal transduction for some biological function through the interaction with SH3-containing proteins.

    Oncogene 2000;19;51;5842-50

  • Stem cell factor induces phosphatidylinositol 3'-kinase-dependent Lyn/Tec/Dok-1 complex formation in hematopoietic cells.

    van Dijk TB, van Den Akker E, Amelsvoort MP, Mano H, Löwenberg B and von Lindern M

    Institute of Hematology, Erasmus University Rotterdam, Rotterdam, The Netherlands. vandijk@hema.fgg.eur.nl

    Stem cell factor (SCF) has an important role in the proliferation, differentiation, survival, and migration of hematopoietic cells. SCF exerts its effects by binding to cKit, a receptor with intrinsic tyrosine kinase activity. Activation of phosphatidylinositol 3'-kinase (PI3-K) by cKit was previously shown to contribute to many SCF-induced cellular responses. Therefore, PI3-K-dependent signaling pathways activated by SCF were investigated. The PI3-K-dependent activation and phosphorylation of the tyrosine kinase Tec and the adapter molecule p62Dok-1 are reported. The study shows that Tec and Dok-1 form a stable complex with Lyn and 2 unidentified phosphoproteins of 56 and 140 kd. Both the Tec homology and the SH2 domain of Tec were identified as being required for the interaction with Dok-1, whereas 2 domains in Dok-1 appeared to mediate the association with Tec. In addition, Tec and Lyn were shown to phosphorylate Dok-1, whereas phosphorylated Dok-1 was demonstrated to bind to the SH2 domains of several signaling molecules activated by SCF, including Abl, CrkL, SHIP, and PLCgamma-1, but not those of Vav and Shc. These findings suggest that p62Dok-1 may function as an important scaffold molecule in cKit-mediated signaling.

    Blood 2000;96;10;3406-13

  • GP2, a GPI-anchored protein in the apical plasma membrane of the pancreatic acinar cell, co-immunoprecipitates with src kinases and caveolin.

    Parker EM, Zaman MM and Freedman SD

    The Pancreas Center, Beth Israel Deaconess Medical Center, Harvard Medical Schlool, Boston, Massachusetts 02215, USA.

    We previously showed that endocytosis at the apical plasma membrane (APM) of the pancreatic acinar cell is activated by the cleavage of GP2, a GPI-linked protein, from the apical cell surface. This endocytic process, as measured by horseradish peroxidase uptake into pancreatic acinar cells, is blocked by the tyrosine kinase inhibitors genistein and tyrphostin B42 as well as by disruption of actin filaments with cytochalasin. This suggests that the cleavage of GP2 from the cell membrane may activate endocytosis through a tyrosine kinase-regulated pathway. However, the mechanism by which GP2 and tyrosine kinases act together to activate endocytosis at the APM remains unknown. In this study, we demonstrate that pp60, p62yes, caveolin, and annexin, which have previously been implicated in endocytosis in other cell lines, were present in high abundance in GPI-enriched membranes by Western blot analysis. pp60, p62yes, and caveolin all co-immunoprecipitated with GP2 except annexin. An 85-kDa protein whose tyrosine-dependent phosphorylation is correlated with the activation of endocytosis in intact acinar cells also was present in these immunoprecipitates. This suggests that in pancreatic acini, GP2 may exist in a complex with src kinases, caveolin, and an 85-kDa phosphorylated substrate to regulate endocytosis at the APM.

    Funded by: NIDDK NIH HHS: R01 DK-52765

    Pancreas 2000;21;3;219-25

  • Collagen, convulxin, and thrombin stimulate aggregation-independent tyrosine phosphorylation of CD31 in platelets. Evidence for the involvement of Src family kinases.

    Cicmil M, Thomas JM, Sage T, Barry FA, Leduc M, Bon C and Gibbins JM

    School of Animal and Microbial Sciences, University of Reading, Whiteknights, Reading, Berkshire RG6 6AJ, United Kingdom.

    Platelet endothelial cell adhesion molecule-1 (CD31) is a 130-kDa glycoprotein receptor present on the surface of platelets, neutrophils, monocytes, certain T-lymphocytes, and vascular endothelial cells. CD31 is involved in adhesion and signal transduction and is implicated in the regulation of a number of cellular processes. These include transendothelial migration of leukocytes, integrin regulation, and T-cell function, although its function in platelets remains unclear. In this study, we demonstrate the ability of the platelet agonists collagen, convulxin, and thrombin to induce tyrosine phosphorylation of CD31. Furthermore, we show that this event is independent of platelet aggregation and secretion and is accompanied by an increase in surface expression of CD31. A kinase capable of phosphorylating CD31 was detected in CD31 immunoprecipitates, and its activity was increased following activation of platelets. CD31 tyrosine phosphorylation was reduced or abolished by the Src family kinase inhibitor PP2, suggesting a role for these enzymes. In accordance with this, each of the Src family members expressed in platelets, namely Fyn, Lyn, Src, Yes, and Hck, was shown to co-immunoprecipitate with CD31. The involvement of Src family kinases in this process was confirmed through the study of mouse platelets deficient in Fyn.

