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• W2029206521 abstract "The cell-surface heparan sulfate proteoglycan syndecan-4 acts in conjunction with the α5β1 integrin to promote the formation of actin stress fibers and focal adhesions in fibronectin (FN)-adherent cells. Fibroblasts seeded onto the cell-binding domain (CBD) fragment of FN attach but do not fully spread or form focal adhesions. Activation of Rho, with lysophosphatidic acid (LPA), or protein kinase C, using the phorbol ester phorbol 12-myristate 13-acetate, or clustering of syndecan-4 with antibodies directed against its extracellular domain will stimulate formation of focal adhesions and stress fibers in CBD-adherent fibroblasts. The distinct morphological differences between the cells adherent to the CBD and to full-length FN suggest that syndecan-4 may influence the organization of the focal adhesion or the activation state of the proteins that comprise it. FN-null fibroblasts (which express syndecan-4) exhibit reduced phosphorylation of focal adhesion kinase (FAK) tyrosine 397 (Tyr397) when adherent to CBD compared with FN-adherent cells. Treating the CBD-adherent fibroblasts with LPA, to activate Rho, or the tyrosine phosphatase inhibitor sodium vanadate increased the level of phosphorylation of Tyr397 to match that of cells plated on FN. Treatment of the fibroblasts with PMA did not elicit such an effect. To confirm that this regulatory pathway includes syndecan-4 specifically, we examined fibroblasts derived from syndecan-4-null mice. The phosphorylation levels of FAK Tyr397 were lower in FN-adherent syndecan-4-null fibroblasts compared with syndecan-4-wild type and these levels were rescued by the addition of LPA or re-expression of syndecan-4. These data indicate that syndecan-4 ligation regulates the phosphorylation of FAK Tyr397 and that this mechanism is dependent on Rho but not protein kinase C activation. In addition, the data suggest that this pathway includes the negative regulation of a protein-tyrosine phosphatase. Our results implicate syndecan-4 activation in a direct role in focal adhesion regulation. The cell-surface heparan sulfate proteoglycan syndecan-4 acts in conjunction with the α5β1 integrin to promote the formation of actin stress fibers and focal adhesions in fibronectin (FN)-adherent cells. Fibroblasts seeded onto the cell-binding domain (CBD) fragment of FN attach but do not fully spread or form focal adhesions. Activation of Rho, with lysophosphatidic acid (LPA), or protein kinase C, using the phorbol ester phorbol 12-myristate 13-acetate, or clustering of syndecan-4 with antibodies directed against its extracellular domain will stimulate formation of focal adhesions and stress fibers in CBD-adherent fibroblasts. The distinct morphological differences between the cells adherent to the CBD and to full-length FN suggest that syndecan-4 may influence the organization of the focal adhesion or the activation state of the proteins that comprise it. FN-null fibroblasts (which express syndecan-4) exhibit reduced phosphorylation of focal adhesion kinase (FAK) tyrosine 397 (Tyr397) when adherent to CBD compared with FN-adherent cells. Treating the CBD-adherent fibroblasts with LPA, to activate Rho, or the tyrosine phosphatase inhibitor sodium vanadate increased the level of phosphorylation of Tyr397 to match that of cells plated on FN. Treatment of the fibroblasts with PMA did not elicit such an effect. To confirm that this regulatory pathway includes syndecan-4 specifically, we examined fibroblasts derived from syndecan-4-null mice. The phosphorylation levels of FAK Tyr397 were