G2Cdb::Gene report

Gene id
G00001300
Gene symbol
PTK2B (HGNC)
Species
Homo sapiens
Description
PTK2B protein tyrosine kinase 2 beta
Orthologue
G00000051 (Mus musculus)

Databases (8)

Gene
ENSG00000120899 (Ensembl human gene)
2185 (Entrez Gene)
19 (G2Cdb plasticity & disease)
PTK2B (GeneCards)
Literature
601212 (OMIM)
Marker Symbol
HGNC:9612 (HGNC)
Protein Expression
3850 (human protein atlas)
Protein Sequence
Q14289 (UniProt)

Synonyms (5)

  • CADTK
  • CAKB
  • PTK
  • PYK2
  • RAFTK

Literature (176)

Pubmed - other

  • Pyk2 inhibition of p53 as an adaptive and intrinsic mechanism facilitating cell proliferation and survival.

    Lim ST, Miller NL, Nam JO, Chen XL, Lim Y and Schlaepfer DD

    Department of Reproductive Medicine, Moores Cancer Center, University of California San Diego, La Jolla, California 92093, USA.

    Pyk2 is a cytoplasmic tyrosine kinase related to focal adhesion kinase (FAK). Compensatory Pyk2 expression occurs upon FAK loss in mice. However, the impact of Pyk2 up-regulation remains unclear. Previous studies showed that nuclear-localized FAK promotes cell proliferation and survival through FAK FERM domain-enhanced p53 tumor suppressor degradation (Lim, S. T., Chen, X. L., Lim, Y., Hanson, D. A., Vo, T. T., Howerton, K., Larocque, N., Fisher, S. J., Schlaepfer, D. D., and Ilic, D. (2008) Mol. Cell 29, 9-22). Here, we show that FAK knockdown triggered p53 activation and G(1) cell cycle arrest in human umbilical vein endothelial cells after 4 days. However, by 7 days elevated Pyk2 expression occurred with a reduction in p53 levels and the release of the G(1) block under conditions of continued FAK knockdown. To determine whether Pyk2 regulates p53, experiments were performed in FAK(-/-)p21(-/-) mouse embryo fibroblasts expressing endogenous Pyk2 and in ID8 ovarian carcinoma cells expressing both Pyk2 and FAK. In both cell lines, Pyk2 knockdown increased p53 levels and inhibited cell proliferation associated with G(1) cell cycle arrest. Pyk2 FERM domain re-expression was sufficient to reduce p53 levels and promote increased BrdUrd incorporation. Pyk2 FERM promoted Mdm2-dependent p53 ubiquitination. Pyk2 FERM effects on p53 were blocked by proteasomal inhibition or mutational-inactivation of Pyk2 FERM nuclear localization. Staurosporine stress of ID8 cells promoted endogenous Pyk2 nuclear accumulation and enhanced Pyk2 binding to p53. Pyk2 knockdown potentiated ID8 cell death upon staurosporine addition. Moreover, Pyk2 FERM expression in human fibroblasts upon FAK knockdown prevented cisplatin-mediated apoptosis. Our studies demonstrate that nuclear Pyk2 functions to limit p53 levels, thus facilitating cell growth and survival in a kinase-independent manner.

    Funded by: NCI NIH HHS: CA102310, R01 CA102310; NHLBI NIH HHS: HL093156, R01 HL093156

    The Journal of biological chemistry 2010;285;3;1743-53

  • Functional activation of proline-rich tyrosine kinase2 (PYK2) in peripheral blood mononuclear cells from patients with systemic lupus erythematosus.

    Wang M, Sun H, Zhang W and Zhang Y

    Department of Rheumatology and Immunology, Provincial Hospital affiliated to Shandong University, Jinan, 250021, China. wmy99wmy99@yahoo.cn

    Background: Systemic lupus erythematosus (SLE) is a representative systemic autoimmune disease characterized by activated T cells and polyclonally activated B cells that produce autoantibodies. Activation of autoreactive T and B cells plays a pivotal role in the pathogenesis of this disease. A role of focal adhesion kinase (FAK) in the pathogenesis has been suggested. Proline-rich tyrosine kinase2 (PYK2) is structurally related to FAK, however, the functional activation of PYK2 in SLE remains unclear. In the present study, we showed that PYK2 is significantly increased and activated in peripheral blood mononuclear cells (PBMCs) of patients with SLE. In addition, we showed the involvement of PYK2 proteins in the up-regulation of CD40L and CTLA4 expression and PBMC proliferation.

    Methods: Freshly isolated PBMCs from 48 SLE patients, 32 patients with rheumatoid arthritis(RA) and 24 healthy individuals were analyzed for the expression and activation of PYK2 by western-blotting and immunocytochemistry. The other isolated PBMCs from patients with this condition were cultured and stimulated with PMA or TyrA9, and then the expression of costimulatory molecules CD40L and CTLA4 was evaluated using flow cytometry, PBMCs proliferation was determined with [3H]-thymidine incorporation (CPM).

    Results: Compared with RA patients and healthy donors, PBMCs from SLE patients expressed more of both the total PYK2 protein and its activated/phosphorylated form. The increase of activated PYK2 protein in SLE PBMCs was correlated with the complication of nephritis and inversly associated the level of serum complements. In active SLE patients, activation of PYK2 in PBMCs is accompanying the increased cell proliferation and the induced expression of costimulatory molecules CD40L and CTLA4.

    Conclusion: Our findings indicate that phosphorylated PYK2 in SLE PBMCs may induce the expression of CD40L and CTLA4, and subsequently the cell proliferation. PYK2 signaling enhances the autoreactive lymphocyte activation and plays an important role in the pathogenesis of SLE.

    BMC musculoskeletal disorders 2009;10;141

  • Identification of 4 new HLA-DR-restricted minor histocompatibility antigens as hematopoietic targets in antitumor immunity.

    Stumpf AN, van der Meijden ED, van Bergen CA, Willemze R, Falkenburg JH and Griffioen M

    Department of Hematology, Leiden University Medical Center, Leiden, The Netherlands.

    Potent graft-versus-leukemia (GVL) effects can be mediated by donor-derived T cells recognizing minor histocompatibility antigens (mHags) in patients treated with donor lymphocyte infusion (DLI) for relapsed hematologic malignancies after HLA-matched allogeneic stem cell transplantation (alloSCT). Donor-derived T cells, however, may not only induce GVL, but also mediate detrimental graft-versus-host disease (GVHD). Because HLA-class II is under noninflammatory conditions predominantly expressed on hematopoietic cells, CD4+ T cells administered late after alloSCT may selectively confer GVL without GVHD. Although a broad range of different HLA-class I-restricted mHags have been identified, the first 2 autosomal HLA-class II-restricted mHags have only recently been characterized. By screening a recombinant bacteria cDNA expression library, we identified 4 new HLA-class II-restricted mHags recognized by CD4+ T cells induced in a patient with relapsed chronic myeloid leukemia who achieved long-term complete remission and experienced only mild GVHD of the skin after DLI. All CD4+ T cells were capable of recognizing the mHags presented by HLA-DR surface molecules on primary hematopoietic cells, but not on skin-derived (cytokine-treated) fibroblasts. The selective recognition of hematopoietic cells as well as the balanced population frequencies and common HLA-DR restriction elements make the novel mHags possible targets for development of immunotherapeutic strategies.

    Blood 2009;114;17;3684-92

  • Analysis of the tyrosine kinome in melanoma reveals recurrent mutations in ERBB4.

    Prickett, Agrawal NS, Wei X, Yates KE, Lin JC, Wunderlich JR, Cronin JC, Cruz P, Rosenberg SA and Samuels Y

    Cancer Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, USA.

    Tyrosine phosphorylation is important in signaling pathways underlying tumorigenesis. We performed a mutational analysis of the protein tyrosine kinase (PTK) gene family in cutaneous metastatic melanoma. We identified 30 somatic mutations affecting the kinase domains of 19 PTKs and subsequently evaluated the entire coding regions of the genes encoding these 19 PTKs for somatic mutations in 79 melanoma samples. We found ERBB4 mutations in 19% of individuals with melanoma and found mutations in two other kinases (FLT1 and PTK2B) in 10% of individuals with melanomas. We examined seven missense mutations in the most commonly altered PTK gene, ERBB4, and found that they resulted in increased kinase activity and transformation ability. Melanoma cells expressing mutant ERBB4 had reduced cell growth after shRNA-mediated knockdown of ERBB4 or treatment with the ERBB inhibitor lapatinib. These studies could lead to personalized therapeutics specifically targeting the kinases that are mutationally altered in individual melanomas.

    Funded by: Intramural NIH HHS: Z01 HG200337-01, Z01 HG200337-02, ZIA HG200337-03

    Nature genetics 2009;41;10;1127-32

  • Expression and activation of proline-rich tyrosine kinase 2 (PYK2) in peripheral blood mononuclear cells from patients with systemic lupus erythematosus.

    Wang M, Zhang W and Zhang Y

    Department of Rheumatology and Immunology, Provincial Hospital affiliated to Shandong University, Jinan, China.

    This study analysed the expression and activation of proline-rich tyrosine kinase 2 (PYK2) in peripheral blood mononuclear cells (PBMCs) from 36 systemic lupus erythematosus (SLE) patients and explored whether activation of PYK2 correlates with disease activity or organ damage in SLE. Samples from 19 patients with rheumatoid arthritis (RA) and 15 healthy individuals were included as controls. There was a significant increase in both total PYK2 protein and its activated/phosphorylated form in PBMCs from patients with SLE, particularly in those with the complication of World Health Organization class IV lupus nephritis. There was a clear correlation between the activation of PYK2 and the level of serum complement, but no relationship was found between the activation of PYK2 and SLE Disease Activity Index (SLEDAI). As previous studies have shown that PYK2 provides important signals during the activation of lymphocytes, PYK2 could be a major contributor to the complex autoimmune pathogenesis of SLE.

    The Journal of international medical research 2009;37;5;1293-300

  • B cell receptor-induced phosphorylation of Pyk2 and focal adhesion kinase involves integrins and the Rap GTPases and is required for B cell spreading.

    Tse KW, Dang-Lawson M, Lee RL, Vong D, Bulic A, Buckbinder L and Gold MR

    Department of Microbiology and Immunology, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.

    Signaling by the B cell receptor (BCR) promotes integrin-mediated adhesion and cytoskeletal reorganization. This results in B cell spreading, which enhances the ability of B cells to bind antigens and become activated. Proline-rich tyrosine kinase (Pyk2) and focal adhesion kinase (FAK) are related cytoplasmic tyrosine kinases that regulate cell adhesion, cell morphology, and cell migration. In this report we show that BCR signaling and integrin signaling collaborate to induce the phosphorylation of Pyk2 and FAK on key tyrosine residues, a modification that increases the kinase activity of Pyk2 and FAK. Activation of the Rap GTPases is critical for BCR-induced integrin activation as well as for BCR- and integrin-induced reorganization of the actin cytoskeleton. We now show that Rap activation is essential for BCR-induced phosphorylation of Pyk2 and for integrin-induced phosphorylation of Pyk2 and FAK. Moreover Rap-dependent phosphorylation of Pyk2 and FAK required an intact actin cytoskeleton as well as actin dynamics, suggesting that Rap regulates Pyk2 and FAK via its effects on the actin cytoskeleton. Importantly B cell spreading induced by BCR/integrin co-stimulation or by integrin engagement was inhibited by short hairpin RNA-mediated knockdown of either Pyk2 or FAK expression and by treatment with PF-431396, a chemical inhibitor that blocks the kinase activities of both Pyk2 and FAK. Thus Pyk2 and FAK are downstream targets of the Rap GTPases that play a key role in regulating B cell morphology.

    The Journal of biological chemistry 2009;284;34;22865-77

  • Prolactin induces chitotriosidase expression in human macrophages through PTK, PI3-K, and MAPK pathways.

    Di Rosa M, Zambito AM, Marsullo AR, Li Volti G and Malaguarnera L

    Department of Biomedical Sciences, University of Catania, Catania, Italy.

    We previously reported that prolactin (PRL) induces chitotriosidase (CHIT-1) mRNA expression in human macrophages. In this investigation we determined the signaling pathways involved in CHIT-1 induction in response to PRL. The CHIT-1 induction PRL-mediated was reduced by wortmannin and LY-294002, inhibitors of phosphatidylinositol 3-kinase (PI3-K) and by genistein an inhibitor of protein tyrosine kinase (PTK). Pre-treatment of macrophages with SB203580, a specific inhibitor of the mitogen-activated kinases (MAPK) p38, or with U0126, an inhibitor of MAPK p44/42, prevented both basal and exogenous PRL-mediated CHIT-1 expression. No significant effects on CHIT-1 induction PRL-mediated were observed with a protein kinase C inhibitor (PKC), rottlerin, or with an Src inhibitor, PP2, or with JAK2 inhibitor, AG490. In addition, PRL induced a phosphorylation of AKT that was prevented both by the two MAPK inhibitors SB203580 and U0126 and by the PI3-K inhibitors wortmannin and LY-294002. In conclusion, our results indicate that PRL up-regulated CHIT-1 expression via PTK, PI3-K, MAPK, and signaling transduction components.

    Journal of cellular biochemistry 2009;107;5;881-9

  • Crystal structures of free and ligand-bound focal adhesion targeting domain of Pyk2.

    Lulo J, Yuzawa S and Schlessinger J

    Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA.

    Focal adhesion targeting (FAT) domains target the non-receptor tyrosine kinases FAK and Pyk2 to cellular focal adhesion areas, where the signaling molecule paxillin is also located. Here, we report the crystal structures of the Pyk2 FAT domain alone or in complex with paxillin LD4 peptides. The overall structure of Pyk2-FAT is an antiparallel four-helix bundle with an up-down, up-down, right-handed topology. In the LD4-bound FAT complex, two paxillin LD4 peptides interact with two opposite sides of Pyk2-FAT, at the surfaces of the alpha1alpha4 and alpha2alpha3 helices of each FAT molecule. We also demonstrate that, while paxillin is phosphorylated by Pyk2, complex formation between Pyk2 and paxillin does not depend on Pyk2 tyrosine kinase activity. These experiments reveal the structural basis underlying the selectivity of paxillin LD4 binding to the Pyk2 FAT domain and provide insights about the molecular details which influence the different behavior of these two closely-related kinases.

    Funded by: NIAMS NIH HHS: P50-AR054086, R01-AR051448, R01-AR051886

    Biochemical and biophysical research communications 2009;383;3;347-52

  • The Pyk2 FERM domain as a target to inhibit glioma migration.

    Loftus JC, Yang Z, Tran NL, Kloss J, Viso C, Berens ME and Lipinski CA

    Mayo Clinic Arizona, 13400 East Shea Boulevard, Scottsdale, AZ 85259, USA. loftus.joseph@mayo.edu

    The invasion of malignant glioma cells into the surrounding normal brain precludes effective clinical treatment. In this report, we investigated the role of the NH(2)-terminal FERM domain in the regulation of the promigratory function of Pyk2. We report that the substitution of residues that constitute a small cleft on the surface of the F3 module of the FERM domain do not significantly alter Pyk2 expression but result in the loss of Pyk2 phosphorylation. A monoclonal antibody, designated 12A10, specifically targeting the Pyk2 FERM domain was generated and recognizes an epitope located on the beta5C-alpha1C surface of the F3 module of the FERM domain. Amino acid substitutions in the F3 module that resulted in the loss of Pyk2 phosphorylation also inhibited the binding of 12A10, suggesting that the 12A10 epitope overlaps a site that plays a role in Pyk2 activity. Conjugation of 12A10 to a membrane transport peptide led to intracellular accumulation and inhibition of glioma cell migration in a concentration-dependent manner. A single chain Fv fragment of 12A10 was stable when expressed in the intracellular environment, interacted directly with Pyk2, reduced Pyk2 phosphorylation, and inhibited glioma cell migration in vitro. Stable intracellular expression of the 12A10 scFv significantly extended survival in a glioma xenograft model. Together, these data substantiate a central role for the FERM domain in regulation of Pyk2 activity and identify the F3 module as a novel target to inhibit Pyk2 activity and inhibit glioma progression.

    Funded by: NCI NIH HHS: CA103956, CA108961, P50 CA108961, R01 CA103956

    Molecular cancer therapeutics 2009;8;6;1505-14

  • A Pyk2-Vav1 complex is recruited to beta3-adhesion sites to initiate Rho activation.

    Gao C and Blystone SD

    Department of Cell and Development Biology, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, USA.

    Integrin alphavbeta3-mediated adhesion of haemopoietic cells to vitronectin results in beta3 tyrosine phosphorylation and Rho activation which is necessary for adhesion. Previously, we have shown that the RhoGEF (Rho guanine-nucleotide-exchange factor) Vav1 could associate indirectly with alphavbeta3 during leucocyte adhesion to vitronectin. In the present study, we have identified the non-receptor tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2) as the adaptor protein that links Vav1 with alphavbeta3. The association of Pyk2 and Vav1 with beta3 relies on the presence of Tyr747 in beta3, the primary site of beta3 phosphorylation. However, association of Pyk2 with Vav1 is independent of beta3 tyrosine phosphorylation. Formation of a Pyk2-Vav1 complex occurs upon cell adhesion and Pro717 of Pyk2 plays a key role in Pyk2 interaction with Vav1. Utilizing purified recombinant proteins, we confirmed the direct interaction between Pyk2 and Vav1 In vitro. Cells transfected with GFP (green fluorescent protein)-Pyk2-P717A demonstrated severely suppressed cytoskeletal reorganization, impaired Vav1 recruitment, decreased Rho GTPase activation and loss of cell adhesion. Using siRNA (small interfering RNA) to specifically reduce Pyk2 levels in cells resulted in disrupted association between Vav1 and beta3 and impaired cell adhesion. These results indicate that Pyk2 is a critical signalling molecule downstream of beta3 integrin tyrosine phosphorylation and mediates Vav1 recruitment to accomplish actin reorganization necessary for adhesion.

    Funded by: NIDDK NIH HHS: DK79884, R01 DK0 1f40 79884-03, R01 DK079884, R01 DK079884-02

    The Biochemical journal 2009;420;1;49-56

  • Pyk2 mediates endothelin-1 signaling via p130Cas/BCAR3 cascade and regulates human glomerular mesangial cell adhesion and spreading.

    Rufanova VA, Alexanian A, Wakatsuki T, Lerner A and Sorokin A

    Division of Nephrology, Department of Medicine, Kidney Disease Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.

    Calcium-regulated non-receptor proline-rich tyrosine kinase 2 (Pyk2) is a critical mediator of endothelin-1 (ET-1) signaling in human glomerular mesangial cells (GMC). We aimed to identify which small G-protein is acting downstream of Pyk2. Dominant interfering Pyk2 construct, termed calcium regulated non kinase (CRNK) or green fluorescent protein (control) were expressed in GMC using adenovirus-mediated gene transfer. ET-1 stimulation resulted in a significant increase of Pyk2 phosphorylation accompanied by GTP-loading of Rap1 and RhoA. CRNK expression inhibited ET-1-induced autophosphorylation of endogenous Pyk2 and diminished Rap1, but not RhoA, activation. The mechanism linking Pyk2 and Rap1 included (1) increased autophosphorylation of Pyk2 associated with p130Cas, (2) augmented p130Cas Y165 and Y249 phosphorylation, and (3) enhanced p130Cas-BCAR3 complex formation. CRNK expression prevented p130Cas phosphorylation and attenuated p130Cas association with BCAR3. Downregulation of endogenous BCAR3 protein expression using an siRNA technique led to a significant decrease in Rap1 activation in response to ET-1. We observed that endogenous Pyk2 was important for GMC adhesion and spreading. Our data suggest that ET-1 stimulated the GTPase Rap1 (but neither RhoA nor Ras) by a mechanism involving Pyk2 activation and recruitment of the p130Cas/BCAR3 complex in GMC.

    Funded by: NCCIH NIH HHS: AT 003984, R41 AT003984; NHLBI NIH HHS: HL 22563, R01 HL022563, R01 HL022563-30; NIDDK NIH HHS: DK 41684, R01 DK041684, R01 DK041684-19

    Journal of cellular physiology 2009;219;1;45-56

  • LPS-induced MCP-1 expression in human microvascular endothelial cells is mediated by the tyrosine kinase, Pyk2 via the p38 MAPK/NF-kappaB-dependent pathway.

    Anand AR, Bradley R and Ganju RK

    Department of Pathology, Ohio State University Medical Center, Columbus, OH 43210, USA.

    Bacterial endotoxin (lipopolysaccharide or LPS) has potent pro-inflammatory properties and acts on many cell types including endothelial cells. Secretion of the CC chemokine, MCP-1 (CCL2) by LPS-activated endothelial cells contributes substantially to the pathogenesis of sepsis. However, the mechanism involved in LPS-induced MCP-1 production in endothelial cells is not well understood. Using human microvascular endothelial cells (HMVEC), we analyzed the involvement of the non-receptor tyrosine kinase, Pyk2, in LPS-mediated MCP-1 production. There was a marked activation of the non-receptor tyrosine kinase, Pyk2, in response to LPS. Inhibition of Pyk2 activity using a pharmacological inhibitor, Tyrphostin A9 significantly attenuated LPS-induced Pyk2 tyrosine phosphorylation, p38 MAP kinase (MAPK) activation, NF-kappaB activation, and MCP-1 expression. Furthermore, specific inactivation of Pyk2 activity by transducing microvascular endoth 12b4 elial cells with catalytically inactive Pyk2 mutant (AAV-Pyk2MT) or Pyk2-specific siRNA significantly blocked LPS-induced MCP-1 production. The supernatants of these LPS-stimulated cells with attenuated Pyk2 activity demonstrated decreased trans-endothelial monocyte migration in comparison to LPS-treated controls, thus confirming the inhibition of functional MCP-1 production. In summary, our data suggest a critical role for the Pyk2 mediated pathway involving p38 MAP kinase and NF-kappaB in LPS-induced MCP-1 production in human microvascular endothelial cells.

    Funded by: NCI NIH HHS: CA109527, R01 CA109527, R01 CA109527-01A1; NHLBI NIH HHS: R01 HL087576, R01 HL087576-01A1; NIAID NIH HHS: AI49140, R01 AI049140

    Molecular immunology 2009;46;5;962-8

  • alpha- and gamma-Protocadherins negatively regulate PYK2.

    Chen J, Lu Y, Meng S, Han MH, Lin C and Wang X

    Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208, USA.

    Genetic studies demonstrate that gamma-protocadherins (PCDH-gamma) are required for the survival and synaptic connectivity in neuronal subpopulations of the central nervous system. However, the intracellular signaling mechanisms for PCDH-gamma are poorly understood. Here, we show that PCDH-gamma binds two tyrosine kinases, PYK2 and focal adhesion kinase (FAK), and interaction with PCDH-gamma inhibits kinase activi 1126 ty. Consistent with this, PYK2 activity is abnormally up-regulated in the Pcdh-gamma-deficient neurons. Overexpression of PYK2 induces apoptosis in the chicken spinal cord. Thus, negative regulation of PYK2 activity by PCDH could contribute to the survival of subsets of neurons. Surprisingly, we found that PCDH-alpha interacts similarly with PYK2 and FAK despite containing a distinct cytoplasmic domain. In neural tissue, PCDH-gamma, together with PCDH-alpha, forms functional complexes with PYK2 and/or FAK. Therefore, the identification of common intracellular effectors for PCDH-gamma and PCDH-alpha suggests that dozens of protocadherins generated by Pcdh-alpha and Pcdh-gamma gene clusters can converge different extracellular signals into common intracellular pathways.

    Funded by: NINDS NIH HHS: 5 R01 NS 051253, R01 NS051253

    The Journal of biological chemistry 2009;284;5;2880-90

  • Up-regulation of proline-rich tyrosine kinase 2 in non-small cell lung cancer.

    Zhang S, Qiu X, Gu Y and Wang E

    Department of Pathology, College of Basic Medical Sciences, China Medical University, and Department of Pathology, the First Affiliated Hospital of China Medical University, Shenyang, China.

    Proline-rich tyrosine kinase 2 (PYK2) is a non-receptor tyrosine kinase, plays different roles in intracellular signaling pathways, that regulates a number of biological processes, such as cell proliferation, differentiation, adhesion and migration, which have been shown to correlate with tumor development and aggression. However, the involvement of PYK2 in human non-small cell lung cancer (NSCLC) has not yet been determined. In the present study, 90 patients with NSCLC (represented by adenocarcinoma and squamous cell carcinoma) were included retrospectively. NSCLC tissues were detected for the expression of PYK2 by immunohistochemistry. Correlation between the expression of PYK2 with the clinicopathological characteristics was analyzed. There were 64% (58 out of 90) of NSCLC patients with higher level of PYK2. Higher expression of PYK2 was significantly correlated with lymph node metastasis (node positive versus node negative, p=0.007). Patients with higher expression of PYK2 had advanced stage of NSCLCs (I+II versus III+IV, p=0.012). Protein level of PYK2 was also examined in 30 of these tumorous samples and matched non-tumorous counterparts by western blotting. PYK2 was apparently up-regulated in NSCLC tissues (tumor versus non-tumor, p=0.000). In the cell studies, extensive expression and activation of PYK2 were both found in higher metastatic BE1 cells. The activity of ERK1/2 in BE1 cells appeared extremely high as well. In conclusion, our results demonstrated that PYK2 is up-regulated in NSCLCs, and the higher expression and activation of PYK2 may play a role in modulating the activity of ERK1/2, and lead to the progression of NSCLC.

    Lung cancer (Amsterdam, Netherlands) 2008;62;3;295-301

  • Focal adhesion kinase-related proline-rich tyrosine kinase 2 and focal adhesion kinase are co-overexpressed in early-stage and invasive ErbB-2-positive breast cancer and cooperate for breast cancer cell tumorigenesis and invasiveness.

    Behmoaram E, Bijian K, J f92 ie S, Xu Y, Darnel A, Bismar TA and Alaoui-Jamali MA

    Department of Pathology, Lady Davis Institute of the Sir Mortimer B. Davis Jewish General Hospital, McGill University, Montreal, Quebec, Canada.

    Early cancer cell migration and invasion of neighboring tissues are mediated by multiple events, including activation of focal adhesion signaling. Key regulators include the focal adhesion kinase (FAK) and FAK-related proline-rich tyrosine kinase 2 (Pyk2), whose distinct functions in cancer progression remain unclear. Here, we compared Pyk2 and FAK expression in breast cancer and their effects on ErbB-2-induced tumorigenesis and the potential therapeutic utility of targeting Pyk2 compared with FAK in preclinical models of breast cancer. Pyk2 is overexpressed in tissues from early and advanced breast cancers and overexpressed with both FAK and epidermal growth factor receptor-2 (ErbB-2) in a subset of breast cancer cases. Down-regulation of Pyk2 in ErbB-2-positive, FAK-proficient, and FAK-deficient cells reduced cell proliferation, which correlated with reduced mitogen-activated protein kinase (MAPK) activity. In contrast, Pyk2 silencing had little impact on cell migration and invasion. In vivo, Pyk2 down-regulation reduced primary tumor growth induced by a metastatic variant of ErbB-2-positive MDA 231 breast cancer cells but had little effect on lung metastases in contrast to FAK down-regulation. Dual reduction of Pyk2 and FAK expression resulted in strong inhibition of both primary tumor growth and lung metastases. Together, these data support the cooperative function of Pyk2 and FAK in breast cancer progression and suggest that dual inhibition of FAK and Pyk2 is an efficient therapeutic approach for targeting invasive breast cancer.

    The American journal of pathology 2008;173;5;1540-50

  • Proline-rich tyrosine kinase 2 (Pyk2) promotes proliferation and invasiveness of hepatocellular carcinoma cells through c-Src/ERK activation.

    Sun CK, Man K, Ng KT, Ho JW, Lim ZX, Cheng Q, CM, Poon RT and Fan ST

    Department of Surgery and Centre for Cancer Research, The University of Hong Kong, Pokfulam, Hong Kong, China.

    The aim of the current study is to elucidate the mechanism of proline-rich tyrosine kinase 2 (Pyk2)-mediated cell proliferation and invasiveness in hepatocellular carcinoma (HCC) cells. Human HCC cell lines PLC and MHCC97L were stably transfected with either full-length Pyk2 or C-terminal non-kinase region of Pyk2 (PRNK). Functional studies on cell proliferation and invasion were conducted in vitro by colony formation assay, adhesion assay, migration assay and wound-healing assay. For the in vivo study, an orthotopic nude mice liver tumor model was applied to investigate the effects of Pyk2 overexpression on tumor growth and metastasis. Overexpression of Pyk2 in PLC cells resulted in an upregulation of colony formation (P = 0.021) and adhesion toward laminin (P = 0.018). Pyk2 promoted wound recovery by stimulation of actin stress fiber polymerization. In the in vivo study, transfection of PRNK in MHCC97L cells significantly decreased tumor volume (P = 0.001) and the incidence of lung metastasis (P = 0.014). Overexpression of Pyk2 promoted the activation of c-Src, formation of Pyk2/c-Src complex and activated the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK)-signaling pathway. Pyk2 upregulated the activation of ERK1/2 that is insensitive to MAPK/ERK kinase (MEK)1/2 inhibition. On the contrary, PRNK overexpression downregulated the activation of c-Src and ERK/MAPK-signaling pathways. Immunofluorescence staining showed that the focal adhesion localization of Pyk2 is a major determinant for c-Src and ERK/MAPK activation. In conclusion, our results showed that Pyk2 promoted cell proliferation and invasiveness by upregulation of the c-Src and ERK/MAPK-signaling pathways.

    Carcinogenesis 2008;29;11;2096-105

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

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

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

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

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

  • Focal adhesion kinase (FAK)-related non-kinase inhibits myofibroblast differentiation through differential MAPK activation in a FAK-dependent manner.

    Ding Q, Gladson CL, Wu H, Hayasaka H and Olman MA

    Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA. qding@uab.edu

    Transforming growth factor (TGF)-beta1 induces fibroblast transdifferentiation to myofibroblasts, a process that requires the involvement of integrin-mediated signaling and focal adhesion kinase (FAK). FAK-related non-kinase (FRNK) is known for its role in inhibiting integrin-mediated cell migration; however, its role in myofibroblast differentiation has not been defined. Here, we report that FRNK abrogates TGF-beta1-induced myofibroblast differentiation in vitro and in vivo. TGF-beta1 can induce alpha-smooth muscle actin (alpha-SMA) expression in the presence or absence of FAK; however, TGF-beta1-induced alpha-SMA expression is reduced (approximately 73%) in FAK-deficient fibroblasts. Although both ERK and p38 MAPK activation is required for maximal TGF-beta1-induced alpha-SMA expression, ERK is the major signaling intermediate in cells that express FAK. In contrast, p38 MAPK is the dominant mediator of TGF-beta1-induced alpha-SMA expression in FAK-deficient cells. FRNK overexpression blocks TGF-beta1-induced ERK or p38 MAPK activation in the presence, and surprisingly, in the absence of FAK. The loss of FRNK was tested in vivo during experimentally induced pulmonary fibrosis in mice. FRNK knock-out mice have a greater increase in alpha-SMA-expressing cells in response to a pulmonary fibrotic stimulus in vivo, as compared with congenic wild type mice. This is the first time that FRNK loss has been shown to modify the pathobiology in any animal disease model. Together, the data demonstrate that FRNK negatively regulates myofibroblast differentiation in vitro and in vivo. These data further suggest that modulation FRNK expression may be a novel avenue for therapeutic intervention in tissue fibrosis.

    Funded by: NHLBI NIH HHS: HL085324, HL58655, R01 HL085324

    The Journal of biological chemistry 2008;283;40;26839-49

  • RhoC promotes metastasis via activation of the Pyk2 pathway in prostate cancer.

    Iiizumi M, Bandyopadhyay S, Pai SK, Watabe M, Hirota S, Hosobe S, Tsukada T, Miura K, Saito K, Furuta E, Liu W, Xing F, Okuda H, Kobayashi A and Watabe K

    Department of Medical Microbiology, Immunology, and Cell Biology, Southern Illinois University School of Medicine, Springfield, Illinois 62702, USA.

    RhoC is a member of the Ras-homologous family of genes which have been implicated in tumorigenesis and tumor progression. However, the exact role of RhoC is controversial and is yet to be clarified. We have examined the effect of RhoC on prostate tumor cells and found that RhoC had no effect on cell proliferation in vitro or on tumor growth in mice. However, RhoC significantly enhanced the metastatic ability of the tumor cells in these animals, suggesting that RhoC affects only the metastasis but not the growth of prostate tumor cells. The results of our immunohistochemical analyses on tumor specimens from 63 patients with prostate cancer indicate that RhoC expression had no significant correlation with Gleason grade. However, the expression of RhoC showed significant positive correlation with both lymph node and distant metastasis, and it was inversely correlated with patient survival. We also found that RhoC significantly augmented the invasion and motility of prostate tumor cells by activating matrix metalloproteinases 2 and 9 (MMP2 and MMP9) in vitro. The results of our antibody array analysis for signal molecules revealed that RhoC significantly activated kinases including mitogen-activated protein kinase (MAPK), focal adhesion kinase (FAK), Akt, and Pyk2. Inhibition of Pyk2 kinase blocked the RhoC-dependent activation of FAK, MAPK, and Akt, followed by the suppression of MMP2 and MMP9. Inhibitors of both MAPK and Akt also significantly blocked the activities of these MMPs. Therefore, our results indicate that RhoC promotes tumor metastasis in prostate cancer by sequential activation of Pyk2, FAK, MAPK, and Akt followed by the up-regulation of MMP2 and MMP9, which results in the stimulation of invasiveness of tumor cells.

    Funded by: NCI NIH HHS: 1R01CA124650, 1R01CA129000, R01 CA124650, R01 CA124650-01A1, R01 CA129000, R01 CA129000-01

    Cancer research 2008;68;18;7613-20

  • Proline-rich tyrosine kinase 2 regulates spreading and migration of eosinophils after beta2-integrin adhesion.

    Zhu X, Boetticher E, Wang L, Duan Y, Learoyd J and Leff AR

    Section of Pulmonary and Critical Care Medicine, Department of Medicine, MC6076, University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637, USA. xzhu@medicine.bsd.uchicago.edu

    We examined the role of proline-rich tyrosine kinase (Pyk) 2 in the spreading and migration of human blood eosinophils after beta(2)-integrin ligation. Western blot analysis showed that Pyk2 was activated by phosphorylation at Y402 after eosinophil adhesion to BSA-coated plates after activation with IL-5, platelet-activating factor (PAF), formyl-met-leu-phe (fMLP), or Mn(2)(+). To determine the role of Pyk2 in regulating eosinophil migration, we used a transducable dominant-negative inhibitor of Pyk2, TAT-mediated protein transduction of dominant-negative C-terminal Pyk2 (TAT-Pyk2-CT), a fusion protein in which TAT peptide was fused to the C-terminal Pyk2. TAT-Pyk2-CT blocked tyrosine phosphorylation of Pyk2 caused by beta(2)-integrin adhesion, but did not block adhesion of eosinophils to plated BSA. TAT-Pyk2-CT also blocked subsequent spreading and migration of eosinophils caused by IL-5, PAF, or fMLP. Spreading eosinophils stained with FITC-conjugated phalloidin showed elongation and formation of multiple fillopodia and lamellipodia, whereas nonspreading eosinophils were smaller and round. Treatment of eosinophils with TAT-Pyk2-CT had no effect on the initial cell polarization, but blocked the formation of fillopodia and lamellipodia in adherent cells. Migration of eosinophils through Transwell plates caused by IL-5, PAF, or fMLP was blocked significantly after inhibition of Pyk2. These data indicate that Pyk2, although not involved in beta(2)-integrin adhesion, causes eosinophil spreading and regulates subsequent chemotactic migration after beta(2)-integrin ligation to endothelial counter ligands. We conclude that Pyk2 is activated by beta(2)-integrin adhesion and is a required signal for eosinophil spreading and subsequent chemotactic migration.