    The Journal of biological chemistry 2000;275;35;27339-47

  • Yes-associated protein 65 localizes p62(c-Yes) to the apical compartment of airway epithelia by association with EBP50.

    Mohler PJ, Kreda SM, Boucher RC, Sudol M, Stutts MJ and Milgram SL

    Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

    We recently showed that the COOH terminus of the cystic fibrosis transmembrane conductance regulator associates with the submembranous scaffolding protein EBP50 (ERM-binding phosphoprotein 50 kD; also called Na(+)/H(+) exchanger regulatory factor). Since EBP50 associates with ezrin, this interaction links the cystic fibrosis transmembrane conductance regulator (CFTR) to the cortical actin cytoskeleton. EBP50 has two PDZ domains, and CFTR binds with high affinity to the first PDZ domain. Here, we report that Yes-associated protein 65 (YAP65) binds with high affinity to the second EBP50 PDZ domain. YAP65 is concentrated at the apical membrane in airway epithelia and interacts with EBP50 in cells. The COOH terminus of YAP65 is necessary and sufficient to mediate association with EBP50. The EBP50-YAP65 interaction is involved in the compartmentalization of YAP65 at the apical membrane since mutant YAP65 proteins lacking the EBP50 interaction motif are mislocalized when expressed in airway epithelial cells. In addition, we show that the nonreceptor tyrosine kinase c-Yes is contained within EBP50 protein complexes by association with YAP65. Subapical EBP50 protein complexes, containing the nonreceptor tyrosine kinase c-Yes, may regulate apical signal transduction pathways leading to changes in ion transport, cytoskeletal organization, or gene expression in epithelial cells.

    Funded by: NCI NIH HHS: CA45757; NHLBI NIH HHS: P50 HL060280; NIDDK NIH HHS: DK50795, P01 DK050795, R29DK50744

    The Journal of cell biology 1999;147;4;879-90

  • CMS: an adapter molecule involved in cytoskeletal rearrangements.

    Kirsch KH, Georgescu MM, Ishimaru S and Hanafusa H

    Laboratory of Molecular Oncology, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA. kirschk@rockvax.rockfeller.edu

    Cas ligand with multiple Src homology (SH) 3 domains (CMS) is an ubiquitously expressed signal transduction molecule that interacts with the focal adhesion protein p130(Cas). CMS contains three SH3 in its NH2 terminus and proline-rich sequences in its center region. The latter sequences mediate the binding to the SH3 domains of p130(Cas), Src-family kinases, p85 subunit of phosphatidylinositol 3-kinase, and Grb2. The COOH-terminal region contains putative actin binding sites and a coiled-coil domain that mediates homodimerization of CMS. CMS is a cytoplasmic protein that colocalizes with F-actin and p130(Cas) to membrane ruffles and leading edges of cells. Ectopic expression of CMS in COS-7 cells resulted in alteration in arrangement of the actin cytoskeleton. We observed a diffuse distribution of actin in small dots and less actin fiber formation. Altogether, these features suggest that CMS functions as a scaffolding molecule with a specialized role in regulation of the actin cytoskeleton.

    Funded by: NCI NIH HHS: CA44356

    Proceedings of the National Academy of Sciences of the United States of America 1999;96;11;6211-6

  • PSD-95 promotes Fyn-mediated tyrosine phosphorylation of the N-methyl-D-aspartate receptor subunit NR2A.

    Tezuka T, Umemori H, Akiyama T, Nakanishi S and Yamamoto T

    Department of Oncology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.

    Fyn, a member of the Src-family protein-tyrosine kinase (PTK), is implicated in learning and memory that involves N-methyl-D-aspartate (NMDA) receptor function. In this study, we examined how Fyn participates in synaptic plasticity by analyzing the physical and functional interaction between Fyn and NMDA receptors. Results showed that tyrosine phosphorylation of NR2A, one of the NMDA receptor subunits, was reduced in fyn-mutant mice. NR2A was tyrosine-phosphorylated in 293T cells when coexpressed with Fyn. Therefore, NR2A would be a substrate for Fyn in vivo. Results also showed that PSD-95, which directly binds to and coclusters with NMDA receptors, promotes Fyn-mediated tyrosine phosphorylation of NR2A. Different regions of PSD-95 associated with NR2A and Fyn, respectively, and so PSD-95 could mediate complex formation of Fyn with NR2A. PSD-95 also associated with other Src-family PTKs, Src, Yes, and Lyn. These results suggest that PSD-95 is critical for regulation of NMDA receptor activity by Fyn and other Src-family PTKs, serving as a molecular scaffold for anchoring these PTKs to NR2A.