lower in FN-adherent syndecan-4-null fibroblasts compared with syndecan-4-wild type and these levels were rescued by the addition of LPA or re-expression of syndecan-4. These data indicate that syndecan-4 ligation regulates the phosphorylation of FAK Tyr397 and that this mechanism is dependent on Rho but not protein kinase C activation. In addition, the data suggest that this pathway includes the negative regulation of a protein-tyrosine phosphatase. Our results implicate syndecan-4 activation in a direct role in focal adhesion regulation. protein kinase C focal adhesion kinase fibronectin cell-binding domain lysophosphatidic acid wild type phosphate-buffered saline phorbol 12-myristate 13-acetate Syndecan-4 is a member of a family of transmembrane heparan sulfate proteoglycans (syndecans 1–4) that are characterized by divergent extracellular domains and short cytoplasmic domains that contain two constant regions separated by a variable region that is unique to each family member (reviewed in Refs. 1Woods A. J. Clin. Invest. 2001; 107: 935-941Crossref PubMed Scopus (112) Google Scholar, 2Woods A., Oh, E.-S. Couchman J.R. Matrix Biol. 1998; 17: 477-483Crossref PubMed Scopus (78) Google Scholar, 3Carey D.J. Biochem. J. 1997; 327: 1-16Crossref PubMed Scopus (604) Google Scholar, 4Bernfield M. Gotte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2319) Google Scholar). Although all members of the syndecan family arose from a single ancestral gene, their expression patterns in tissues and during development are highly regulated (3Carey D.J. Biochem. J. 1997; 327: 1-16Crossref PubMed Scopus (604) Google Scholar, 4Bernfield M. Gotte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2319) Google Scholar, 5Elenius K. Jalkanen M. J. Cell Sci. 1994; 107: 2975-2982PubMed Google Scholar). The terminal four amino acids (EFYA) of the cytoplasmic domain of all syndecan family members compose a binding site for the PDZ-containing proteins: synbindin, syntenin, CASK/LIN-2, and synectin (6Ethell I.M. Hagihara K. Miura Y. Irie F. Yamaguchi Y. J. Cell Biol. 2000; 151: 53-68Crossref PubMed Scopus (104) Google Scholar, 7Hsueh Y.-P. Sheng M. J. Neurosci. 1999; 19: 7415-7425Crossref PubMed Google Scholar, 8Gao Y., Li, M. Chen W. Simons M. J. Cell. Physiol. 2000; 184: 373-379Crossref PubMed Scopus (152) Google Scholar, 9Grootjans J.J. Zimmermann P. Reekmans G. Smets A. Degeest G. Durr J. David G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13683-13688Crossref PubMed Scopus (345) Google Scholar, 10Cohen D.J. Woods D.F. Marfatia S.M. Walther Z. Chishti A.H. Anderson J.M. Wood D.F. J. Cell Biol. 1998; 142: 129-138Crossref PubMed Scopus (321) Google Scholar). Unlike other family members, syndecan-4 binds protein kinase C-α (PKC-α)1 through the intermediary phosphatidylinositol bisphosphate (11Horowitz A. Murakami M. Gao Y. Simons M. Biochemistry. 1999; 38: 15871-15877Crossref PubMed Scopus (78) Google Scholar, 12Oh E.-S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 8133-8136Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar) at the variable region and the cytoplasmic protein syndesmos through both the variable and the membrane-proximal constant regions (13Baciu P.C. Saoncella S. Lee S.H. Denhez F. Leuthardt D. Goetinck P.F. J. Cell Sci. 2000; 113: 315-324Crossref PubMed Google Scholar). Syndecans-1, -2, and -4 have been shown to bind extracellular matrix proteins (14Lebakken C.S. McQuade K.J. Rapraeger A.C. Exp. Cell Res. 2000; 259: 315-325Crossref PubMed Scopus (22) Google Scholar, 15Utani A. Nomizu M. Matsuura H. Kato K. Kobayashi T. Takeda U. Aota S. Nielsen P.K. Shinkai H. J. Biol. Chem. 2001; 276: 28779-28788Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 16Woods A. Longley R.L. Tumova S. Couchman J.R. Arch. Biochem. Biophys. 