    Funded by: NHLBI NIH HHS: HL-85779, HL-92328

    American journal of respiratory cell and molecular biology 2008;39;3;263-9

  • The tyrosine kinase Pyk2 mediates lipopolysaccharide-induced IL-8 expression in human endothelial cells.

    Anand AR, Cucchiarini M, Terwilliger EF and Ganju RK

    Department of Pathology, Ohio State University Medical Center, Columbus, OH 43210, USA.

    Secretion of proinflammatory cytokines by LPS activated endothelial cells contributes substantially to the pathogenesis of sepsis. However, the mechanism involved in this process is not well understood. In the present study, we determined the role of a nonreceptor proline-rich tyrosine kinase, Pyk2, in LPS-induced IL-8 (CXCL8) production in endothelial cells. First, we observed a marked activation of Pyk2 in response to LPS. Furthermore, inhibition of Pyk2 activity in these cells by transduction with the catalytically inactive Pyk2 mutant, transfection with Pyk2-specific small interfering RNA, or treatment with Tyrphostin A9 significantly blocked LPS-induced IL-8 production. The supernatants of LPS-stimulated cells exhibiting attenuated Pyk2 activity blocked transendothelial neutrophil migration in comparison to the supernatants of LPS-treated controls, thus confirming the inhibition of functional IL-8 production. Investigations into the molecular mechanism of this pathway revealed that LPS activates Pyk2 leading to IL-8 production through the TLR4. In addition, we identified the p38 MAPK pathway to be a critical step downstre 1f40 am of Pyk2 during LPS-induced IL-8 production. Taken together, these results demonstrate a novel role for Pyk2 in LPS-induced IL-8 production in endothelial cells.

    Funded by: NCI NIH HHS: CA109527, R01 CA109527; NHLBI NIH HHS: R01 HL087576; NIAID NIH HHS: AI49140, R01 AI049140

    Journal of immunology (Baltimore, Md. : 1950) 2008;180;8;5636-44

  • Essential role of Pyk2 and Src kinase activation in neuropeptide-induced proliferation of small cell lung cancer cells.

    Roelle S, Grosse R, Buech T, Chubanov V and Gudermann T

    1Institut für Pharmakologie und Toxikologie, Philipps-Universität Marburg, Marburg, Germany.

    Neuropeptide hormones like bombesin/gastrin-releasing peptide, galanin or bradykinin, acting via auto and paracrine growth loops, represent the principal mitogens of small cell lung cancer (SCLC). These mitogenic neuropeptides activate G(q/11)-coupled receptors which stimulate phospholipase Cbeta activity, followed by rises of the intracellular calcium concentration ([Ca2+](i)) and activation of protein kinase C (PKC). We report here that proline-rich tyrosine kinase Pyk2 is highly expressed in SCLC cells and provides a functional link between neuropeptide-induced increases in [Ca2+](i) and tumor cell proliferation. Activation of Pyk2 and its association with Src kinases critically depends on the elevation of [Ca2+](i), but is independent of PKC. Src kinase activities are crucial for neuropeptide-mediated GTP-loading of Ras and activation of extracellular signal-regulated kinases in SCLC cells. Pyk2 and Src 1f40 kinases essentially contribute to anchorage-independent proliferation of SCLC cells. Inhibition of either Pyk2 or Src kinases by lentiviral RNAi or pharmacological inhibition with PP2, respectively, attenuated basal and neuropeptide-elicited survival and proliferation of SCLC cells in liquid culture and in soft agar. Thus, neuropeptides stimulate anchorage-independent survival and proliferation of SCLC cells via pathways involving Pyk2 and Src kinases. Therefore, Ca2+-induced Pyk2/Src complex formation may be a rewarding molecular target for novel therapeutic strategies in SCLC cells.

    Oncogene 2008;27;12;1737-48

  • The atypical Rho GTPase Wrch1 collaborates with the nonreceptor tyrosine kinases Pyk2 and Src in regulating cytoskeletal dynamics.

    Ruusala A and Aspenström P

    Ludwig Institute for Cancer Research, Biomedical Center, Uppsala University, SE-751 24 Uppsala, Sweden.

    The Cdc42-like GTPase Wnt responsive Cdc42 homolog 1 (Wrch1) has several atypical features; it has an N-terminal proline-rich extension that confers binding to SH3 domains, and it harbors an extremely high intrinsic nucleotide exchange activity, which overrides the normal GTPase activity. As a result, Wrch1 resides mainly in the active, GTP-loaded conformation under normal cellular conditions. We have previously shown that ectopic expression of Wrch1 in fibroblasts resulted in an altered cell morphology visible as a formation of filopodia, a loss of stress fibers, and a reduction in focal adhesions. Here, we show that Wrch1 binds to the nonreceptor tyrosine kinase Pyk2. The interaction required Wrch1 to be in a GTP conformation and also required an intact N-terminal proline-rich extension as well as an intact effector loop. Wrch1 requires Pyk2 in imposing the cytoskeletal effects, seen as the formation of filopodia, since treatment of cells with a Pyk2-specific small interfering RNA abrogated this response. Interestingly, we found that the presence and activity of Src were needed for the formation of a Wrch1-Pyk2 complex as well as for the Wrch1-induced formation of filopodia. We propose a model in which Pyk2 and Src function to coordinate the Wrch1-dependent effects on cytoskeletal dynamics.

    Molecular and cellular biology 2008;28;5;1802-14

  • Induction of proline-rich tyrosine kinase2 (Pyk2) through C/EBPbeta is involved in PMA-induced monocyte differentiation.

    Park MH, Park SY and Kim Y

    Department of Molecular Biology, College of Natural Sciences, Pusan National University, Pusan 609-735, Republic of Korea.

    Proline-rich tyrosine kinase2 (Pyk2) is a cytoplasmic tyrosine kinase related to focal adhesion kinase. Pyk2 expression has been known to be restricted to neuronal and hematopoietic cells and Pyk2 tyrosine phosphorylation and its kinase activity is important for the function of monocytes/macrophages. In NB4 acute promyelocytic leukemia cells, the expression of Pyk2 was increased in parallel with differentiation, and inhibited by PD98059, indicating Pyk2 expression is regulated through MAPK/ERK pathway. Dominant-negative kinase-deficient mutant of Pyk2 reduced the differentiation of NB4 cells in response to phorbol 12-mystate 13-acetate. Transcription factor CCAAT enhancer-binding protein (C/EBP) beta was required to induce Pyk2 expression in promoter analysis. These results suggest that Pyk2 is induced and involved in monocyte differentiation and C/EBPbeta is a critical regulator of the Pyk2 expression.

    FEBS letters 2008;582;3;415-22

  • Identification of a functional protein kinase Cbeta promoter polymorphism in humans related to insulin resistance.

    Osterhoff MA, Heuer S, Pfeiffer M, Tasic J, Kaiser S, Isken F, Spranger J, Weickert MO, Möhlig M and Pfeiffer AF

    German Institute of Human Nutrition, Potsdam-Rehbruecke (DIfE), Department of Clinical Nutrition (KLE), Arthur-Scheunert-Allee 155, D-14558 Nuthetal, Germany. martino@dife.de

    Protein kinase Cbeta (PKCbeta) is known to inhibit insulin production in beta-cells and to support insulin action in skeletal muscle. We therefore searched for functional polymorphisms among already known genetic variants in the PKCbeta promoter and investigated their relation to glucose metabolism in humans. We found that the gene variant in the PKCbeta promoter at position -546 signif 1f40 icantly reduced promoter activity in functional assays (P<0.05). Human subjects carrying this variant had a 3.5-fold decrease in PKCbeta2-protein expression in their thrombocytes (P=0.006). Additionally, we tested whether this variant affects parameters of glucose metabolism using 1012 humans included into the MeSyBePo study (Metabolic Syndrome Berlin Potsdam). The -546 variant was highly significant associated with increased homeostasis model assessment for insulin resistance (HOMA-IR, P=0.009) in the cohort. This association was accompanied by significantly increased fasting insulin concentrations in carriers of the homozygous polymorphism (P=0.021). Our results suggest that the -546 polymorphism in the PKCbeta promoter reduces promoter activity, which leads to a decreased expression of PKCbeta2 and subsequently is associated with decreased peripheral insulin-dependent glucose uptake.

    Molecular genetics and metabolism 2008;93;2;210-5

  • Pyk2/ERK 1/2 mediate Sp1- and c-Myc-dependent induction of telomerase activity by epidermal growth factor.

    Bermudez Y, Yang H, Cheng JQ and Kruk PA

    Department of Pathology and Cell Biology, University of South Florida, Tampa, Florida 33612, USA.

    Epidermal growth factor (EGF) promotes growth of normal ovarian surface as well as malignant ovarian epithelial cells. Further, EGF receptors are present on both normal and malignant ovarian surface epithelial cells and they are often constitutively activated in many cancers. Since telomerase confers cellular immortalization and survival through increased cellular proliferation, we sought to investigate the potential role of EGF to regulate telomerase activity in normal and ovarian cancer cells. While exogenous EGF failed to activate telomerase in normal ovarian surface epithelial cells, in cancer cells we herein report that: exogenous EGF activates telomerase activity and human telomerase reverse transcriptase gene (hTERT) transcription; EGF-induced telomerase activity is ERK 1/2-dependent; EGF targets Sp1 and c-Myc binding sites within the core region of the hTERT promoter; and proline-rich tyrosine kinase 2 (Pyk2) is a key mediator of EGF-mediated telomerase activity. Together, these data show that dysregulation of EGF signaling may promote cancer cell survival through up-regulation of telomerase activity.

    Growth factors (Chur, Switzerland) 2008;26;1;1-11

  • PKB and the mitochondria: AKTing on apoptosis.

    Parcellier A, Tintignac LA, Zhuravleva E and Hemmings BA

    Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland.

    Cellular homeostasis depends upon the strict regulation of responses to external stimuli, such as signalling cascades triggered by nutrients and growth factors, and upon cellular metabolism. One of the major molecules coordinating complex signalling pathways is protein kinase B (PKB), a serine/threonine kinase also known as Akt. The number of substrates known to be phosphorylated by PKB and its interacting partners, as well as our broad understanding of how PKB is implicated in responses to growth factors, metabolic pathways, proliferation, and cell death via apoptosis is constantly increasing. Activated by the insulin/growth factor-phosphatidylinositol 3-kinase (PI3K) cascade, PKB triggers events that promote cell survival and prevent apoptosis. It is also now widely accepted that mitochondria are not just suppliers of ATP, but that they participate in regulatory and signalling events, responding to multiple physiological inputs and genetic stresses, and regulate both cell proliferation and death. Thus, mitochondria are recognized as important players in apoptotic events and it is logical to predict some form of interplay with PKB. In this review, we will summarize mechanisms by which PKB mediates its anti-apoptotic activities in cells and survey recent developments in understanding mitochondrial dynamics and their role during apoptosis.

    Cellular signalling 2008;20;1;21-30

  • Focal adhesion kinase signaling controls cyclic tensile strain enhanced collagen I-induced osteogenic differentiation of human mesenchymal stem cells.

    Ward DF, Williams WA, Schapiro NE, Weber GL, Christy SR, Salt M, Klees RF, Boskey A and Plopper GE

    Department of Biology, Rensselaer Polytechnic Institute, Troy, NY 12180-3596, USA.

    Focal adhesion kinase (FAK) is a key integrator of integrin-mediated signals from the extracellular matrix to the cytoskeleton and downstream signaling molecules. FAK is activated by phosphorylation at specific tyrosine residues, which then stimulate downstream signaling including the ERK1/2 pathway, leading to a variety of cellular responses. In this study, we examined the effects of FAK point mutations at tyrosine residues (Y397, Y925, Y861, and Y576/7) on osteogenic differentiation of human mesenchymal stem cells exposed to collagen I and cyclic tensile strain. Our results demonstrate that FAK signaling emanating from Y397, Y925, and to a lesser extent Y576/7, but not from Y861, controls osteogenic differentiation through an ERK1/2 pathway, as measured by expression levels of key osteogenesis marker genes and subsequent matrix mineralization. These data indicate that FAK is a critical decision maker in extracellular matrix/strain-enhanced osteogenic differentiation.

    Funded by: NIBIB NIH HHS: 5R01EB002197

    Molecular & cellular biomechanics : MCB 2007;4;4;177-88

  • ErbB-2 via PYK2 upregulates the adhesive ability of androgen receptor-positive human prostate cancer cells.

    Yuan TC, Lin FF, Veeramani S, Chen SJ, Earp HS and Lin MF

    Department of Biochemistry and Molecular Biology, College of Medicine, University of Nebraska Medical Center, Omaha, NE 68198-5870, USA.

    Aberrant regulation in the adhesive ability of cancer cells is closely associated with their metastatic activity. In this study, we examine the role of ErbB-2 in regulating the adhesive ability of androgen receptor (AR)-positive human prostate cancer (PCa) cells, the major cell population of PCa. Utilizing different LNCaP and MDA PCa2b cells as model systems, we found that ErbB-2 activity was correlated with PYK2 activity and adhesive ability in those cells. Increased ErbB-2 expression or activity in LNCaP C-33 cells enhanced PYK2 activation and cell adhesion, while the high PYK2 activity and the rapid adhesion of LNCaP C-81 cells were decreased by diminishing ErbB-2 expression or activity. Knockdown studies revealed the predominant role of ErbB-2 in regulating LNCaP C-81 cell adhesion. Coimmunoprecipitation showed that C-81 cells had increased interaction between ErbB-2 and PYK2. Elevated ErbB-2 activity in LNCaP cells correlated with increased ERK/MAPK activity and enhanced adhesive ability, which were abolished by the expression of K457A-PYK2 mutant or the treatment of PD98059, a MEK inhibitor. In summary, our data suggested that ErbB-2, via PYK2-ERK/MAPK, upregulates the adhesive ability of AR-positive human PCa cells.

    Funded by: NCI NIH HHS: CA88184

    Oncogene 2007;26;54;7552-9

  • Protein tyrosine kinase 2beta as a candidate gene for hypertension.

    Kamide K, Kokubo Y, Fukuhara S, Hanada H, Yang J, Kada A, Nagura J, Takiuchi S, Horio T, Kawano Y, Okayama A, Tomoike H and Miyata T

    Divisions of Hypertension, National Cardiovascular Center, Suita, Osaka, Japan. kamide@hsp.ncvc.go.jp

    Protein tyrosine kinase 2beta (PTK2B) is a member of the focal adhesion kinase family and is activated by angiotensin II through Ca2+-dependent pathways. An evidence exists that PTK2B is involved in cell growth, vascular contraction, inflammatory responses, and salt and water retention through activation of the angiotensin II type 1 receptor. To examine the contribution of PTK2B, we sequenced the PTK2B gene using 48 patients with hypertension, identified 62 genetic polymorphisms, and genotyped six representative single nucleotide polymorphisms in population-based case-control samples from 3655 Japanese individuals (1520 patients with hypertension and 2135 controls). Multivariate logistic regression analysis after adjustments for age, body mass index, present illness (hyperlipidemia and diabetes mellitus), and lifestyle (smoki 1f40 ng and drinking) showed -22A>G to have an association with hypertension in men (AA vs. AG+GG: odds ratio=1.27; 95% confidence interval: 1.02-1.57; P=0.030). Another polymorphism, 53484A>C (K838T), in linkage disequilibrium with -22A>G showed a marginal association with hypertension in men (AA vs. AC+CC: odds ratio=1.25; 95% confidence interval: 0.99-1.57; P=0.059). Diastolic blood pressure was 1.6 mmHg higher in men with the AC+CC genotype of 53484A>C than those with the AA genotype (P=0.003), after adjustments for the same factors. These polymorphisms are in linkage disequilibrium with others in a range of 113 kb in PTK2B. The intracellular distribution of the recombinant PTK2B protein and that of the mutant protein with T838 were indistinguishable even after angiotensin II stimulation, both proteins localizing at a focal point in the peripheral area in the cells. Thus, a haplotype in PTK2B may play a role in essential hypertension in Japanese.

    Pharmacogenetics and genomics 2007;17;11;931-9

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

    Allingham MJ, van Buul JD and Burridge K

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

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

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

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

  • The significance of proline-rich tyrosine kinase2 (Pyk2) on hepatocellular carcinoma progression and recurrence.

    Sun CK, Ng KT, Sun BS, Ho JW, Lee TK, Ng I, Poon RT, Lo CM, Liu CL, Man K and Fan ST

    Centre of Cancer Research and Department of Surgery, The University of Hong Kong, Pokfulam, Hong Kong, China.

    Understanding the precise molecular mechanisms that trigger liver cancer cell migration and invasion could develop novel therapeutic strategies targeting cancer cell invasion to increase the sensitivity to current treatment modalities. In the current study, 49 patients with hepatocellular carcinoma (HCC) were included prospectively. Liver tumour and adjacent non-tumour tissues were detected for the expression of Proline-rich tyrosine kinase 2 (Pyk2), focal adhesion kinase (FAK), ezrin and fibronectin at protein and/or gene levels. Correlation between the expressions of Pyk2/FAK with the clinical pathological data was analysed. Protein expression of Pyk2 was also examined in a nude mice orthotopic liver tumour model with higher metastatic potential. There were 59% (29 out of 49) and 57% (28 out of 49) of HCC patients with higher levels of Pyk2 and FAK protein/gene expression, respectively. We observed a positive correlation between the protein and gene expression levels of Pyk2 and FAK (P=0.000, r=0.875). Overexpression of Pyk2 and FAK was significantly correlated with shorter disease-free survival. Patients with higher levels of Pyk2/FAK had larger tumour size and advanced Edmonson grading. In the animal studies, Pyk2 overexpression was found in infiltrative tumour cells and lung metastatic nodules. In conclusion, overexpression of Pyk2 and FAK was found in nearly 60% of HCC patients and was significantly correlated with poor prognosis. The significance of Pyk2 in HCC invasiveness was confirmed by animal studies.

    British journal of cancer 2007;97;1;50-7

  • Breaking new ground to build bone.

    Kingsley LA, Chirgwin JM and Guise TA

    Division of Endocrinology and Metabolism, Department of Medicine, University of Virginia, Charlottesville, VA 22908, USA.

    Proceedings of the National Academy of Sciences of the United States of America 2007;104;26;10753-4

  • Proline-rich tyrosine kinase 2 regulates osteoprogenitor cells and bone formation, and offers an anabolic treatment approach for osteoporosis.

    Buckbinder L, Crawford DT, Qi H, Ke HZ, Olson LM, Long KR, Bonnette PC, Baumann AP, Hambor JE, Grasser WA, Pan LC, Owen TA, Luzzio MJ, Hulford CA, Gebhard DF, Paralkar VM, Simmons HA, Kath JC, Roberts WG, Smock SL, Guzman-Perez A, Brown TA and Li M

    Pfizer Global Research and Development, Groton, CT 06340, USA. leonard.buckbinder@pfizer.com

    Bone is accrued and maintained primarily through the coupled actions of bone-forming osteoblasts and bone-resorbing osteoclasts. Cumulative in vitro studies indicated that proline-rich tyrosine kinase 2 (PYK2) is a positive mediator of osteoclast function and activity. However, our investigation of PYK2-/- mice did not reveal evidence supporting an essential function for PYK2 in osteoclasts either in vivo or in culture. We find that PYK2-/- mice have high bone mass resulting from an unexpected increase in bone formation. Consistent with the in vivo findings, mouse bone marrow cultures show that PYK2 deficiency enhances differentiation and activity of osteoprogenitor cells, as does expressing a PYK2-specific short hairpin RNA or dominantly interfering proteins in human mesenchymal stem cells. Furthermore, the daily administration of a small-molecule PYK2 inhibitor increases bone formation and protects against bone loss in ovariectomized rats, an established preclinical model of postmenopausal osteoporosis. In summary, we find that PYK2 regulates the differentiation of early osteoprogenitor cells across species and that inhibi 10f8 tors of the PYK2 have potential as a bone anabolic approach for the treatment of osteoporosis.

    Proceedings of the National Academy of Sciences of the United States of America 2007;104;25;10619-24

  • Interaction of Pyk2 and PTP-PEST with leupaxin in prostate cancer cells.

    Sahu SN, Nunez S, Bai G and Gupta A

    Dept. of Biomedical Sciences, Dental School, University of Maryland, Baltimore, MD 21201, USA.

    We have identified the presence of leupaxin (LPXN), which belongs to the paxillin extended family of focal adhesion-associated adaptor proteins, in prostate cancer cells. Previous studies have demonstrated that LPXN is a component of the podosomal signaling complex found in osteoclasts, where LPXN was found to associate with the protein tyrosine kinases Pyk2 and c-Src and the cytosolic protein tyrosine phosphatase-proline-, glutamate-, serine-, and threonine-rich sequence (PTP-PEST). In the current study, LPXN was detectable as a 50-kDa protein in PC-3 cells, a bone-derived metastatic prostate cancer cell line. In PC-3 cells, LPXN was also found to associate with Pyk2, c-Src, and PTP-PEST. A siRNA-mediated inhibition of LPXN resulted in decreased in vitro PC-3 cell migration. A recombinant adenoviral-mediated overexpression of LPXN resulted in an increased association of Pyk2 with LPXN, whereas a similar adenoviral-mediated overexpression of PTP-PEST resulted in decreased association of Pyk2 and c-Src with LPXN. The overexpression of LPXN in PC-3 cells resulted in increased migration, as assessed by in vitro Transwell migration assays. On the contrary, the overexpression of PTP-PEST in PC-3 cells resulted in decreased migration. The overexpression of LPXN resulted in increased activity of Rho GTPase, which was decreased in PTP-PEST-overexpressing cells. The increase in Rho GTPase activity following overexpression of LPXN was inhibited in the presence of Y27632, a selective inhibitor of Rho GTPase. In conclusion, our data demonstrate that LPXN forms a signaling complex with Pyk2, c-Src, and PTP-PEST to regulate migration of prostate cancer cells.

    Funded by: NIAMS NIH HHS: AR-47942

    American journal of physiology. Cell physiology 2007;292;6;C2288-96

  • The role of proline-rich protein tyrosine kinase 2 in differentiation-dependent signaling in human epidermal keratinocytes.

    Schindler EM, Baumgartner M, Gribben EM, Li L and Efimova T

    Division of Dermatology, Department of Internal Medicine, Washington University School of Medicine, St Louis, Missouri 63110, USA.

    Non-receptor tyrosine kinase proline-rich protein tyrosine kinase 2 (Pyk2) functions as an integrator of multiple signaling pathways involved in the regulation of fundamental cellular processes. Pyk2 expression, regulation, and functions in skin have not been examined. Here we investigated the expression and subcellular localization of Pyk2 in human epidermis and in primary human keratinocytes, and studied the mechanisms of Pyk2 activation by differentiation-inducing stimuli, and the role of Pyk2 as a regulator of keratinocyte differentiation. We demonstrate that Pyk2 is abundantly expressed in skin keratinocytes. Notably, the endogenous Pyk2 protein is predominantly localized in keratinocyte nuclei throughout all layers of healthy human epidermis, and in cultured human keratinocytes. Pyk2 is activated by treatment with keratinocyte-differentiating agents, 12-O-tetradecanoylphorbol-13-acetate and calcium via a mechanism that requires intracellular calcium release and functional protein kinase C (PKC) and Src activities. Particularly, differentiation-promoting PKC delta and PKC eta elicit Pyk2 activation. Our data show that Pyk2 increases promoter activity and endogenous protein levels of involucrin, a marker of keratinocyte terminal differentiation. This regulation is associated with increased expression of Fra-1 and JunD, activator protein-1 transcription factors known to be required for involucrin expression. Altogether, these results provide insights into Pyk2 signaling in epidermis and reveal a novel role for Pyk2 in regulation of keratinocyte differentiation.

    The Journal of investigative dermatology 2007;127;5;1094-106

  • 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

  • RAFTK/Pyk2 regulates EGF-induced PC12 cell spreading and movement.

    Park SY, Li H and Avraham S

    Division of Experimental Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Harvard Institutes of Medicine, 4 Blackfan Circle, Boston, MA 02215, USA.

    The protein tyrosine kinase RAFTK, also termed Pyk2, is a member of the focal adhesion kinase (FAK) subfamily. In this report, we show the role of RAFTK in neuroendocrine PC12 cells upon epidermal growth factor (EGF) stimulation. Following EGF treatment, we observed that RAFTK was tyrosine-phosphorylated in a time- and dose-dependent manner, while FAK was constitutively phosphorylated and primarily regulated by cell adhesion. Moreover, we found that RAFTK associated with the phosphorylated EGF receptor (EGFR) upon EGF stimulation. RAFTK phosphorylation was mediated primarily through PLCgamma-IP3-Ca(2+) signaling and partially through PI3-Kinase. Furthermore, overexpression of PRNK, a specific dominant-negative construct of RAFTK, was sufficient to block EGF-induced cell spreading and movement. Paxillin, a key modulator of the actin cytoskeleton and an RAFTK substrate, was also phosphorylated following EGF treatment. EGF induced a dynamic reorganization of RAFTK and paxillin at neuronal adhesion sites, with the specific localization of paxillin at the inner juxtaposition of RAFTK. Additionally, we observed that RAFTK associated with the scaffold protein c-Cbl and mediated its phosphorylation. Our data demonstrate that while FAK mediated cell adhesion, RAFTK was localized at the cytoplasm where it mediated inside-out signaling through intracellular Ca(2+), thus leading to cell spreading and movement upon EGF stimulation.

    Funded by: NHLBI NIH HHS: HL 080699

    Cellular signalling 2007;19;2;289-300

  • PYK2 mediates anti-apoptotic AKT signaling in response to benzo[a]pyrene diol epoxide in mammary epithelial cells.

    Burdick AD, Ivnitski-Steele ID, Lauer FT and Burchiel SW

    The University of New Mexico College of Pharmacy Toxicology Program, 1 University of New Mexico Albuquerque, NM, USA.

    Polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (BaP), are known mammary carcinogens in rodents and may be involved in human breast cancer. The carcinogenicity of BaP has been partially attributed to the formation of the BaP diol epoxide (BPDE), which has been shown to stably bind DNA and act as an initiator. BaP is a complete carcinogen, but the mechanisms for tumor promotion are less well characterized. Previous studies have demonstrated that BPDE enhanced anti-apoptotic signaling through Akt; however, mechanisms for Akt activation by BPDE are not well defined. In the current studies, we found that BPDE increased intracellular Ca2+ concentration in the human mammary epithelial cell line MCF-10A. A peak in Ca2+ concentration at 20 min was followed by increased phosphorylation of Pyk2 at Tyr881 and increased total tyrosine phosphorylation of the epidermal growth factor receptor (EGFR). Consistent with activation of the EGFR, Akt and ERK1/2 phosphorylation was detected in MCF-10A cells treated with BPDE. Pharmacological methods to prevent Ca2+ elevation and EGFR activity, and small-interfering RNA against Pyk2, prevented Akt phosphorylation by BPDE, which suggested that Ca2+, Pyk2 and EGFR activation lay upstream of Akt. In addition, we found that BPDE increased p53 activity and apoptosis in MCF-10A; however, transient transfection of constitutively active Akt attenuated both BPDE-dependent apoptosis and p53 activity. In contrast, apoptosis was enhanced by inhibitors of phosphatidyl inositol 3-kinase (PI3-K). This work demonstrates a novel mechanism for Akt activation by BPDE that occurs through increased Ca2+ concentration, and implicates Ca2+, Pyk2, EGFR and Akt as a potential pathway by which BPDE can inhibit apoptosis and act as a promoter of carcinogenesis.

    Funded by: NIEHS NIH HHS: P30 ES-012072, R01 ES07259

    Carcinogenesis 2006;27;11;2331-40

  • de Amicis F, M, Kisslinger A, Calì G, Chieffi P, Andò S, Mancini FP and Tramontano D

    Dipartimento Farmaco-Biologico, Università della Calabria Arcavacata, Cosenza, Italy.

    Although prostate carcinoma is an aggressive cancer preferentially metastasizing to the bones, many prostate tumors remain localized and confined to the prostate indefinitely. Prediction of the behavior of anatomically localized and moderately differentiated prostate tumors remains difficult because of lack of prognostic markers. Cell motility is an important step in the progression of epithelial tumor toward invasive metastatic carcinomas and changes in the expression and function of adhesion molecules contribute to the acquisition of a more malignant phenotype. Proline-rich tyrosine kinase 2 (Pyk2) is implicated in regulating the organization of actin cytoskeleton, a process critical for cell migration, mitosis, and tumor metastasis. In this report, we investigated whether Pyk2 played a role in the acquisition of an aggressive phenotype in prostate cell. Data reported here demonstrate that loss of Pyk2 kinase function results in induction of cell motility and migration in EPN cells, a line of non-transformed epithelial cells derived from human normal prostate tissue. Changes in motility and migration of prostate cells were associated with changes in the expression of several proteins involved in cell adhesion and reorganization of actin cytoskeleton. Ablation of Pyk2 kinase activity caused a dramatic decrease of the expression of E-cadherin and IRS1 and an increase of the expression of alpha5-integrin. In addition, a massive reorganization of actin cytoskeleton was observed. Our data indicate that Pyk2 plays a central role in the mechanism that regulate cell-cell and cell-substrate interaction and lack of its kinase activity induces prostate cells to acquire a malignant, migrating phenotype.

    Journal of cellular physiology 2006;209;1;74-80

  • Focal adhesion kinase is involved in type III group B streptococcal invasion of human brain microvascular endothelial cells.

    Shin S, Paul-Satyaseela M, Maneesh PS, Lee JS, Romer LH and Kim KS

    Division of Pediatric Infectious Diseases, Johns Hopkins University School of Medicine, 600 North Wolfe Street, Park 256, Baltimore, MD 21287, USA.

    Group B streptococcus (GBS), the leading cause of neonatal meningitis, has been shown to invade human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. GBS invasion of HBMEC has been shown to require the host cell actin cytoskeleton rearrangements. The present study examined the mechanisms underlying actin cytoskeleton rearrangements that are involved in type III GBS invasion of HBMEC. We showed that type III GBS invasion was inhibited by genistein, a general tyrosine kinase inhibitor (mean 54% invasion decrease at 100 microM), and LY294002, a phosphatidylinositol 3 (PI3) kinase inhibitor (mean 70% invasion decrease at 50 microM), but not by PP2, an inhibitor of the Src family tyrosine kinases. We subsequently showed that the focal adhesion kinase (FAK) was the one of the host proteins tyrosine phosphorylated by type III GBS. Over-expression of a dominant negative form of 1f40 the FAK C-terminal domain significantly decreased type III GBS invasion of HBMEC (mean 51% invasion decrease). In addition, we showed that FAK phosphorylation correlated with its association of paxillin, an adapter protein of actin filament, and PI3-kinase subunit p85. This is the first demonstration that FAK phosphorylation and its association with paxillin and PI3 kinase play a key role in type III GBS invasion of HBMEC.

    Microbial pathogenesis 2006;41;4-5;168-73

  • Expression of FAK-related non-kinase (FRNK) coincides with morphological change in the early stage of cell adhesion.

    Nagoshi Y, Yamamoto G, Irie T and Tachikawa T

    Department of Oral Pathology and Diagnosis, Showa University School of Dentistry, 1-5-8 Hatanodai, Tokyo, 142-8555, Japan. syura@dent.showa-u.ac.jp

    Focal adhesion kinase (FAK), a protein tyrosine kinase, has recently been suggested to play a role in signal transduction through integrins. In fact, FAK is involved in cell proliferation and cell motility by performing signal transduction through integrins. FAK-related non-kinase (FRNK) has been found to be an inhibitor of FAK. As the expression level of FRNK in the cell is very low, the study of FRNK has been preferentially performed by gene overexpression, up to the present, and the role of constitutive FRNK in cells remains unclear. We hypothesized that FRNK is involved in the adhesion of cells to the extracellular matrix (ECM) and investigated the expression of FRNK by time kinetic analysis shortly after cell seeding. We found that FRNK expression was significantly increased in the cells during the early stage of cell adhesion to the ECM. These data indicated that FRNK plays an important role in cell adhesion during the very early stages of cell culture.

    Medical molecular morphology 2006;39;3;154-60

  • Novel role for integrin-linked kinase in modulation of coxsackievirus B3 replication and virus-induced cardiomyocyte injury.

    Esfandiarei M, Suarez A, Amaral A, Si X, Rahmani M, Dedhar S and McManus BM

    The James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research/Providence Health Care Research Institute, Vancouver, British Columbia, Canada.

    Viral myocarditis is a major cause of sudden cardiac death in children and young adults. Among viruses, coxsackievirus B3 (CVB3) is the most common agent for myocarditis. Recently, more consideration has been given to the role of signaling pathways in pathogenesis of enteroviral myocarditis, providing new platform for identifying a new potential therapeutic target for this, so far, incurable disease. Previously, we reported on the role of the protein kinase-B/Akt in CVB3 replication and virus-induced cell injury. Here, we report on regulation of virus-induced Akt activation by the integrin-linked kinase in infected mouse cardiomyocytes and HeLa cells. This study also presents the first observation that inhibition of ILK in CVB3-infected cells significantly improves the viability of infected cells, while blocking viral replication and virus release. Complementary experiments using a constitutively active form of Akt1 revealed that the observed protective effect of ILK inhibition is dependent on the associated downregulation of virus-induced Akt activation. To our knowledge, this is the first report of such beneficial effects of ILK inhibition in a viral infection model and conveys new insights in our efforts to characterize a novel therapeutic target for treatment of enteroviral myocarditis.

    Circulation research 2006;99;4;354-61

  • Phosphorylation of focal adhesion kinase (FAK) on Ser732 is induced by rho-dependent kinase and is essential for proline-rich tyrosine kinase-2-mediated phosphorylation of FAK on Tyr407 in response to vascular endothelial growth factor.

    Le Boeuf F, Houle F, Sussman M and Huot J

    Le Centre de Recherche en Cancérologie de l'Université Laval, Québec, Québec G1R-2J6, Canada.

    Focal adhesion kinase (FAK) is phosphorylated on tyrosine and serine residues after cell activation. In the present work, we investigated the relationship between tyrosine and serine phosphorylation of FAK in promoting endothelial cell migration in response to vascular endothelial growth factor (VEGF). We found that VEGF induces the activation of the Rho-dependent kinase (ROCK) downstream from vascular endothelial growth factor receptor (VEGFR) 2. In turn, activated ROCK directly phosphorylates FAK on Ser732. Proline-rich tyrosine kinase-2 (Pyk2) is also activated in response to VEGF. Its activation requires the clustering of integrin alphavbeta3 and triggers directly the phosphorylation of Tyr407 within FAK, an event necessary for cell migration. Interestingly, ROCK-mediated phosphorylation of Ser732 is essential for Pyk2-dependent phosphorylation of Tyr407, because the latter is abrogated in cells expressing a FAK mutant that is nonphosphorylatable on Ser732. We suggest that VEGF elicits the activation of the VEGFR2-ROCK pathway, leading to phosphorylation of Ser732 within FAK. In turn, phosphorylation of Ser732 would change the conformation of FAK, making it accessible to Pyk2 activated in response to its association with integrin beta3. Then, activated Pyk2 triggers the phosphorylation of FAK on Tyr407, promoting cell migration.