    Proceedings of the National Academy of Sciences of the United States of America 1999;96;2;435-40

  • Autophosphorylation of Src and Yes blocks their inactivation by Csk phosphorylation.

    Sun G, Sharma AK and Budde RJ

    Department of Neuro-Oncology, University of Texas, MD Anderson Cancer Center, Houston 77030, USA.

    Csk phosphorylates Src family protein tyrosine kinases on a tyrosine residue near their C-terminus and downregulates their activity. We previously observed that this regulation requires a stoichiometric ratio of Csk:Src in a time-independent manner. In this report we examined this unusual kinetic behavior and found it to be caused by Src autophosphorylation. First, pre-incubation of Src with ATP-Mg led to time-dependent autophosphorylation of Src, activation of its kinase activity and loss of its ability to be inactivated by Csk. However, the autophosphorylated Src can still be phosphorylated by Csk. The SH2 binding site for phospho-Tyr of this hyperactive and doubly phosphorylated form of Src is not accessible. Second, dephosphorylation of autophosphorylated Src by protein tyrosine phosphatase 1B allowed Src to be inactivated by Csk. Third, protein tyrosine phosphatase 1B preferentially dephosphorylates the Src autophosphorylation site and allows for Src regulation by Csk. Finally, Yes, another member of the Src family, was also only partially inactivated when a sub-stoichiometric amount of Csk was used. Mutation of the tyrosine autophosphorylation site of Yes to a phenylalanine resulted in a mutant Yes enzyme that can be fully inactivated by a sub-stoichiometric amount of Csk in a time-dependent manner. These results demonstrate that Csk phosphorylation inactivates Src and Yes only when they are not previously autophosphorylated and Src autophosphorylation can block the inactivation by Csk phosphorylation. This conclusion suggests a dynamic model for the regulation of the Src family protein tyrosine kinases, which is discussed in the context of previously reported observations on the regulation of Src family protein tyrosine kinases.

    Funded by: NCI NIH HHS: CA16672, CA53617

    Oncogene 1998;17;12;1587-95

  • Complex formation between EphB2 and Src requires phosphorylation of tyrosine 611 in the EphB2 juxtamembrane region.

    Zisch AH, Kalo MS, Chong LD and Pasquale EB

    The Burnham Institute, La Jolla, California 92037, USA.

    The cellular components of the neuronal signaling pathways of Eph receptor tyrosine kinases are only beginning to be elucidated. Here we show that in vivo tyrosine phosphorylation sites of the Eph receptors EphA3, EphA4, and EphB2 in embryonic retina serve as binding sites for the Src-homology 2 (SH2) domain of Src kinase. Furthermore, tyrosine-phosphorylated EphB2 was detected in Src immunoprecipitates from transfected Cos cells, indicating that EphB2 and Src can physically associate. Interestingly, a form of Src with reduced electrophoretic mobility and increased tyrosine phosphorylation was detected in Cos cells expressing tyrosine-phosphorylated EphB2, suggesting a functional interaction between EphB2 and Src. Yeast two-hybrid analysis in conjunction with site-directed mutagenesis demonstrated that phosphorylated tyrosine 611 in the juxtamembrane region of EphB2 is crucial for the interaction with the SH2 domain of Src. In contrast, binding of the carboxy-terminal SH2 domain of phospholipase Cgamma was not abolished upon mutation of tyrosine 611 in EphB2. Phosphopeptide mapping of autophosphorylated full-length EphB2, and wild-type and tyrosine to phenylalanine mutants of the EphB2 cytoplasmic domain fused to LexA, showed tyrosine 611 in the sequence motif YEDP as a major site of autophosphorylation in EphB2. Our mutational analysis also indicated that tyrosines 605 and 611 are important for EphB2 kinase activity. We propose Src kinase as a downstream effector that mediates the neuron's response to Eph receptor activation.

    Funded by: NEI NIH HHS: EY105576; NICHD NIH HHS: HD25938

    Oncogene 1998;16;20;2657-70

  • STK/RON receptor tyrosine kinase mediates both apoptotic and growth signals via the multifunctional docking site conserved among the HGF receptor family.

    Iwama A, Yamaguchi N and Suda T

    Department of Cell Differentiation, Kumamoto University School of Medicine, Japan.