2000; 374: 66-72Crossref PubMed Scopus (194) Google Scholar). However, syndecan-4 is the only family member to localize to sites of cell-matrix adhesions (17Woods A. Couchman J.R. Mol. Biol. Cell. 1994; 5: 183-192Crossref PubMed Scopus (278) Google Scholar). Comparison of the localization of syndecan-4 with the focal adhesion marker protein vinculin suggests that syndecan-4 does not localize to newly formed contacts but with more established adhesion sites (18Baciu P.C. Goetinck P.F. Mol. Biol. Cell. 1995; 6: 1503-1513Crossref PubMed Scopus (120) Google Scholar). Focal adhesions are macromolecular complexes that localize to sites of closest contact (10–15 nm) between cells and the underlying extracellular matrix substrate (reviewed in Refs. 19Burridge K. Chrzanowska-Wodnicka M. Annu. Rev. Cell Dev. Biol. 1996; 12: 463-519Crossref PubMed Scopus (1659) Google Scholar, 20Zamir E. Geiger B. J. Cell Sci. 2001; 114: 3583-3590Crossref PubMed Google Scholar, 21Petit V. Thiery J.P. Biol. Cell. 2000; 92: 477-494Crossref PubMed Scopus (275) Google Scholar). Focal adhesions are composed of transmembrane receptors (primarily syndecan-4 and members of the integrin superfamily), structural molecules (such as actin, talin, tensin, vinculin, and α-actinin), and signaling molecules (i.e. focal adhesion kinase (FAK), PKC, and Src). Focal adhesions, therefore, serve not only as structural supports but also as signaling conduits between the actin cytoskeleton and the surrounding environment of the cell. The generation of focal adhesions in fibronectin (FN)-adherent cells is dependent on the ligation of two different transmembrane receptors: integrins and syndecan-4. Fibroblasts seeded on the cell-binding domain (CBD) of FN (which contains only the integrin-binding RGD sequence) will attach but not form focal adhesions or actin stress fibers (22Woods A. Couchman J.R. Johansson S. Hook M. EMBO J. 1986; 5: 665-670Crossref PubMed Scopus (322) Google Scholar, 23Bloom L. Ingham K.C. Hynes R.O. Mol. Biol. Cell. 1999; 10: 1521-1536Crossref PubMed Scopus (121) Google Scholar, 24Saoncella S. Echtermeyer F. Denhez F. Nowlen J.K. Mosher D.F. Robinson S.D. Hynes R.O. Goetinck P.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2805-2810Crossref PubMed Scopus (335) Google Scholar). The addition of an antibody against the extracellular domain of syndecan-4 stimulates focal adhesion and stress fiber formation in cells plated on the CBD (24Saoncella S. Echtermeyer F. Denhez F. Nowlen J.K. Mosher D.F. Robinson S.D. Hynes R.O. Goetinck P.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2805-2810Crossref PubMed Scopus (335) Google Scholar). The syndecan-4 signal can be bypassed in CBD-adherent fibroblasts by directly stimulating the small GTPase Rho with lysophosphatidic acid (LPA) (24Saoncella S. Echtermeyer F. Denhez F. Nowlen J.K. Mosher D.F. Robinson S.D. Hynes R.O. Goetinck P.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2805-2810Crossref PubMed Scopus (335) Google Scholar). These data indicate that syndecan-4 acts in cooperation with the α5β1 integrin to direct focal adhesion formation and that the action of syndecan-4 is through a Rho-dependent mechanism (24Saoncella S. Echtermeyer F. Denhez F. Nowlen J.K. Mosher D.F. Robinson S.D. Hynes R.O. Goetinck P.