    Molecular biology of the cell 2006;17;8;3508-20

  • Activation pathways of alpha4beta1 integrin leading to distinct T-cell cytoskeleton reorganization, Rac1 regulation and Pyk2 phosphorylation.

    Maqueda A, Moyano JV, Gutiérrez-López MD, Ovalle S, Rodríguez-Frade JM, Cabañas C and Garcia-Pardo A

    Departamento de Inmunología, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.

    Alpha4beta1 integrin is highly expressed in lymphocytes and is essential in hematopoiesis, extravasation, and the inflammatory response. Alpha4beta1 can be activated by intracellular signals elicited upon T-cell activation by phorbol esters, CD3 crosslinking, or certain chemokine/receptor interactions (inside-out activation). Divalent cations or certain anti-beta1 mAbs (i.e., TS2/16) can also bind and activate integrins directly (outside-in activation). In both cases, activation results in increased adhesion and/or affinity for ligands. It is not known if these various stimuli produce the same or different post-adhesion events. To address this, we have studied the cytoskeleton organization and intracellular signaling following activation of 41 in Jurkat cells and in human T-lymphoblasts. Treatment with Mn2+, alpha-CD3 mAb or the chemokine SDF-1alpha followed by attachment to the fibronectin fragment H89 or the endothelial molecule VCAM-1 (alpha4beta1 ligands), resulted in cell polarization and migration. In contrast, activation with PMA or TS2/16 induced cell spreading and strong adherence. Video microscopy and Transwell analyses confirmed these results, which correlated with different resistance to detachment under flow. Activation of the small GTPase RhoA or transfection with the constitutively active mutants V14RhoA or V12Rac1, abolished the alpha4beta1-induced cell polarization but did not affect cell spreading. Moreover, Rac1 activity was distinctly modulated by agents that induce a polarized or spread phenotype. The tyrosine kinase Pyk2 was highly phosphorylated upon induction of cell polarity but not during cell spreading. These results reveal novel properties of alpha4beta1 integrin, namely the ability to trigger two types of T-cell cytoskeletal response with different signaling requirements.

    Journal of cellular physiology 2006;207;3;746-56

  • Src-family kinases mediate an outside-in signal necessary for beta2 integrins to achieve full activation and sustain firm adhesion of polymorphonuclear leucocytes tethered on E-selectin.

    Totani L, Piccoli A, Manarini S, Federico L, Pecce R, Martelli N, Cerletti C, Piccardoni P, Lowell CA, Smyth SS, Berton G and Evangelista V

    Laboratory of Vascular Biology and Pharmacology, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro, Italy. totani@negrisud.it

    In cell suspensions subjected to high-shear rotatory motion, human PMN (polymorphonuclear cells) adhered to E-selectin-expressing CHO (Chinese-hamster ovary) cells (CHO-E), and formed homotypic aggregates when challenged by E-selectin-IgG fusion protein, by a mechanism that involved beta2 integrins. Both heterotypic and homotypic PMN adhesion was accompanied by tyrosine phosphorylation of a 110 kDa protein (P110). This event was prevented by blocking anti-(beta2 integrin) antibodies and by inhibitors of Src-family kinases, suggesting that it was part of an 'outside-in' signalling that was initiated by integrin engagement. Interestingly, Src-family kinase inhibitors prevented beta2-integrin-mediated (i) homotypic PMN adhesion triggered by E-selectin-IgG, (ii) heterotypic CHO-E/PMN adhesion in mixed-cell suspensions, and (iii) firm adhesion of PMN to CHO-E monolayers under physiological flow. Similarly to PMN treated with Src-family kinase inhibitors, PMN from hck-/-fgr-/- and hck-/-fgr-/-lyn-/- mice showed significant impairment of beta2-integrin-mediated adhesion to CHO-E. Moreover, the expression of beta2 integrin activation epitopes at the sites of cell-cell contact in CHO-E/PMN conjugates was abolished by Src-family kinase inhibitors. One component of P110 was identified as the FAK (focal adhesion kinase) Pyk2 (proline-rich tyrosine kinase 2), which was phosphorylated in a beta2 integrin- and Src-family-kinase-dependent manner. Thus, Src-family kinases, and perhaps Pyk2, mediate a signal necessary for beta2 integrin function in PMN tethered by E-selectin.

    Funded by: NHLBI NIH HHS: HL070304, HL074219, HL78663, K08 HL070304, R01 HL074219, R01 HL078663; NIDDK NIH HHS: DK064183, R21 DK064183

    The Biochemical journal 2006;396;1;89-98

  • DNA sequence and analysis of human chromosome 8.

    Nusbaum C, Mikkelsen TS, Zody MC, Asakawa S, Taudien S, Garber M, Kodira CD, Schueler MG, Shimizu A, Whittaker CA, Chang JL, Cuomo CA, Dewar K, FitzGerald MG, Yang X, Allen NR, Anderson S, Asakawa T, Blechschmidt K, Bloom T, Borowsky ML, Butler J, Cook A, Corum B, DeArellano K, DeCaprio D, Dooley KT, Dorris L, Engels R, Glöckner G, Hafez N, Hagopian DS, Hall JL, Ishikawa SK, Jaffe DB, Kamat A, Kudoh J, Lehmann R, Lokitsang T, Macdonald P, Major JE, Matthews CD, Mauceli E, Menzel U, Mihalev AH, Minoshima S, Murayama Y, Naylor JW, Nicol R, Nguyen C, O'Leary SB, O'Neill K, Parker SC, Polley A, Raymond CK, Reichwald K, Rodriguez J, Sasaki T, Schilhabel M, Siddiqui R, Smith CL, Sneddon TP, Talamas JA, Tenzin P, Topham K, Venkataraman V, Wen G, Yamazaki S, Young SK, Zeng Q, Zimmer AR, Rosenthal A, Birren BW, Platzer M, Shimizu N and Lander ES

    Broad Institute of MIT and Harvard, 320 Charles St, Cambridge, Massachusetts 02141, USA. chad@broad.mit.edu

    The International Human Genome Sequencing Consortium (IHGSC) recently completed a sequence of the human genome. As part of this project, we have focused on chromosome 8. Although some chromosomes exhibit extreme characteristics in terms of length, gene content, repeat content and fraction segmentally duplicated, chromosome 8 is distinctly typical in character, being very close to the genome median in each of these aspects. This work describes a finished sequence and gene catalogue for the chromosome, which represents just over 5% of the euchromatic human genome. A unique feature of the chromosome is a vast region of approximately 15 megabases on distal 8p that appears to have a strikingly high mutation rate, which has accelerated in the hominids relative to other sequenced mammals. This fast-evolving region contains a number of genes related to innate immunity and the nervous system, including loci that appear to be under positive selection--these include the major defensin (DEF) gene cluster and MCPH1, a gene that may have contributed to the evolution of expanded brain size in the great apes. The data from chromosome 8 should allow a better understanding of both normal and disease biology and genome evolution.

    Funded by: Medical Research Council: G0000107

    Nature 2006;439;7074;331-5

  • FAK and PYK2 interact with SAP90/PSD-95-Associated Protein-3.

    Bongiorno-Borbone L, Kadaré G, Benfenati F and Girault JA

    INSERM, U536, Paris, France.

    Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2) are two related non-receptor tyrosine kinases highly expressed in brain. Although they are both involved in synaptic plasticity, little is known about their specific neuronal partners. Using a yeast two-hybrid screen and GST pull-down assays we show that SAPAP3 (SAP90/PSD-95-Associated Protein-3) interacts with FAK (residues 676-840) and PYK2. The three proteins partly co-distribute in the same sucrose gradient fractions as the post-synaptic density protein PSD-95 and Src. Our results suggest that SAPAP3 is an anchoring protein for FAK and PYK2 in post-synaptic densities and may contribute to the synaptic function of these tyrosine kinases.

    Biochemical and biophysical research communications 2005;337;2;641-6

  • Semaphorin 4D/plexin-B1 induces endothelial cell migration through the activation of PYK2, Src, and the phosphatidylinositol 3-kinase-Akt pathway.

    Basile JR, Afkhami T and Gutkind JS

    Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA. sg39v@nih.gov

    Semaphorins are cell surface and secreted proteins that provide axonal guidance in neuronal tissues and regulate cell motility in many cell types. They act by binding a family of transmembrane receptors known as plexins, which belong to the c-Met family of scatter factor receptors but lack an intrinsic tyrosine kinase domain. Interestingly, we have recently shown that Plexin-B1 is highly expressed in endothelial cells and that its activation by Semaphorin 4D elicits a potent proangiogenic response (J. R. Basile, A. Barac, T. Zhu, K. L. Guan, and J. S. Gutkind, Cancer Res. 64:5212-5224, 2004). In searches for the underlying molecular mechanism, we observed that Semaphorin 4D-stimulated endothelial cell migration requires the activation of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway. Surprisingly, we found that Plexin-B1 stimulates PI3K-Akt through the activation of an intracellular tyrosine kinase cascade that involves the sequential activation of PYK2 and Src. This results in the tyrosine phosphorylation of Plexin-B1, the rapid recruitment of a multimeric signaling complex that includes PYK2, Src, and PI3K to Plexin-B1 and the activation of Akt. These findings suggest that Plexin-B1 may achieve its numerous physiological functions through the direct activation of intracellular tyrosine kinase cascades.

    Molecular and cellular biology 2005;25;16;6889-98

  • Critical role of proline-rich tyrosine kinase 2 in reversion of the adhesion-mediated suppression of reactive oxygen species generation by human neutrophils.

    Zhao T and Bokoch GM

    Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037, USA. tzhao@scripps.edu

    Neutrophils act as the first line of innate immune defense against invading microorganisms during infection and inflammation. The tightly regulated production of reactive oxygen species (ROS) through activation of NADPH oxidase is a major weapon used by neutrophils and other phagocytic leukocytes to combat such pathogens. Cellular adhesion signals play important physiological roles in regulating the activation of NADPH oxidase and subsequent ROS formation. We previously showed that the initial suppression of the oxidase response of chemoattractant-stimulated adherent neutrophils is mediated via inhibition of Vav1-induced activation of the NADPH oxidase regulatory GTPase Rac2 by adhesion signals. In this study we show that prior exposure of neutrophils to a number of cytokines and inflammatory mediators, including TNF-alpha, GM-CSF, and platelet-activating factor, overcomes the adhesion-mediated suppression of ROS formation. Proline-rich tyrosine kinase 2 (pyk2) activity is enhanced under these conditions, correlating with the restoration of Vav1 and Rac2 activities. Both dominant negative pyk2 and a pyk2-selective inhibitor prevented restoration of ROS production induced by TNF-alpha, GM-CSF, and platelet-activating factor, and this loss of pyk2 activity resulted in decreased Vav1 tyrosine phosphorylation and subsequent Rac2 activation. Our studies identify pyk2 as a critical regulatory component and a molecular switch to overcome the suppression of leukocyte oxidant generation by cell adhesion. This activity constitutes a mechanism by which cytokines might lead to rapid elimination of invading pathogens by adherent neutrophils under normal conditions or enhance tissue damage in pathological states.

    Funded by: NHLBI NIH HHS: HL48008

    Journal of immunology (Baltimore, Md. : 1950) 2005;174;12;8049-55

  • Proline-rich tyrosine kinase 2 (Pyk2) mediates vascular endothelial-cadherin-based cell-cell adhesion by regulating beta-catenin tyrosine phosphorylation.

    van Buul JD, Anthony EC, Fernandez-Borja M, Burridge K and Hordijk PL

    Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, 1066 CX Amsterdam, The Netherlands.

    Vascular endothelial-cadherin (VE-cadherin) controls endothelial cell-cell adhesion and preserves endothelial integrity. In order to maintain endothelial barrier function, VE-cadherin function is tightly regulated through mechanisms that involve protein phosphorylation and cytoskeletal dynamics. Here, we show that loss of VE-cadherin function results in intercellular gap formation and a drop in electrical resistance of monolayers of primary human endothelial cells. Detailed analysis revealed that loss of endothelial cell-cell adhesion, induced by VE-cadherin-blocking antibodies, is preceded by and dependent on a rapid activation of Rac1 and increased production of reactive oxygen species. Moreover, VE-cadherin-associated beta-catenin is tyrosine-phosphorylated upon loss of cell-cell contact. Finally, the redox-sensitive proline-rich tyrosine kinase 2 (Pyk2) is activated and recruited to cell-cell junctions following the loss of VE-cadherin homotypic adhesion. Conversely, the inhibition of Pyk2 activity in endothelial cells by the expression of CRNK (CADTK/CAKbeta-related non-kinase), an N-terminal deletion mutant that acts in a dominant negative fashion, not only abolishes the increase in beta-catenin tyrosine phosphorylation but also prevents the loss of endothelial cell-cell contact. These results implicate Pyk2 in the reduced cell-cell adhesion induced by the Rac-mediated production of ROS through the tyrosine phosphorylation of beta-catenin. This signaling is initiated upon loss of VE-cadherin function and is important for our insight in the modulation of endothelial integrity.

    Funded by: NHLBI NIH HHS: HL45100

    The Journal of biological chemistry 2005;280;22;21129-36

  • The tyrosine kinase pyk2 promotes migration and invasion of glioma cells.

    Lipinski CA, Tran NL, Menashi E, Rohl C, Kloss J, Bay RC, Berens ME and Loftus JC

    Mayo Clinic Scottsdale, Scottsdale, AZ 85259, USA.

    Glioblastoma multiforme is extraordinarily aggressive due to the propensity of cells to migrate away from the tumor core into the surrounding normal brain. In this report, we investigated the role of proline-rich tyrosine kinase 2 (Pyk2) and FAK with regard to influencing glioma cell phenotypes. Expression of Pyk2 stimulated glioma cell migration, whereas expression of FAK inhibited glioma cell migration and stimulated cell cycle progression. Pyk2 autophosphorylation was necessary, but not sufficient, to stimulate cellular migration. The N-terminal domain of Pyk2 is required for stimulation of migration as an N-terminally deleted variant of Pyk2 failed to stimulate migration, whereas expression of an autonomous Pyk2 N-terminal domain inhibited cell migration. Substitution of the C-terminal domain of Pyk2 with the corresponding domain of FAK stimulated cell migration as effectively as wild-type Pyk2; however, substitution of the N-terminal domain of Pyk2 with that of FAK inhibited cell migration, substantiating that the N-terminal domain of Pyk2 was required to stimulate mig 5a8 ration. Silencing of Pyk2 expression by RNA interference significantly inhibited glioma migration. Cell migration was restored on re-expression of Pyk2, but expression of FAK in Pyk2 knockdown cells failed to restore migration. We conclude that Pyk2 plays a central role in the migratory behavior of glioblastomas.

    Neoplasia (New York, N.Y.) 2005;7;5;435-45

  • Tyrosine phosphorylation of beta3 integrin provides a binding site for Pyk2.

    Butler B and Blystone SD

    Department of Cell and Developmental Biology, SUNY Upstate Medical University, Syracuse, New York 13210, USA.

    Integrins expressed on leukocytes possess the ability to maintain themselves in a non-adhesive state, thus preventing unwarranted adhesion and uncontrolled inflammation. Leukocyte adhesion is regulated through the modulation of integrin receptors such as alpha(V)beta(3). Firm adhesion to the extracellular matrix and directed cellular motility requires the reorganization of the actin cytoskeleton. The ability of beta(3) to recruit signaling and scaffolding molecules to propagate alpha(V)beta(3) -mediated signals is regulated in part by the phosphorylation of the beta(3) cytoplasmic tail. The identities of integrin-associated signaling molecules within alpha(V)beta(3) podosomes and in particular the proximal binding partners of the beta(3) cytoplasmic tail are not completely known. Here we show that alpha(V)beta(3) ligation induces Pyk2-Tyr-402 phosphorylation and its association with the beta(3) cytoplasmic tail in a beta(3)-Tyr-747 phosphorylation-dependent manner. Pyk2 binding to the beta(3) cytoplasmic tail is direct and dependent upon Pyk2-Tyr-402 and beta(3) -Tyr-747 phosphorylations. These data identify Pyk2 as a phosphorylated beta(3) binding partner, providing a potential structural and signaling platform to achieve alpha(V)beta(3) -mediated remodeling of the actin cytoskeleton.

    Funded by: NIAID NIH HHS: K02AI57384, R01AI40602

    The Journal of biological chemistry 2005;280;15;14556-62

  • Ephrin-A1 binding to CD4+ T lymphocytes stimulates migration and induces tyrosine phosphorylation of PYK2.

    Aasheim HC, Delabie J and Finne EF

    Department of Immunology and Pathology, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway. h.c.asheim@labmed.uio.no

    Eph receptors, the largest subfamily of receptor tyrosine kinases, and their ephrin ligands are important mediators of cell-cell communication regulating cell attachment, shape, and mobility. Here we demonstrate that CD4+ T lymphocytes express the EphA1 and EphA4 receptors and that these cells bind the ligand ephrin-A1. Further we show ephrin-A1 expression in vivo on high endothelial venule (HEV) endothelial cells. Ephrin-A1 binding to CD4+ T cells stimulates both stromal cell-derived factor 1alpha (SDF-1alpha)- and macrophage inflammatory protein 3beta (MIP3beta)-mediated chemotaxis. In line with the increased chemotactic response, increased actin polymerization is observed in particular with the combination of ephrin-A1 and SDF-1alpha. Signaling through EphA receptors induces intracellular tyrosine phosphorylation. In particular, proline-rich tyrosine kinase 2 (PYK2) is phosphorylated on tyrosine residues 402 and 580. Ephrin-A1-induced chemotaxis and intracellular tyrosine phosphorylation, including EphA1 and Pyk2, was inhibited by Tyrphostin-A9. In conclusion, ligand engagement of EphA receptors on CD4+ T cells stimulates chemotaxis, induces intracellular tyrosine phosphorylation, and affects actin polymerization. This, together with our finding that ephrin-A1 is expressed by HEV endothelial cells, suggests a role for Eph receptors in transendothelial migration.

    Blood 2005;105;7;2869-76

  • Angiotensin II stimulated transcription of cyclooxygenase II is regulated by a novel kinase cascade involving Pyk2, MEKK4 and annexin II.

    Derbyshire ZE, Halfter UM, Heimark RL, Sy TH and Vaillancourt RR

    Department of Pharmacology and Toxicology, University of Arizona, Tucson, AZ 85721, USA.

    Although it is known that MEKK4 regulates MKK6, and p38 MAP kinase, extracellular stimuli that activate the serine/threonine kinase, MEKK4, are unknown. The aim of this study was then to identify stimuli that regulate MEKK4. By using recombinant MEKK4, as bait to attract interacting proteins, the calcium binding protein, annexin II, was identified by mass spectrometry as interacting with MEKK4, suggesting that MEKK4 might be regulated by calcium. A calcium-dependent interaction between MEKK4 and annexin II was observed when MEKK4 was immunoprecipitated from rat aortic smooth muscle cells that were treated with angiotensin II. Additional studies using recombinant MEKK4 in a Far-Western immunoblot identified a protein of 120 kDa as interacting directly with MEKK4. Prior studies indicated that MEKK4 was phosphorylated on tyrosine in vivo, and in fact, Pyk2 interacts with MEKK4 in an angiotensin II dependent manner in rat aortic smooth muscle cells. Pyk2 phosphorylates MEKK4 in vitro and Pyk2-dependent phosphorylation further regulates MEKK4-dependent phosphorylation of MKK6. Finally, dominant-negative MEKK4 inhibits angiotensin II mediated transcription of a luciferase reporter construct containing the cyclooxygenase II promoter, demonstrating that MEKK4 functions in a calcium-dependent manner as a substrate for Pyk2 and regulates transcription of cyclooxygenase II.

    Funded by: NIA NIH HHS: AG19710; NIEHS NIH HHS: ES12007, P30 ES06694, P42 ES04940

    Molecular and cellular biochemistry 2005;271;1-2;77-90

  • Tyrosine phosphorylation of PYK2 mediates heregulin-induced glioma invasion: novel heregulin/HER3-stimulated signaling pathway in glioma.

    van der Horst EH, Weber I and Ullrich A

    Department of Molecular Biology, Max Planck Institute for Biochemistry, Munich, Germany.

    Receptor tyrosine kinases of the EGFR family transmit extracellular signals that control diverse cellular functions such as proliferation, differentiation and survival. Signaling function of a member of this family, HER3, is believed to be impaired due to deviations in its kinase consensus motifs. Here we address the functional role and signaling mechanisms of HER3. HER3 preferentially forms heterodimers with HER2 inducing the most potent mitogenic signal among EGFR family members. Our data show that in a glioma-derived cell line the cytoplasmic tyrosine kinase PYK2 is constitutively associated with HER3 and that stimulation with Heregulin results in PYK2 tyrosine phosphorylation. HER3, but not HER2, mediates the phosphorylation of the C-terminal region of PYK2 to promote a mitogenic response through activation of the MAPK pathway. A central role of PYK2 in signaling downstream of HER3 is substantiated by the demonstration that expression of a dominant-negative PYK2-KM construct abrogates the Heregulin-induced MAPK activity and inhibits the invasive potential of glioma cells. These results suggest a novel Heregulin/HER3-stimulated signaling pathway in glioblastoma-derived cell lines that involves phosphorylation of PYK2 and mediates invasiveness of glioma cells.

    International journal of cancer 2005;113;5;689-98

  • Src phosphorylation of Alix/AIP1 modulates its interaction with binding partners and antagonizes its activities.

    Schmidt MH, 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

  • 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

  • Focal adhesion kinase in netrin-1 signaling.

    Ren XR, Ming GL, Xie Y, Hong Y, Sun DM, Zhao ZQ, Feng Z, Wang Q, Shim S, Chen ZF, Song HJ, Mei L and Xiong WC

    Department of Pathology, University of Alabama, Birmingham, Alabama 35294, USA.

    Netrins are a family of secreted molecules that are important for axonal outgrowth and guidance in the developing nervous system. However, the signaling mechanisms that lie immediately downstream of netrin receptors remain poorly understood. Here we report that the netrin receptor DCC (deleted in colorectal cancer) interacts with the focal adhesion kinase (FAK), a kinase implicated in regulating cell adhesion and migration. FAK was expressed in developing brains and was localized with DCC in cultured neurons. Netrin-1 induced FAK and DCC tyrosine phosphorylation. Disruption of FAK signaling abolished netrin-1-induced neurite outgrowth and attractive growth cone turning. Taken together, these results indicate a new signaling mechanism for DCC, in which FAK is activated upon netrin-1 stimulation and mediates netrin-1 function; they also identify a critical role for FAK in axon navigation.

    Nature neuroscience 2004;7;11;1204-12

  • Phosphatidylinositol-3-kinase activation mediates proline-rich tyrosine kinase 2 phosphorylation and recruitment to beta1-integrins in human CD34+ cells.

    Melikova S, Dylla SJ and Verfaillie CM

    Stem Cell Institute, University of Minnesota, Minneapolis, Minnesota, USA.

    Objective: beta1-integrins mediate hematopoietic stem and progenitor cell homing and retention in the bone marrow (BM) and inhibit hematopoietic proliferation and differentiation. Having no intrinsic kinase activity, integrins recruit intracellular kinases, such as the focal adhesion kinase (FAK) or the related proline-rich tyrosine kinase 2 (PYK2), to initiate signal transduction. Phosphatidylinositol-3-kinase (PI3K), which is involved in beta1-integrin signaling in many cell types, is physically and functionally associated with FAK in anchorage-dependent cells. Because PYK2 is the principal focal adhesion kinase expressed in primary human CD34+ cells, we assessed its functional relationship with PI3K in CD34+ cells in response to integrin engagement.

    Methods: beta1-integrins on primary mobilized peripheral blood CD34+ cells and CD34+ KG1A cells were engaged by adhesion to fibronectin (FN) or by cross-linking with an anti-beta1 integrin antibody, respectively. PI3K activity and PYK2 phosphorylation were then assessed in the presence or absence of the PI3K inhibitor, wortmannin. Association between PI3K, PYK2, and the beta1-integrin subunit were also evaluated in co-immunoprecipitation experiments.

    Results: beta1-integrin engagement induced PI3K activation, which was required for, and temporally preceded, PYK2 phosphorylation, indicating that PI3K lies upstream of PYK2 in CD34+ cells. Furthermore, although PYK2 and PI3K were constitutively associated, interaction of the PYK2/PI3K complex with beta1-integrins required prior integrin engagement and PI3K activation.

    Conclusion: Activation of PI3K following beta1-integrin engagement on human CD34+ cells results in subsequent phosphorylation of PYK2, and is required for the recruitment of the PI3K/PYK2 complex to beta1-integrins at the cell surface.

    Funded by: NCI NIH HHS: CA09138; NHLBI NIH HHS: R01 HL-49930; NIDDK NIH HHS: R01 DK-53673

    Experimental hematology 2004;32;11;1051-6

  • 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 cytoplasmic tyrosine kinase Pyk2 as a novel effector of fibroblast growth factor receptor 3 activation.

    Meyer AN, Gastwirt RF, Schlaepfer DD and Donoghue DJ

    Department of Chemistry and Biochemistry, Center for Molecular Genetics, University of California-San Diego, La Jolla, CA 92093-0367, USA.

    Activating mutations within fibroblast growth factor receptor 3 (FGFR3), a receptor tyrosine kinase, are responsible for human skeletal dysplasias including achondroplasia and the neonatal lethal syndromes thanatophoric dysplasia types I and II. Several of these same FGFR3 mutations have also been identified somatically in human cancers, including multiple myeloma, bladder carcinoma, and cervical cancer. The molecular pathways exploited by FGFR3 to stimulate abnormal proliferation during neoplasia are unclear. The nonreceptor protein-tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2) has been shown previously to regulate apoptosis in multiple myeloma cells. Here we describe a novel interaction between FGFR3 and Pyk2, mediated by the juxtamembrane domain of FGFR3 and the kinase domain of Pyk2. Within the FGFR family, Pyk2 also interacted significantly with FGFR2. Overexpression of Pyk2 alone led to its spontaneous activation and tyrosine phosphorylation, resulting in activation of Stat5B, indicated by the reporter GFP-Stat5B. These effects were completely dependent upon Tyr(402), the autophosphorylation site of Pyk2, which allows recruitment of Src family members for further activating phosphorylations at other sites on Pyk2. In the presence of activated FGFR3, the activation of Pyk2 itself became independent of Tyr(402), indicating that FGFR3 activation circumvents the requirement for c-Src recruitment at Tyr(402) of Pyk2. We also examined the role of the tyrosine phosphatase Shp2 in antagonizing Pyk2 activation. Taken together, these results suggest that signaling pathways regulated by FGFR3 may converge with Pyk2-dependent pathways to provide maximal activation of Stat5B.

    Funded by: NCI NIH HHS: R01 CA 90900; NIGMS NIH HHS: R01 GM 65490

    The Journal of biological chemistry 2004;279;27;28450-7

  • The epidermal growth factor receptor (EGFR) and proline rich tyrosine kinase 2 (PYK2) are involved in tissue factor dependent factor VIIa signalling in HaCaT cells.

    Wiiger MT and Prydz H

    The Biotechnology Centre of Oslo, University of Oslo, Gaustadalleen 21, 0349 Oslo, Norway.

    Binding of the coagulation protease factor VIIa to its receptor Tissue Factor (TF) induces intracellular signals in several cell types including HaCaT keratinocytes. TF belongs to the cytokine receptor family, but is most likely not alone in transferring the complete TF/FVIIa signal over the plasma membrane. The protease activated receptor PAR2 is involved in factor VIIa and factor Xa signal transduction. Our results indicate that the epidermal growth factor receptor (EGFR) and the proline rich tyrosine kinase 2 (PYK2) participate in TF/FVIIa signalling as formation of the TF/FVIIa complex increased the phosphorylation of these proteins. Both FVIIa protease activity and available TF were necessary for generation of the signal. Increased tyrosine phosphorylation of the EGFR was observed following TF/FVIIa complex formation on the cell surface. The EGFR kinase inhibitor tyrphostin AG1478 abrogated the TF/FVIIa-complex induced MAP kinase activation and mRNA increase of egr-1, heparin-binding EGF, and interleukin-8 following FVIIa addition. Using specific antibodies, increased phosphorylation of PYK2 tyrosine residues 402 and 580 was observed. The first site is the major autophosphorylation site and the docking site for Src family kinases. The second site is important for the kinase activity. The Src family kinase Yes and the tyrosine phosphatase SHP-2 were detected in immunoprecipitates using either anti-PYK2 or anti-EGFR antibodies. Their coprecipitation with EGFR increased in the presence of FVIIa. Moreover, the coprecipitation of EGFR and PYK2 increased with FVIIa stimulation. Together, these data suggest that EGFR, PYK2, Yes, and SHP-2 are involved in transduction of the TF/FVIIa signal possibly via transactivation of the EGF receptor.

    Thrombosis and haemostasis 2004;92;1;13-22

  • Differential regulation of cell migration and proliferation through proline-rich tyrosine kinase 2 in endothelial cells.

    Kuwabara K, Nakaoka T, Sato K, Nishishita T, Sasaki T and Yamashita N

    Department of Advanced Medical Science, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

    Proline-rich tyrosine kinase 2 (Pyk2), a member of the focal adhesion kinase family, is thought to act as a key component in vasculogenesis and angiogenesis. Therefore, we studied the effect of mutant Pyk2 expression on the migration and proliferation in endothelial cells (ECs). Two types of mutant Pyk2 were examined by adenovirus vectors AxCA-Pyk2K457A, expressing a kinase inactive mutant, and AxCA-Pyk2Y402F, expressing a tyrosine autophosphorylation site mutant, in addition to AxCA-Pyk2, expressing wild-type Pyk2. Migration of ECs infected with AxCA-Pyk2Y402F increased to a level similar to that of ECs infected with AxCA-Pyk2. The size of effect was dependent on the amount of applied adenoviruses within the range of 3-30 multiplicity of infection. In contrast, AxCA-Pyk2K457A infection did not show any significant effect on cell migration. Western blotting showed that both phosphorylation of Pyk2 Y(881) and association of p130(Cas) with Pyk2 were enhanced in ECs infected with AxCA-Pyk2Y402F as well as with AxCA-Pyk2, but not in ECs infected with AxCA-Pyk2K457A. Therefore, signaling mediated by Pyk2 Y(881) and p130(Cas) may be involved in the migration of ECs infected either with AxCA-Pyk2Y402F or with AxCA-Pyk2. In proliferation assay, AxCA-Pyk2 infection suppressed EC proliferation significantly; however, neither AxCA-Pyk2Y402F nor AxCA-Pyk2K457A showed such an inhibitory effect. Thus, the two Pyk2 mutants revealed that Pyk2 signaling differentially regulates cell migration and proliferation pathways.

    Endocrinology 2004;145;7;3324-30

  • Recruitment of Pyk2 and Cbl to lipid rafts mediates signals important for actin reorganization in growing neurites.

    Haglund K, Ivankovic-Dikic I, Shimokawa N, Kruh GD and Dikic I

    Institute for Biochemistry II, Building 75, Goethe University Medical School, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany.

    Protein tyrosine kinase Pyk2 and multifunctional adaptor protein Cbl are implicated in the regulation of the cytoskeleton in several cell types. We report that Pyk2 and Cbl form a signaling complex that is translocated to lipid rafts and is enriched in growth cones of differentiating PC12 cells following growth factor stimulation. We found that Pyk2 and Cbl interacted with the adaptor protein ArgBP2, which also bound to flotillin-1, a component of lipid raft microdomains. These interactions contributed to recruitment of the Pyk2/Cbl complex to lipid raft compartments. In addition, Pyk2, Cbl and ArgBP2 were found co-localized with actin in axons and growth cones of differentiated PC12 cells. Moreover, co-expression of Pyk2, ArgBP2 and Cbl facilitated growth factor-induced formation of lamellipodia at the tip of neurites. Formation of these growth cone lamellipodia was dependent on intact lipid rafts and the Cbl-associated effectors Crk and phosphatidylinositol 3 (PI 3)-kinase. Our results indicate that recruitment of Pyk2/Cbl complexes to lipid rafts participates in growth factor-induced regulation of the actin cytoskeleton in growing neurites.

    Journal of cell science 2004;117;Pt 12;2557-68

  • SRC-dependent outside-in signalling is a key step in the process of autoregulation of beta2 integrins in polymorphonuclear cells.

    Piccardoni P, Manarini S, Federico L, Bagoly Z, Pecce R, Martelli N, Piccoli A, Totani L, Cerletti C and Evangelista V

    Laboratory of Vascular Biology and Pharmacology, Consorzio Mario Negri Sud, Via Nazionale 1, 66030, Santa Maria Imbaro, Italy.

    In human PMN (polymorphonuclear cells), challenged by P-selectin, the beta2-integrin Mac-1 (macrophage antigen-1) promoted the activation of the SRC (cellular homologue of Rous sarcoma virus oncogenic protein) family members HCK (haematopoietic cell kinase) and LYN (an SRC family protein tyrosine kinase) and phosphorylation of a P-110 (110 kDa protein). SRC kinase activity in turn was necessary for macrophage antigen-1-mediated adhesion [Piccardoni, Sideri, Manarini, Piccoli, Martelli, de Gaetano, Cerletti and Evangelista (2001) Blood 98, 108-116]. This suggested that an SRC-dependent outside-in signalling strengthens the beta2-integrin interaction with the ligand. To support this hypothesis further, in the present study, we used the monoclonal antibody KIM127 or manganese to lock beta2 integrins in a high-affinity state, and homotypic PMN adhesion was analysed to monitor beta2-integrin adhesive function. KIM127 or manganese induced PMN homotypic adhesion and P-110 phosphorylation. Both these processes were abolished by blocking antibodies against the common beta2 chain, by a combination of antibodies against alphaL and alphaM or by inhibitors of SRC activity. Confocal microscopy showed that activation epitopes were expressed by beta2 integrins co-localized with patches of F-actin at the adhesion sites. Blockade of SRC kinases or of actin polymerization prevented clustering of activated integrins as well as F-actin accumulation. FACS analysis showed that SRC inhibitors modified neither basal nor manganese-induced KIM127 binding. An SRC-dependent outside-in signalling initiated by beta2 integrins was also required for adhesion triggered by interleukin-8. These results confirm the hypothesis that an SRC-dependent outside-in signalling triggered by high affinity and ligand binding is necessary to stabilize beta2-integrin-mediated adhesion. Allowing clustering of activated integrins, SRC might link the high-affinity with the high-avidity state. Proline-rich tyrosine kinase-2 appears to be involved in this process.

    The Biochemical journal 2004;380;Pt 1;57-65

  • Pyk2 amplifies epidermal growth factor and c-Src-induced Stat3 activation.

    Shi CS and Kehrl JH

    B Cell Molecular Immunology Section, Laboratory of Immunoregulation, NIAID, National Institutes of Health, Bethesda, Maryland 20892-1876, USA.