    STK/RON tyrosine kinase, a member of the hepatocyte growth factor (HGF) receptor family, is a receptor for macrophage-stimulating protein (MSP). To examine the STK/RON signalling pathway, we generated STK/ RON transfectants showing opposite features in growth. STK/RON-expressing Ba/F3 pro-B cells (BaF/STK) exhibited MSP-dependent growth, whereas STK/ RON-expressing mouse erythroleukaemia cells (MEL/ STK) displayed MSP-induced apoptosis. This apoptosis was accompanied by the prolonged activation of c-Jun N-terminal kinase (JNK), which has recently been implicated in the initiation of apoptosis. Co-immunoprecipitation analyses showed that autophosphorylated STK/RON associated with PLC-gamma, P13-kinase, Shc and Grb2 in both transfectants. However, major tyrosine-phosphorylated proteins, p61 and p65, specifically associated with STK/RON in MEL/STK cells. Mutations at two C-terminal tyrosine residues, Y1330 and Y1337, in the counterpart of the multifunctional docking site of the HGF receptor abolished both MSP-induced growth and apoptosis. Analyses of these mutants and in vitro association revealed that signalling proteins including p61 and p65 directly bound to the phosphotyrosines in the multifunctional docking site. These results demonstrate that positive or negative signals toward cell growth are generated through the multifunctional docking site and suggest the involvement of p61 and p65 as well as JNK in apoptosis. Our findings provide the first evidence for apoptosis via a receptor tyrosine kinase.

    The EMBO journal 1996;15;21;5866-75

  • Complex formation of JAK2 with PP2A, P13K, and Yes in response to the hematopoietic cytokine interleukin-11.

    Fuhrer DK and Yang YC

    Department of Medicine (Hematology/Oncology), Indiana University School of Medicine, Indianapolis 46202, USA.

    Interleukin-11 is a stromal derived cytokine important in hematopoiesis. IL-11 intracellular signaling travels through cytoplasmic kinases of the Janus family. How JAKs accomplish the multiple functions of IL-11 has not been determined and until recently only a few associated downstream proteins have been identified. We present evidence here for the IL-11 induced association of PP2A, P13K, and Yes to JAK2. Reciprocal immunoprecipitations support the mutual involvement of these signaling components in IL-11 mediated signal transduction. This novel finding of JAK2/PP2A binding and release may have relevance to many serine/threonine regulated mechanisms such as P13K, Stat, and MAPK activation. These associations support a model of JAK2 as a protein kinase docking protein of IL-11 signal transduction that may be applicable to other gp130 and JAK signal transduction systems.

    Funded by: NHLBI NIH HHS: R01HL48819; NIDDK NIH HHS: R01DK43105

    Biochemical and biophysical research communications 1996;224;2;289-96

  • Characterization of a novel protein-binding module--the WW domain.

    Sudol M, Chen HI, Bougeret C, Einbond A and Bork P

    Laboratory of Molecular Oncology, Rockefeller University, New York, NY 10021, USA.

    We have identified, characterized and cloned human, mouse and chicken cDNA of a novel protein that binds to the Src homology domain 3 (SH3) of the Yes proto-oncogene product. We subsequently named it YAP for Yes-associated protein. Analysis of the YAP sequence revealed a protein module that was found in various structural, regulatory and signaling molecules. Because one of the prominent features of this sequence motif is the presence of two conserved tryptophans (W), we named it the WW domain. Using a functional screen of a cDNA expression library, we have identified two putative ligands of the WW domain of YAP which we named WBP-1 and WBP-2. Peptide sequence comparison between the two partial clones revealed a homologous proline-rich region. Binding assays and site-specific mutagenesis have shown that the proline-rich motif binds with relatively high affinity and specificity to the WW domain of YAP, with a preliminary consensus that is different from the SH3-binding PXXP motif. This suggests that the WW domain has a role in mediating protein-protein interactions via proline-rich regions, similar but distinct from Src homology 3 (SH3) domains. Based on this finding, we hypothesize that additional protein modules exist and that they could be isolated using proline-rich peptides as functional probes.

    Funded by: NCI NIH HHS: CA01605; NIGMS NIH HHS: 5T32GM07739-16

    FEBS letters 1995;369;1;67-71

  • Autophosphorylation activity and association with Src family kinase of Sky receptor tyrosine kinase.

    Toshima J, Ohashi K, Iwashita S and Mizuno K

    Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan.

    "Sky" is a putative receptor tyrosine kinase predominantly expressed in the brain. Sky, like Axl/Ufo/Ark and c-Eyk, has an extracellular domain composed of two immunoglobulin-like domains and two fibronectin type III domains. Immunoblot analysis using an antibody raised against a C-terminal peptide of Sky identified a 98-kDa Sky protein in COS cells transfected with sky cDNA (COS/sky cells). A 98-kDa protein in the immunoprecipitates with anti-Sky antibody was autophosphorylated on tyrosine, by in vitro kinase reaction. When the lysates of COS/sky cells were immunoprecipitated with anti-Sky antibody and immunoblotted with an anti-phosphotyrosine antibody, a 60-kDa phosphotyrosine-containing protein, in addition to the tyrosine-phosphorylated Sky, was detected. Using the anti-Src antibody, which is reactive to Src, Fyn and Yes, we obtained evidence for an association between the Src family tyrosine kinase and the tyrosine-phosphorylated Sky receptor. These results suggest that the Src family kinase may play an important role in signal transduction of the Sky receptor.