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2805-2810Crossref PubMed Scopus (335) Google Scholar). The generation of syndecan-4-null mice demonstrated no initial obvious phenotype and showed, surprisingly, that cells seeded onto FN will form stress fibers and focal adhesions in the absence of syndecan-4 (25Ishiguro K. Kadomatsu K. Kojima T. Muramatsu H. Tsuzuki S. Nakamura E. Kusugami K. Saito H. Muramatsu T. J. Biol. Chem. 2000; 275: 5249-5252Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar,26Echtermeyer F. Streit M. Wilcox-Adelman S. Saoncella S. Denhez F. Detmar M. Goetinck P. J. Clin. Invest. 2001; 107: R9-R14Crossref PubMed Scopus (352) Google Scholar). These data point to another cell-surface heparan sulfate proteoglycan that can compensate for the absence of syndecan-4. Treatment of CBD-adherent syndecan-4-null fibroblasts with antibodies to syndecan-4 do not form focal adhesions or stress fibers although wild type fibroblasts do, suggesting that the syndecan-4 signaling pathway can be selectively activated and does not function in the syndecan-4-null cells (25Ishiguro K. Kadomatsu K. Kojima T. Muramatsu H. Tsuzuki S. Nakamura E. Kusugami K. Saito H. Muramatsu T. J. Biol. Chem. 2000; 275: 5249-5252Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Interestingly, further studies have documented that syndecan-4-null mice do not respond to physiological insults as well as their wild type counterparts implying that syndecan-4 may be important in combating “stress situations” (26Echtermeyer F. Streit M. Wilcox-Adelman S. Saoncella S. Denhez F. Detmar M. Goetinck P. J. Clin. Invest. 2001; 107: R9-R14Crossref PubMed Scopus (352) Google Scholar, 27Ishiguro K. Kadomatsu K. Kojima T. Muramatsu H. Iwase M. Yoshikai Y. Yanada M. Yamamoto K. Matsushita T. Nishimura M. Kusugami K. Saito H. Muramatsu T. J. Biol. Chem. 2001; 276: 47483-47488Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 28Ishiguro K. Kadomatsu K. Kojima T. Muramatsu H. Matsuo S. Kusugami K. Saito H. Muramatsu T. Lab. Invest. 2001; 81: 509-516Crossref PubMed Scopus (37) Google Scholar). Syndecan-4-null mice exhibit a delay in wound healing and this deficiency appears to be due to an impairment in cell migration that can also be demonstrated in in vitro migration assays (26Echtermeyer F. Streit M. Wilcox-Adelman S. Saoncella S. Denhez F. Detmar M. Goetinck P. J. Clin. Invest. 2001; 107: R9-R14Crossref PubMed Scopus (352) Google Scholar). Impaired cell migration may be because of the inability of cells to either generate enough force to propel themselves over an underlying substrate or to disengage established adhesion contacts to promote new adhesions (29Palecek S.P. Loftus J.C. Ginsberg M.H. Lauffenburger D.A. Horwitz A.F. Nature. 1997; 385: 537-540Crossref PubMed Scopus (1189) Google Scholar). Although many focal adhesion-associated proteins are involved in cell migration, the tyrosine kinase FAK has been shown to be intimately involved in focal adhesion turnover (30Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Aizawa S. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1586) Google Scholar, 31Ren X.D. Kiosses W.B. Sieg D.J. Otey C.A. Schlaepfer D.D. Schwartz M.A. J. Cell Sci. 2000; 113: 3673-3678Crossref PubMed Google Scholar). Loss of FAK is associated with decreased cell migration and increased focal adhesion size (30Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Aizawa S. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1586) Google Scholar, 32Ilic D. Kanazawa S. Furuta Y. Yamamoto T. Aizawa S. Exp. Cell Res. 1996; 222: 298-303Crossref PubMed Scopus (62) Google Scholar, 33Sieg D.J. Ilic D. Jones K.C. Damsky C.H. Hunter T. Schlaepfer D.D. EMBO J. 1998; 17: 5933-5947Crossref PubMed Scopus (289) Google Scholar, 34Owen J.D. Ruest P.J. Fry D.W. Hanks S.K. Mol. Cell. Biol. 1999; 19: 4806-4818Crossref PubMed Scopus (341) Google Scholar), whereas overexpression of FAK increases cell migration (34Owen J.D. Ruest P.J. Fry D.W. Hanks S.K. Mol. Cell. Biol. 1999; 19: 4806-4818Crossref PubMed Scopus (341) Google Scholar, 35Cary L.A. Chang J.F. Guan J.