    Signal transducers and activators of transcription factors (STATs) mediate many of the cellular responses that occur following cytokine, growth factor, and hormone signaling. STATs are activated by tyrosine and serine phosphorylation, which normally occurs as a tightly regulated process. Dysregulated STAT activity may facilitate oncogenesis, as constitutively activated STATs have been found in many human tumors as well as in v-abl- and v-src-transformed cell lines. Pyk2 is a member of the focal adhesion kinase family and can be activated by c-Src, epidermal growth factor receptor (EGFR), Janus kinase 1, tyrosine kinases, and G-protein-coupled receptor signaling. Although Pyk2 has been implicated in Janus kinase-dependent activation of MAPK and Stat1, no role for Pyk2 in the activation of other STAT proteins has been ascribed. Here, we provide evidence that Pyk2, along with c-Src, facilitates EGFR-mediated Stat3 activation. Pyk2 expression in HeLa cells induces Stat3 reporter gene activation and Stat3 phosphorylation on amino acid residues Tyr-705 and Ser-727. Together Pyk2 and c-Src potently activate Stat3, and Pyk2 enhances Stat3-induced cell proliferation. Moreover, the expression of a dominant negative version of Pyk2 impairs c-Src-induced Stat3 activation and cell proliferation. The treatment of A431 cells with EGF results in the recruitment of c-Src, Pyk2, and Stat3 to the EGFR and the phosphorylation of c-Src, Pyk2, and Stat3. Expression of constructs for dominant negative forms of either Pyk2 or c-Src impair EGF-induced Stat3 phosphorylation. These results indicate that Pyk2 facilitates EGFR- and c-Src-mediated Stat3 activation, thereby implicating Pyk2 activation as a potential co-mediator in triggering Stat3-induced oncogenesis.

    The Journal of biological chemistry 2004;279;17;17224-31

  • Integrin engagement-induced inhibition of human myelopoiesis is mediated by proline-rich tyrosine kinase 2 gene products.

    Dylla SJ, Deyle DR, Theunissen K, Padurean AM and Verfaillie CM

    Stem Cell Institute and Department of Medicine, University of Minnesota, Minneapolis, Minn, USA.

    Objective: Hematopoietic progenitor proliferation and differentiation are inhibited by integrin engagement of fibronectin (FN). Focal adhesion kinases have been shown to mediate intracellular signaling from integrins, and we recently demonstrated that gene expression and pre-mRNA splicing of the focal adhesion kinase, PYK2, is abnormal in CD34(+) cells from chronic myelogenous leukemia (CML) patients. Here we investigated whether PYK2 gene products mediate integrin signaling in hematopoietic stem and progenitor cells.

    Methods: Cord blood CD34(+) cells were retrovirally transduced with vectors encoding Pyk2H, Pyk2, or the dominant negative-acting, kinase-deficient, C-terminal PYK2 fragment, PRNK, and myeloid proliferation and differenti 1284 ation was assessed using colony-forming cell (CFC), long-term culture-initiating cell (LTC-IC), and liquid culture assays.

    Results: CD34(+) cells overexpressing Pyk2H or Pyk2 generated 50% less colony-forming unit granulocyte/macrophage (CFU-GM) than eGFP-transduced controls. Although the number of CFC generated by PRNK-expressing cells was unchanged, LTC-IC were significantly reduced. Culture of CD34(+) cells on FN significantly reduced the generation of mature myeloid cells vs those cultured on BSA-coated wells, and could be overcome by addition of SCF. As is observed when integrins are engaged, overexpression of either Pyk2H or Pyk2 decreased committed myeloid progenitor proliferation and differentiation; however, SCF could not override this inhibition. Finally, as is observed when integrins are not engaged, PRNK-mediated inhibition of endogenous Pyk2H resulted in integrin-nonresponsive proliferation and differentiation of myeloid precursors and accelerated differentiation of primitive hematopoietic progenitors.

    Conclusion: These studies indicate that PYK2 gene products mediate integrin-induced signals that regulate myelopoiesis.

    Funded by: NCI NIH HHS: CA09138; NHLBI NIH HHS: R01 HL-49930; NIDDK NIH HHS: R01 DK-53673

    Experimental hematology 2004;32;4;365-74

  • p210BCR/ABL-induced alteration of pre-mRNA splicing in primary human CD34+ hematopoietic progenitor cells.

    Salesse S, Dylla SJ and Verfaillie CM

    Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA.

    Chronic myelogenous leukemia (CML) is a malignancy of the human hematopoietic stem cell (HSC) caused by the p210BCR/ABL oncoprotein. Although alternative splicing of pre-mRNA is a critical determinant of a cell's protein repertoire, it has not been associated with CML pathogenesis. We identified a BCR/ABL-dependent increase in expression of multiple genes involved in pre-mRNA splicing (eg SRPK1, RNA Helicase II/Gu, and hnRNPA2/B1) by subtractive hybridization of cDNA from p210BCR/ABL-eGFP vs eGFP-transduced umbilical cord blood CD34+ cells. beta1-integrin signaling is important to HSC maintenance and proliferation/differentiation, and is abnormal in CML. As an example of how changes in pre-mRNA processing might contribute to CML pathogenesis, we observed alternative splicing of a gene for a beta1-integrin-responsive nonreceptor tyrosine kinase (PYK2), resulting in increased expression of full-length Pyk2 in BCR/ABL-containing cells. Treatment of p210BCR/ABL-positive cells with the Abl-specific tyrosine kinase inhibitor STI571 reverted PYK2 splicing to a configuration more consistent with normal cells, and correlated with decreased expression of BCR/ABL-induced proteins involved in pre-mRNA processing. Whether altered PYK2 splicing contributes to CML pathogenesis remains undetermined; however, we propose that generic changes in pre-mRNA splicing as a result of p210BCR/ABL kinase activity may contribute to CML pathogenesis.

    Funded by: NCI NIH HHS: CA09138; NHLBI NIH HHS: R01 HL-49930; NIDDK NIH HHS: R01 DK-53673

    Leukemia 2004;18;4;727-33

  • Vascular endothelial growth factor-mediated activation of p38 is dependent upon Src and RAFTK/Pyk2.

    McMullen M, Keller R, Sussman M and Pumiglia K

    Center for Cell Biology and Cancer Research, Albany Medical College, Albany NY, USA.

    Vascular endothelial growth factor (VEGF) induces activation of p38 mitogen-activated protein kinase (MAPK) in primary endothelial cells and may be critical for VEGF-induced angiogenesis. We investigated the molecular basis for p38 activation in response to VEGF. The expression of a C-terminal splice variant of FAK, FRNK, had no affect on VEGF-induced activation of p38; however, expression of a dominant-negative RAFTK/Pyk2 mutant led to a decrease in the activation of p38, but had no affect on extracellular signal-regulated kinase (ERK). Since calcium regulates RAFTK/Pyk2, we investigated its role in p38 activity. Preincubation with EGTA suppressed p38 activation, while calcium ionophore induced p38 activity. Inhibition of phospholipase C (PLC) resulted in complete inhibition of ERK, while having no affect on p38 activity. These data suggested a bifurcation in the regulation of MAPKs that occurs at the level of PLC and RAFTK/Pyk2 activation. Src family kinases interact with RAFTK/Pyk2. Inhibition of Src by either pharmacological or genetic means decreased p38 activity. Finally, we found that both Src and RAFTK/Pyk2 were essential for endothelial cell migration. These data identified a novel regulatory network involving extracellular calcium, RAFTK/Pyk2, Src and p38. This signaling network appears to be critical for VEGF-induced endothelial cell migration.

    Funded by: NCI NIH HHS: R01 CA081419, R01-CA-81419; NHLBI NIH HHS: T32-HL-07194

    Oncogene 2004;23;6;1275-82

  • Phosphatidylinositol triphosphate kinase-dependent and c-jun NH2-terminal kinase-dependent induction of telomerase by calcium requires Pyk2.

    Alfonso-De Matte MY and Kruk PA

    Department of Pathology, University of South Florida and the H. Lee Moffitt Cancer Center, Tampa, Florida 33612, USA.

    Calcium signaling has been linked to activation of Pyk2, a calcium-dependent, focal adhesion kinase-related, non-receptor tyrosine kinase. Signaling via Pyk2 can activate c-jun NH(2)-terminal kinase (JNK). Calcium has also been shown to activate phosphatidylinositol triphosphate kinase and/or JNK. Here, we show that calcium signaling in ovarian surface epithelial cells not only induces telomerase activity via JNK but also activates Pyk2. Moreover, telomerase activation by Pyk2 requires JNK activation. In contrast, a kinase-deficient Pyk2 construct failed to activate either JNK or telomerase. Finally, we demonstrate that Pyk2 is capable of driving the human telomerase reverse transcriptase promoter, resulting in telomerase activation. These data suggest a novel role of Pyk2 for telomerase regulation.

    Cancer research 2004;64;1;23-6

  • Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays.

    Johnson JM, Castle J, Garrett-Engele P, Kan Z, Loerch PM, Armour CD, Santos R, Schadt EE, Stoughton R and Shoemaker DD

    Rosetta Inpharmatics LLC, Merck & Co., Inc., 12040 115th Avenue N.E., Kirkland, WA 98034, USA. jason_johnson@merck.com

    Alternative pre-messenger RNA (pre-mRNA) splicing plays important roles in development, physiology, and disease, and more than half of human genes are alternatively spliced. To understand the biological roles and regulation of alternative splicing across different tissues and stages of development, systematic methods are needed. Here, we demonstrate the use of microarrays to monitor splicing at every exon-exon junction in more than 10,000 multi-exon human genes in 52 tissues and cell lines. These genome-wide data provide experimental evidence and tissue distributions for thousands of known and novel alternative splicing events. Adding to previous studies, the results indicate that at least 74% of human multi-exon genes are alternatively spliced.

    Science (New York, N.Y.) 2003;302;5653;2141-4

  • Taniyama Y, Weber DS, Rocic P, Hilenski L, Akers ML, Park J, Hemmings BA, Alexander RW and Griendling KK

    Department of Medicine, Division of Cardiology, Emory University School of Medicine, 1639 Pierce Drive, Atlanta, GA 30322, USA.

    3-Phosphoinositide-dependent protein kinase 1 (PDK1) is a signal integrator that activates the AGC superfamily of serine/threonine kinases. PDK1 is phosphorylated on tyrosine by oxidants, although its regulation by agonists that stimulate G-protein-coupled receptor signaling pathways and the physiological consequences of tyrosine phosphorylation in this setting have not been fully identified. We found that angiotensin II stimulates the tyrosine phosphorylation of PDK1 in vascular smooth muscle in a calcium- and c-Src-dependent manner. The calcium-activated tyrosine kinase Pyk2 acts as a scaffold for Src-dependent phosphorylation of PDK1 on Tyr9, which permits phosphorylation of Tyr373 and -376 by Src. This critical function of Pyk2 is further supported by the observation that Pyk2 and tyrosine-phosphorylated PDK1 colocalize in focal adhesions after angiotensin II stimulation. Importantly, infection of smooth muscle cells with a Tyr9 mutant of PDK1 inhibits angiotensin II-induced tyrosine phosphorylation of paxillin and focal adhesion formation. These observations identify a novel interaction between PDK1 and Pyk2 that regulates the integrity of focal adhesions, which are major compartments for integrating signals for cell growth, apoptosis, and migration.

    Funded by: NHLBI NIH HHS: HL 38206, HL 58000, HL 60728, P01 HL058000, R01 HL038206, R01 HL060728, R37 HL038206

    Molecular and cellular biology 2003;23;22;8019-29

  • Macrophage activation through CCR5- and CXCR4-mediated gp120-elicited signaling pathways.

    Lee C, Liu QH, Tomkowicz B, Yi Y, Freedman BD and Collman RG

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

    Macrophages are major targets for infection by human immunodeficiency virus type 1 (HIV-1). In addition to their role as productive viral reservoirs, inappropriate activation of infected and uninfected macrophages appears to contribute to pathogenesis. HIV-1 infection requires initial interactions between the viral envelope surface glycoprotein gp120, the cell-surface protein CD4, and a chemokine receptor CCR5 or CXCR4. Besides their role in HIV-1 entry, CCR5 and CXCR4 are G protein-coupled receptors that can activate multiple intracellular signaling pathways. HIV-1 gp120 has been shown to activate signaling pathways through the chemokine receptors in several cell types including lymphocytes, neurons, and astrocytes. In some cell types, these consequences may cause cellular injury. In this review, we highlight our data demonstrating diverse signaling events that occur in primary human macrophages in response to gp120/chemokine receptor interactions. These responses include K+, Cl-, and nonselective cation currents, intracellular Ca2+ increases, and activation of several kinases including the focal adhesion-related tyrosine kinase Pyk2, mitogen-activated protein kinases (MAPK), and phosphoinositol-3 kinase. Activation of the MAPK leads to gp120-induced expression of chemokines such as monocyte chemoattractant protein-1 and macrophage-inflammatory protein-1beta and the proinflammatory cytokine tumor necrosis factor alpha. These responses establish a complex cytokine network, which may enhance or suppress HIV-1 replication. In addition, dysregulation of macrophage function by gp120/chemokine receptor signaling may contribute to local inflammation and injury and further recruit additional inflammatory and/or target cells. Targeting these cellular signaling pathways may have benefit in controlling inflammatory sequelae of HIV infection such as in neurological disease.

    Funded by: NIAID NIH HHS: R01 AI060921

    Journal of leukocyte biology 2003;74;5;676-82

  • Identification and characterization of a novel Pyk2/related adhesion focal tyrosine kinase-associated protein that inhibits alpha-synuclein phosphorylation.

    Takahashi T, Yamashita H, Nagano Y, Nakamura T, Ohmori H, Avraham H, Avraham S, Yasuda M and Matsumoto M

    Department of Clinical Neuroscience and Therapeutics, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3 Kasumi, Hiroshima, Japan 734-8551.

    alpha-Synuclein is a presynaptic protein involved in the pathogenesis of several neurodegenerative diseases, such as Parkinson's disease. Pyk2/related adhesion focal tyrosine kinase (RAFTK) tyrosine kinase is an upstream regulator of Src family kinases in the central nervous system that is involved in alpha-synuclein phosphorylation. The present study reports the cloning and characterization of a novel adaptor protein, Pyk2/RAFTK-associated protein (PRAP), that specifically binds to Pyk2/RAFTK and inhibits alpha-synuclein tyrosine phosphorylation. PRAP contains a coiled-coil domain, a pleckstrin homology domain, and a SH3 domain; the SH3 domain binds to the proline-rich domain of Pyk2/RAFTK. PRAP was observed to be present throughout the brain, including substantia nigra dopaminergic neurons, in which it localized to the cytoplasm. PRAP was found to function as a substrate for Src family kinases, such as c-Src or Fyn, but not for Pyk2/RAFTK. Hyperosmotic stress induced phosphorylation of tyrosine 125 of alpha-synuclein via Pyk2/RAFTK, which acted through Src family kinases. Such phosphorylation was inhibited by PRAP expression, suggesting that PRAP negatively regulates alpha-synuclein phosphorylation following cell stress. In conclusion, PRAP functions as a downstream target for Pyk2/RAFTK and plays a role in alpha-synuclein phosphorylation.

    The Journal of biological chemistry 2003;278;43;42225-33

  • Vascular endothelial growth factor regulates focal adhesion assembly in human brain microvascular endothelial cells through activation of the focal adhesion kinase and related adhesion focal tyrosine kinase.

    Avraham HK, Lee TH, Koh Y, Kim TA, Jiang S, Sussman M, Samarel AM and Avraham S

    Division of Experimental Medicine and Hematology/Oncology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02115, USA. savraham@bidmc.harvard.edu

    Vascular endothelial growth factor (VEGF) plays a significant role in blood-brain barrier breakdown and angiogenesis after brain injury. VEGF-induced endothelial cell migration is a key step in the angiogenic response and is mediated by an accelerated rate of focal adhesion complex assembly and disassembly. In this study, we identified the signaling mechanisms by which VEGF regulates human brain microvascular endothelial cell (HBMEC) integrity and assembly of focal adhesions, complexes comprised of scaffolding and signaling proteins organized by adhesion to the extracellular matrix. We found that VEGF treatment of HBMECs plated on laminin or fibronectin stimulated cytoskeletal organization and increased focal adhesion sites. Pretreating cells with VEGF antibodies or with the specific inhibitor SU-1498, which inhibits Flk-1/KDR receptor phosphorylation, blocked the ability of VEGF to stimulate focal adhesion assembly. VEGF induced the coupling of focal adhesion kinase (FAK) to integrin alphavbeta5 and tyrosine phosphorylation of the cytoskeletal components paxillin and p130cas. Additionally, FAK and related adhesion focal tyrosine kinase (RAFTK)/Pyk2 kinases were tyrosine-phosphorylated by VEGF and found to be important for focal adhesion sites. Overexpression of wild type RAFTK/Pyk2 increased cell spreading and the migration of HBMECs, whereas overexpression of catalytically inactive mutant RAFTK/Pyk2 markedly suppressed HBMEC spreading ( approximately 70%), adhesion ( approximately 82%), and migration ( approximately 65%). Furthermore, blocking of FAK by the dominant-interfering mutant FRNK (FAK-related non-kinase) significantly inhibited HBMEC spreading and migration and also disrupted focal adhesions. Thus, these studies define a mechanism for the regulatory role of VEGF in focal adhesion complex assembly in HBMECs via activation of FAK and RAFTK/Pyk2.

    Funded by: NCI NIH HHS: CA096805, CA76226; NHLBI NIH HHS: HL51456; NINDS NIH HHS: NS39558

    The Journal of biological chemistry 2003;278;38;36661-8

  • Protein kinase B is obligatory for follicle-stimulating hormone-induced granulosa cell differentiation.

    Zeleznik AJ, Saxena D and Little-Ihrig L

    Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA. zeleznik@pitt.edu

    Although FSH receptors are linked to the cAMP second messenger system, additional intracellular signaling pathways appear to be required for the induction of aromatase and the LH receptor during granulosa cell differentiation. We employed adenovirus vectors to modulate specific intracellular signaling systems in undifferentiated granulosa cells to identify the signaling pathway(s) that may be involved in the FSH-mediated induction of aromatase and the LH receptor. Expression of either the constitutively activated human LH receptor D578H or the constitutively active human G(s)alpha Q227L resulted in increased cAMP production without increasing aromatase activity or mRNA levels for the LH receptor. To explore the contributions of other pathways, we expressed the constitutively activated forms MAPK kinase (MEK) and protein kinase B (PKB). Neither MEK nor PKB alone increased estrogen or progesterone production by undifferentiated granulosa cells. Stimulation of granulosa cells by FSH in the presence of the constitutively active PKB, but not MEK, led to an amplification of FSH-induced aromatase and LH receptor mRNA levels, whereas a dominant negative PKB vector completely abolished the actions of FSH. The expression of the constitutively active PKB in combination with the constitutively active LH receptor D578H, the constitutively active G(s)alpha Q227L, or 8-bromo-cAMP led to an induction of aromatase as well as LH receptor mRNA comparable to that seen in cells stimulated with FSH alone. These results demonstrate that PKB is an essential component of the FSH-mediated granulosa cell differentiation and that both PKB and G(s)alpha signaling pathways are required.

    Funded by: NICHD NIH HHS: HD-16842

    Endocrinology 2003;144;9;3985-94

  • The tyrosine kinase Pyk2 regulates Arf1 activity by phosphorylation and inhibition of the Arf-GTPase-activating protein ASAP1.

    Kruljac-Letunic A, Moelleken J, Kallin A, Wieland F and Blaukat A

    Institute of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, D-69120 Heidelberg, Germany.

    Proline-rich tyrosine kinase 2 (Pyk2), a non-receptor tyrosine kinase structurally related to focal adhesion kinase, has been implicated in the regulation of mitogen-activated protein kinase cascades and ion channels, the induction of apoptosis, and in the modulation of the cytoskeleton. In order to understand how Pyk2 signaling mediates these diverse cellular functions, we performed a yeast two-hybrid screening using the C-terminal part of Pyk2 that contains potential protein-protein interaction sites as bait. A prominent binder of Pyk2 identified by this method was the Arf-GTPase-activating protein ASAP1. Pyk2-ASAP1 interaction was confirmed in pull-down as well as in co-immunoprecipitation experiments, and contact sites were mapped to the proline-rich regions of Pyk2 and the SH3 domain of ASAP1. Pyk2 directly phosphorylates ASAP1 on tyrosine residues in vitro and increases ASAP1 tyrosine phosphorylation when co-expressed in HEK293T cells. Phosphorylation of tyrosine 308 and 782 affects the phosphoinositide binding profile of ASAP1, and fluorimetric Arf-GTPase assays with purified proteins revealed an inhibition of ASAP1 GTPase-activating protein activity by Pyk2-mediated tyrosine phosphorylation. We therefore provide evidence for a functional interaction between Pyk2 and ASAP1 and a regulation of ASAP1 and hence Arf1 activity by Pyk2-mediated tyrosine phosphorylation.

    The Journal of biological chemistry 2003;278;32;29560-70

  • Paxillin binding to the alpha 4 integrin subunit stimulates LFA-1 (integrin alpha L beta 2)-dependent T cell migration by augmenting the activation of focal adhesion kinase/proline-rich tyrosine kinase-2.

    Rose DM, Liu S, Woodside DG, Han J, Schlaepfer DD and Ginsberg MH

    Division of Rheumatology, Allergy, and Immunology, Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA. drose@vapop.ucsd.edu

    Engagement of very late Ag-4 (integrin alpha(4)beta(1)) by ligands such as VCAM-1 markedly stimulates leukocyte migration mediated by LFA-1 (integrin alpha(L)beta(2)). This form of integrin trans-regulation in T cells requires the binding of paxillin to the alpha(4) integrin cytoplasmic domain. This conclusion is based on the abolition of trans-regulation in Jurkat T cells by an alpha(4) mutation (alpha(4)(Y991A)) that disrupts paxillin binding. Furthermore, cellular expression of an alpha(4)-binding fragment of paxillin that blocks the alpha(4)-paxillin interaction, selectively blocked VCAM-1 stimulation of alpha(L)beta(2)-dependent cell migration. The alpha(4)-paxillin association mediates trans-regulation by enhancing the activation of tyrosine kinases, focal adhesion kinase (FAK) and/or proline-rich tyrosine kinase-2 (Pyk2), based on two lines of evidence. First, disruption of 1120 the paxillin-binding site in the alpha(4) tail resulted in much less alpha(4)beta(1)-mediated phosphorylation of Pyk2 and FAK. Second, transfection with cDNAs encoding C-terminal fragments of Pyk2 and FAK, which block the function of the intact kinases, blocked alpha(4)beta(1) stimulation of alpha(L)beta(2)-dependent migration. These results define a proximal protein-protein interaction of an integrin cytoplasmic domain required for trans-regulation between integrins, and establish that augmented activation of Pyk2 and/or FAK is an immediate signaling event required for the trans-regulation of integrin alpha(L)beta(2) by alpha(4)beta(1).

    Funded by: NHLBI NIH HHS: HL48728, HL59007; NIAMS NIH HHS: AR27214, P30 AR47360

    Journal of immunology (Baltimore, Md. : 1950) 2003;170;12;5912-8

  • Activation and nuclear translocation of PKCdelta, Pyk2 and ERK1/2 by gonadotropin releasing hormone in HEK293 cells.

    Farshori PQ, Shah BH, Arora KK, Martinez-Fuentes A and Catt KJ

    Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Building 49, Room 6A-36, Bethesda, MD 20892-4510, USA.

    The mechanism of agonist-induced activation of Pyk2 and its relationship with ERK1/2 phosphorylation was analyzed in HEK293 cells stably expressing the gonadotropin releasing hormone (GnRH) receptor. GnRH stimulation caused rapid and sustained phosphorylation of ERK1/2 and Pyk2 that was accompanied by their nuclear translocation. Pyk2 was also localized on cell membranes and at focal adhesions. Dominant negative Pyk2 (PKM) had no effect on GnRH-induced ERK1/2 phosphorylation and c-fos expression. These actions of GnRH on ERK1/2 and Pyk2 were mimicked by activation of protein kinase C (PKC) and were abolished by its inhibition. GnRH caused translocation of PKCalpha and delta, but not of epsilon, iota and lambda, to the cell membrane, as well as phosphorylation of Raf at Ser338, a major site in the activation of MEK/ERK1/2. Stimulation of HEK293 cells by EGF caused marked ERK1/2 phosphorylation that was attenuated by the selective EGFR receptor (EGF-R) kinase inhibitor, AG1478. However, GnRH-induced ERK1/2 activation was independent of EGF-R activation. These results indicate that activation of PKC is responsible for GnRH-induced phosphorylation of both ERK1/2 and Pyk2, and that Pyk2 activation does not contribute to GnRH signaling. Moreover, GnRH-induced phosphorylation of ERK1/2 and expression of c-fos in HEK293 cells is independent of Src and EGF-R transactivation, and is mediated through the PKC/Raf/MEK cascade.

    The Journal of steroid biochemistry and molecular biology 2003;85;2-5;337-47

  • Interaction of the tyrosine kinase Pyk2 with the N-methyl-D-aspartate receptor complex via the Src homology 3 domains of PSD-95 and SAP102.

    Seabold GK, Burette A, Lim IA, Weinberg RJ and Hell JW

    Department of Pharmacology, University of Wisconsin, Madison, Wisconsin 53706-1532, USA.

    The protein-tyrosine kinase Pyk2/CAKbeta/CADTK is a key activator of Src in many cells. At hippocampal synapses, induction of long term potentiation requires the Pyk2/Src signaling pathway, which up-regulates the activity of N-methyl-d-aspartate-type glutamate receptors. Because localization of protein kinases close to their substrates is crucial for effective phosphorylation, we investigated how Pyk2 might be recruited to the N-methyl-d-aspartate receptor complex. This interaction is mediated by PSD-95 and its homolog SAP102. Both proteins colocalize with Pyk2 at postsynaptic dendritic spines in the cerebral cortex. The proline-rich regions in the C-terminal half of Pyk2 bind to the SH3 domain of PSD-95 and SAP102. The SH3 and guanylate kinase homology (GK) domain of PSD-95 and SAP102 interact intramolecularly, but the physiological significance of this interaction has been unclear. We show that Pyk2 effectively binds to the Src homology 3 (SH3) domain of SAP102 only when the GK domain is removed from the SH3 domain. Characterization of PSD-95 and SAP102 as adaptor proteins for Pyk2 fills a critical gap in the understanding of the spatial organization of the Pyk2-Src signaling pathway at the postsynaptic site and reveals a physiological function of the intramolecular SH3-GK domain interaction in SAP102.

    Funded by: NIA NIH HHS: AG17502; NINDS NIH HHS: NS35563, NS39444

    The Journal of biological chemistry 2003;278;17;15040-8

  • Evidence for a role of protein tyrosine kinases in cell death induced by gp120 in CHP100 neuroblastoma cells.

    Russo R, Navarra M, Rotiroti D and Di Renzo G

    Department of Pharmacobiological Sciences, Faculty of Pharmacy, University 'Magna Graecia' of Catanzaro & ISN-CNR, Complesso 'Nini; Barbieri', 88021 Roccelletta di Borgia, Catanzaro, Italy.

    HIV-1 coat protein gp120 is able to kill neuronal cells in culture. Here we address the possible role of protein tyrosine kinases (PTKs) in gp120-induced neurotoxicity using the CHP100 human neuroblastoma cell line as experimental model. For this purpose, the effect of specific PTK inhibitors like genistein, herbimycin A and lavendustin A was evaluated on CHP100 cell death elicited by the viral protein. Here we report that genistein (1-10 microM) significantly reduced the cytotoxic effects induced by gp120 (10 pM). The same protective action was offered by a pre-treatment with herbimycin A (0.1-1 microM) or lavendustin A (1-10 microM). Conversely, daidzein (1-100 microM), a genistein analogue devoid of PTK inhibitory properties, failed to reduce CHP100 cell death caused by gp120. These findings suggest that PTKs can be involved in the signal transduction cascade by which the glycoprotein induces neurotoxicity.

    Toxicology letters 2003;139;2-3;207-11

  • Proline-rich tyrosine kinase 2 and Rac activation by chemokine and integrin receptors controls NK cell transendothelial migration.

    Gismondi A, Jacobelli J, Strippoli R, Mainiero F, Soriani A, Cifaldi L, Piccoli M, Frati L and Santoni A

    Department of Experimental Medicine and Pathology, Istituto Pasteur-Fondazione Cenci Bolognetti, University of Rome La Sapienza, Rome, Italy. angela.gismondi@uniroma1.it

    Protein tyrosine kinase activation is an important requisite for leukocyte migration. Herein we demonstrate that NK cell binding to endothelium activates proline-rich tyrosine kinase 2 (Pyk-2) and the small GTP binding protein Rac that are coupled to integrin and chemokine receptors. Chemokine-mediated, but not integrin-mediated, Pyk-2 and Rac activation was sensitive to pretreatment of NK cells with pertussis toxin, a pharmacological inhibitor of G(i) protein-coupled receptors. Both Pyk-2 and Rac are functionally involved in chemokine-induced NK cell migration through endothelium or ICAM-1 or VCAM-1 adhesive p 1f40 roteins, as shown by the use of recombinant vaccinia viruses encoding dominant negative mutants of Pyk-2 and Rac. Moreover, we found that Pyk-2 is associated with the Rac guanine nucleotide exchange factor Vav, which undergoes tyrosine phosphorylation upon integrin triggering. Finally, we provide direct evidence for the involvement of Pyk-2 in the control of both chemokine- and integrin-mediated Rac activation. Collectively, our results indicate that Pyk-2 acts as a receptor-proximal link between integrin and chemokine receptor signaling, and the Pyk-2/Rac pathway plays a pivotal role in the control of NK cell transendothelial migration.

    Journal of immunology (Baltimore, Md. : 1950) 2003;170;6;3065-73

  • Interaction between liprin-alpha and GIT1 is required for AMPA receptor targeting.

    Ko J, Kim S, Valtschanoff JG, Shin H, Lee JR, Sheng M, Premont RT, Weinberg RJ and Kim E

    Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea.

    Liprin-alpha is a multidomain protein that interacts with the LAR family of receptor protein tyrosine phosphatases and the GRIP/ABP family of AMPA receptor-interacting proteins. Previous studies have indicated that liprin-alpha regulates the development of presynaptic active zones and that the association of liprin-alpha with GRIP is required for postsynaptic targeting of AMPA receptors. However, the underlying molecular mechanisms are not well understood. Here we report that liprin-alpha directly interacts with GIT1, a multidomain protein with GTPase-activating protein activity for the ADP-ribosylation factor family of small GTPases known to regulate protein trafficking and the actin cytoskeleton. Electron microscopic analysis indicates that GIT1 distributes to the region of postsynaptic density (PSD) as well as presynaptic active zones. GIT1 is enriched in PSD fractions and forms a complex with liprin-alpha, GRIP, and AMPA receptors in brain. Expression of dominant-negative constructs interfering with the GIT1-liprin-alpha interaction leads to a selective and marked reduction in the dendritic and surface clustering of AMPA receptors in cultured neurons. These results suggest that the GIT1-liprin-alpha interaction is required for AMPA receptor targeting and that GIT1 may play an important role in the organization of presynaptic and postsynaptic multiprotein complexes.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2003;23;5;1667-77

  • Regulation of PGC-1 promoter activity by protein kinase B and the forkhead transcription factor FKHR.

    Daitoku H, Yamagata K, Matsuzaki H, Hatta M and Fukamizu A

    Center for Tsukuba Advanced Research Alliance, Institute of Applied Biochemistry, University of Tsukuba, Tennoudai 1-1-1, Tsukuba, Ibaraki 305-8577, Japan.

    Peroxisome proliferator-activated receptor-gamma coactivator-1 (PGC-1) plays a major role in mediating hepatic gluconeogenesis in response to starvation, during which PGC-1 is induced by the cyclic AMP response element binding protein. Although it is observed that insulin counteracts PGC-1 transcription, the mechanism by which insulin suppresses the transcription of PGC-1 is still unclear. Here, we show that forkhead transcription factor FKHR contributes to mediating the effects of insulin on PGC-1 promoter activity. Reporter assays demonstrate that insulin suppresses the basal PGC-1 promoter activity and that coexpression of protein kinase (PK)-B mimics the effect of insulin in HepG2 cells. Insulin response sequences (IRSs) are addressed in the PGC-1 promoter as the direct target for FKHR in vivo. Coexpression of FKHR stimulates the PGC-1 promoter activity via interaction with the IRSs, while coexpression of FKHR (3A), in which the three putative PKB site df8 s in FKHR are mutated, mainly abolishes the suppressive effect of PKB. Whereas deletion of the IRSs prevents the promoter stimulation by FKHR, that activity is still partially inhibited by insulin. These results indicate that signaling via PKB to FKHR can partly account for the effect of insulin to regulate the PGC-1 promoter activity via the IRSs.

    Diabetes 2003;52;3;642-9

  • A novel hematopoietic adaptor protein, Chat-H, positively regulates T cell receptor-mediated interleukin-2 production by Jurkat cells.

    Sakakibara A, Hattori S, Nakamura S and Katagiri T

    Division of Biochemistry and Cellular Biology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8502, Japan.

    Chat (Cas/HEF1-associated signal transducer) is a novel adaptor protein with an N-terminal Src homology-2 domain and C-terminal Cas/HEF1 association domain. We report here the molecular cloning of Chat-H, the hematopoietic isoform of Chat. Chat-H has an extended N-terminal domain besides the known Chat domain structures, suggesting a unique function of Chat-H in hematopoietic cells. Jurkat transfectants overexpressing Chat-H show a marked increase in interleukin-2 production after costimulation of T cell receptor and CD28. The degree of JNK activation is enhanced substantially in the Chat-H transfectants upon costimulation. The Src homology-2 domain mutant of Chat-H loses this signal modulating activity. Expression of the Cas/HEF1 association domain mutant exhibits a dominant negative effect on both JNK activation and interleukin-2 production. We further found that Chat-H forms a complex with Pyk2H and enhances its tyrosine 402 phosphorylation, an up-regulator of the JNK pathway. These results suggest that Chat-H positively controls T cell function via integrating the costimulatory signals.

    The Journal of biological chemistry 2003;278;8;6012-7

  • Regulation of the formation of osteoclastic actin rings by proline-rich tyrosine kinase 2 interacting with gelsolin.

    Wang Q, Xie Y, Du QS, Wu XJ, Feng X, Mei L, McDonald JM and Xiong WC

    Department of Neurobiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

    Osteoclast activation is important for bone remodeling and is altered in multiple bone disorders. This process requires cell adhesion and extensive actin cytoskeletal reorganization. Proline-rich tyrosine kinase 2 (PYK2), a major cell adhesion-activated tyrosine kinase in osteoclasts, plays an important role in regulating this event. The mechanisms by which PYK2 regulates actin cytoskeletal organization and osteoclastic activation remain largely unknown. In this paper, we provide evidence that PYK2 directly interacts with gelsolin, an actin binding, severing, and capping protein essential for osteoclastic actin cytoskeletal organization. The interaction is mediated via the focal adhesion-targeting domain of PYK2 and an LD motif in gelsolin's COOH terminus. PYK2 phosphorylates gelsolin at tyrosine residues and regulates gelsolin bioactivity, including decreasing gelsolin binding to actin monomer and increasing gelsolin binding to phosphatidylinositol lipids. In addition, PYK2 increases actin polymerization at the fibroblastic cell periphery. Finally, PYK2 interacts with gelsolin in osteoclasts, where PYK2 activation is required for the formation of actin rings. Together, our results suggest that PYK2 is a regulator of gelsolin, revealing a novel PYK2-gelsolin pathway in regulating actin cytoskeletal organization in multiple cells, including osteoclasts.