    Biochemical and biophysical research communications 1995;209;2;656-63

  • Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor.

    Waltenberger J, Claesson-Welsh L, Siegbahn A, Shibuya M and Heldin CH

    Ludwig Institute for Cancer Research, Uppsala Branch, Sweden.

    Vascular endothelial growth factor (VEGF) is a homodimeric peptide growth factor which binds to two structurally related tyrosine kinase receptors denoted Flt1 and KDR. In order to compare the signal transduction via these two receptors, the human Flt1 and KDR proteins were stably expressed in porcine aortic endothelial cells. Binding analyses using 125I-VEGF revealed Kd values of 16 pM for Flt1 and 760 pM for KDR. Cultured human umbilical vein endothelial (HUVE) cells were found to express two distinct populations of binding sites with affinities similar to those for Flt1 and KDR, respectively. The KDR expressing cells showed striking changes in cell morphology, actin reorganization and membrane ruffling, chemotaxis and mitogenicity upon VEGF stimulation, whereas Flt1 expressing cells lacked such responses. KDR was found to undergo ligand-induced autophosphorylation in intact cells, and both Flt1 and KDR were phosphorylated in vitro in response to VEGF, however, KDR much more efficiently than Flt1. Neither the receptor-associated activity of phosphatidylinositol 3'-kinase nor tyrosine phosphorylation of phospholipase C-gamma were affected by stimulation of Flt1 or KDR expressing cells, and phosphorylation of GTPase activating protein was only slightly increased. Members of the Src family such as Fyn and Yes showed an increased level of phosphorylation upon VEGF stimulation of cells expressing Flt1 but not in cells expressing KDR. The maximal responses in KDR expressing porcine aortic endothelial cells were obtained at higher VEGF concentrations as compared to HUVE cells, i.e. in the presence of Flt1. This difference could possibly be explained by the formation of heterodimeric complexes between KDR and Flt1, or other molecules, in HUVE cells.

    The Journal of biological chemistry 1994;269;43;26988-95

  • Src-related protein tyrosine kinases are physically associated with the surface antigen CD36 in human dermal microvascular endothelial cells.

    Bull HA, Brickell PM and Dowd PM

    Department of Medicine University College London Medical School, Rayne Institute, UK.

    Src-related cytoplasmic PTKs are physically and functionally associated with cell surface receptors and are involved in signal transduction. In this paper we report the identification of src-related proteins p59fyn, pp60c-src and p62yes in human microvascular endothelial cells cultured from normal human skin and their physical association with the thrombospondin receptor CD36. Such an association represents a potential signalling pathway by which thrombospondin may regulate angiogenesis.

    FEBS letters 1994;351;1;41-4

  • Identification of two juxtamembrane autophosphorylation sites in the PDGF beta-receptor; involvement in the interaction with Src family tyrosine kinases.

    Mori S, Rönnstrand L, Yokote K, Engström A, Courtneidge SA, Claesson-Welsh L and Heldin CH

    Ludwig Institute for Cancer Research Uppsala, Sweden.

    Two novel sites of autophosphorylation were localized to the juxtamembrane segment of the human platelet-derived growth factor (PDGF) beta-receptor. To evaluate the importance of these phosphorylation sites, receptor mutants were made in which Tyr579, Tyr581 or both were replaced with phenylalanine residues; the receptor mutants were stably expressed in porcine aortic endothelial cells. Compared with the wild-type receptor, the Y579F and Y581F mutants were less able to mediate association with and activation of the Src family tyrosine kinases. The ability of these phosphorylation sites to mediate directly the binding of the Src family proteins was also demonstrated by using phosphotyrosine-containing synthetic peptides representing the juxtamembrane sequence of the receptor. Both the Y579F and Y581F mutants were similar to the wild-type receptor with regard to their protein tyrosine kinase activity and ability to induce mitogenicity in response to PDGF-BB. A conclusive evaluation of the role of the Src family members in signal transduction could, however, not be made since our attempt to prevent completely the association by mutation of both Tyr579 and Tyr581, resulted in loss of kinase activity and was therefore not informative. The present data, together with previous observations, demonstrate a high degree of specificity in the interaction between different autophosphorylation sites in the PDGF beta-receptor and downstream components in the signal transduction pathway.

    The EMBO journal 1993;12;6;2257-64

  • Activation of Src family kinases by colony stimulating factor-1, and their association with its receptor.

    Courtneidge SA, Dhand R, Pilat D, Twamley GM, Waterfield MD and Roussel MF

    Differentiation Programme, European Molecular Biology Laboratory, Heidelberg, Germany.