-L. J. Cell Sci. 1996; 109: 1787-1794Crossref PubMed Google Scholar, 36Sieg D.J. Hauck C.R. Schlaepfer D.D. J. Cell Sci. 1999; 112: 2677-2691Crossref PubMed Google Scholar). FAK serves as both a scaffolding and signaling protein in the focal adhesion. A nonreceptor tyrosine kinase, FAK, is composed of a central catalytic domain flanked by two noncatalytic regions (37Zachary I. Int. J. Biochem. Cell Biol. 1997; 29: 929-934Crossref PubMed Scopus (90) Google Scholar). FAK contains multiple tyrosine residues that, upon their phosphorylation, are capable of binding proteins containing SH2 domains. Tyrosine 397 (Tyr397) is a critical phosphorylation site that results from autophosphorylation upon antibody-induced clustering of cell-surface integrins or cell adhesion to extracellular matrix proteins (such as collagen, fibronectin, and vitronectin) (Refs. 38Schaller M.D. Hildebrand J.D. Shannon J.D. Fox J.W. Vines R.R. Parsons J.T. Mol. Cell. Biol. 1994; 14: 1680-1688Crossref PubMed Scopus (1116) Google Scholar and39Miyamoto S. Teramoto H. Coso O.A. Gutkind J.S. Burbelo P.D. Akiyama S.K. Yamada K.M. J. Cell Biol. 1995; 131: 791-805Crossref PubMed Scopus (1104) Google Scholar and reviewed in Refs. 40Otey C.A. Int. Rev. Cytol. 1996; 167: 161-183Crossref PubMed Google Scholar and 41Schlaepfer D.D. Hauck C.R. Sieg D.J. Prog. Biophys. Mol. Biol. 1999; 71: 435-478Crossref PubMed Scopus (1033) Google Scholar). FAK-mediated cell migration is dependent on phosphorylation of FAK Tyr397 (35Cary L.A. Chang J.F. Guan J.-L. J. Cell Sci. 1996; 109: 1787-1794Crossref PubMed Google Scholar). The phosphorylated form of Tyr397 binds Src (38Schaller M.D. Hildebrand J.D. Shannon J.D. Fox J.W. Vines R.R. Parsons J.T. Mol. Cell. Biol. 1994; 14: 1680-1688Crossref PubMed Scopus (1116) Google Scholar), the p85 subunit of phosphatidylinositol 3-kinase (42Chen H.-C. Appeddu P.A. Isoda H. Guan J.-L. J. Biol. Chem. 1996; 271: 26329-26334Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar), phospholipase Cγ1 (43Zhang X. Chattopadhyay A., Ji, Q.-S. Owen J.D. Ruest P.J. Carpenter G. Hanks S.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9021-9026Crossref PubMed Scopus (158) Google Scholar), the adaptor proteins Grb7 (44Han D.C. Guan J.-L. J. Biol. Chem. 1999; 274: 24425-24430Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar) and Shc (45Schlaepfer D.D. Jones K.C. Hunter T. Mol. Cell. Biol. 1998; 18: 2571-2585Crossref PubMed Scopus (357) Google Scholar), and the phosphatase PTEN (46Tamura M., Gu, J. Danen E.H. Takino T. Miyamoto S. Yamada K.M. J. Biol. Chem. 1999; 274: 20693-20703Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). The unlikely possibility that all of these proteins bind FAK simultaneously suggests that distinct FAK-containing signaling complexes probably exist within the focal adhesion (47Schaller M.D. Biochim. Biophys. Acta. 2001; 1540: 1-21Crossref PubMed Scopus (506) Google Scholar). The phosphorylation of the remaining tyrosine residues (407, 576, 577, 861, and 925) occurs in a Src-dependent manner (48Calalb M.B. Polte T.R. Hanks S.K. Mol. Cell. Biol. 1995; 15: 954-963Crossref PubMed Google Scholar, 49Calalb M.B. Zhang X. Polte T.R. Hanks S.K. Biochem. Biophys. Res. Commun. 1996; 228: 662-668Crossref PubMed Scopus (193) Google Scholar, 50Schlaepfer D.D. Hanks S.K. Hunter T. van der Geer P. Nature. 