    Funded by: NIAMS NIH HHS: AR43225, AR46031, AR47830, AR48120, P30 AR046031, P30AR46031, R01 AR043225, R01 AR047830, R01 AR048120; NINDS NIH HHS: NS40480, R01 NS040480

    The Journal of cell biology 2003;160;4;565-75

  • Negative regulation of mixed lineage kinase 3 by protein kinase B/AKT leads to cell survival.

    Barthwal MK, Sathyanarayana P, Kundu CN, Rana B, Pradeep A, Sharma C, Woodgett JR and Rana A

    Division of Molecular Cardiology, Cardiovascular Research Institute, The Texas A&M University System Health Science Center, College of Medicine, Temple 76504, USA.

    Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that activates c-jun N-terminal kinase (JNK) and can induce cell death in neurons. By contrast, the activation of phosphatidylinositol 3-kinase and AKT/protein kinase B (PKB) acts to suppress neuronal apoptosis. Here, we report a functional interaction between MLK3 and AKT1/PKBalpha. Endogenous MLK3 and AKT1 interact in HepG2 cells, and this interaction is regulated by insulin. The interaction domain maps to the C-terminal half of MLK3 (amino acids 511-847), an 1f40 d this region also contains a putative AKT phosphorylation consensus sequence. Endogenous JNK, MKK7, and MLK3 kinase activities in HepG2 cells are significantly attenuated by insulin treatment, whereas the phosphatidylinositol 3-kinase inhibitors LY294002 and wortmannin reversed the effect. Finally, MLK3-mediated JNK activation is inhibited by AKT1. AKT phosphorylates MLK3 on serine 674 both in vitro and in vivo. Furthermore, the expression of activated AKT1 inhibits MLK3-mediated cell death in a manner dependent on serine 674 phosphorylation. Thus, these data provide the first direct link between MLK3-mediated cell death and its regulation by a cell survival signaling protein, AKT1.

    Funded by: NIGMS NIH HHS: GM55853

    The Journal of biological chemistry 2003;278;6;3897-902

  • Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry.

    Salomon AR, Ficarro SB, Brill LM, Brinker A, Phung QT, Ericson C, Sauer K, Brock A, Horn DM, Schultz PG and Peters EC

    Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121, USA.

    The reversible phosphorylation of tyrosine residues is an important mechanism for modulating biological processes such as cellular signaling, differentiation, and growth, and if deregulated, can result in various types of cancer. Therefore, an understanding of these dynamic cellular processes at the molecular level requires the ability to assess changes in the sites of tyrosine phosphorylation across numerous proteins simultaneously as well as over time. Here we describe a sensitive approach based on multidimensional liquid chromatography/mass spectrometry that enables the rapid identification of numerous sites of tyrosine phosphorylation on a number of different proteins from human whole cell lysates. We used this methodology to follow changes in tyrosine phosphorylation patterns that occur over time during either the activation of human T cells or the inhibition of the oncogenic BCR-ABL fusion product in chronic myelogenous leukemia cells in response to treatment with STI571 (Gleevec). Together, these experiments rapidly identified 64 unique sites of tyrosine phosphorylation on 32 different proteins. Half of these sites have been documented in the literature, validating the merits of our approach, whereas motif analysis suggests that a number of the undocumented sites are also potentially involved in biological pathways. This methodology should enable the rapid generation of new insights into signaling pathways as they occur in states of health and disease.

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;2;443-8

  • Han H, Fuortes M and Nathan C

    Department of Microbiology and Immunology, Graduate Programs in Immunology, Weill Medical College of Cornell University, New York, NY 10021, USA.

    Transduction of Tat-tagged fusion proteins confirmed a hypothesis based on pharmacologic inhibitors (Fuortes, M., M. Melchior, H. Han, G.J. Lyon, and C. Nathan. 1999. J. Clin. Invest. 104:327-335) that proline-rich tyrosine kinase (Pyk2) plays a critical role in the activation of adherent human neutrophils, and allowed an analysis of individual Pyk2 domains not possible with chemical inhibitors. Acting as a dominant negative, the COOH terminus of Pyk2 fused to a Tat peptide (Tat-CT), but not other regions of Pyk2, specifically inhibited the respiratory burst of cells responding to tumor necrosis factor (TNF), Salmonella, or Listeria, while sparing responses induced by phorbol ester. Tat-CT suppressed TNF-triggered cell spreading and the phosphorylation of endogenous Pyk2 and the associated tyrosine kinase Syk without blocking the ability of neutrophils to degranulate and kill bacteria. Thus, separate signals control the respiratory burst and degranulation, and a normal rate of killing of some bacteria can be sustained by granule products in conjunction with a minimal residual respiratory burst. Inhibition of select inflammatory functions without impairment of antibacterial activity may commend the Pyk2 pathway as a potential target for antiinflammatory therapy.

    Funded by: NIAID NIH HHS: R01 AI046382, T32 AI007621

    The Journal of experimental medicine 2003;197;1;63-75

  • HIV-1 gp120 chemokine receptor-mediated signaling in human macrophages.

    Freedman BD, Liu QH, Del Corno M and Collman RG

    Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine, PA 19104, USA. bruce@vet.upenn.edu

    The chemokine receptors CCR5 and CXCR4 serve as the cellular receptors in conjunction with CD4 for HIV-1 entry and infection of target cells. Although the virus has subverted these molecules for its own use, their natural function is to respond to activation and migration signals delivered by extracellular chemokines. A principal research objective of our laboratory is to understand the consequences of virus-chemokine receptor interactions for cellular function, as well as for entry and infection. We hypothesized that CXCR4-using (X4) and CCR5-using (R5) HIV-1 strains might elicit signals through the chemokine receptors that result in aberrant function and/or regulate virus entry or postentry steps of infection. We have focused on primary human macrophages, which express both CXCR4 and CCR5, because macrophages are a principal target for HIV-1 in vivo, inappropriate macrophage activation appears to play a major role in the pathogenesis of certain sequelae of AIDS, such as HIV encephalopathy, and macrophage infection is regulated at several steps subsequent to entry in ways that are linked to envelope- receptor interactions. This review summarizes our recent findings regarding the mechanisms of chemokine-receptor signaling in macrophages, the role of viral envelope glycoproteins in eliciting macrophage signals, and how these activation pathways may participate in macrophage infection and affect cell functions apart from infection.

    Funded by: NIAID NIH HHS: R01 AI060921

    Immunologic research 2003;27;2-3;261-76

  • HIV Nef inhibits T cell migration.

    Choe EY, Schoenberger ES, Groopman JE and Park IW

    Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115, USA.

    Nef is a viral regulatory protein of the human immunodeficiency virus (HIV) that has been shown to contribute to disease progression. Among its putative effects on T cell functions are the down-regulation of CD4 and major histocompatibility class I surface molecules. These effects occur in part via Nef interactions with intracellular signaling molecules. We sought to better characterize the effects of HIV Nef on T cell function by examining chemotaxis in response to stromal cell-derived factor-1alpha (SDF-1alpha) as well as CXCR4 signaling molecules. Here, we report the novel observation that HIV Nef inhibited chemotaxis in response to SDF-1alpha in both Jurkat T cells and primary peripheral CD4+ T lymphocytes. Our data indicate that HIV Nef altered critical downstream molecules in the CXCR4 pathway, including focal adhesion kinases. These findings suggest that HIV Nef may blunt the T cell response to chemokines. Because T lymphocyte migration is an integral component of host defense, HIV Nef may thereby contribute to the pathogenesis of AIDS.

    Funded by: NHLBI NIH HHS: HL53745, HL61940; NIDA NIH HHS: DA5008

    The Journal of biological chemistry 2002;277;48;46079-84

  • Binding of protein kinase B to the plakin family member periplakin.

    van den Heuvel AP, de Vries-Smits AM, van Weeren PC, Dijkers PF, de Bruyn KM, Riedl JA and Burgering BM

    Laboratory of Physiological Chemistry and Centre for Biomedical Genetics, University Medical Center Utrecht, Stratenum, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands.

    The serine/threonine kinase protein kinase B (PKB/c-Akt) acts downstream of the lipid kinase phosphoinositide 3-kinase (PI3K) and functions as an essential mediator in many growth-factor-induced cellular responses such as cell cycle regulation, cell survival and transcriptional regulation. PI3K activation generates 3'-phosphorylated phosphatidylinositol lipids (PtdIns3P) and PKB activation requires PtdIns3P-dependent membrane translocation and phosphorylation by upstream kinases. However PKB activation and function is also regulated by interaction with other proteins. Here we show binding of PKB to periplakin, a member of the plakin family of cytolinker proteins. Interaction between PKB and periplakin was mapped to part of the pleckstrin homology (PH) domain of PKB, which is probably not involved in lipid binding, and indeed binding to periplakin did not affect PKB activation. We therefore investigated the possibility that periplakin may act as a scaffold or localization signal for PKB. In cells endogenous periplakin localizes to different cellular compartments, including plasma membrane, intermediate filament structures, the nucleus and mitochondria. Overexpression of the C-terminal part of periplakin, encompassing the PKB binding region, results in predominant intermediate filament localization and little nuclear staining. This also resulted in inhibition of nuclear PKB signalling as indicated by inhibition of PKB-dependent Forkhead transcription factor regulation. These results suggest a possible role for periplakin as a localization signal in PKB-mediated signalling.

    Journal of cell science 2002;115;Pt 20;3957-66

  • Calcitonin induces dephosphorylation of Pyk2 and phosphorylation of focal adhesion kinase in osteoclasts.

    Zhang Z, Neff L, Bothwell AL, Baron R and Horne WC

    Department of Cell Biology, New Haven, CT 06520-8044, USA.

    Calcitonin induces the association and tyrosine phosphorylation of focal adhesion kinase (FAK), paxillin, and HEF1 in HEK-293 cells that overexpress the calcitonin receptor (C1a-HEK), but the hormone's effect on these adhesion-related proteins in osteoclasts is not known. We therefore studied the effect of calcitonin on the tyrosine phosphorylation and subcellular distribution of paxillin, HEF1, FAK, and Pyk2, a FAK-related tyrosine kinase, in osteoclasts. Osteoclasts expressed both Pyk2 and FAK, with Pyk2 much more highly expressed. The two tyrosine kinases and paxillin were prominently associated with small punctate structures that were most densely clustered in the region of the peripheral F-actin-rich ring. Some of the punctate structures stained either for Pyk2 alone or FAK alone. Treatment with calcitonin disrupted the actin ring and induced the loss of the peripheral staining of paxillin, Pyk2, and FAK. In calcitonin-treated osteoclast-like cells, the tyrosine phosphorylation of paxillin and FAK increased, whereas the tyrosine phosphorylation of Pyk2 decreased. Calcitonin also induced increased phosphorylation of Erk1 and Erk2 in osteoclasts, as it did in the C1a-HEK cells. The unexpected dephosphorylation of Pyk2 correlated with decreased phosphorylation of Tyr(402), the autophosphorylation site of Pyk2. The calcitonin-induced dephosphorylation of Pyk2 was not observed in C1a-HEK cells transfected with Pyk2, suggesting that the reduced phosphorylation seen in osteoclasts may be specific to these cells. Treatment of osteoclast-like cells with 12-phorbol 13-myristate acetate increased the tyrosine phosphorylation of both Pyk2 and FAK, and calphostin C, an inhibitor of protein kinase C, blocked calcitonin-stimulated FAK phosphorylation. Increasing intracellular calcium with ionomycin caused a decrease in the tyrosine phosphorylation of Pyk2 and the loss of the actin ring in a manner similar to the effect of calcitonin. Ionomycin had no effect on FAK tyrosine phosphorylation. Calcitonin (CT)-induced changes in Pyk2, FAK, and Erk1/2 phosphorylation were independent of c-Src.

    Funded by: NIAMS NIH HHS: AR-42927, AR-46032; NIDCR NIH HHS: DE-04724

    Bone 2002;31;3;359-65

  • Transforming growth factor-beta 2 is a transcriptional target for Akt/protein kinase B via forkhead transcription factor.

    Samatar AA, Wang L, Mirza A, Koseoglu S, Liu S and Kumar CC

    Department of Tumor Biology, Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA.

    Tumors evade cell death by constitutively activating cell survival pathways and suppressing intrinsic death machinery. Activation of cell survival pathways leads to transcriptional repression of genes associated with cell death and activation of ones promoting anti-apoptosis. Akt/protein kinase B phosphorylates forkhead transcription factors and prevents their nuclear localization, leading to repression of genes involved in apoptosis, such as Fas ligand (FasL). Using bioinformatic approaches, we have identified three consensus sequences for forkhead transcription factor binding in transforming growth factor beta2 (TGF-beta2) promoter. TGF-beta inhibits cell proliferation and induces apoptosis in many cell types, and acquisition of TGF-beta resistance is linked to tumorigenesis. In this study, we show that activated Akt down-regulates TGF-beta2 promoter, and sequences within the promoter that are related to consensus forkhead binding sites are necessary for repression. Forkhead factor FKHRL1 binds in vitro to the three consensus sequences and can activate TGF-beta2 promoter in normal and Akt-transformed cell lines. In human breast and pancreatic tumors, activated Akt expression correlated with down-regulation of TGF-beta 2 mRNA levels. A number of tumor cells expressing activated Akt were responsive to TGF-beta addition, indicating the presence of an intact TGF-beta-signaling pathway. These results suggest that repression of TGF-beta 2 promoter activity in cells expressing activated Akt may play a role in promoting tumorigenesis and escape from the growth-inhibitory and/or apoptotic effects of TGF-beta.

    The Journal of biological chemistry 2002;277;31;28118-26

  • TCR engagement induces proline-rich tyrosine kinase-2 (Pyk2) translocation to the T cell-APC interface independently of Pyk2 activity and in an immunoreceptor tyrosine-based activation motif-mediated fashion.

    Sancho D, Montoya MC, Monjas A, Gordón-Alonso M, Katagiri T, Gil D, Tejedor R, Alarcón B and Sánchez-Madrid F

    Servicio de Inmunología, Hospital de la Princesa, Madrid, Spain.

    The relocation of kinases in T lymphocytes during their cognate interaction with APCs is essential for lymphocyte activation. We found that the proline-rich tyrosine kinase-2 (Pyk2) is rapidly translocated to the T cell-APC contact area upon T cell-specific recognition of superantigen-pulsed APCs. Stimulation with anti-CD3-coated latex microspheres was sufficient for Pyk2 reorientation, and the coengagement of CD28 boosted Pyk2 redistribution. Nevertheless, Pyk2 translocation did not result in its recruitment to lipid rafts. Two results support that Pyk2 translocation was independent of its kinase activity. First, Lck activity was required for TCR-induced Pyk2 translocation, but not for TCR-induced Pyk2 activation. Second, a kinase-dead Pyk2 mutant was equally translocated upon TCR triggering. In addition, Lck activity alone was insufficient to induce Pyk2 reorientation and activation, requiring the presence of at least one intact immunoreceptor tyrosine-based activation motif (ITAM). Despite the dependence on functional Lck and on phosphorylated ITAM for Pyk2 translocation, the ITAM-binding tyrosine kinase zeta-associated protein 70 (ZAP-70) was not essential. All these data suggest that, by translocating to the vicinity of the immune synapse, Pyk2 could play an essential role in T cell activation and polarized secretion of cytokines.

    Journal of immunology (Baltimore, Md. : 1950) 2002;169;1;292-300

  • Csk homologous kinase associates with RAFTK/Pyk2 in breast cancer cells and negatively regulates its activation and breast cancer cell migration.

    McShan GD, Zagozdzon R, Park SY, Zrihan-Licht S, Fu Y, Avraham S and Avraham H

    Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, Boston, MA 02115, USA.

    Our recent observations indicated that RAFTK (also termed Pyk2 and CAK-beta) participated in intracellular signaling upon heregulin (HRG) stimulation and promoted breast carcinoma invasion. Furthermore, studies from our group indicate that the Csk homologous kinase (CHK), a member of the Csk family, directly associates with HER2/Neu and down-regulates HER2/Neu-mediated Src kinase activation in breast cancer cells upon heregulin stimulation. Since activation of RAFTK is associated with the activity of Src family kinases, we analyzed whether CHK is capable of opposing HRG-induced activation of RAFTK. Stimulation of human T47D breast cancer cells with HRG induced the tyrosine phosphorylation of RAFTK and its association with CHK in vitro and in vivo. This interaction was mediated through the Src binding site (amino acid residue at 402) of RAFTK and the SH2 domain of CHK. RAFTK phosphorylation downstream of the activated HER2/Neu was greatly reduced in the presence of CHK. Maximal inhibition of RAFTK phosphorylation by CHK required the kinase activity of CHK. Furthermore, CHK inhibited the tyrosine phosphorylation of the focal adhesion-associated protein, paxillin, and inhibited HRG-induced T47D breast cancer cell migration. These findings indicate the role of CHK as a negative regulator in HRG- and RAFTK-mediated intracellular signaling in breast cancer cells.

    Funded by: NCI NIH HHS: CA76226, CA76772; NHLBI NIH HHS: HL5 1f40 1456, HL55445

    International journal of oncology 2002;21;1;197-205

  • Activation of Pyk2/RAFTK induces tyrosine phosphorylation of alpha-synuclein via Src-family kinases.

    Nakamura T, Yamashita H, Nagano Y, Takahashi T, Avraham S, Avraham H, Matsumoto M and Nakamura S

    Department of Clinical Neuroscience and Therapeutics, Hiroshima University Graduate School of Biomedical Sciences, Japan.

    alpha-Synuclein (alpha S) is a neuronal protein that has been implicated in the pathogenesis of Parkinson's disease. The present report demonstrates that the protein tyrosine kinase Pyk2/RAFTK is involved in cell stress-induced tyrosine phosphorylation of alpha S. Hyperosmotic stress induced tyrosine phosphorylation of alpha S via Pyk2/RAFTK at tyrosine residue 125. Pyk2/RAFTK-mediated phosphorylation of alpha S was primarily achieved with Src-family kinases. In addition, osmotic stress-induced phosphorylation of alpha S was dependent on Pyk2/RAFTK activation. Accordingly, such results indicate that Pyk2/RAFTK lies upstream of Src-family kinases in the signaling cascade by which osmotic stress induces tyrosine phosphorylation of alpha S.

    FEBS letters 2002;521;1-3;190-4

  • Suppression of androgen receptor transactivation by Pyk2 via interaction and phosphorylation of the ARA55 coregulator.

    Wang X, Yang Y, Guo X, Sampson ER, Hsu CL, Tsai MY, Yeh S, Wu G, Guo Y and Chang C

    George Whipple Laboratory for Cancer Research, Department of Urology and the Cancer Center, University of Rochester, Rochester, New York 14642, USA.

    The proline-rich tyrosine kinase 2 (Pyk2) was first identified as a key kinase linked to the MAP kinase and JNK signaling pathways that play important roles in cell growth and adhesion. The linkage between Pyk2 and the androgen receptor (AR), an important transcription factor in prostate cancer progression, however, remains unclear. Here we report that using the full-length androgen receptor-associated protein, ARA55, coregulator as bait, we were able to isolate an ARA55-interacting protein, Pyk2, and demonstrated that Pyk2 could repress AR transactivation via inactivation of ARA55. This inactivation may result from the direct phosphorylation of ARA55 by Pyk2 at tyrosine 43, impairing the coactivator activity of ARA55 and/or sequestering ARA55 to reduce its interaction with AR. Our finding that Pyk2 can indirectly modulate AR function via interaction and/or phosphorylation of ARA55 not only expands the role of Pyk2 in AR-mediated prostate cancer growth but also strengthens the role of ARA55 as an AR coregulator.

    Funded by: NCI NIH HHS: CA71570

    The Journal of biological chemistry 2002;277;18;15426-31

  • Vascular endothelial growth factor-induced migration of multiple myeloma cells is associated with beta 1 integrin- and phosphatidylinositol 3-kinase-dependent PKC alpha activation.

    Podar K, Tai YT, Lin BK, Narsimhan RP, Sattler M, Kijima T, Salgia R, Gupta D, Chauhan D and Anderson KC

    Jerome Lipper Multiple Myeloma Research Center/Dana-Farber Cancer Institute and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA.

    In multiple myeloma (MM), migration is necessary for the homing of tumor cells to bone marrow (BM), for expansion within the BM microenvironment, and for egress into the peripheral blood. In the present study we characterize the role of vascular endothelial growth factor (VEGF) and beta(1) integrin (CD29) in MM cell migration. We show that protein kinase C (PKC) alpha is translocated to the plasma membrane and activated by adhesion of MM cells to fibronectin and VEGF. We identify beta(1) integrin modulating VEGF-triggered MM cell migration on fibronectin. We show that transient enhancement of MM cell adhesion to fibronectin triggered by VEGF is dependent on the activity of both PKC and beta(1) integrin. Moreover, we demonstrate that PKC alpha is constitutively associated with beta(1) integrin. These data are consistent with PKC alpha-dependent exocytosis of activated beta(1) integrin to the plasma membrane, where its increased surface expression mediates binding to fibronectin; conversely, catalytically active PKC alpha-driven internalization of beta(1) integrin results in MM cell de-adhesion. We show that the regulatory subunit of phosphatidylinositol (PI) 3-kinase (p85) is constitutively associated with FMS-like tyrosine kinase-1 (Flt-1). VEGF stimulates activation of PI 3-kinase, and both MM cell adhesion and migration are PI 3-kinase-dependent. Moreover, both VEGF-induced PI 3-kinase activation and beta(1) integrin-mediated binding to fibronectin are required for the recruitment and activation of PKC alpha. Time-lapse phase contrast video microscopy (TLVM) studies confirm the importance of these signaling components in VEGF-triggered MM cell migration on fibronectin.

    Funded by: PHS HHS: P0-1 78378

    The Journal of biological chemistry 2002;277;10;7875-81

  • Signal transduction of physiological concentrations of vasopressin in A7r5 vascular smooth muscle cells. A role for PYK2 and tyrosine phosphorylation of K+ channels in the stimulation of Ca2+ spiking.

    Byron KL and Lucchesi PA

    Department of Medicine, Cardiovascular Institute, Loyola University Chicago, 2160 South First Avenue, Maywood, IL 60153, USA. kbyron@lumc.edu

    The signal transduction pathway linking physiological concentrations of [Arg(8)]vasopressin (AVP) to an increase in frequency of Ca(2+) spiking was examined in confluent cultures of A7r5 vascular smooth muscle cells. Immunoprecipitation/Western blot studies revealed a robust increase in tyrosine phosphorylation of the non-receptor tyrosine kinase, PYK2, in A7r5 cells treated with 4beta-phorbol 12-myristate 13-acetate or ionomycin. 100 pm AVP also induced PYK2 tyrosine phosphorylation, and this effect was inhibited by protein kinase C inhibitors Ro-31-8220 (1-10 microm) or chelerythrine chloride (1-20 microm). In fura-2-loaded A7r5 cells, the stimulation of Ca(2+) spiking by 100 pm AVP or 1 nm 4beta-phorbol 12-myristate 13-acetate was completely blocked by PP2 (10 microm, a Src family kinase inhibitor). Salicylate (20 mm, recently identified as a PYK2 inhibitor) and the tyrosine kinase inhibitor, tyrphostin A47 (50 microm), but not its inactive analog, tyrphostin A63, also blocked AVP-stimulated Ca(2+) spiking. PYK2 phosphorylation was inhibited by both PP2 and salicylate, whereas tyrphostin A47 failed to inhibit PYK2 tyrosine phosphorylation. ERK1/2 kinases did not appear to be involved because 1) 100 pm AVP did not appreciably increase ERK1/2 phosphorylation and U-0126 (2.5 microm) did not inhibit AVP-stimulated Ca(2+) spiking; and 2) epidermal growth factor (10 nm) robustly stimulated ERK1/2 phosphorylation but did not induce Ca(2+) spiking. Delayed rectifier K(+) channels may mediate the PYK2 activity because Kv1.2 channel protein co-immunoprecipitated with PYK2 and tyrosine phosphorylation of Kv1.2 was stimulated by AVP and inhibited by Ro-31-8220, PP2, and salicylate but not tyrphostin A47. Our findings are consistent with a role for PYK2 and phosphorylation of K(+) channels in the stimulation of Ca(2+) spiking by physiological concentrations of AVP.

    Funded by: NHLBI NIH HHS: R01 HL 60164, R29 HL 56046

    The Journal of biological chemistry 2002;277;9;7298-307

  • Proline-rich tyrosine kinase 2 and focal adhesion kinase are involved in different phases of platelet activation by vWF.

    Canobbio I, Lova P, Sinigaglia F, Balduini C and Torti M

    Department of Biochemistry, University of Pavia, Italy.

    Stimulation of human platelets with von Willebrand factor (vWF) induces the rapid tyrosine phosphorylation of several proteins, but very little is known on the tyrosine kinases involved in this process. In the present work, we investigated and compared the activation of two related tyrosine kinases expressed in platelets: the proline-rich tyrosine kinase 2 (Pyk2) and the focal adhesion kinase (FAK). Both kinases were tyrosine phosphorylated upon vWF interaction with glycoprotein Ib-IX-V complex, but with different mechanisms. Tyrosine phosphorylation of FAK was totally dependent on thromboxane A2 production, and was inhibited by the integrin alphaIIbeta3 antagonist RGDS peptide. Moreover, chelation of intracellular calcium or inhibition of protein kinase C (PKC) totally blocked vWF-induced tyrosine phosphorylation of FAK, indicating that this event is downstream phospholipase A2 and phospholipase C activation. By contrast, tyrosine phosphorylation of Pyk2 was only partially reduced by aspirin and RGDS, and was not affected by either calcium chelation or PKC inhibition, suggesting that activation of this kinase does not require phospholipase-mediated signalling. Both FAK and Pyk2 translocated to the cytoskeleton upon vWF stimulation of human platelets by a mechanism depending on agonist-induced actin polymerisation. Prevention of cytoskeletal relocation of Pyk2 and FAK by cytochalasin D totally blocked vWF-induced tyrosine phosphorylation of both kinases. Finally, phosphorylation of Pyk2 induced by vWF, but not by thrombin, was inhibited by piceatannol, suggesting that this kinase lies downstream Syk. These results demonstrate that both Pyk2 and FAK are involved in platelet stimulation by vWF, but indicate that only Pyk2 may play a role in the early signal transduction events activated by ligand binding to glycoprotein Ib-IX-V.

    Thrombosis and haemostasis 2002;87;3;509-17

  • Human endothelial Pyk2 is expressed in two isoforms and associates with paxillin and p130Cas.

    Keogh RJ, Houliston RA and Wheeler-Jones CP

    Department of Veterinary Basic Sciences, Royal Veterinary College, Royal College Street, London, NW1 0TU, United Kingdom.

    Proline-rich kinase 2 (Pyk2) is a non-receptor tyrosine kinase belonging to the focal adhesion kinase family. Many stimuli can initiate phosphorylation and activation of Pyk2 but its specific activators and downstream targets are still largely unidentified and little is known of the mechanisms or role of Pyk2 activation in endothelial cells. In human umbilical vein endothelial cells (HUVEC), we show that (1) Pyk2 is phosphorylated on tyrosine residues 402, 580, and 881 in response to stimulation with G-protein-coupled receptor agonists (GPCAs), vascular endothelial growth factor, and the cytokine interleukin-1alpha; (2) HUVEC express mRNA for two isoforms of Pyk2 which do not appear to be regulated transcriptionally by GPCAs, growth factors, or cytokines; and (3) Pyk2 is localised to the cytosol and associates through its C-terminus with the cytoskeletal protein paxillin and the adapter molecule p130Cas in phosphorylation-independent interactions. These results demonstrate that Pyk2 is rapidly activated and associates with structural and adapter proteins suggesting that it is an important kinase involved in mediating acute responses in endothelium.

    Biochemical and biophysical research communications 2002;290;5;1470-7

  • Multiple phosphorylation of alpha-synuclein by protein tyrosine kinase Syk prevents eosin-induced aggregation.

    Negro A, Brunati AM, Donella-Deana A, Massimino ML and Pinna LA

    Dipartimento di Chimica Biologica and Centro di Studio delle Biomembrane del C.N.R., University of Padova, Padova, Italy.

    The presence of aggregated alpha-synuclein molecules is a common denominator in a variety of neurodegenerative disorders. Here, we show that alpha-synuclein (alpha-syn) is an outstanding substrate for the protein tyrosine kinase p72syk (Syk), which phosphorylates three tyrosyl residues in its C-terminal domain (Y-125, Y-133, and Y-136), as revealed from experiments with mutants where these residues have been individually or multiply replaced by phenylalanine. In contrast, only Y-125 is phosphorylated by Lyn and c-Fgr. Eosin-induced multimerization is observed with wild-type alpha-syn, either phosphorylated or not by Lyn, and with all its Tyr to Phe mutants but not with the protein previously phosphorylated by Syk. Syk-mediated phosphorylation also counteracts alpha-syn assembly into filaments as judged from the disappearance of alpha-syn precipitated upon centrifugation at 100,000 x g. We also show that Syk and alpha-syn colocalize in the brain, and upon cotransfection in Chinese hamster ovary cells, alpha-syn becomes Tyr-phosphorylated by Syk. Moreover, Syk and alpha-syn interact with each other as judged from the mammalian two-hybrid system approach. These data suggest that Syk or tyrosine kinase(s) with similar specificity may play an antineurodegenerative role by phosphorylating a-syn, thereby preventing its aggregation.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2002;16;2;210-2

  • Nuclear translocation of cell adhesion kinase beta/proline-rich tyrosine kinase 2.

    Aoto H, Sasaki H, Ishino M and Sasaki T

    Department of Biochemistry, Cancer Research Institute, Sapporo Medical University, Japan. aoto@sapmed.ac.jp

    Cell adhesion kinase beta (CAKbeta/PYK2) is a protein-tyrosine kinase of the focal adhesion kinase (FAK) family. Whereas FAK predominantly localizes at focal adhesions, CAK beta localizes at the perinuclear region in fibroblasts. Here we expressed in cultured cells two point mutants of CAKbeta, P717A and P859A, each of which had lost one of its two PXXP motifs, the ligand sequence for SH3 domains, found at the CAKbeta C-terminal region. We observed a remarkable change in the subcellular distribution of the P859A mutant; while that of the P717A mutant was the same as the wild type. The P859A mutant localized exclusively in the cell nucleus in all cell lines examined. Wild-type CAKbeta also accumulated in the nucleus when cells were treated with an inhibitor of the nuclear export of proteins. These results indicate that CAK beta shuttles between the cytoplasm and the nucleus. On nuclear accumulation of P859A-CAKbeta, a CAKbeta-binding protein, Hic-5, also accumulated in the nucleus. P859A-CAKbeta and co-expressed Hic-5 formed nuclear speckles, in which one other CAK beta-binding protein, p130(Cas), was also concentrated. These findings on nuclear translocation of CAK beta imply that CAKbeta may regulate nuclear processes such as transcription, particularly because Hic-5 was recently shown to be a coactivator of nuclear receptors.

    Cell structure and function 2002;27;1;47-61

  • Requirement of Ca(2+) and PKCdelta for Janus kinase 2 activation by angiotensin II: involvement of PYK2.

    Frank GD, Saito S, Motley ED, Sasaki T, Ohba M, Kuroki T, Inagami T and Eguchi S

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

    In vascular smooth muscle cells, angiotensin II (AngII) stimulates association of its G protein-coupled AngII type 1 (AT(1)) receptor with Janus kinase 2 (JAK2), resulting in the activation of signal transducer and activator of transcription proteins. Although the association and activation of subsequent signal transducer and activator of transcription proteins appear to prerequire JAK2 activation, the signaling mechanism by which the AT(1) receptor activates JAK2 remains uncertain. Here, we have examined the signaling mechanism required for JAK2 activation by AngII in vascular smooth muscle cells. We found that AngII, through the AT(1) receptor, rapidly stimulated JAK2 phosphorylation at Tyr(1007/1008), the critical sites for the kinase activation. By using selective agonists and inhibitors, we demonstrated that PLC and its derived signaling molecules, phosphatidylinositol triphosphate/Ca(2+) and diacylglycerol/PKC, were essential for AngII-induced JAK2 phosphorylation. The PKC isoform required for JAK2 activation appears to be PKCdelta since a selective PKCdelta but not PKCalpha/beta inhibitor and dominant-negative PKCdelta overexpression inhibited JAK2 activation. We further examined a link between JAK2 and a Ca(2+)/PKC-sensitive tyrosine kinase, PYK2. We found that PYK2 activation by AngII requires PKCdelta, and that PYK2 associates with JAK2 constitutively. Moreover, transfection of two distinct PYK2 dominant-negative mutants markedly inhibited AngII-induced JAK2 activation. From these data we conclude that AT(1)-derived signaling molecules, specifically Ca(2+) and PKCdelta, participate in AngII-induced JAK2 activation through PYK2. These data provide a new mechanistic insight by which the hormone AngII exerts its cytokine-like actions in mediating vascular remodeling.

    Funded by: NHLBI NIH HHS: HL-03320, HL-58205; NIDDK NIH HHS: DK-20593

    Molecular endocrinology (Baltimore, Md.) 2002;16;2;367-77

  • Proline-rich tyrosine kinase 2 regulates proliferation and differentiation of prostate cells.

    Picascia A, Stanzione R, Chieffi, Kisslinger A, Dikic I and Tramontano D

    Dipartimento di Biologia e Patologia Molecolare e Cellulare 'Luigi Califano', Centro di Endocrinologia ed Oncologia Sperimentale del CNR, Universita degli Studi di Napoli, 'Federico II' Via Pansini 5, 80131, Naples, Italy.

    Proline-rich tyrosin 860 e kinase 2 (Pyk2) expression in prostate epithelium inversely correlated with degree of malignancy of prostate cancers, thus the role of Pyk2 in the regulation of prostate cells proliferation and differentiation was investigate in PC3 cells. Pyk2 can be activated by canonic stimuli such as tumor necrosis factoralpha and lysophosphatidic acid (LPA) in PC3 cells, in addition, LPA stimulated Pyk2 phosphorylation also induced extracellular signal-regulated kinase 1 and 2 activation in these cells. Proliferation of PC3 cell clones (PC3-PKM) expressing a dominant negative kinase-defective Pyk2 mutant is consistently decreased in respect to that of wild type PC3 cells. In addition, PC3-PKM clones underwent total block cell proliferation upon treatment with dibutyryl cAMP. Finally, in the presence of sustained levels of intracellular cAMP, PC3-PKM cells, but not wild type PC3 cells, acquired a neuron-like morphology. Taken together our results suggest that Pyk2 plays a role in the regulation of prostate cell proliferation and, more interestingly, its expression may represents a sensitive marker of prostate state of differentiation.

    Molecular and cellular endocrinology 2002;186;1;81-7

  • Increased expression of cell adhesion kinase beta in human and rat crescentic glomerulonephritis.

    Takagi C, Ueki K, Ikeuchi H, Kuroiwa T, Kaneko Y, Tsukada Y, Maezawa A, Mitaka T, Sasaki T and Nojima Y

    Third Department of Internal Medicine, Gunma University School of Medicine, Maebashi, Japan.

    American journal of kidney diseases : the official journal of the National Kidney Foundation 2002;39;1;174-82

  • Integrins and cell signaling in chondrocytes.