    The receptor for the macrophage colony stimulating factor-1 (CSF-1R) is a transmembrane glycoprotein with intrinsic tyrosine kinase activity. CSF-1 stimulation promotes the growth of cells of the macrophage lineage and of fibroblasts engineered to express CSF-1R. We show that CSF-1 stimulation resulted in activation of three Src family kinases, Src, Fyn and Yes. Concomitant with their activation, all three Src family kinases were found to associate with the ligand-activated CSF-1 receptor. These interactions were also demonstrated in SF9 insect cells co-infected with viruses encoding the CSF-1 receptor and Fyn, and the isolated SH2 domain of Fyn was capable of binding the CSF-1R in vitro. Analysis of mutant CSF-1Rs revealed that the 'kinase insert' (KI) domain of CSF-1R was not required for interactions with Src family kinases, but that mutation of one of the receptor autophosphorylation sites, Tyr809, reduced both their binding and enzymatic activation. Because fibroblasts expressing this receptor mutant are unable to form colonies in semi-solid medium or to grow in chemically defined medium in the presence of CSF-1, the Src family kinases may play a physiological role in the mitogenic response to CSF-1.

    Funded by: NCI NIH HHS: CA-56819

    The EMBO journal 1993;12;3;943-50

  • Chromosomal reassignment: YACs containing both YES1 and thymidylate synthase map to the short arm of chromosome 18.

    Silverman GA, Kuo WL, Taillon-Miller P and Gray JW

    Department of Pediatrics, Joint Program in Neonatology, Harvard Medical School, Boston, Massachusetts 02115.

    The YES1 proto-oncogene was mapped previously to human chromosome band 18q21.3 by using isotopic in situ hybridization. Using yeast artificial chromosomes (YACs) as probes and fluorescence in situ hybridization, a strong signal was detected in the region corresponding to 18p11.3. Restriction digests confirmed that the YACs contained the YES1 gene and not other cross-hybridizing, protein-tyrosine kinases. In addition, these YACs were found to contain another 18p11.32 gene, thymidylate synthase. These genes were less than 50 kb apart. Collectively, these data suggest that YES1 maps to 18p11.32 rather than to 18q21.3.

    Funded by: NICHD NIH HHS: HD17665, HD8475

    Genomics 1993;15;2;442-5

  • p21rasGAP association with Fyn, Lyn, and Yes in thrombin-activated platelets.

    Cichowski K, McCormick F and Brugge JS

    Howard Hughes Medical Institute, Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia 19104-6148.

    Activation of platelets by thrombin and other physiological agonists leads to a dramatic increase in tyrosine phosphorylation of multiple cellular proteins (Ferrell, J. E., and Martin, G. S. (1988) Mol. Cell. Biol. 8, 3606-3610; Golden, A., and Brugge, J. S. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 901-905; Nakamura, S., and Yamamura, H. (1989) J. Biol. Chem. 264, 7089-7091). To date, none of the tyrosine kinases that are involved in platelet activation, nor the substrates that are phosphorylated in response to agonists, have been identified. A "kinase trapping" strategy, designed to take advantage of the stability of known tyrosine kinase-substrate interactions, was employed to address both issues. p21rasGAP antibodies were used to examine the phosphorylated state of GAP in agonist-treated platelets and to isolate potential GAP-kinase complexes. We show that GAP and two proteins of 59 and 68 kDa are phosphorylated on tyrosine after thrombin stimulation and that three Src-related protein tyrosine kinases, Fyn, Lyn and Yes, are associated with GAP in complexes, detectable only after agonist stimulation. The thrombin-dependent detection of these kinases in GAP immunoprecipitates suggests that thrombin may either induce the formation of these complexes or activate kinases that are associated with GAP prior to, or following, agonist stimulation. This approach of "trapping" kinases bound to their substrates will be useful in identifying non-receptor tyrosine kinases involved in signaling pathways. Furthermore, although GAP phosphorylation has been previously implicated in growth factor signaling pathways, this is the first example of its involvement downstream from a G-protein-coupled receptor.

    Funded by: NCI NIH HHS: CA47572

    The Journal of biological chemistry 1992;267;8;5025-8

  • The p60c-src family of protein-tyrosine kinases: structure, regulation, and function.

    Brickell PM

    Department of Biochemistry and Molecular Biology, University College and Middlesex School of Medicine, London, U.K.

    In 1911, Peyton Rous reported that a fibrosarcoma could be transmitted between chickens in a cell-free extract of the tumor. The transmissible agent, Rous sarcoma virus (RSV), transforms cells by virtue of the presence within its genome of a viral oncogene, v-src, which is derived from a normal cellular gene that has been picked up, or transduced, by the virus. This cellular proto-oncogene, c-src, encodes a protein, p60c-src, which has the ability to phosphorylate proteins on tyrosine residues. Studies of RSV were thus directly responsible for the discovery of cellular proto-oncogenes and of protein-tyrosine kinases, discoveries which have been fundamental in shaping our ideas about cellular growth control. In spite of this, the normal biological role of p60c-src is still unclear and it remains impossible to provide a full answer to the question of how RSV causes tumors. It is clear, however, that c-src is the prototype of a family of at least 8 closely related genes encoding protein tyrosine kinases, the other family members being blk, c-fgr, fyn, hck, lck, lyn, and c-yes. The purpose of this review is to outline our current knowledge of the structure, expression pattern, and function of each of the members of the c-src gene family and to describe recent data which begins to explain how these proteins interact with other cellular proteins to control cell behavior. The evidence for involvement of these proteins in oncogenesis is also discussed.