1994; 372: 786-791Crossref PubMed Scopus (1445) Google Scholar, 51Schlaepfer D.D. Hunter T. Mol. Cell. Biol. 1996; 16: 5623-5633Crossref PubMed Scopus (400) Google Scholar). Phosphotyrosines 576 and 577 are required for maximal FAK kinase activity (48Calalb M.B. Polte T.R. Hanks S.K. Mol. Cell. Biol. 1995; 15: 954-963Crossref PubMed Google Scholar) and phosphotyrosine 925 acts as a binding site for the Grb2 adaptor protein leading to activation of the Ras pathway (50Schlaepfer D.D. Hanks S.K. Hunter T. van der Geer P. Nature. 1994; 372: 786-791Crossref PubMed Scopus (1445) Google Scholar). Fibroblasts treated with LPA, which directly stimulates the small GTPase Rho, show increases in FAK phosphorylation and its subsequent localization to focal adhesions (52Kumagai N. Morii N. Fujisawa K. Yoshimasa T. Nakao K. Narumiya S. FEBS Lett. 1993; 329: 273-276Crossref PubMed Scopus (70) Google Scholar, 53Chrzanowska-Wodnicka M. Burridge K. J. Cell Sci. 1994; 107: 3643-3654PubMed Google Scholar, 54Ridley A.J. Hall A. EMBO J. 1994; 13: 2600-2610Crossref PubMed Scopus (440) Google Scholar, 55Barry S.T. Critchley D.R. J. Cell Sci. 1994; 107: 2033-2045Crossref PubMed Google Scholar, 56Flinn H.M. Ridley A.J. J. Cell Sci. 1996; 109: 1133-1141PubMed Google Scholar, 57Rodriguez-Fernandez J.L. Rozengurt E. J. Biol. Chem. 1998; 273: 19321-19328Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Closer examination of early adhesion events documented initial FAK phosphorylation occurring in a Rho-independent manner followed by Rho-mediated FAK phosphorylation (58Clark E.A. King W.G. Brugge J.S. Symons M. Hynes R.O. J. Cell Biol. 1998; 142: 573-586Crossref PubMed Scopus (533) Google Scholar). Recently, Ren et al. (31Ren X.D. Kiosses W.B. Sieg D.J. Otey C.A. Schlaepfer D.D. Schwartz M.A. J. Cell Sci. 2000; 113: 3673-3678Crossref PubMed Google Scholar) demonstrated that FAK-null cells exhibit constitutive activation of Rho and this activity level is inversely correlated with focal adhesion turnover. They reintroduced FAK to the deficient cells and showed that Rho activity was restored to normal levels. This suggests that FAK is responsible for the transient inhibition of Rho during early cell spreading (31Ren X.D. Kiosses W.B. Sieg D.J. Otey C.A. Schlaepfer D.D. Schwartz M.A. J. Cell Sci. 2000; 113: 3673-3678Crossref PubMed Google Scholar). All of these studies indicate that reciprocal interactions may occur between FAK and Rho to facilitate cell spreading and focal adhesion and stress fiber formation. General FAK phosphorylation levels increase during early cell spreading (59Burridge K. Turner C.E. Romer L.H. J. Cell Biol. 1992; 119: 893-903Crossref PubMed Scopus (1182) Google Scholar) but maximal phosphorylation requires both the integrin-binding and heparin-binding domains of FN (60Jeong J. Han I. Lim Y. Kim J. Park I. Woods A. Couchman J.R. Oh E.S. Biochem. J. 2001; 356: 531-537Crossref PubMed Scopus (32) Google Scholar). As syndecan-4 binds the heparin-binding domain of FN (61Tumova S. Woods A. Couchman J.R. J. Biol. Chem. 2000; 275: 9410-9417Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) and has been shown to act in a Rho-dependent manner to influence focal adhesions and actin stress fibers (24Saoncella S. Echtermeyer F. Denhez F. Nowlen J.K. Mosher D.F. Robinson S.D. Hynes R.O. Goetinck P.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2805-2810Crossref PubMed Scopus (335) Google Scholar), we were interested in determining what effect syndecan-4 signaling might have on the autophosphorylation site of FAK. We now demonstrate that increased phosphorylation of FAK Tyr397 is dependent on syndecan-4 ligation, and that the syndecan-4 signal may be superseded by direct activation of Rho. Three types of cells were used in this study. Fibronectin-null cells which express syndecan-4 (24Saoncella S. Echtermeyer F. Denhez F. Nowlen J.K. Mosher D.F. Robinson S.D. Hynes R.O. Goetinck P.