    Loeser RF

    Department of Internal Medicine, Section of Rheumatology, Rush Medical College of Rush-Presbyterian-St. Luke's Medical Center, Chicago, IL 60612, USA. rloeser@rush.edu

    Integrins are adhesion receptor heterodimers that transmit information from the extracellular matrix (ECM) to the cell through activation of cell signaling pathways. Chondrocytes express several members of the integrin family including alpha5beta1 which is the primary chondrocyte receptor for fibronectin. Cell signaling mediated through integrins regulates several chondrocyte functions including differentiation, matrix remodeling, responses to mechanical stimulation and cell survival. Integrin-mediated activation of members of the mitogen-activated protein kinase family likely plays a key role in transmitting signals regulating chondrocyte gene expression. Upstream mediators of mitogen-activated protein kinase (MAP kinase) activation include focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (pyk2) which are both expressed by chondrocytes. A better understanding of chondrocyte integrin signaling is needed to define the mechanisms by which the ECM regulates chondrocyte function.

    Biorheology 2002;39;1-2;119-24

  • Role of tyrosine phosphorylation in ligand-independent sequestration of CXCR4 in human primary monocytes-macrophages.

    Wang J, Guan E, Roderiquez G, Calvert V, Alvarez R and Norcross MA

    Laboratory of Gene Regulation, Division of Therapeutic Proteins, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, USA. wangj@cber.fda.gov

    The chemokine stromal cell-derived factor (SDF)-1 and its receptor, CXCR4, play important roles in human immunodeficiency virus type 1 (HIV-1) pathophysiology, leukocyte trafficking, inflammation, hematopoiesis, embryogenesis, angiogenesis, and cancer metastasis. The effects of cytokines on the regulation of CXCR4 function were investigated in human primary monocytes-macrophages. The expression of functional CXCR4 on the cell surface was demonstrated by the detection of ligand-induced Ca(2+) mobilization, chemotaxis, and ligand-induced receptor endocytosis. Surface CXCR4 expression was down-regulated by cytokines interleukin-4 (IL-4), IL-13, and granulocyte-macrophage colony-stimulating factor (GM-CSF) and up-regulated by IL-10 and transforming growth factor-beta 1. Down-regulation was mediated post-translationally, in the absence of protein degradation, through an endocytotic mechanism. In contrast to SDF-1 alpha-induced CXCR4 endocytosis, cytokine-induced endocytosis of this receptor was independent of actin filament polymerization. GM-CSF increased the expression of G protein-coupled receptor kinase 3 (GRK3), beta-arrestin-1, Pyk2, and focal adhesion kinase (FAK). Cytokine treatment also increased the total and tyrosine-specific phosphorylation of CXCR4 as well as the phosphorylation of FAK on tyrosine 397. It also induced the formation of GRK3.CXCR4 or FAK.CXCR4 complexes. Infection of macrophages by primary R5X4 and X4 isolates of HIV-1 was inhibited by IL-4, IL-13, and GM-CSF, an effect that was associated with down-regulation of surface CXCR4 expression. These data indicate that ligand-dependent and ligand-independent endocytoses of CXCR4 are mediated by different mechanisms. Cytokine-induced endocytosis of chemokine receptors may be of therapeutic value in HIV-1 infection, inflammation, tumor metastasis, and defective hematopoiesis.

    The Journal of biological chemistry 2001;276;52;49236-43

  • Nephrocystin interacts with Pyk2, p130(Cas), and tensin and triggers phosphorylation of Pyk2.

    Benzing T, Gerke P, Höpker K, Hildebrandt F, Kim E and Walz G

    Renal Division, University Hospital Freiburg, 79106 Freiburg, Germany; and Children's Hospital, and Molecular Medicine, University of Freiburg, 79106 Freiburg, Germany.

    Juvenile nephronophthisis type 1 is caused by mutations of NPHP1, the gene encoding for nephrocystin. The function of nephrocystin is presently unknown, but the presence of a Src homology 3 domain and its recently described interaction with p130(Cas) sugg f4e est that nephrocystin is part of the focal adhesion signaling complex. We generated a nephrocystin-specific antiserum and analyzed the interaction of native nephrocystin with endogenous proteins. Immunoprecipitation of nephrocystin revealed that nephrocystin forms protein complexes with p130(Cas), proline-rich tyrosine kinase 2 (Pyk2), and tensin, indicating that these proteins participate in a common signaling pathway. Expression of nephrocystin resulted in phosphorylation of Pyk2 on tyrosine 402 as well as activation of downstream mitogen-activated protein kinases, such as ERK1 and ERK2. Our findings suggest that nephrocystin helps to recruit Pyk2 to cell matrix adhesions, thereby initiating phosphorylation of Pyk2 and Pyk2-dependent signaling. A lack of functional nephrocystin may compromise Pyk2 signaling in a subset of renal epithelial cells.

    Proceedings of the National Academy of Sciences of the United States of America 2001;98;17;9784-9

  • NMDA receptor activation results in tyrosine phosphorylation of NMDA receptor subunit 2A(NR2A) and interaction of Pyk2 and Src with NR2A after transient cerebral ischemia and reperfusion.

    Liu Y, Zhang G, Gao C and Hou X

    Research Center for Biochemistry and Molecular Biology, Xuzhou Medical College, 84 West Huai-hai Road, Xuzhou, 221002, Jiangsu, China.

    Transient ischemia increases tyrosine phosphorylation of N-methyl-D-aspartate (NMDA) receptor. Several tyrosine kinases are involved in this process. In this study, effect of ischemia and reperfusion (I/R) on tyrosine phosphorylation of NMDA receptor subunit 2A (NR2A) and the interaction of two tyrosine kinases, Src and Pyk2, with NR2A was investigated. Four-vessel occlusion was used to produce transient (15 min) cerebral ischemia in SD rats. Tyrosine phosphorylation of NR2A in hippocampus was enhanced after 15 min of reperfusion and reached its peak level at 6 h of reperfusion. The increase sustained for at least 24 h. Src and Pyk2 co-immunoprecipitated with NR2A and the binding increased after I/R, which also reached a peak at 6 h of reperfusion. Besides, Src and Pyk2 were activated after I/R. These increases were prevented by ketamine, a selective NMDA receptor antagonist, which was administered to the SD rats 20 min before ischemia. Moreover, Src and Pyk2 coprecipitated with each other. These data show that NR2A, Src and Pyk2 might form a protein complex in vivo and the interaction suggests a possible mechanism of signal transduction in the postischemic hippocampus.

    Brain research 2001;909;1-2;51-8

  • MIP-1alpha induces activation of phosphatidylinositol-3 kinase that associates with Pyk-2 and is necessary for B-cell migration.

    Rumsey LM, Teague RM, Benedict SH and Chan MA

    Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045, USA.

    The chemokine macrophage inflammatory protein-1 alpha [MIP-1alpha] causes migration of B cells and also induces changes in antibody secretion. However, the signal transduction pathways leading to these phenotypic changes remain undefined. We have identified a signal transduction pathway initiated by MIP-1alpha in B cells. Here we report that stimulation of tonsil B cells with MIP-1alpha induces phosphatidylinositol 3-kinase [PI3-K] activation. Kinase activity was transient with peak induction occurring withi 1f40 n 2.5 to 5 min after stimulation and was dose-dependent. In addition stimulation with MIP-1alpha induces tyrosine phosphorylation of the proline-rich tyrosine kinase Pyk-2. Immunoprecipitation analysis showed a constitutive association between Pyk-2 and PI3-K and pretreatment of MIP-1alpha-stimulated B cells with wortmannin, a specific inhibitor of PI3-K, resulted in a loss of PI3-K activity. The PI3-K inhibitor wortmannin prevented B cells from migrating in response to MIP-1alpha. Hence, PI3-K and Pyk-2 seem to be components of a signal transduction pathway induced by stimulation of B cells with MIP-1alpha, and this pathway may play a role in B-cell migration.

    Experimental cell research 2001;268;1;77-83

  • Podosomes in osteoclast-like cells: structural analysis and cooperative roles of paxillin, proline-rich tyrosine kinase 2 (Pyk2) and integrin alphaVbeta3.

    Pfaff M and Jurdic P

    Ecole Normale Supérieure de Lyon, France. martin.pfaff@ujf-grenoble.fr

    Macrophages and osteoclasts develop unique contact sites with the extracellular matrix called podosomes. Podosomes have been associated with migratory and invasive cell characteristics, but a basic mechanism outlining their function is lacking. We have used chicken and human monocytes differentiating in vitro into osteoclast-like cells in the presence of RANKL-ODF to study these cytoskeletal structures. During the differentiation process, podosomes are redistributed from the cell body in early macrophages to the cell periphery in increasingly spread and multinucleated cells expressing high levels of integrin alphaVbeta3. Immunofluorescence with anti-phosphotyrosine antibodies revealed increased tyrosine-phosphorylation at the basal tips of these podosomes. RANKL-ODF treatment reinforced the peripheral location of podosomes and initiated their partial fusion to larger F-actin-containing structures that displayed reduced levels of tyrosine phosphorylation. Paxillin and the FAK-related kinase Pyk2 colocalized with integrin alphaVbeta3 in the juxtamembrane region surrounding individual podosomes. In lysates of macro 1f40 phages and differentiated osteoclasts both paxillin and Pyk2 associated with synthetic and recombinant polypeptides containing the C-terminal region of the integrin beta3 cytoplasmic domain. These in vitro interactions were direct and they were abolished by substitutions in the beta3 integrin peptides known to disrupt integrin function in vivo. The marked adhesion-dependent tyrosinephosphorylation of Pyk2 and paxillin however did not detectably alter their interaction with beta3 tail peptides in cell lysates. Our results provide novel insight into the molecular architecture and the phosphorylation dynamics in podosomes. Moreover, they outline a novel potential mechanism for the recruitment of paxillin and Pyk2 to beta3 integrin-dependent cell contacts.

    Journal of cell science 2001;114;Pt 15;2775-86

  • Inhibition of the catalytic activity of cell adhesion kinase beta by protein-tyrosine phosphatase-PEST-mediated dephosphorylation.

    Lyons PD, Dunty JM, Schaefer EM and Schaller MD

    Department of Cell and Developmental Biology and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA.

    Protein-tyrosine phosphatase (PTP)-PEST is a cytoplasmic tyrosine phosphatase that can bind and dephosphorylate the focal adhesion-associated proteins p130(CAS) and paxillin. Focal adhesion kinase (FAK) and cell adhesion kinase beta (CAKbeta)/PYK2/CADTK/RAFTK are protein-tyrosine kinases that can colocalize with, bind to, and induce tyrosine phosphorylation of p130(CAS) and paxillin. Thus, we considered the possibility that these kinases might be substrates for PTP-PEST. Using a combination of substrate-trapping assays and overexpression of PTP-PEST in mammalian cells, CAKbeta was found to be a substrate for PTP-PEST. Both the major autophosphorylation site of CAKbeta (Tyr(402)) and activation loop tyrosine residues, Tyr(579) and Tyr(580), were targeted for dephosphorylation by PTP-PEST. Dephosphorylation of CAKbeta by PTP-PEST dramatically inhibited CAKbeta kinase activity. In contrast, FAK was a poor substrate for PTP-PEST, and treatment with PTP-PEST had no effect on FAK kinase activity. Tyrosine phosphorylation of paxillin, which is greatly enhanced by CAKbeta overexpression, was dramatically reduced upon coexpression of PTP-PEST. Finally, endogenous PTP-PEST and endogenous CAKbeta were found to localize to similar cellular compartments in epithelial and smooth muscle cells. These results suggest that CAKbeta is a substrate of PTP-PEST and that FAK is a poor PTP-PEST substrate. Further, PTP-PEST can negatively regulate CAKbeta signaling by inhibiting the catalytic activity of the kinase.

    Funded by: NIGMS NIH HHS: GM57943

    The Journal of biological chemistry 2001;276;26;24422-31

  • 3-Hydroxy-3-methylglutaryl-CoA reductase inhibitors block calcium-dependent tyrosine kinase Pyk2 activation by angiotensin II in vascular endothelial cells. involvement of geranylgeranylation of small G protein Rap1.

    No authors listed

    Departments of Biochemistry and Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.

    We recently reported the calcium-dependent activation of tyrosine kinase Pyk2 by angiotensin II (Ang II) in pulmonary vein endothelial cells (PVEC). Since Pyk2 has no calcium binding domain, and neither Ca(2+) nor Ca(2+)/calmodulin directly activates Pyk2, it is not clear how Ca(2+) transduces the signal to activate Pyk2, a key tyrosine kinase, in the early events of Ang II signaling. In the present study, we investigated the mechanism of the calcium-dependent activation of Pyk2 in response to Ang II by using 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors and isoprenoid intermediates in PVEC. We have obtained substantial evidence indicating that Ang II activates Pyk2 through calcium-mediated activation of the geranylgeranylated small G protein Rap1 and the Rap1 association with Pyk2. Thus, the small G protein Rap1 is an intermediary signaling molecule linking Ang II-induced calcium signal to Pyk2 activation in PVEC. In addition, our results indicate that 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, a class of cholesterol-lowering drugs, could interrupt Ang II signaling independent of cholesterol lowering in endothelial cells.

    Funded by: NHLBI NIH HHS: HL-58205

    The Journal of biological chemistry 2001;276;19;15761-7

  • Monocyte chemoattractant protein 1 causes differential signalling mediated by proline-rich tyrosine kinase 2 in THP-1 cells.

    Yamasaki M, Arai H, Ashida N, Ishii K and Kita T

    Department of Geriatric Medicine, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.

    Monocyte chemoattractant protein 1 (MCP-1) has a crucial role in atherogenesis and inflammation. However, MCP-1-mediated signalling pathways in monocytes have not been fully elucidated. In the present study we investigated the role of tyrosine kinases such as proline-rich tyrosine kinase 2 (Pyk2) in MCP-1-mediated signal transduction in the monocytic cell line THP-1. Pyk2 was tyrosine phosphorylated very quickly after stimulation with MCP-1. We found that Lyn, Shc and paxillin were also tyrosine phosphorylated by MCP-1. We examined the association of these molecules by immunoprecipitation and immunoblot analysis. The association of Pyk2 with Lyn was dependent on stimulation with MCP-1 and on tyrosine phosphorylation of Pyk2. Phosphorylation of p38 was also dependent on tyrosine phosphorylation of Pyk2. However, the association of Pyk2 with paxillin and Grb2 was not affected by stimulation with MCP-1. Phosphorylation of ERK (extracellular-signal-regulated protein kinase) was not affected by overexpression of kinase-negative Pyk2. Our results indicate that Pyk2 forms a complex with paxillin, Grb2 and Lyn in THP-1 cells. However, Pyk2 is not always involved in MCP-1-mediated signalling pathways.

    The Biochemical journal 2001;355;Pt 3;751-6

  • Carbachol stimulates TYR phosphorylation and association of PKCdelta and PYK2 in pancreas.

    Wrenn RW

    Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, Georgia 30912-2000, USA. rwrenn@mail.mcg.edu

    Carbachol treatment resulted in increased phosphorylation on tyrosine of PKCdelta immunoprecipitated from rat pancreatic acinar cells. The Ca2+-dependent tyrosine kinase PYK2 coimmunoprecipitated with PKCdelta from carbachol-exposed cells and also exhibited increased tyrosine phosphorylation. Tyrosine phosphorylation of both PKCdelta and PYK2 was concentration-dependent with res 1a54 pect to carbachol, and rapid, reaching maximal levels by 5 min of treatment. Exposure of acinar cells to phorbol myristate acetate (PMA), a phorbol ester activator of PKCdelta, also resulted in increased phosphorylation of PKCdelta and PYK2 isolated using anti-PKCdelta immunoprecipitation. These results are suggestive of a physical and functional interaction between PKCdelta and PYK2 following muscarinic stimulation in the pancreatic acinar cell.

    Biochemical and biophysical research communications 2001;282;4;882-6

  • Regulation of CDC42 GTPase by proline-rich tyrosine kinase 2 interacting with PSGAP, a novel pleckstrin homology and Src homology 3 domain containing rhoGAP protein.

    Ren XR, Du QS, Huang YZ, Ao SZ, Mei L and Xiong WC

    Department of Pathology and Cell Adhesion and Matrix Center, Pathology, and Physical Medicine and Rehabilitation, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.

    Proline-rich tyrosine kinase 2 (PYK2), a tyrosine kinase structurally related to focal adhesion kinase (FAK), is implicated in regulating cytoskeletal organization. However, mechanisms by which PYK2 participates in and regulates cytoskeletal organization remain largely unknown. Here we report identification of PSGAP, a novel protein that interacts with PYK2 and FAK and contains multiple domains including a pleckstrin homology domain, a rhoGTPase-activating protein domain, and a Src homology 3 domain. PYK2 interacts with PSGAP Src homology 3 domain via the carboxyl-terminal proline-rich sequence. PSGAP is able to increase GTPase activity of CDC42 and RhoA in vitro and in vivo. Remarkably, PYK2, but not FAK, can activate CDC42 via inhibition of PSGAP-mediated GTP hydrolysis of CDC42. Moreover, PSGAP is localized at cell periphery in fibroblasts in a pleckstrin homology domain-dependent manner. Over expression of PSGAP in fibroblasts results in reorganization of cytoskeletal structures and changes of cellular morphology, which requires rhoGTPase-activating activity. Taken together, our results suggest that PSGAP is a signaling protein essential for PYK2 regulation of cytoskeletal organization via Rho family GTPases.

    Funded by: NINDS NIH HHS: NS34062, NS40480, R01 NS040480

    The Journal of cell biology 2001;152;5;971-84

  • Activated Fyn phosphorylates alpha-synuclein at tyrosine residue 125.

    Nakamura T, Yamashita H, Takahashi T and Nakamura S

    Third Department of Internal Medicine, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minamiku, Hiroshima, 734-8551, Japan.

    alpha-Synuclein is a presynaptic protein of unknown function that has been implicated in the pathogenesis of several neurodegenerative diseases, including Parkinson's and Alzheimer's diseases. To gain insight into the functions of alpha-synuclein, we sought protein kinases that phosphorylate alpha-synuclein in the central nervous system. In contrast to Lyn, PYK2, FAK, MAPK/ERK1, SAPK/JNK, and Cdk5, only Fyn could phosphorylate alpha-synuclein. In addition, A30P and A53T mutations did not affect the phosphorylation of alpha-synuclein by Fyn. Mutation analysis revealed that activated Fyn phosphorylates specifically tyrosine residue 125 of alpha-synuclein. The distribution of alpha-synuclein and Fyn expression was similar in various parts of the brain and was colocalized in subcellular structures. Since Fyn regulates various signal transduction pathways in the central nervous system and plays an essential role in the neuronal cell differentiation, survival, and plasticity, results of this paper indicate that phosphorylation of alpha-synuclein might be involved in one of the Fyn-mediated signaling pathways in neuronal cells.

    Biochemical and biophysical research communications 2001;280;4;1085-92

  • Outside-in signaling pathway linked to CD146 engagement in human endothelial cells.

    Anfosso F, Bardin N, Vivier E, Sabatier F, Sampol J and Dignat-George F

    INSERM EMI 00-19 Physiopathologie de l'Endothélium, UFR Pharmacie, Université de la Mediterranée, 13385 Marseille, France. anfosso@pharmacie.univ-mrs.fr

    CD146 (S-Endo 1 Ag or MUC18) is a transmembrane glycoprotein expressed on endothelial cells on the whole vascular tree. CD146 is located at the intercellular junction where it plays a role in the cohesion of the endothelial monolayer. CD146 engagement initiates an outside-in signaling pathway involving the protein tyrosine kinases FYN and FAK as well as paxillin. Here we report that CD146 engagement by its specific monoclonal antibody in human umbilical vein endothelial cells induces a Ca(2+) influx that is sensitive to thapsigargin and EGTA treatment, indicating that CD146 engagement initiates a store-operated calcium mobilization. In addition, biochemical and pharmacological analysis revealed that CD146 engagement initiates the tyrosine phosphorylation of phospholipase C-gamma, Pyk2, and p130(Cas). Pharmacological inhibition of Ca(2+) flux with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acetoxymethyl ester and EGTA indicated that an increase in Ca(2+) is required for Pyk2 and p130(Cas) tyrosine phosphorylation. Moreover, a complex association was observed between Pyk2, p130(Cas), and paxillin. These results indicate that CD146 is coupled to a FYN-dependent pathway that triggers Ca(2+) flux via phospholipase C-gamma activation leading subsequently to the tyrosine phosphorylation of downstream targets such as Pyk2, p130(Cas), FAK, and paxillin. In addition to its role in cell-cell adhesion, CD146 is a signaling molecule involved in the dynamics of actin cytoskeleton rearrangement.

    The Journal of biological chemistry 2001;276;2;1564-9

  • Cbl associates with Pyk2 and Src to regulate Src kinase activity, alpha(v)beta(3) integrin-mediated signaling, cell adhesion, and osteoclast motility.

    Sanjay A, Houghton A, Neff L, DiDomenico E, Bardelay C, Antoine E, Levy J, Gailit J, Bowtell D, Horne WC and Baron R

    Department of Cell Biology and Orthopedics, Yale University School of Medicine, New Haven, Connecticut 06510, USA.

    The signaling events downstream of integrins that regulate cell attachment and motility are only partially understood. Using osteoclasts and transfected 293 cells, we find that a molecular complex comprising Src, Pyk2, and Cbl functions to regulate cell adhesion and motility. The activation of integrin alpha(v)beta(3) induces the [Ca(2+)](i)-dependent phosphorylation of Pyk2 Y402, its association with Src SH2, Src activation, and the Src SH3-dependent recruitment and phosphorylation of c-Cbl. Furthermore, the PTB domain of Cbl is shown to bind to phosphorylated Tyr-416 in the activation loop of Src, the autophosphorylation site of Src, inhibiting Src kinase activity and integrin-mediated adhesion. Finally, we show that deletion of c Src or c-Cbl leads to a decrease in osteoclast migration. Thus, binding of alpha(v)beta(3) integrin induces the formation of a Pyk2/Src/Cbl complex in which Cbl is a key regulator of Src kinase activity and of cell adhesion and migration. These findings may explain the osteopetrotic phenotype in the Src(-/-) mice.

    Funded by: NIAMS NIH HHS: AR42927, R01 AR042927

    The Journal of cell biology 2001;152;1;181-95

  • Glucose activates mitogen-activated protein kinase (extracellular signal-regulated kinase) through proline-rich tyrosine kinase-2 and the Glut1 glucose transporter.

    Bandyopadhyay G, Sajan MP, Kanoh Y, Standaert, Burke TR, Quon MJ, Reed BC, Dikic I, Noel, Newgard CB and Farese R

    J. A. Haley Veterans' Hospital Research Service, and Department of Internal Medicine, University of South Florida College of Medicine, Tampa, Florida 33612, USA.

    Glucose serves as both a nutrient and regulator of physiological and pathological processes. Presently, we found that glucose and certain sugars rapidly activated extracellular signal-regulated kinase (ERK) by a mechanism that was: (a) independent of glucose uptake/metabolism and protein kinase C but nevertheless cytochalasin B-inhibitable; (b) dependent upon proline-rich tyrosine kinase-2 (PYK2), GRB2, SOS, RAS, RAF, and MEK1; and (c) amplified by overexpression of the Glut1, but not Glut2, Glut3, or Glut4, glucose transporter. This amplifying effect was independent of glucose uptake but dependent on residues 463-468, IASGFR, in the Glut1 C terminus. Accordingly, glucose effects on ERK were amplified by expression of Glut4/Glut1 or Glut2/Glut1 chimeras containing IASGFR but not by Glut1/Glut4 or Glut1/Glut2 chimeras lacking these residues. Also, deletion of Glut1 residues 469-492 was without effect, but mutations involving serine 465 or arginine 468 yielded dominant-negative forms that inhibited glucose-dependent ERK activation. Glucose stimulated the phosphorylation of tyrosine residues 402 and 881 in PYK2 and binding of PYK2 to Myc-Glut1. Our findings suggest that: (a) glucose activates the GRB2/SOS/RAS/RAF/MEK1/ERK pathway by a mechanism that requires PYK2 and residues 463-468, IASGFR, in the Glut1 C terminus and (b) Glut1 serves as a sensor, transducer, and amplifier for glucose signaling to PYK2 and ERK.

    Funded by: NIDDK NIH HHS: 2R01DK38079-09A1

    The Journal of biological chemistry 2000;275;52;40817-26

  • SHP2 mediates the protective effect of interleukin-6 against dexamethasone-induced apoptosis in multiple myeloma cells.

    Chauhan D, Pandey P, Hideshima T, Treon S, Raje N, Davies FE, Shima Y, Tai YT, Rosen S, Avraham S, Kharbanda S and Anderson KC

    Department of Adult Oncology, Dana-Farber Cancer Institute, Division of Experimental Medicine and Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine and Harvard Medical School, Boston, Massachusetts 02115, USA.

    Our previous studies have shown that activation of a related adhesion focal tyrosine kinase (RAFTK) (also known as Pyk2) is required for dexamethasone (Dex)-induced apoptosis in multiple myeloma (MM) cells and that human interleukin-6 (IL-6), a known growth and survival factor for MM cells, blocks both RAFTK activation and apoptosis induced by Dex. However, the mechanism whereby IL-6 inhibits Dex-induced apoptosis is undefined. In this study, we demonstrate that protein-tyrosine phosphatase SHP2 mediates this protective effect. We show that IL-6 triggers selective activation of SHP2 and its association with RAFTK in Dex-treated MM cells. SHP2 interacts with RAFTK through a region other than its Src homology 2 domains. We demonstrate that RAFTK is a direct substrate of SHP2 both in vitro and in vivo, and that Tyr(906) in the C-terminal domain of RAFTK mediates its interaction with SHP2. Moreover, overexpression of dominant negative SHP2 blocked the protective effect of IL-6 against Dex-induced apoptosis. These findings demonstrate that SHP2 mediates the anti-apoptotic effect of IL-6 and suggest SHP2 as a novel therapeutic target in MM.

    Funded by: NCI NIH HHS: CA 50947, CA 75216

    The Journal of biological chemistry 2000;275;36;27845-50

  • Pyk2 and Syk participate in functional activation of granulocytic HL-60 cells in a different manner.

    Miura Y, Tohyama Y, Hishita T, Lala A, De Nardin E, Yoshida Y, Yamamura H, Uchiyama T and Tohyama K

    Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

    The roles of the protein tyrosine kinases Pyk2 (also called RAFTK or CAK beta) and Syk in the process of functional activation of human myeloid cells were examined. During granulocytic differentiation of HL-60 cells with dimethyl sulfoxide (DMSO), the amounts of Pyk2 and beta2 integrin increased, whereas the amount of Syk was abundant before differentiation and did not change during differentiation. When the granulocytic cells were stimulated with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), tyrosine phosphorylation of Pyk2 occurred promptly and subsequent association of Pyk2 with beta2 integrin was detected. In contrast, Syk was not tyrosine phosphorylated by fMLP stimulation but constitutively associated with beta2 integrin. Stimulation with fMLP also caused the alteration of beta2 integrin to an activated form, a finding that was confirmed by the observation of fMLP-induced cell attachment on fibrinogen-coated dishes and inhibition of this attachment by pretreatment with anti-beta2 integrin antibody. Cell attachment to fibrinogen caused the enhanced tyrosine phosphorylation of Pyk2 and the initial tyrosine phosphorylation of Syk, which was also inhibited by pretreatment with anti-beta2 integrin antibody. In vitro kinase assays revealed that Pyk2 and Syk represented kinase activities to induce tyrosine phosphorylation of several molecules in the anti-beta2 integrin immunoprecipitates of the attached cells. These results showed that Pyk2 is involved in the functional activation of granulocytic cells in 2 signaling pathways: an fMLP receptor-mediated "inside-out" signaling pathway that might cause beta2 integrin activation and a subsequent beta2 integrin-mediated "outside-in" signaling pathway. Syk was activated in relation to cell attachment to fibrinogen as a result of "outside-in" signaling, although it was already associated with beta2 integrin before fMLP stimulation. (Blood. 2000;96:1733-1739)

    Blood 2000;96;5;1733-9

  • G13alpha-mediated PYK2 activation. PYK2 is a mediator of G13alpha -induced serum response element-dependent transcription.

    Shi CS, Sinnarajah S, Cho H, Kozasa T and Kehrl JH

    BCell Molecular Immunology Section, Laboratory of Immunoregulation, NIAID, National Institutes of Health, Bethesda, Maryland 20892-1876, USA.

    G(12)alpha/G(13)alpha transduces signals from G-protein-coupled receptors to stimulate growth-promoting pathways and the early response gene c-fos. Within the c-fos promoter lies a key regulatory site, the serum response element (SRE). Here we show a critical role for the tyrosine kinase PYK2 in muscarinic receptor type 1 and G(12)alpha/G(13)alpha signaling to an SRE reporter gene. A kinase-inactivate form of PYK2 (PYK2 KD) inhibits muscarinic receptor type 1 signaling to the SRE and PYK2 itself triggers SRE reporter gene activation through a RhoA-dependent pathway. Placing PYK2 downstream of G-protein activation but upstream of RhoA, the expression of PYK2 KD blocks the activation of an SRE reporter gene by GTPase-deficient forms of G(12)alpha or G(13)alpha but not by RhoA. The GTPase-deficient form of G(13)alpha triggers PYK2 kinase activity and PYK2 tyrosine phosphorylation, and co-expression of the RGS domain of p115 RhoGEF inhibits both responses. Finally, we show that in vivo G(13)alpha, although not G(12)alpha, readily associates with PYK2. Thus, G-protein-coupled receptors via G(13)alpha activation can use PYK2 to link to SRE-dependent gene expression.

    The Journal of biological chemistry 2000;275;32;24470-6

  • Protein-tyrosine kinase Pyk2 is involved in interleukin-2 production by Jurkat T cells via its tyrosine 402.

    Katagiri T, Takahashi T, Sasaki T, Nakamura S and Hattori S

    Division of Biochemistry and Cellular Biology, National Institute of Neuroscience, Kodaira, Tokyo 187-8502, Japan. katagiri@ncnp.go.jp

    We established Jurkat transfectants that overexpress Pyk2 or its mutants, K457A (lysine 457 was mutated to alanine), Pyk2-Y402F (tyrosine 402 to phenylalanine), and Pyk2-Y881F to investigate the role of Pyk2 in T cell activation. Pyk2 as well as kinase-inactive Pyk2-K457A, was phosphorylated at tyrosine residues 402, 580, and 881 upon T cell antigen receptor cross-linking, indicating that these residues are phosphorylated by other tyrosine kinase(s). However, no tyrosine phosphorylation of Pyk2-Y402F was detected while more than 60% of the tyrosine phosphorylation was observed in Pyk2-Y881F. Pyk2-Y402F inhibited the activation of endogenous Pyk2. The degree of activation of both c-Jun NH(2)-terminal kinase and p38 mitogen-activated protein kinase but not extracellular signal-regulated protein kinase after concurrent ligation of T cell antigen receptor and CD28 was reduced by more than 50% in the clones expressing Pyk2-Y402F. Consistent with this inhibition, IL-2 production was significantly diminished in the Pyk2-Y402F-expressing clones. Furthermore, we found that Pyk2, when overexpressed, associates with Zap70 and Vav. Taken together, these findings suggest that Pyk2 is involved in the activation of T cells through its tyrosine 402.

    The Journal of biological chemistry 2000;275;26;19645-52

  • Beta-chemokine receptor CCR5 signals through SHP1, SHP2, and Syk.

    Ganju RK, Brubaker SA, Chernock RD, Avraham S and Groopman JE

    Division of Experimental Medicine, Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115, USA. rganju@caregroup.harvard.edu

    The beta-chemokine receptor CCR5 has been shown to modulate cell migration, proliferation, and immune functions and to serve as a co-receptor for the human immunodeficiency virus. We and others have shown that CCR5 activates related adhesion focal tyrosine kinase (RAFTK)/Pyk2/CAK-beta. In this study, we further characterize the signaling molecules activated by CCR5 upon binding to its cognate ligand, macrophage inflammatory protein-1beta (MIP1beta). We observed enhanced tyrosine phosphorylation of the phosphatases SHP1 and SHP2 upon MIP1beta stimulation of CCR5 L1.2 transfectants and T-cells derived from peripheral blood mononuclear cells. Furthermore, we observed that SHP1 associated with RAFTK. However, using a dominant-negative phosphatase-binding mutant of RAFTK (RAFTK(m906)), we found that RAFTK does not mediate SHP1 or SHP2 phosphorylation. SHP1 and SHP2 also associated with the adaptor protein Grb2 and the Src-related kinase Syk. Pretreatment of CCR5 L1.2 transfectants or T-cells with the phosphatase inhibitor orthovanadate markedly abolished MIP1beta-induced chemotaxis. Syk was also activated upon MIP1beta stimulation of CCR5 L1.2 transfectants or T-cells and associated with RAFTK. Overexpression of a dominant-negative Src-binding mutant of RAFTK (RAFTK(m402)) significantly attenuated Syk activation, whereas overexpression of wild-type RAFTK enhanced Syk activity, indicating that RAFTK acts upstream of CCR5-mediated Syk activation. Taken together, these results suggest that MIP1beta stimulation mediated by CCR5 induces the formation of a signaling complex consisting of RAFTK, Syk, SHP1, and Grb2.

    Funded by: NCI NIH HHS: CA76950; NHLBI NIH HHS: HL53745, HL61940

    The Journal of biological chemistry 2000;275;23;17263-8

  • Thrombin-stimulated phosphatidylinositol 3-kinase activity in platelets is associated with activation of PYK2 tyrosine kinase: activation of both enzymes is aggregation independent.

    Sayed MR, Sheid MP, Stevens CM and Duronio V

    Department of Medicine, University of British Columbia and Vancouver Hospital and Health Sciences Centre, Jack Bell Research Centre, Vancouver, British Columbia, Canada.

    In this study, we investigated the activation of a new member of the focal adhesion kinase family of tyrosine kinases, the proline-rich tyrosine kinase, or PYK2, in platelets. We show that PYK2 is tyrosine phosphorylated and its activity is increased during early stages of platelet aggregation. This activation coincided with increased association of phosphatidylinositol (PI) 3-kinase and PYK2, as determined by both anti-PI 3-kinase and anti-PYK2 immunoprecipitates. However, under basal conditions, some association of PYK2 and PI 3-kinase was consistently observed, even though little or no tyrosine phosphorylated PYK2 could be detected. In addition, both increased PI 3-kinase activity and increased PYK2 activity could be detected in immu 1f40 noprecipitates following thrombin stimulation. All of these events were unaffected by blocking platelet aggregation with arginine-glycine-aspartate-serine (RGDS) peptide, which interferes with binding of the platelet integrin alpha(IIb)beta(3) to fibrinogen. Neither was the activation of the PYK2 kinase activity affected by blocking PI 3-kinase activity. These results support a model in which PYK2 is associated with PI 3-kinase in unstimulated platelets and following activation of platelets, there is an increase in tyrosine phosphorylation of PYK2, increased PYK2 activity, and increased association of PYK2 with PI 3-kinase, which may contribute to the increase in PI 3-kinase activity. All of these were found to be early events independent of subsequent platelet aggregation.

    Journal of cellular physiology 2000;183;3;314-20

  • Carbachol-stimulated transactivation of epidermal growth factor receptor and mitogen-activated protein kinase in T(84) cells is mediated by intracellular Ca2+, PYK-2, and p60(src).