    Critical reviews in oncogenesis 1992;3;4;401-46

  • Two additional protein-tyrosine kinases expressed in human lung: fourth member of the fibroblast growth factor receptor family and an intracellular protein-tyrosine kinase.

    Holtrich U, Bräuninger A, Strebhardt K and Rübsamen-Waigmann H

    Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus, Frankfurt, Federal Republic of Germany.

    The expression of protein-tyrosine kinases (PTKs; ATP:protein-tyrosine O-phosphotransferase, EC 2.7.1.112) was studied in normal human lung and various tumors by PCR followed by molecular cloning and sequence analysis. Six known PTKs (YES, FGR, LYN, HCK, PDGFB-R, and CSF1-R), as well as two additional members of this enzyme family, were detected in lung. One of the newly discovered sequences appears to represent a group of cytosolic PTKs. The cDNA sequence of the second unknown PTK revealed that it is a fourth member of the fibroblast growth factor receptor family. It was therefore called TKF (tyrosine kinase related to fibroblast growth factor receptor). Among a wide variety of cells and tissues tested, including human lymphocytes and macrophages, TKF was only found expressed in lung. Apart from normal lung, TKF expression could be demonstrated in some tumors of lung origin, but also in malignancies not derived from lung tissues. As fibroblast growth factors are generally involved in a variety of functions such as mitogenesis, angiogenesis, and wound healing, the specific expression of a receptor-related gene in lung only may point to yet another special function of this group of proteins.

    Proceedings of the National Academy of Sciences of the United States of America 1991;88;23;10411-5

  • Membrane glycoprotein IV (CD36) is physically associated with the Fyn, Lyn, and Yes protein-tyrosine kinases in human platelets.

    Huang MM, Bolen JB, Barnwell JW, Shattil SJ and Brugge JS

    Howard Hughes Medical Institute, University of Pennsylvania, School of Medicine, Philadelphia 19104.

    Activation of platelets with thrombin and other agonists causes a rapid increase in the phosphorylation of multiple proteins on tyrosine. To identify candidate protein-tyrosine kinases (PTKs; EC 2.7.1.112) that may be responsible for these phosphorylation events, we analyzed the expression of seven Src-family PTKs and examined the association of these kinases with known platelet membrane glycoproteins. Five Src-related PTKs were detected in platelets: pp60SRC, pp60FYN, pp62YES, pp61HCK, and two LYN products of Mr 54,000 and 58,000. The Fgr and Lck PTKs were not detected. Although strict comparative quantification of protein levels was not possible, pp60SRC was detected at higher levels than any of the other kinases. In addition, glycoprotein IV (GPIV, CD36), one of the major platelet membrane glycoproteins, was associated in a complex with the Fyn, Yes, and Lyn proteins in platelet lysates. Similar complexes were also found in two GPIV-expressing cell lines, C32 melanoma cells and HEL cells. Since PTKs appear to be involved in stimulus-response coupling at the plasma membrane, these results suggest that ligand interaction with GPIV may activate signaling pathways that are triggered by tyrosine phosphorylation.

    Funded by: NCI NIH HHS: CA27951

    Proceedings of the National Academy of Sciences of the United States of America 1991;88;17;7844-8

  • Differential expression of p62c-yes in normal, hyperplastic and neoplastic human epidermis.

    Krueger J, Zhao YH, Murphy D and Sudol M

    Laboratory for Investigative Dermatology, Rockefeller University, New York, NY 10021-6399.

    The protein product of the c-yes proto-oncogene, p62c-yes, is highly expressed in a variety of mammalian cell types, including neurons, spermatozoa, platelets, and epithelial cells. In order to understand the function of p62c-yes in epithelial cells, the expression and localization of p62c-yes was studied in cultured human epidermal keratinocytes and in normal, hyperplastic, and neoplastic human epidermis. Human keratinocytes in culture produce a single 4kb c-yes mRNA and a 62kd protein product, p62c-yes, which is active as a protein tyrosine kinase. Using affinity-purified antibodies generated to the amino-terminus of the human c-yes protein, the expression of p62c-yes was localized to keratinocytes in the basal epidermal layer of normal neonatal and adult epidermis. There was a marked reduction in expression of p62c-yes by suprabasal keratinocytes undergoing progressive differentiation. By immunofluorescence microscopy, p62c-yes was localized to the plasma membrane and to a perinuclear cytoplasmic area in cultured keratinocytes. The apparent association of p62c-yes with plasma membranes was particularly evident in suprabasal keratinocytes from hyperplastic epidermis. Neoplastic keratinocytes in basal cell carcinomas showed a marked reduction in p62c-yes expression compared to normal basal keratinocytes in epidermis or to proliferating cultured keratinocytes. Thus the expression of p62c-yes by one epithelial cell type, the keratinocyte, is altered by cellular differentiation and neoplastic transformation. Keratinocytes provide a normal epithelial cell model in which the biochemical function of p62c-yes can be studied.