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2805-2810Crossref PubMed Scopus (335) Google Scholar) and fibroblasts derived from newborn littermates that were either wild type (+/+) or null (−/−) for the syndecan-4 core protein gene. These cells are referred to as FN-null and syndecan-4-WT or syndecan-4-null, respectively. All cell types were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen). For experimental assays, 1.5 × 105 cells/100-mm tissue culture dish were seeded in serum overnight and subsequently serum starved for 18 h. The cells were washed three times with phosphate-buffered saline minus calcium chloride and magnesium chloride (PBS, Invitrogen) containing 0.5 mm EDTA and lifted from the dishes with a 1:1 dilution of PBS/EDTA and 0.25% trypsin/EDTA (Invitrogen). The tissue culture plates and glass coverslips used for the experiments were coated with either 30 μg/ml FN (BD Biosciences, San Jose, CA) or 10 μg/ml CBD (Invitrogen) diluted in PBS overnight at 4 °C. The plates and coverslips were then washed with PBS, blocked with 1% bovine serum albumin for 60 min at room temperature, and given a final wash before the cells were seeded. The fibroblasts were allowed to attach and spread for 3 h in serum-free Dulbecco’s modified Eagle’s medium. Following this incubation the cell medium was replaced with serum-free medium containing either 500 ng/ml LPA (Sigma), 250 nmphorbol myristate acetate (PMA) (Sigma), 0.5 mm sodium vanadate (Sigma), or medium alone for 30 (for the FN-null cells) or 60 min (for the syndecan-4-WT and syndecan-4-null cells). All experiments were done on cultures that were 50% confluent. Syndecan-4-null fibroblasts were transiently transfected with the rat syndecan-4 cDNA cloned in pcDNA3.1 Hygro (Invitrogen) or pcDNA3.1 Hygro alone using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's protocol. Detection of protein expression was ascertained by heparitinase I treating the transfected cell lysates following a protocol by Rapraeger and Ott (62Rapraeger A.C. Ott V.L. J. Biol. Chem. 1998; 273: 35291-35298Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Syndecan-4 was detected using the MS-4-E polyclonal antibody (24Saoncella S. Echtermeyer F. Denhez F. Nowlen J.K. Mosher D.F. Robinson S.D. Hynes R.O. Goetinck P.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2805-2810Crossref PubMed Scopus (335) Google Scholar). Fibroblasts were placed on ice and washed twice with cold PBS containing calcium chloride and magnesium chloride and 1 mm sodium vanadate. The cells were lysed with an extraction buffer containing 25 mm β-glycerolphosphate (pH 7.3), 10 mm EDTA, 2 mm EGTA, 0.1 m NaCl, 1% Triton X-100, 10 mm β-mercaptoethanol, 0.2 mm sodium vanadate, 1 mm benzamidine, 0.1 mm phenymethylsulfonyl fluoride, 2 μg/ml leupeptin, 1 μm pepstatin A, and 1 μg/ml aprotinin (63Xu F. Zhao Z.J. Exp. Cell Res. 2001; 262: 49-58Crossref PubMed Scopus (16) Google Scholar). The lysates were centrifuged at 14,000 rpm for 20 min (4 °C) to remove insoluble material and protein concentration was determined. Equal protein concentrations were resolved on 12% SDS-PAGE and transferred electrophoretically to polyvinylidene difluoride membranes. The membranes were blocked with 5% bovine serum albumin for 2 h at room temperature and incubated with primary antibodies diluted in 1% bovine serum