    Keely SJ, Calandrella SO and Barrett KE

    Department of Medicine, University of California, San Diego, School of Medicine, San Diego, California 92103, USA. skeely@ucsd.edu

    Ca(2+)-dependent agonists, such as carbachol (CCh), stimulate epidermal growth factor receptor (EGFR) transactivation and mitogen-activated protein kinase activation in T(84) intestinal epithelial cells. This pathway constitutes an antisecretory mechanism by which CCh-stimulated chloride secretion is limited. Here, we investigated mechanisms underlying CCh-stimulated epidermal growth factor receptor (EGFR) transactivation. Thapsigargin (TG, 2 microM) stimulated EGFR and extracellular signal-regulated kinase (ERK) phosphorylation in T(84) cells. Inhibition of either EGFR or ERK activation, w 1f40 ith tyrphostin AG1478 (1 microM) and PD 98059 (20 microM), respectively, potentiated chloride secretory responses to TG, as measured by changes in short-circuit current (I(sc)) across T(84) cells. CCh (100 microM) stimulated tyrosine phosphorylation and association of the Ca(2+)-dependent tyrosine kinase, PYK-2, with the EGFR, which was inhibited by the Ca(2+) chelator, BAPTA (20 microM). The calmodulin inhibitor, fluphenazine (50 microM) inhibited CCh-stimulated PYK-2 association with the EGFR and phosphorylation of EGFR and ERK. CCh also induced tyrosine phosphorylation of p60(src) and association of p60(src) with both PYK-2 and the EGFR. The Src family kinase inhibitor, PP2 (20 nM-20 microM) attenuated CCh-stimulated EGFR and ERK phosphorylation and potentiated chloride secretory responses to CCh. We conclude that CCh-stimulated transactivation of the EGFR is mediated by a pathway involving elevations in intracellular Ca(2+), calmodulin, PYK-2, and p60(src). This pathway represents a mechanism that limits CCh-stimulated chloride secretion across intestinal epithelia.

    Funded by: NIDDK NIH HHS: DK28305

    The Journal of biological chemistry 2000;275;17;12619-25

  • Suppression of Pyk2 kinase and cellular activities by FIP200.

    Ueda H, Abbi S, Zheng C and Guan JL

    Cancer Biology Laboratories, Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA.

    Proline-rich tyrosine kinase 2 (Pyk2) is a cytoplasmic tyrosine kinase implicated to play a role in several intracellular signaling pathways. We report the identification of a novel Pyk2-interacting protein designated FIP200 (FAK family kinase-interacting protein of 200 kD) by using a yeast two-hybrid screen. In vitro binding assays and coimmunoprecipitation confirmed association of FIP200 with Pyk2, and similar assays also showed FIP200 binding to FAK. However, immunofluorescent staining indicated that FIP200 was predominantly localized in the cytoplasm. FIP200 bound to the kinase domain of Pyk2 and inhibited its kinase activity in in vitro kinase assays. FIP200 also inhibited the kinase activity of the Pyk2 isolated from SYF cells (deficient in Src, Yes, and Fyn expression) and the Pyk2 mutant lacking binding site for Src, suggesting that it regulated Pyk2 kinase directly rather than affecting the associated Src family kinases. Consistent with its inhibitory effect in vitro, FIP200 inhibited activation of Pyk2 and Pyk2-induced apoptosis in intact cells, which correlated with its binding to Pyk2. Finally, activation of Pyk2 by several biological stimuli correlated with the dissociation of endogenous FIP200-Pyk2 complex, which provided further support for inhibition of Pyk2 by FIP200 in intact cells. Together, these results suggest that FIP200 functions as an inhibitor of Pyk2 via binding to its kinase domain.

    Funded by: NIGMS NIH HHS: GM48050, GM52890, R01 GM048050, R01 GM052890

    The Journal of cell biology 2000;149;2;423-30

  • Interleukin (IL)-7 induces rapid activation of Pyk2, which is bound to Janus kinase 1 and IL-7Ralpha.

    Benbernou N, Muegge K and Durum SK

    Intramural Research Support Program, SAIC Frederick, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702, USA.

    Interleukin-7 (IL-7) receptor signaling begins with activation of the Janus tyrosine kinases Jak1 and Jak3, which are associated with the receptor complex. To identify potential targets of these kinases, we examined Pyk2 (a member of the focal adhesion kinase family) using an IL-7-dependent murine thymocyte line, D1. We demonstrate that stimulation of D1 (or normal pro-T) cells by IL-7 rapidly increased tyrosine phosphorylation and enzymatic activity of Pyk2, with kinetics slightly lagging that of Jak1 and Jak3 phosphorylation. Conversely, IL-7 withdrawal resulted in a marked decrease of Pyk2 phosphorylation. Pyk2 was found to be physically associated with Jak1 prior to IL-7 stimulation and to increase its association with IL-7Ralpha chain following IL-7 stimulation. Pyk2 appeared to be involved in cell survival, because antisense Pyk2 accelerated the cell death process. Activation of Pyk2 via the muscarinic and nicotinic receptors using carbachol or via intracellular Ca(2+) rise using ionomycin/phorbol myristate acetate promoted survival in the absence of IL-7. These data support a role for Pyk2 in coupling Jak signaling to the trophic response.

    Funded by: NCI NIH HHS: N01-CO-56000

    The Journal of biological chemistry 2000;275;10;7060-5

  • Identification of a novel protein complex containing annexin VI, Fyn, Pyk2, and the p120(GAP) C2 domain.

    Chow A, AJ and Gawler DJ

    School of Biomedical Sciences, University of Leeds, Woodhouse Lane, Leeds, UK.

    p120(GAP) (RasGAP) has been proposed to function as both an inhibitor and effector of Ras. Previously we have shown that RasGAP contains a C2 domain which mediates both Ca(2+)-dependent membrane association and protein-protein interactions. Specifically, three proteins have been isolated in a complex with the C2 domain of RasGAP; these are the Ca(2+)-dependent lipid binding protein annexin VI (p70) and two previously unidentified proteins, p55 and p120. Here we provide evidence that p55 is the Src family kinase Fyn and p120 is the focal adhesion kinase family member Pyk2. In addition, in vitro binding assays indicate that Fyn, but not Pyk2 binds directly to annexin VI. Finally, co-immunoprecipitation studies in Rat-1 fibroblasts confirm that Fyn, Pyk2, annexin VI and RasGAP can form a protein complex in mammalian cells.

    FEBS letters 2000;469;1;88-92

  • RAFTK/Pyk2 tyrosine kinase mediates the association of p190 RhoGAP with RasGAP and is involved in breast cancer cell invasion.

    Zrihan-Licht S, Fu, Settleman J, Schinkmann K, Shaw L, Keydar I, Avraham S and Avraham H

    Division of Experimental Medicine, Beth Israel Deaconess M 158 edical Center, Harvard Institutes of Medicine, 4 Blackfan Circle, Boston, Massachusetts, MA 02115, USA.

    Focal adhesions and actin cytoskeleton are involved in cell growth, shape and movement and in tumor invasion. Mitogen-induced changes in actin cytoskeleton are accompanied by changes in the tyrosine phosphorylation of several focal adhesion proteins. In this study, we have investigated the role of RAFTK, a cytoplasmic tyrosine kinase related to focal adhesion kinase (FAK), in heregulin-mediated signal transduction in breast cancer cells. Stimulation of T47D cells with heregulin (HRG) induced the tyrosine phosphorylation of RAFTK and the formation of a multiprotein complex. Analyses of the members of the HRG-stimulated complex revealed that RAFTK is associated with p190 RhoGAP (p190), RasGAP and ErbB-2, and plays an essential role in mediating the tyrosine phosphorylation of p190 by Src. Mutation of the Src binding site within RAFTK (402) abolished the phosphorylation of p190. In addition, upon HRG stimulation of T47D cells, association of ErbB-2 with RAFTK was observed and found to be indirect and mediated by Src. Expression of wild-type RAFTK (WT) significantly increased MDA-MB-435 and MCF-7 breast cancer cell invasion, while expression of the kinase-mutated RAFTK-R457 (KM) or the Src binding site mutant RAFTK (402) did not affect this cell invasion. Furthermore, HRG leads to the activation of MAP kinase which is mediated by RAFTK. These findings indicate that RAFTK serves as a mediator and an integration point between the GAP proteins and HRG-mediated signaling in breast cancer cells, and implicate RAFTK involvement in the MAP kinase pathway and in breast cancer cell invasion.

    Funded by: NCI NIH HHS: CA76226; NHLBI NIH HHS: HL51456, HL55455

    Oncogene 2000;19;10;1318-28

  • Negative regulation of PYK2/related adhesion focal tyrosine kinase signal transduction by hematopoietic tyrosine phosphatase SHPTP1.

    Kumar S, Avraham S, Bharti A, Goyal J, Pandey P and Kharbanda S

    Department of Adult Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.

    Related adhesion focal tyrosine kinase (RAFTK) (also known as PYK2) is a cytoplasmic tyrosine kinase related to the focal adhesion kinase (FAK) p125(FAK). RAFTK is rapidly phosphorylated on tyrosine residues in response to various stimuli, such as tumor necrosis factor-alpha, changes in osmolarity, elevation in intracellular calcium concentration, lysophosphatidic acid, and bradykinin. Overexpression of RAFTK induces activation of c-Jun amino-terminal kinase (also known as stress-activated protein kinase), mitogen-activated protein kinase (MAPK), and p38 MAPK. The present studies demonstrate that RAFTK binds constitutively to the protein tyrosine phosphatase SHPTP1. In contrast to PTP1B, overexpression of wild-type SHPTP1 blocks tyrosine phosphorylation of RAFTK. The results further demonstrate that RAFTK is a direct substrate of SHPTP1 in vitro. Moreover, treatment of PC12 cells with bradykinin is associated with inhibition in tyrosine phosphorylation of RAFTK in the presence of SHPTP1. Furthermore, in contrast to the phosphatase-dead SHPTP1 C453S mutant, overexpression of wild-type SHPTP1 blocks interaction of RAFTK with the SH2-domain of c-Src and inhibits RAFTK-mediated MAPK activation. Significantly, cotransfection of RAFTK with SHPTP1 did not inhibit RAFTK-mediated c-Jun amino-terminal kinase activation. Taken together, these findings suggest that SHPTP1 plays a negative role in PYK2/RAFTK signaling by dephosphorylating RAFTK.

    Funded by: NCI NIH HHS: CA75216

    The Journal of biological chemistry 1999;274;43;30657-63

  • Molecular cloning of a docking protein, BRDG1, that acts downstream of the Tec tyrosine kinase.

    Ohya K, Kajigaya S, Kitanaka A, Yoshida K, Miyazato A, Yamashita Y, Yamanaka T, Ikeda U, Shimada K, Ozawa K and Mano H

    Department of Molecular Biology, Jichi Medical School, Kawachi-gun, Tochigi 329-0498, Japan.

    Tec, Btk, Itk, Bmx, and Txk constitute the Tec family of protein tyrosine kinases (PTKs), a family with the distinct feature of containing a pleckstrin homology (PH) domain. Tec acts in signaling pathways triggered by the B cell antigen receptor (BCR), cytokine receptors, integrins, and receptor-type PTKs. Although upstream regulators of Tec family kinases are relatively well characterized, little is known of the downstream effectors of these enzymes. The yeast two-hybrid system has identified several proteins that interact with the kinase domain of Tec, one of which is now revealed to be a previously unknown docking protein termed BRDG1 (BCR downstream signaling 1). BRDG1 contains a proline-rich motif, a PH domain, and multiple tyrosine residues that are potential target sites for Src homology 2 domains. In 293 cells expressing recombinant BRDG1 and various PTKs, Tec and Pyk2, but not Btk, Bmx, Lyn, Syk, or c-Abl, induced marked phosphorylation of BRDG1 on tyrosine residues. BRDG1 was also phosphorylated by Tec directly in vitro. Efficient phosphorylation of BRDG1 by Tec required the PH and SH2 domains as well as the kinase domain of the latter. Furthermore, BRDG1 was shown to participate in a positive feedback loop by increasing the activity of Tec. BRDG1 transcripts are abundant in the human B cell line Ramos, and the endogenous protein underwent tyrosine phosphorylation in response to BCR stimulation. BRDG1 thus appears to function as a docking protein acting downstream of Tec in BCR signaling.

    Proceedings of the National Academy of Sciences of the United States of America 1999;96;21;11976-81

  • Adaptor proteins Grb2 and Crk couple Pyk2 with activation of specific mitogen-activated protein kinase cascades.

    Blaukat A, Ivankovic-Dikic I, Grönroos E, Dolfi F, Tokiwa G, Vuori K and Dikic I

    Ludwig Institute for Cancer Research, Box 595, Husargatan 3, Uppsala S-75124, Sweden.

    The protein tyrosine kinase Pyk2 acts as an upstream regulator of mitogen-activated protein (MAP) kinase cascades in response to numerous extracellular signals. The precise molecular mechanisms by which Pyk2 activates distinct MAP kinase pathways are not yet fully understood. In this report, we provide evidence that the protein tyrosine kinase Src and adaptor proteins Grb2, Crk, and p130Cas act as downstream mediators of Pyk2 leading to the activation of extracellular signal-regulated kinase (ERK) and c-Jun amino-terminal kinase (JNK). Pyk2-induced activation of Src is necessary for phosphorylation of Shc and p130Cas and their association with Grb2 and Crk, respectively, and for the activation of ERK and JNK cascades. Expression of a Grb2 mutant with a deletion of the amino-terminal Src homology 3 domain or the carboxyl-terminal tail of Sos strongly reduced Pyk2-induced ERK activation, with no apparent effect on JNK activity. Grb2 with a deleted carboxyl-terminal Src homology 3 domain partially blocked Pyk2-induced ERK and JNK pathways, whereas expression of dominant interfering mutants of p130Cas or Crk specifically inhibited JNK but not ERK activation by Pyk2. Taken together, our data reveal specific pathways that couple Pyk2 with MAP kinases: the Grb2/Sos complex connects Pyk2 to the activation of ERK, whereas adaptor proteins p130Cas and Crk link Pyk2 with the JNK pathway.

    Funded by: NCI NIH HHS: CA71560

    The Journal of biological chemistry 1999;274;21;14893-901

  • Tyrosine kinase Pyk2 mediates G-protein-coupled receptor regulation of the Ewing sarcoma RNA-binding protein EWS.

    Felsch JS, Lane WS and Peralta EG

    Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, Massachusetts, 02138, USA. felsch@fas. harvard.edu.

    Ewing family tumors result from the effects of chromosomal translocations that fuse the Ewing sarcoma (EWS) gene to various genes encoding transcription factors. The resulting chimeric EWS fusion proteins are transcriptional activators with transforming potential that have received much study. By contrast, the cellular function of somatic EWS remains obscure. EWS belongs to a family of RNA-binding proteins thought to play role in RNA synthesis or processing. Here, we show that EWS interacts with Pyk2, a protein tyrosine kinase implicated in a variety of signal transduction processes. G-protein-coupled receptor signaling and other stimuli of Pyk2 kinase activity significantly block the interaction between EWS and Pyk2. Furthermore, as assessed by sucrose gradient centrifugation, EWS partitions with dense ribosome-containing fractions in a manner that is enhanced by signaling from the G-protein-coupled m1 muscarinic acetylcholine receptor (mAChR). We conclude that extranuclear EWS is a previously unrecognized target of G-protein-coupled receptor regulation.

    Current biology : CB 1999;9;9;485-8

  • Human glomerular epithelial cell express CD4 and interaction with gp120 protein promotes PYK2 tyrosine phosphorylation.

    Kapasi AA, Franki N, Ding G and Singhal PC

    Department of Medicine, Long Island Jewish Medical Center, New Hyde Park, New York 11040, USA.

    Focal segmental glomerulosclerosis (FSGS) is the predominant glomerular lesion in patients with HIV infection. Visceral glomerular epithelial cell (vGEC) injury is a key feature of this glomerular lesion. However, the exact mechanism of HIV-1-induced vGEC injury is not clear. We studied the presence of CD4 (HIV-1 receptor) in vGECs. vGECs were cultured from human kidneys and used during the 5th to 10th passages. Immunocytochemical studies were carried out to visualize CD4 receptors in these cells. Protein and RNA were extracted from vGECs and renal cortical tissues. Western and Northern blots were generated and probed for the expression of CD4. To determine the downstream effect of ligand receptor interaction, vGECs were treated either with variable concentrations of HIV-1 gp120 protein (0.001 to 0.1 microg/ml) for 1 min or with a fixed dose of gp120 protein (0.01 microg/ml) for variable time periods (0 to 10 min), and at the end of the incubation period, tyrosine phosphorylation of pyk2 was studied. Immunocytochemical studies showed the presence of CD4 receptors in vGECs. Western and Northern blot studies confirmed the presence of CD4 expression in these cells. gp120 protein promoted vGEC tyrosine phosphorylation of pyk2 in a dose- and time-dependent manner. The present study provides a mechanistical insight for the role of HIV-1 in the development of glomerular injury in patients with HIV infection.

    Funded by: NIDA NIH HHS: R01 DA 12111

    Molecular cell biology research communications : MCBRC 1999;1;2;140-3

  • A CD4-independent interaction of human immunodeficiency virus-1 gp120 with CXCR4 induces their cointernalization, cell signaling, and T-cell chemotaxis.

    Missé D, Cerutti M, Noraz N, Jourdan P, Favero J, Devauchelle G, Yssel H, Taylor N and Veas F

    Laboratoire d'Immunologie Rétrovirale et Moléculaire, Institut de Recherches pour le Développement, Montpellier, France.

    The gp120 envelope glycoprotein of human immunodeficiency virus-1 (HIV-1) interacts with the CXCR4 chemokine receptor, but it is not known whether gp120 activates CXCR4-mediated signaling cascades in the same manner as its natural ligand, SDF1alpha. We assessed the effects of wild-type gp120 and a mutant gp120 that interacts with CXCR4 but not CD4 on CD4(-)/CXCR4(+) cells and CD4(+)/CXCR4(+) cells, respectively. Under both experimental conditions, the interaction of CXCR4 and gp120 resulted in their CD4-independent cointernalization. Both molecules were translocated into early endosomes, whereas neither protein could be detected in late endosomes. Binding of gp120 to CXCR4 resulted in a CD4-independent phosphorylation of Pyk2 and an induction of chemotactic activity, demonstrating that this interaction has functional consequences. Interestingly, however, whereas SDF1alpha activated the ERK/MAP kinase pathway, this cascade was not induced by gp120. Together, these results suggest that the pathology of HIV-1 infection may be modulated by the distinct signal transduction pathway mediated by gp120 upon its interaction with CXCR4.

    Blood 1999;93;8;2454-62

  • Identification of a new Pyk2 target protein with Arf-GAP activity.

    Andreev J, Simon JP, Sabatini DD, Kam J, Plowman G, Randazzo PA and Schlessinger J

    Department of Pharmacology, New York University Medical Center, New York, New York 10016, USA.

    Protein tyrosine kinase Pyk2 is activated by a variety of G-protein-coupled receptors and by extracellular signals that elevate intracellular Ca2+ concentration. We have identified a new Pyk2 binding protein designated Pap. Pap is a multidomain protein composed of an N-terminal alpha-helical region with a coiled-coil motif, followed by a pleckstrin homology domain, an Arf-GAP domain, an ankyrin homology region, a proline-rich region, and a C-terminal SH3 domain. We demonstrate that Pap forms a stable complex with Pyk2 and that activation of Pyk2 leads to tyrosine phosphorylation of Pap in living cells. Immunofluorescence experiments demonstrate that Pap is localized in the Golgi apparatus and at the plasma membrane, where it is colocalized with Pyk2. In addition, in vitro recombinant Pap exhibits strong GTPase-activating protein (GAP) activity towards the small GTPases Arf1 and Arf5 and weak activity towards Arf6. Addition of recombinant Pap protein to Golgi preparations prevented Arf-dependent generation of post-Golgi vesicles in vitro. Moreover, overexpression of Pap in cultured cells reduced the constitutive secretion of a marker protein. We propose that Pap functions as a GAP for Arf and that Pyk2 may be involved in regulation of vesicular transport through its interaction with Pap.

    Molecular and cellular biology 1999;19;3;2338-50

  • Identification of a novel family of targets of PYK2 related to Drosophila retinal degeneration B (rdgB) protein.

    Lev S, Hernandez J, Martinez R, Chen A, Plowman G and Schlessinger J

    Sugen, Inc., South San Francisco, California 94080, USA.

    The protein tyrosine kinase PYK2 has been implicated in signaling pathways activated by G-protein-coupled receptors, intracellular calcium, and stress signals. Here we describe the molecular cloning and characterization of a novel family of PYK2-binding proteins designated Nirs (PYK2 N-terminal domain-interacting receptors). The three Nir proteins (Nir1, Nir2, and Nir3) bind to the amino-terminal domain of PYK2 via a conserved sequence motif located in the carboxy terminus. The primary structures of Nirs reveal six putative transmembrane domains, a region homologous to phosphatidylinositol (PI) transfer protein, and an acidic domain. The Nir proteins are the human homologues of the Drosophila retinal degeneration B protein (rdgB), a protein implicated in the visual transduction pathway in flies. We demonstrate that Nirs are calcium-binding proteins that exhibit PI transfer activity in vivo. Activation of PYK2 by agents that elevate intracellular calcium or by phorbol ester induce tyrosine phosphorylation of Nirs. Moreover, PYK2 and Nirs exhibit similar expression patterns in several regions of the brain and retina. In addition, PYK2-Nir complexes are detected in lysates prepared from cultured cells or from brain tissues. Finally, the Nir1-encoding gene is located at human chromosome 17p13.1, in proximity to a locus responsible for several human retinal diseases. We propose that the Nir and rdgB proteins represent a new family of evolutionarily conserved PYK2-binding proteins that play a role in the control of calcium and phosphoinositide metabolism downstream of G-protein-coupled receptors.

    Molecular and cellular biology 1999;19;3;2278-88

  • Identification of myosin II as a binding protein to the PH domain of protein kinase B.

    Tanaka M, Konishi H, Touhara K, Sakane F, Hirata M, Ono Y and Kikkawa U

    Department of Biology, Faculty of Science, Kobe University, Kobe, 657-8501, Japan.

    Myosin II was identified as a binding protein to the pleckstrin homology (PH) domain of protein kinase B (PKB) in CHO cell extract by using the glutathione S-transferase-fusion protein as a probe. When myosin II purified from rabbit skeletal muscle was employed, myosin II was shown to bind almost exclusively to the PH domain of PKB among the PH domain fusion proteins examined. The purified myosin II bound to the PH domain of PKB with a Kd value of 1.1 x 10(-7) M. Studies with a series of truncated molecules indicated that the whole structure of the PH domain is required for the binding of myosin II, and the binding to the PH domain was inhibited by phosphatidylinositol 4,5-bisphosphate. These results suggest that myosin II is a specific binding protein to the PH domain of particular proteins including PKB.

    Biochemical and biophysical research communications 1999;255;1;169-74

  • Characterization of a focal adhesion protein, Hic-5, that shares extensive homology with paxillin.

    Thomas SM, Hagel M and Turner CE

    Department of Medicine, Cancer Biology Program, Beth Israel Deaconess Medical Center/Harvard Medical School, Boston, MA 02215, USA. sthomas@bidmc.harvard.edu.

    Paxillin is a focal adhesion scaffolding protein which was originally identified as a substrate of the oncogenic tyrosine kinase, v-src. Paxillin has been proposed to be involved in regulation of focal adhesion dynamics. Two alternatively spliced mouse paxillin cDNAs were cloned and in the process, a paxillin-related protein, Hic-5, was also identified. Cloning and characterization of Hic-5 indicates that this protein shares extensive homology with paxillin. Although Hic-5 was originally characterized as a TGF-beta-inducible gene and proposed to be a transcription factor involved in senescence, the studies here demonstrate that Hic-5 is localized to focal adhesion in REF52 cells and can interact with the focal adhesion proteins, Fak, Frnk, and vinculin. In addition, like paxillin, Hic-5 can bind to a negative regulator of Src PTKs, csk but does not bind to the adaptor protein Crk. Like paxillin, localization of this protein to focal adhesions is mediated primarily by the LIM domains; however, sequences outside the LIM domains also play a minor role in focal adhesion targeting. These results suggest that Hic-5 like paxillin could be involved in regulation of focal adhesion dynamics and raise the possibility that Hic-5 and paxillin could have overlapping or opposing functions in the overall regulation of cell growth and differentiation.

    Funded by: NCI NIH HHS: CA75621; NIGMS NIH HHS: GM47607

    Journal of cell science 1999;112 ( Pt 2);181-90

  • Signaling through focal adhesion kinase.

    Schlaepfer DD, Hauck CR and Sieg DJ

    Scripps Research Institute, Department of Immunology, La Jolla, CA 92037, USA. dschlaep@scripps.edu

    Integrin receptor binding to extracellular matrix proteins generates intracellular signals via enhanced tyrosine phosphorylation events that are important for cell growth, survival, and migration. This review will focus on the functions of the focal adhesion kinase (FAK) protein-tyrosine kinase (PTK) and its role in linking integrin receptors to intracellular signaling pathways. FAK associates with several different signaling proteins such as Src-family PTKs, p130Cas, Shc, Grb2, PI 3-kinase, and paxillin. This enables FAK to function within a network of integrin-stimulated signaling pathways leading to the activation of targets such as the ERK and JNK/mitogen-activated protein kinase pathways. Focus will be placed on the structural domains and sites of FAK tyrosine phosphorylation important for FAK-mediated signaling events and how these sites are conserved in the FAK-related PTK, Pyk2. We will review what is known about FAK activation by integrin receptor-mediated events and also non-integrin stimuli. In addition, we discuss the emergence of a consensus FAK substrat 1f40 e phosphorylation sequence. Emphasis will also be placed on the role of FAK in generating cell survival signals and the cleavage of FAK during caspase-mediated apoptosis. An in-depth discussion will be presented of integrin-stimulated signaling events occurring in the FAK knockout fibroblasts (FAK-) and how these cells exhibit deficits in cell migration. FAK re-expression in the FAK- cells confirms the role of this PTK in the regulation of cell morphology and in promoting cell migration events. In addition, these results reinforce the potential role for FAK in promoting an invasive phenotype in human tumors.

    Funded by: NCI NIH HHS: R29 CA75240; PHS HHS: H1-07195-21

    Progress in biophysics and molecular biology 1999;71;3-4;435-78

  • Dot far-western blot analysis of relative binding affinities of the Src homology 3 domains of Efs and its related proteins.

    Ohba T, Ishino M, Aoto H and Sasaki T

    Cancer Research Institute, Sapporo Medical University School of Medicine, South-1, West-17, Sapporo, Chuo-Ku, 060-8556, Japan.

    The Src homology 3 (SH3) domains are a modular structure of about 60 amino acid residues found in many proteins important in signal transduction. Each SH3 domain has a binding specificity to sequences containing a PXXP motif in ligand proteins. We found that a focal adhesion kinase (FAK)-related protein, cell adhesion kinase beta (CAKbeta), was bound in vitro by the SH3 domain of embryonal Fyn-associated substrate (Efs), a docking protein structurally related to p130Cas (Cas) and HEF1. Here, we employed a dot far-Western blotting technique to evaluate the affinity and specificity of the binding by the SH3 domains of Efs and its related proteins. The SH3 domains and their ligands were prepared as glutathione S-transferase fusion proteins, and one of the binding components was immobilized on membranes while the other was labeled with 32P to use as a probe. The amount of the bound probe was determined by autoradiography using an imaging plate and a bioimaging analyzer. A competitive binding assay showed that Efs, compared with Cas and HEF1, had a SH3 domain with a lower relative affinity to CAKbeta and FAK and with a preference to interact with FAK rather than CAKbeta. Our assay based on dot far-Western blotting is a simple and sensitive method to evaluate fine differences in the binding affinity of SH3-mediated interactions.

    Analytical biochemistry 1998;262;2;185-92

  • Human immunodeficiency virus tat modulates the Flk-1/KDR receptor, mitogen-activated protein kinases, and components of focal adhesion in Kaposi's sarcoma cells.

    Ganju RK, Munshi N, Nair BC, Liu ZY, Gill P and Groopman JE

    Divisions of Experimental Medicine and Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, Boston, Massachusetts 02115, USA.

    Kaposi's sarcoma (KS) spindle cell growth and spread have been reported to be modulated by various cytokines as well as the human immunodeficiency virus (HIV) gene product Tat. Recently, HIV-1 Tat has been shown to act like a cytokine and bind to the Flk-1/KDR receptor for the vascular endothelial growth factor A (VEGF-A), which is expressed by KS cells. We have characterized signal transduction pathways stimulated by HIV-1 Tat upon its binding to surface receptors on KS cells. We observed that stimulation in KS 38 spindle cells resulted in tyrosine phosphorylation and activation of the Flk-1/KDR receptor. We also report that HIV-1 Tat treatment enhanced the phosphorylation and association of proteins found in focal adhesions, such as the related adhesion focal tyrosine kinase RAFTK, paxillin, and p130(cas). Further characterization revealed the activation of mitogen-activated protein kinase, c-Jun amino-terminal kinase (JNK), and Src kinase. HIV-1 Tat contains a basic domain which can interact with growth factor tyrosine kinase receptors and a classical RGD sequence which may bind to and activate the surface integrin receptors for fibronectin and vitronectin. We observed that stimulation of KS cells with basic as well as RGD sequence-containing Tat peptides resulted in enhanced phosphorylation of RAFTK and activation of MAP kinase. These studies reveal that Tat stimulation activates a number of signal transduction pathways that are associated with cell growth and migration.

    Funded by: NHLBI NIH HHS: HL 43510, HL 53745, HL 55187, R01 HL053745

    Journal of virology 1998;72;7;6131-7

  • Leupaxin is a novel LIM domain protein that forms a complex with PYK2.

    Lipsky BP, Beals CR and Staunton DE

    ICOS Corporation, Bothell, Washington 98021, USA.

    We have identified a novel cytoplasmic protein, leupaxin, that is preferentially expressed in hematopoietic cells and is most homologous to the focal adhesion protein, paxillin. Leupaxin possesses two types of protein interaction domains. There are four carboxyl-terminal LIM domains in leupaxin that share 70% amino acid identity and 80% similarity with those in paxillin. Paxillin LIM domains mediate localization to focal contacts. In the amino-terminal region of leupaxin there are three short stretches of approximately 13 amino acids that share 70-90% similarity with paxillin LD motifs. Paxillin LD motifs have been implicated in focal adhesion kinase (FAK) and vinculin binding resulting in the localization of FAK to focal adhesions. Leupaxin is expressed in cell types, such as macrophage, that lack FAK. We demonstrate here that leupaxin associates with a second FAK family member, PYK2. As leupaxin and PYK2 are both preferentially expressed in leukocytes they may therefore form a cell type-specific signaling complex. We also demonstrate that leupaxin is a substrate for a tyrosine kinase in lymphoid cells and thus may function in and be regulated by tyrosine kinase activity. Leupaxin is thus a phosphotyrosine protein with LD and LIM binding motifs most homologous to paxillin that may assemble and regulate PYK2 signaling complexes in leukocytes.

    The Journal of biological chemistry 1998;273;19;11709-13

  • Activation of protein tyrosine kinase PYK2 by the m1 muscarinic acetylcholine receptor.

    Felsch JS, Cachero TG and Peralta EG

    Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.

    Several G protein-coupled receptors are known to direct the tyrosine phosphorylation, and in some cases the activation, of diverse tyrosine kinases. Using a stable cell line approach, we characterize the activation of PYK2, a tyrosine kinase structurally related to focal adhesion kinase, by the G protein-coupled m1 muscarinic acetylcholine receptor. We find that PYK2 tyrosine kinase activity is critical for the m1 receptor-stimulated tyrosine phosphorylation of PYK2. Furthermore, we identify two tyrosine residues that are subject to phosphorylation in response to muscarinic signaling and show that this phosphorylation induces two cytosolic proteins, c-Src and Grb2, to bind to PYK2. This is the first demonstration of the significance played by distinct PYK2 tyrosine residues in G protein-coupled signaling to this kinase. By comparison, though m1 receptors induce the tyrosine phosphorylation of the cytoskeletal protein paxillin, the association of paxillin with PYK2 is unaffected by muscarinic signaling. We also provide evidence that PYK2 specifically phosphorylates the carboxyl-terminal cytosolic portion of the potassium channel Kv1.2 in a manner regulated by the m1 receptor. These results delineate molecular events attending the m1 muscarinic receptor stimulation of this tyrosine kinase and establish PYK2 as an effector of the m1 muscarinic receptor in the regulation of multiple cell functions.

    Proceedings of the National Academy of Sciences of the United States of America 1998;95;9;5051-6

  • A calcium-dependent tyrosine kinase splice variant in human monocytes. Activation by a two-stage process involving adherence and a subsequent intracellular signal.

    Li X, Hunter D, Morris J, Haskill JS and Earp HS

    University of North Carolina Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.

    Freshly isolated human monocytes do not express p125(FAK) but upon adherence to substrata activate the highly related calcium-dependent tyrosine kinase (CADTK), also known as Pyk2, CAKbeta, RAFTK, and FAK2. The monocyte CADTK was 5 kDa smaller than protein from epithelial cells; isolation and sequencing of the monocyte CADTK cDNA revealed a predicted 42-amino acid deletion between the two proline-rich domains of the enzyme. The nucleic acid sequence suggests that the deletion is caused by alternative RNA splicing. This species was also found in T and B lymphocytes and appears to be the predominant form of cytoskeletal associated tyrosine kinase in non-neoplastic, circulating, hematopoietic cells. CADTK was not activated when monocytes maintained in suspension were treated with agents that produce an intracellular calcium (thapsigargin) or protein kinase C (phorbol 12-myristate 13-acetate) signal including a chemokine, RANTES, that binds to the HIV co-receptor, CCK5. In contrast, monocyte adherence to tissue culture plastic-stimulated CADTK tyrosine phosphorylation, a process that was enhanced by thapsigargin, phorbol 12-myristate 13-acetate, and RANTES but that was completely blocked by preincubation with cytochalasin D. When compared with plastic, adherence to fibronectin- or collagen-coated surfaces produced only minimal CADTK activation but permitted significant stimulation by added thapsigargin. These data suggest that in a cell type that lacks p125(FAK), CADTK plays an early role in post-adherence signaling. Its activation involves two stages, cytoskeletal engagement, which is permissive, and co-stimulatory signals (calcium or protein kinase C) generated by extensive cell surface engagement, agonists, or inflammatory chemokines.

    The Journal of biological chemistry 1998;273;16;9361-4

  • Pyk2 is a downstream mediator of the IL-2 receptor-coupled Jak signaling pathway.

    Miyazaki T, Takaoka A, Nogueira L, Dikic I, Fujii H, Tsujino S, Mitani Y, Maeda M, Schlessinger J and Taniguchi T

    Department of Immunology, Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, 113, Japan.

    Many cytokines transmit signals to t ac0 he cell interior through activation of receptor-associated, Janus family protein tyrosine kinases (Jak PTKs). The interleukin-2 receptor (IL-2R) is associated with the Jak1 and Jak3 PTKs, and ligand-induced activation of these PTKs is essential for lymphocyte proliferation. Here, the nonreceptor PTK, Pyk2, was found to be activated following IL-2 stimulation in a Jak-dependent manner. Furthermore, physical association was detected between endogenous Pyk2 and Jak3, and a dominant interfering mutant of Pyk2 inhibited IL-2-induced cell proliferation without affecting Stat5 activation. Collectively, these results suggest that Pyk2 is a newly identified component of the Jak-mediated IL-2 signaling pathway.