    Funded by: NCRR NIH HHS: M01-RR00102, RR07065-23; NIAMS NIH HHS: AR07525; ...

    Oncogene 1991;6;6;933-40

  • Expression of cellular-yes protein in mammalian tissues.

    Zhao YH, Krueger JG and Sudol M

    Laboratory of Molecular Oncology, Rockefeller University, New York, New York 10021.

    Expression of the c-yes proto-oncogene in normal mammalian tissues was studied by immunohistochemistry and by immunoprecipitation assays using affinity purified anti-yes IgG. The antibody was raised against the amino-terminal domain of the human c-yes protein expressed in bacteria as a trpE-yes fusion protein. We detected p62c-yes in a variety of rat and human tissues but its relative level of expression varied significantly among different cell types. Certain epithelial cells, platelets and spermatid acrosomes showed the highest levels of p62c-yes. Intracellular localization of p62c-yes in epithelial cells differed among organs performing different physiological functions. The overall pattern of expression suggests that p62c-yes may be involved in numerous cellular pathways.

    Funded by: NCI NIH HHS: CA45757

    Oncogene 1990;5;11;1629-35

  • Characterization of cDNA clones for the human c-yes gene.

    Sukegawa J, Semba K, Yamanashi Y, Nishizawa M, Miyajima N, Yamamoto T and Toyoshima K

    Three c-yes cDNA clones were obtained from poly(A)+ RNA of human embryo fibroblasts. Sequence analysis of the clones showed that they contained inserts corresponding to nearly full-length human c-yes mRNA, which could encode a polypeptide of 543 amino acids with a relative molecular weight (Mr) of 60,801. The predicted amino acid sequence of the protein has no apparent membrane-spanning region or suspected ligand binding domain and closely resembles pp60c-src. Comparison of the sequences of c-yes and v-yes revealed that the v-yes gene contains most of the c-yes coding sequence except the region encoding its extreme carboxyl terminus. The region missing from the v-yes protein is the part that is highly conserved in cellular gene products of the protein-tyrosine kinase family.

    Molecular and cellular biology 1987;7;1;41-7

  • Nakahara memorial lecture. Non-receptor type protein-tyrosine kinases closely related to src and yes compose a multigene family.

    Toyoshima K, Semba K, Nishizawa M, Yamanashi Y, Sukegawa J, Miyajima N and Yamamoto T

    Department of Oncology, University of Tokyo, Japan.

    We have determined the total coding sequence of human c-yes, a non-receptor type protein-tyrosine kinase gene, and found that the c-yes gene closely resembles the c-src gene. Recently, two new genes, syn and lyn, were found to encode proteins closely related to the yes product. In addition, we also determined the partial sequence of fgr. These genes together with lck reported by two American groups have very closely related structures and are thought to compose a closely related group of non-receptor type protein-tyrosine kinases. Partial analysis of the structures of these genes indicated that they have identical splicing junctions at all sites so far examined. On the other hand, the erbB-1/EGF (epidermal growth factor) receptor gene and the erbB-2/neu gene have completely different splicing junctions from those of the above gene group even in the kinase domain, although these genes also have protein kinase activity specific for tyrosine residues and the erbB-1 and -2 genes share splicing sites. These results suggest that the genes of the group of six non-receptor type kinases and those of the erbB-1 and erbB-2 gene group are descendants evolved by duplication of two distinct ancestor genes and are members of two distinct multi-gene families. The genes coding for protein kinases may be members of a super-family including multiple distinct gene families.

    Princess Takamatsu symposia 1986;17;11-20

  • Location of the c-yes gene on the human chromosome and its expression in various tissues.

    Semba K, Yamanashi Y, Nishizawa M, Sukegawa J, Yoshida M, Sasaki M, Yamamoto T and Toyoshima K

    Analysis of DNA from human embryo fibroblasts showed that ten Eco RI fragments were hybridizable with the Yamaguchi sarcoma virus oncogene (v-yes). Four of the Eco RI fragments were assigned to chromosome 18 and one to chromosome 6. There was evidence for multiple copies of yes-related genes in the human genome; however, only a single RNA species, 4.8 kilobases in length, was related to yes in various cells.

    Science (New York, N.Y.) 1985;227;4690;1038-40

Gene lists (6)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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