    Genes & development 1998;12;6;770-5

  • Beta-chemokine receptor CCR5 signals via the novel tyrosine kinase RAFTK.

    Ganju RK, Dutt P, Wu L, Newman W, Avraham H, Avraham S and Groopman JE

    Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.

    Chemokine receptors are coupled to G-proteins and their activation results in prominent changes in cell migration and growth. The downstream signaling pathways that mediate these effects of chemokines are largely uncharacterized. Macrophage inflammatory protein 1 beta (MIP 1 beta) binding to its cognate receptor CCR5 resulted in activation of the related adhesion focal tyrosine kinase (RAFTK), with subsequent activation of the cytoskeletal protein paxillin and the down-stream transcriptional activators, c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) and p38 mitogen-activated protein (MAP) kinase. Inhibition of RAFTK by a dominant-negative kinase mutant markedly attenuated JNK/ SAPK activity. Thus, RAFTK appears to provide a functional "bridge" for the transmission of CCR5 receptor signaling to the cytoskeleton and nucleus, primary sites of chemotaxis and growth regulation.

    Funded by: NHLBI NIH HHS: HL 43510, HL 53745, HL 55187; ...

    Blood 1998;91;3;791-7

  • Differential regulation of Pyk2 and focal adhesion kinase (FAK). The C-terminal domain of FAK confers response to cell adhesion.

    Zheng C, Xing Z, Bian ZC, Guo C, Akbay A, Warner L and Guan JL

    Department of Pathology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA.

    Pyk2 is a recently described cytoplasmic tyrosine kinase that is related to focal adhesion kinase (FAK) and can be activated by a variety of stimuli that elevate intracellular calcium. In this report, we showed that Pyk2 and FAK tyrosine phosphorylation are regulated differentially by integrin-mediated cell adhesion and soluble factors both in rat aortic smooth muscle cells, which express endogenous Pyk2 and FAK, and in transfected Chinese hamster ovary cells. We also found that Pyk2 is diffusely present throughout the cytoplasm, while FAK is localized in focal contacts as expected, suggesting that the different localization may account for their differential regulation. By analyzing a chimeric protein contain N-terminal and kinase domains of Pyk2 and C-terminal domain of FAK, we provided evidence that the distinctive C-terminal domains of Pyk2 and FAK were responsible for their differential regulation by integrins and soluble stimuli as well as their subcellular localization. Finally, we correlated FAK, Pyk2, and the chimeric protein binding to talin, but not paxillin, with their regulation by integrins and focal contact localization. These results demonstrate that the distinctive C-terminal domain of Pyk2 and FAK confer their differential regulation by different subcellular localization and association with the cytoskeletal protein talin.

    Funded by: NCI NIH HHS: T32CA09682; NIGMS NIH HHS: R01GM52890

    The Journal of biological chemistry 1998;273;4;2384-9

  • Cell adhesion kinase beta forms a complex with a new member, Hic-5, of proteins localized at focal adhesions.

    Matsuya M, Sasaki H, Aoto H, Mitaka T, Nagura K, Ohba T, Ishino M, Takahashi S, Suzuki R and Sasaki T

    Department of Biochemistry, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-Ku, Sapporo 060, Japan.

    Cell adhesion kinase beta (CAKbeta/PYK2) is the second protein-tyrosine kinase of the focal adhesion kinase subfamily. We identified a cDNA that encodes a CAKbeta-binding protein. This cDNA clone encodes the human homologue of Hic-5, the cDNA of which was cloned in 1994 as transforming growth factor beta1- and hydrogen peroxide-inducible mRNA. We found that Hic-5 exclusively localized at focal adhesions in a rat fibroblast line, WFB. This localization of Hic-5 was confirmed in WFB cells expressing Myc-tagged Hic-5. The amino acid sequence of Hic-5 is highly similar to that of paxillin in the four LD motifs as well as in the four contiguous LIM domains. The Hic-5 N-terminal domain directly associated in vitro with the extreme C-terminal region (residue 801 to the end) of CAKbeta. CAKbeta was coimmunoprecipitated with Hic-5 from the WFB cell lysate. The coimmunoprecipitation of CAKbeta with Hic-5 was markedly inhibited by the addition of the extreme C-terminal region of CAKbeta. Coimmunoprecipitation of Hic-5 with CAKbeta, which was shown in COS-7 cells doubly transfected with cDNA constructs of CAKbeta and Myc-tagged Hic-5, was lost when the CAKbeta amino acid residues 741-903 were deleted. Hic-5 was tyrosine-phosphorylated in Src-transformed 3Y1 cells and in cells treated with pervanadate. Hic-5 associated with CAKbeta was selectively tyrosine-phosphorylated in WFB cells exposed to hypertonic osmotic stress. These results indicate that Hic-5 is a paxillin-related component of focal adhesions and binds to CAKbeta, implying possible involvement of Hic-5 in the downstream signaling of CAKbeta.

    The Journal of biological chemistry 1998;273;2;1003-14

  • Signal transduction due to HIV-1 envelope interactions with chemokine receptors CXCR4 or CCR5.

    Davis CB, Dikic I, Unutmaz D, Hill CM, Arthos J, Siani MA, Thompson DA, Schlessinger J and Littman DR

    Division of Molecular Pathogenesis, Skirball Institute for Biomolecular Medicine, NYU Medical Center 10016, USA. davis@saturn.med.nyu.edu

    Infection with HIV-1 requires expression of CD4 and the chemokine receptors CXCR4 or CCR5 at the target cell surface. Engagement of these receptors by the HIV-1 envelope glycoprotein is essential for membrane fusion, but may additionally activate intracellular signaling pathways. In this study, we demonstrate that chemokines and HIV-1 envelope glycoproteins from both T-tropic and macrophage-tropic strains rapidly induce tyrosine phosphorylation of the protein tyrosine kinase Pyk2. The response requires CXCR4 and CCR5 to be accessible on the cell surface. The results presented here provide the first evidence for activation of an intracellular signaling event that can initiate multiple signaling pathways as a consequence of contact between HIV-1 and chemokine receptors.

    The Journal of experimental medicine 1997;186;10;1793-8

  • Cloning and characterization of novel CIS family genes.

    Masuhara M, Sakamoto H, Matsumoto A, Suzuki R, Yasukawa H, Mitsui K, Wakioka T, Tanimura S, Sasaki A, Misawa H, Yokouchi M, Ohtsubo M and Yoshimura A

    Institute of Life Science, Kurume University, Japan.

    Biochemical and biophy 738 sical research communications 1997;239;2;439-46

  • Characterization of the novel focal adhesion kinase RAFTK in hematopoietic cells.

    Avraham S and Avraham H

    Division of Experimental Medicine, Harvard Institutes of Medicine-BIDMC, Boston, MA 02115, USA.

    Protein tyrosine kinases (PTKs) mediate signals that respond to many pivotal cellular functions. Tyrosine phosphorylation, controlled by the coordinated actions of protein tyrosine phosphatases (PTPs) and PTKs, is a critical control mechanism for various physiological processes, including cell growth, differentiation, metabolism, cell cycle regulation and cytoskeleton function. The focal adhesion kinase (FAK) is a widely expressed non-receptor tyrosine kinase that is implicated in integrin-mediated signaling and plays a role in signal transduction pathways mediating cell adhesion, motility and anchorage-independent growth. Recently, we and others have identified a novel protein tyrosine kinase termed RAFTK, (also known as Pyk2 or Cak-beta), which is related to FAK. This review describes the role of RAFTK in various signaling cascades mainly in reference to hematopoietic cell lineages.

    Funded by: NHLBI NIH HHS: HL51456, HL55445

    Leukemia & lymphoma 1997;27;3-4;247-56

  • The related adhesion focal tyrosine kinase differentially phosphorylates p130Cas and the Cas-like protein, p105HEF1.

    Astier A, Manié SN, Avraham H, Hirai H, Law SF, Zhang Y, Golemis EA, Fu Y, Druker BJ, Haghayeghi N, Freedman AS and Avraham S

    Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA.

    The related adhesion focal tyrosine kinase (RAFTK) is tyrosine-phosphorylated following beta1 integrin or B cell antigen receptor stimulation in human B cells. Two substrates that are tyrosine-phosphorylated following integrin ligation in B cells are p130(Cas) and the Cas family member human enhancer of filamentation 1 (HEF1), both of which can associate with RAFTK. In this report we observed that RAFTK was involved in the phosphorylation of these two proteins. While a catalytically active RAFTK was required for both p130(Cas) and HEF1, phosphorylation of p130(Cas), but not of HEF1, was dependent on an intact autophosphorylation site (Tyr402) on RAFTK. To determine if RAFTK phosphorylated p130(Cas) and HEF1 directly or through an intermediate, we assayed the ability of RAFTK and of a Tyr402 mutant to phosphorylate purified HEF1 and p130(Cas) domains. RAFTK was able to phosphorylate the substrate domains of both p130(Cas) and HEF1, but only the C-terminal domain of p130(Cas). Furthermore, Tyr402, which mediates the binding of RAFTK to c-Src kinase, was required for the phosphorylation of the C-terminal domain of p130(Cas). These data suggest that RAFTK itself is sufficient for HEF1 phosphorylation, whereas a cooperation between RAFTK and Src kinases is required for the complete phosphorylation of p130(Cas).

    Funded by: NCI NIH HHS: CA55207, CA66996; NHLBI NIH HHS: HL55445; ...

    The Journal of biological chemistry 1997;272;32;19719-24

  • Tyrosine phosphorylation of the related adhesion focal tyrosine kinase in megakaryocytes upon stem cell factor and phorbol myristate acetate stimulation and its association with paxillin.

    Hiregowdara D, Avraham H, Fu Y, London R and Avraham S

    Division of Experimental Medicine, Beth Israel Deaconess Medical Center (West Campus), Harvard Medical School, Boston, Massachusetts 02215, USA.

    We have characterized signaling pathways involving the related adhesion focal tyrosine kinase (RAFTK, also known as PYK2 or CAK-beta) in CMK human megakaryocytic cells. Stem cell factor, which potentiates the growth of megakaryocytes and their progenitors, and phorbol myristate acetate, which causes differentiation of megakaryocytic cell lines, induced the tyrosine phosphorylation of RAFTK but not of focal adhesion kinase. Stimulation of CMK cells with stem cell fact 1d48 or resulted in an increase in the autophosphorylation and kinase activity of RAFTK. Phosphorylation of RAFTK under these conditions was mediated by a protein kinase C-dependent pathway. Cytochalasin D, which disrupts the cytoskeleton, abolished the phosphorylation of RAFTK upon phorbol myristate acetate and stem cell factor stimulation, indicating that RAFTK association with the actin cytoskeleton appears to be critical for its phosphorylation. In addition, we observed an association of RAFTK with paxillin, a 68-kDa cytoskeleton protein. Using in vitro binding assays, RAFTK and paxillin were shown to bind directly through the C-terminal proline-rich domain. Transient overexpression of a dominant-negative mutant of RAFTK inhibited significantly the tyrosine phosphorylation of paxillin upon phorbol myristate acetate stimulation. These observations indicate that RAFTK might play an important role in the phosphorylation of signaling pathways within the focal adhesions and that RAFTK participates in signaling events that link signals from the cell surface to the cytoskeleton. Furthermore, this study suggests that RAFTK might be involved in megakaryocyte proliferation and differentiation.

    Funded by: NHLBI NIH HHS: HL51456, HL55445

    The Journal of biological chemistry 1997;272;16;10804-10

  • Tyrosine phosphorylation of Pyk2 is selectively regulated by Fyn during TCR signaling.

    Qian D, Lev S, van Oers NS, Dikic I, Schlessinger J and Weiss A

    Department of Medicine, University of California, San Francisco 94143, USA.

    The Src family protein tyrosine kinases (PTKs), Lck and Fyn, are coexpressed in T cells and perform crucial functions involved in the initiation of T cell antigen receptor (TCR) signal transduction. However, the mechanisms by which Lck and Fyn regulate TCR signaling are still not completely understood. One important question is whether Lck and Fyn have specific targets or only provide functional redundancy during TCR signaling. We have previously shown that Lck plays a major role in the tyrosine phosphorylation of the TCR-zeta chain and the ZAP-70 PTK. In an effort to identify the targets that are specifically regulated by Fyn, we have studied the tyrosine phosphorylation of Pyk2, a recently discovered new member of the focal adhesion kinase family PTK. We demonstrated that Pyk2 was rapidly tyrosine phosphorylated following TCR stimulation. TCR-induced tyrosine phosphorylation of Pyk2 was selectively dependent on Fyn but not Lck. Moreover, in heterologous COS-7 cells, coexpression of Pyk2 with Fyn but not Lck resulted in substantial increases in Pyk2 tyrosine phosphorylation. The selective regulation of Pyk2 tyrosine phosphorylation by Fyn in vivo correlated with the preferential phosphorylation of Pyk2 by Fyn in vitro. Our results demonstrate that Pyk2 is a specific target regulated by Fyn during TCR signaling.

    The Journal of experimental medicine 1997;185;7;1253-9

  • RAFTK, a novel member of the focal adhesion kinase family, is phosphorylated and associates with signaling molecules upon activation of mature T lymphocytes.

    Ganju RK, Hatch WC, Avraham H, Ona MA, Druker B, Avraham S and Groopman JE

    Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.

    The related adhesion focal tyrosine kinase (RAFTK), a recently discovered member of the focal adhesion kinase family, has previously been reported to participate in signal transduction in neuronal cells, megakaryocytes, and B lymphocytes. We have found that RAFTK is constitutively expressed in human T cells and is rapidly phosphorylated upon the activation of the T cell receptor (TCR). This activation also results in an increase in the autophosphorylation and kinase activity of RAFTK. After its stimulation, there was an increase in the association of the src cytoplasmic tyrosine kinase Fyn and the adapter protein Grb2. This association was mediated through the SH2 domains of Fyn and Grb2. RAFTK also co-immunoprecipitates with the SH2 domain of Lck and with the cytoskeletal protein paxillin through its COOH-terminal proline-rich domain. The tyrosine phosphorylation of RAFTK after T cell receptor-mediated stimulation was reduced by the pretreatment of cells with cytochalasin D, suggesting the role of the cytoskeleton in this process. These observations indicate that RAFTK participates in T cell receptor signaling and may act to link signals from the cell surface to the cytoskeleton and thereby affect the host immune response.

    Funded by: NHLBI NIH HHS: HL 43510-07, HL 53745-02, HL 55187-01, R01 HL053745

    The Journal of experimental medicine 1997;185;6;1055-63

  • Involvement of p130(Cas) and p105(HEF1), a novel Cas-like docking protein, in a cytoskeleton-dependent signaling pathway initiated by ligation of integrin or antigen receptor on human B cells.

    Manié SN, Beck AR, Astier A, Law SF, Canty T, Hirai H, Druker BJ, Avraham H, Haghayeghi N, Sattler M, Salgia R, Griffin JD, Golemis EA and Freedman AS

    Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA.

    The Crk-associated substrate p130(Cas) (Cas) and the recently described human enhancer of filamentation 1 (HEF1) are two proteins with similar structure (64% amino acid homology), which are thought to act as "docking" molecules in intracellular signaling cascades. Both proteins contain an N-terminal Src homology (SH), three domain and a cluster of SH2 binding motifs. Here we show that ligation of either beta1 integrin or B cell antigen receptor (BCR) on human tonsillar B cells and B cell lines promoted tyrosine phosphorylation of HEF1. In contrast, Cas tyrosine phosphorylation was observed in certain B cell lines but not in tonsillar B cells, indicating a more general role for HEF1 in B cell signaling. Interestingly, pretreatment of tonsillar B cells with cytochalasin B dramatically reduced both integrin- and BCR-induced HEF1 phosphorylation, suggesting that some component of the BCR-mediated signaling pathway is closely linked with a cytoskeletal reorganization. Both HEF1 and Cas were found to complex with the related adhesion focal tyrosine kinase (RAFTK), and when tyrosine phosphorylated, with the adapter molecule CrkL. In addition, the two molecules were detected in p53/56(Lyn) immunoprecipitates, and Lyn kinase was found to specifically bind the C-terminal proline-rich sequence of Cas in an in vitro binding assay. These associations implicate HEF1 and Cas as important components in a cytoskeleton-linked signaling pathway initiated by ligation of beta1 integrin or BCR on human B cells.

    Funded by: NCI NIH HHS: CA55207, CA60821, CA66996; ...

    The Journal of biological chemistry 1997;272;7;4230-6

  • The related adhesion focal tyrosine kinase is tyrosine-phosphorylated after beta1-integrin stimulation in B cells and binds to p130cas.

    Astier A, Avraham H, Manie SN, Groopman J, Canty T, Avraham S and Freedman AS

    Division of Hematologic Malignancies, Dana-Farber Cancer Institute, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA.

    Integrin ligation initiates intracellular signaling events, among which are the activation of protein tyrosine kinases. The related adhesion focal tyrosine kinase (RAFTK), also known as PYK2 and CAKbeta, is a tyrosine kinase that is homologous to the focal adhesion kinase (FAK) p125FAK. The structure of RAFTK is similar to p125FAK in that it lacks a transmembrane region, does not contain Src homology 2 or 3 domains, and has a proline-rich region in its C terminus. Here we report that RAFTK is a target for beta1-integrin-mediated tyrosine phosphorylation in both transformed and normal human B cells. Ligation of the B cell antigen receptor also induced tyrosine phosphorylation of RAFTK. Phosphorylation of RAFTK following integrin- or B cell antigen receptor-mediated stimulation was decreased by prior treatment of cells with cytochalasin B, indicating that this process was at least partially cytoskeleton-dependent. One of the tyrosine-phosphorylated substrates after integrin stimulation in fibroblasts is p130cas, which can associate with p125FAK. RAFTK also interacted constitutively with p130cas in B cells, since p130cas was detected in RAFTK immunoprecipitates. Although the function of RAFTK remains unknown, these data suggest that RAFTK may have a significant function in integrin-mediated signaling pathways in B cells.

    Funded by: NCI NIH HHS: CA55207, CA66996; NHLBI NIH HHS: HL51456; ...

    The Journal of biological chemistry 1997;272;1;228-32

  • Activation of a novel calcium-dependent protein-tyrosine kinase. Correlation with c-Jun N-terminal kinase but not mitogen-activated protein kinase activation.

    Yu H, Li X, Marchetto GS, Dy R, Hunter D, Calvo B, Dawson TL, Wilm M, Anderegg RJ, Graves LM and Earp HS

    Department of Medicine, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina 27599, USA.

    Many G protein-coupled receptors (e.g. that of angiotensin II) activate phospholipase Cbeta, initially increasing intracellular calcium and activating protein kinase C. In the WB and GN4 rat liver epithelial cell lines, agonist-induced calcium signals also stimulate tyrosine phosphorylation and subsequently increase the activity of c-Jun N-terminal kinase (JNK). We have now purified the major calcium-dependent tyrosine kinase (CADTK), and by peptide and nucleic acid sequencing identified it as a rat homologue of human PYK2. CADTK/PYK2 is most closely related to p125(FAK) and both enzymes are expressed in WB and GN4 cells. Angiotensin II, which only slightly increases p125(FAK) tyrosine phosphorylation in GN4 cells, substantially increased CADTK tyrosine autophosphorylation and kinase activity. Agonists for other G protein-coupled receptors (e.g. LPA), or those increasing intracellular calcium (thapsigargin), also stimulated CADTK. In comparing the two rat liver cell lines, GN4 cells exhibited approximately 5-fold greater angiotensin II- and thapsigargin-dependent CADTK activation than WB cells. Although maximal JNK activation by stress-dependent pathways (e.g. UV and anisomycin) was equivalent in the two cell lines, calcium-dependent JNK activation was 5-fold greater in GN4, correlating with CADTK activation. In contrast to JNK, the thapsigargin-dependent calcium signal did not activate mitogen-activated protein kinase and Ang II-dependent mitogen-activated protein kinase activation was not correlated with CADTK activation. Finally, while some stress-dependent activators of the JNK pathway (NaCl and sorbitol) stimulated CADTK, others (anisomycin, UV, and TNFalpha) did not. In summary, cells expressing CADTK/PYK2 appear to have two alternative JNK activation pathways: one stress-activated and the othe 1f40 r calcium-dependent.

    Funded by: NIDDK NIH HHS: DK 31683

    The Journal of biological chemistry 1996;271;47;29993-8

  • A role for Pyk2 and Src in linking G-protein-coupled receptors with MAP kinase activation.

    Dikic I, Tokiwa G, Lev S, Courtneidge SA and Schlessinger J

    Department of Pharmacology, New York University Medical Center, 10016, USA.

    The mechanisms by which mitogenic G-protein-coupled receptors activate the MAP kinase signalling pathway are poorly understood. Candidate protein tyrosine kinases that link G-protein-coupled receptors with MAP kinase include Src family kinases, the epidermal growth factor receptor, Lyn and Syk. Here we show that lysophosphatidic acid (LPA) and bradykinin induce tyrosine phosphorylation of Pyk2 and complex formation between Pyk2 and activated Src. Moreover, tyrosine phosphorylation of Pyk2 leads to binding of the SH2 domain of Src to tyrosine 402 of Pyk2 and activation of Src. Transient overexpression of a dominant interfering mutant of Pyk2 or the protein tyrosine kinase Csk reduces LPA- or bradykinin-induced activation of MAP kinase. LPA- or bradykinin-induced MAP kinase activation was also inhibited by overexpression of dominant interfering mutants of Grb2 and Sos. We propose that Pyk2 acts with Src to link Gi- and Gq-coupled receptors with Grb2 and Sos to activate the MAP kinase signalling pathway in PC12 cells.

    Nature 1996;383;6600;547-50

  • The Ca2+-dependent lipid binding domain of P120GAP mediates protein-protein interactions with Ca2+-dependent membrane-binding proteins. Evidence for a direct interaction between annexin VI and P120GAP.

    Davis AJ, Butt JT, Walker JH, Moss SE and Gawler DJ

    Department of Pharmacology, University of Leeds, Leeds LS2 9JT, United Kingdom.

    The CaLB domain is a 43-amino acid sequence motif found in a number of functionally diverse signaling proteins including three Ras-specific GTPase activating proteins (GAPs). In the Ras GTPase activating protein, P120(GAP), this domain has the ability to confer membrane association in response to intracellular Ca2+ elevation. Here we have isolated three proteins, p55, p70, and p120, which interact with the P120(GAP) CaLB domain in vitro. We identify p70 as the Ca2+-dependent phospholipid-binding protein annexin VI. Using co-immunoprecipitation studies, we have shown that the interaction between P120(GAP) and annexin VI is also detectable in rat fibroblasts, suggesting that this interaction may have a physiological role in vivo. Thus, the CaLB domain in P120(GAP) appears to have the ability to direct specific protein-protein interactions with Ca2+-dependent membrane-associated proteins. In addition, annexin VI is known to have tumor suppressor activity. Therefore, it is possible that the interaction of annexin VI with P120(GAP) may be important in the subsequent modulation of p21(ras) activity.

    The Journal of biological chemistry 1996;271;40;24333-6

  • Precise localization of the human gene encoding cell adhesion kinase beta (CAK beta/PYK2) to chromosome 8 at p21.1 by fluorescence in situ hybridization.

    Inazawa J, Sasaki H, Nagura K, Kakazu N, Abe T and Sasaki T

    Department of Hygiene, Kyoto Prefectural University of Medicine, Japan.

    Cell adhesion kinase beta (CAK beta) is the second protein-tyrosine kinase of the focal adhesion kinase subfamily with large N- and C-domains in addition to the central kinase domain. The cDNA of the human CAK beta has been cloned and used as a probe for the assignment of this gene by fluorescence in situ hybridization. CAK beta is sublocalized on chromosome 8p21.1, a locus frequently involved in allelic losses in colorectal cancers and prostate carcinomas.

    Human genetics 1996;98;4;508-10

  • Molecular cloning and assignment of FAK2, a novel human focal adhesion kinase, to 8p11.2-p22 by nonisotopic in situ hybridization.

    Herzog H, Nicholl J, Hort YJ, Sutherland GR and Shine J

    Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney, New 1f40 South Wales, Australia.

    Genomics 1996;32;3;484-6

  • Identification and characterization of a novel related adhesion focal tyrosine kinase (RAFTK) from megakaryocytes and brain.

    Avraham S, London R, Fu Y, Ota S, Hiregowdara D, Li J, Jiang S, Pasztor LM, White RA, Groopman JE et al.

    Deaconess Hospital, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215, USA.

    We have isolated a cDNA encoding a novel human intracytoplasmic tyrosine kinase, termed RAFTK (for a related adhesion focal tyrosine kinase). In addition, we have cloned and characterized the murine homolog of the human RAFTK cDNA. Comparison of the deduced amino acid sequences of human RAFTK and murine Raftk cDNAs revealed 95% homology, indicating that RAFTK is highly conserved between these species. The RAFTK cDNA clone, encoding a polypeptide of 1009 amino acids, has closest homology (48% identity, 65% similarity) to the focal adhesion kinase (pp125FAK). Comparison of the deduced amino acid sequences also indicates that RAFTK, like pp125FAK, lacks a transmembrane region, myristylation sites, and SH2 and SH3 domains. In addition, like pp125FAK, RAFTK contains a kinase domain flanked by large N-terminal (426 residues) and C-terminal (331 residues) domains, and the C-terminal region contains a predicted proline-rich stretch of residues. In fetal tissues, RAFTK expression was abundant in brain, and low levels were observed in lung and liver. In adult tissues, it was less restricted, indicating that RAFTK expression is developmentally up-regulated. Expression of RAFTK was also observed in human CD34+ marrow cells, primary bone marrow megakaryocytes, platelets, and various areas of brain. The human RAFTK gene was assigned to human chromosome 8 using genomic DNAs from human/rodent somatic cell hybrid lines. The mouse Raftk gene was mapped to chromosome 14, closely linked to gonadotropin-releasing hormone. Using specific antibodies for RAFTK, a approximately 123-kDa protein from the human megakaryocytic CMK cell line was immunoprecipitated. Treatment of the megakaryocytic CMK cells with thrombin caused a rapid induction of tyrosine phosphorylation of RAFTK protein. The structural features of RAFTK suggest that it is a member of the focal adhesion kinase gene family and may participate in signal transduction in human megakaryocytes and brain as well as in other cell types.

    Funded by: NHLBI NIH HHS: HL46668, HL51456

    The Journal of biological chemistry 1995;270;46;27742-51

  • Cloning and characterization of cell adhesion kinase beta, a novel protein-tyrosine kinase of the focal adhesion kinase subfamily.

    Sasaki H, Nagura K, Ishino M, Tobioka H, Kotani K and Sasaki T

    Department of Biochemistry, Sapporo Medical University School of Medicine, Japan.

    A second protein-tyrosine kinase (PTK) of the focal adhesion kinase (FAK) subfamily, cell adhesion kinase beta (CAK beta), was identified by cDNA cloning. The rat CAK beta is a 115.7-kDa PTK that contains N- and C-terminal domains of 418 and 330 amino acid residues besides the central kinase domain. The rat CAK beta has a homology with mouse FAK over their entire lengths except for the extreme N-terminal 88 residues and shares 45% overall sequence identity (60% identical in the catalytic domain), which indicates that CAK beta is a protein structurally related to but different from FAK. The CAK beta gene is less evenly expressed in a variety of rat organs than the FAK gene. Anti-CAK beta antibody immunoprecipitated a 113-kDa protein from rat brain, 3Y1 fibroblasts, and COS-7 cells transfected with CAK 12d0 beta cDNA. The tyrosine-phosphorylated state of CAK beta was not reduced on trypsinization, nor enhanced in response to plating 3Y1 cells onto fibronectin. CAK beta localized to sites of cell-to-cell contact in COS-7 transfected with CAK beta cDNA, in which FAK was found at the bottom of the cells. Thus, CAK beta is a PTK possibly participating in the signal transduction regulated by cell-to-cell contacts.

    The Journal of biological chemistry 1995;270;36;21206-19

  • Protein tyrosine kinase PYK2 involved in Ca(2+)-induced regulation of ion channel and MAP kinase functions.

    Lev S, Moreno H, Martinez R, Canoll P, Peles E, Musacchio JM, Plowman GD, Rudy B and Schlessinger J

    Department of Pharmacology, New York University Medical Center, New York 10016, USA.

    The protein tyrosine kinase PYK2, which is highly expressed in the central nervous system, is rapidly phosphorylated on tyrosine residues in response to various stimuli that elevate the intracellular calcium concentration, as well as by protein kinase C activation. Activation of PYK2 leads to modulation of ion channel function and activation of the MAP kinase signalling pathway. PYK2 activation may provide a mechanism for a variety of short- and long-term calcium-dependent signalling events in the nervous system.

    Nature 1995;376;6543;737-45

  • Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity: a role for Src family kinases.

    Calalb MB, Polte TR and Hanks SK

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

    Focal adhesion kinase (FAK) is a widely expressed nonreceptor protein-tyrosine kinase implicated in integrin-mediated signal transduction pathways and in the process of oncogenic transformation by v-Src. Elevation of FAK's phosphotyrosine content, following both cell adhesion to extracellular matrix substrata and cell transformation by Rous sarcoma virus, correlates directly with an increased kinase activity. To help elucidate the role of FAK phosphorylation in signal transduction events, we used a tryptic phosphopeptide mapping approach to identify tyrosine sites of phosphorylation responsive to both cell adhesion and Src transformation. We have identified four tyrosines, 397, 407, 576, and 577, which are phosphorylated in mouse BALB/3T3 fibroblasts in an adhesion-dependent manner. Tyrosine 397 has been previously recognized as the major site of FAK autophosphorylation. Phosphorylation of tyrosines 407, 576, and 577, which are previously unrecognized sites, is significantly elevated in the presence of c-Src in vitro and v-Src in vivo. Tyrosines 576 and 577 lie within catalytic subdomain VIII--a region recognized as a target for phosphorylation-mediated regulation of protein kinase activity. We found that maximal kinase activity of FAK immune complexes requires phosphorylation of both tyrosines 576 and 577. Our results indicate that phosphorylation of FAK by Src (or other Src family kinases) is an important step in the formation of an active signaling complex.

    Molecular and cellular biology 1995;15;2;954-63

  • A non-receptor tyrosine kinase that inhibits the GTPase activity of p21cdc42.

    Manser E, Leung T, Salihuddin H, Tan L and Lim L

    Institute of Molecular and Cell Biology, National University of Singapore.

    The Ras-related Rho subfamily of GTP-binding proteins (p21s), which includes Rho, Rac and Cdc42Hs, is implicated in different aspects of cytoskeletal organization. These proteins behave like Ras (p21ras) in that their active GTP-bound form is inactivated by intrinsic hydrolysis of the nucleotide gamma-phosphate, which can be stimulated by GTPase-activating proteins (GAPs). We have previously shown that there is a diversity of GAPs t 1e5a hat recognize this subfamily, including n-chimaerin, which is enriched in the hippocampus; we also detected proteins that bind these p21 proteins and seem to inhibit GTP hydrolysis. We now report the characterization of a hippocampal complementary DNA encoding a tyrosine kinase that specifically binds Cdc42Hs in its GTP-bound form. This binding is mediated by a unique sequence of 47 amino acids C-terminal to an SH3 domain and inhibits both the intrinsic and GAP-stimulated GTPase activity of Cdc42Hs. Our findings indicate that there may be a regulatory mechanism that sustains the GTP-bound active form of Cdc42Hs and which is directly linked to a tyrosine phosphorylation pathway.

    Nature 1993;363;6427;364-7

  • Molecular cloning and characterisation of a novel putative protein-serine kinase related to the cAMP-dependent and protein kinase C families.

    Coffer PJ and Woodgett JR

    Ludwig Institute for Cancer Research, London, England.

    Highly degenerate oligonucleotide primers designed from regions conserved between protein-serine kinases have been used specifically to amplify human epithelial (HeLa) cDNA by the polymerase chain reaction (PCR). Of several novel cDNA fragments encoding putative kinases thus isolated, one was further characterised. Screening of human fibroblast and bovine brain cDNA libraries with the PCR fragment yielded several clones with an open reading frame of 479 amino acids containing all of the conserved sequence motifs of protein-serine kinases. The predicted protein was most similar to the protein kinase C (PKC)/cAMP-dependent protein kinase (PKA) families and its gene has thus been termed pkb. Expression of the pkb gene is general but highest in brain, heart and lung. Translation of pkb RNA in vitro generated a 57-kDa protein (PKB) recognised by antisera raised to a bacterially expressed PKB/TrpE fusion protein. Transfection of COS cells with the kinase cDNA resulted in the synthesis of a 60-kDa protein which was partially purified by Mono Q anion-exchange chromatography. Column fractions containing PKB-immunoreactive protein exhibited elevated histone H1 kinase activity compared with similar fractions from control cells, demonstrating the enzymatic activity of this protein kinase.

    European journal of biochemistry 1991;201;2;475-81

  • The calcium-dependent activity of large-conductance, calcium-activated K+ channels is enhanced by Pyk2- and Hck-induced tyrosine phosphorylation.

    Ling S, Sheng JZ and Braun AP

    Dept. of Pharmacology and Therapeutics, Faculty of Medicine, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta, Canada T2N 4N1.

    Recent results showing that large-conductance, calcium-activated K(+) (BK(Ca)) channels undergo direct tyrosine phosphorylation in the presence of c-Src tyrosine kinase have suggested the involvement of these channels in Src-mediated signaling pathways. Given the important role for c-Src in integrin-mediated signal transduction, we have examined the potential regulation of BK(Ca) channels by proline-rich tyrosine kinase 2 (Pyk2), a calcium-sensitive tyrosine kinase activated upon integrin stimulation. Transient coexpression of murine BK(Ca) channels with either wild-type Pyk2 or hematopoietic cell kinase (Hck), a Src-family kinase, led to an enhancement of BK(Ca) channel activity over the range of 1-10 microM free calcium, whereas coexpression with catalytically inactive forms of either kinase did not significantly alter BK(Ca) gating compared with channels expressed alone. In the presence of either wild-type Pyk2 or Hck, BK(Ca) alpha-subunits were found to undergo tyrosine phosphorylation, as determined by immunoprecipitation and Western blotting strategies. However, tyrosine phosphorylation of the BK(Ca) alpha-subunit was not detected for channels expressed alone or together with inactive forms of either Pyk2 or Hck. Interestingly, wild-type, but not inactive, Pyk2 was also present in BK(Ca) channel immunoprecipitates, suggesting that Pyk2 may coassociate with the BK(Ca) channel complex after phosphorylation. Collectively, the observed modulation and phosphorylation of BK(Ca) channels by Pyk2 and a Src-family kinase may reflect a general cellular mechanism by which G protein-coupled receptor and/or integrin activation leads to the regulation of membrane ion channels.

    American journal of physiology. Cell physiology 200;287;3;C698-706

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
L00000015 G2C Homo sapiens Human NRC Human orthologues of mouse NRC adapted from Collins et al (2006) 186
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000034 G2C Homo sapiens Pocklington H3 Human orthologues of cluster 3 (mouse) from Pocklington et al (2006) 30
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
© 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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