albumin overnight at 4 °C. The monoclonal phosphotyrosine antibody directly conjugated to horseradish peroxidase (clone PY20) was purchased from BD Transduction Laboratories (Lexington, KY). The polyclonal FAK and phosphospecific FAK Tyr397 antibodies were obtained from Upstate Biotechnology Inc. (Lake Placid, NY) and the α-actinin antibody was purchased from Sigma. After washing the blots, secondary antibodies conjugated to horseradish peroxidase were added for 60 min at room temperature. The membranes were subsequently washed again and detection of signal was obtained using the West Pico enhanced chemiluminescent kit according to the manufacturer’s instructions (Pierce). Figures are representative results of experiments that were performed at least three times. Syndecan-4-WT and syndecan-4-null cells were seeded on glass coverslips at a concentration of 9,000 cells/well of a 24-well plate for 3 h in serum-free medium and then incubated for an additional 60 min in the presence or absence of 500 ng/ml LPA or 250 nm PMA or an additional 2 h in the presence or absence of 15 μg/ml C3 exotransferase (List Biological Laboratories, Campbell, CA). The cells were fixed with 4% formaldehyde for 15 min, permeabilized for 10 min with cold 0.5% Triton X-100, and blocked with 2 mg/ml bovine serum albumin for 20 min at room temperature. The cells were then incubated with a monoclonal FAK antibody (clone 4.47, Upstate Biotechnology Inc.; 1:50) or polyclonal phosphospecific FAK Tyr397 antibodies (Upstate Biotechnology Inc.; 1:50) and a monoclonal vinculin antibody (clone hVIN-1, Sigma; 1:400) diluted in 2 mg/ml bovine serum albumin for 60 min at 37 °C. For experiments in which the monoclonal antibodies to FAK and vinculin were to be used, the vinculin antibody was directly conjugated to fluorescein isothiocyanate using a protein labeling kit (Pierce) and incubated with the cells subsequent to incubation of the FAK primary antibody and the appropriate secondary antibody to prevent cross-reactivity. All reagents and antibodies were diluted in Small’s buffer (64Small J.V. Celis J.E. J. Cell Sci. 1978; 31: 393-409PubMed Google Scholar). The cells were washed for 60 min with Small’s buffer and then incubated with the corresponding secondary antibodies conjugated to either fluorescein or Cy5 (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 60 min at 37 °C. The cells received final washes for 1 h, and were mounted on slides and visualized using a Leica TCS NT4D confocal imaging system with a ×40 oil-immersion lens (Leica, Heidelberg, Germany). During double labeling experiments there was no evidence of bleed-through between channels. Cell images were processed using Adobe Photoshop software. Images represent typical staining patterns from multiple experiments. Syndecan-4-null and WT fibroblasts were seeded on FN-coated 150-mm tissue culture dishes at a concentration of 1.6 × 106 cells/dish. Following adhesion and incubation with LPA, as described above, the cells were processed using the Rho Activation Assay kit (purchased from Upstate Biotechnology Inc.) according to manufacturer’s instructions. Briefly, cell lysates were incubated with beads conjugated to the Rho-binding domain of the Rhotekin protein (which only binds GTP-bound Rho (65Reid T. Furuyashiki T. Ishizaki T. Watanabe G. Watanabe N. Fujisawa K. Morii N. Madaule P. Narumiya S. J. Biol. Chem. 1996; 271: 13556-13560Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar)) for 45 min at 4 °C. The beads were then washed three times and the samples were resolved on a 16% SDS-PAGE, transferred to polyvinylidene difluoride, and incubated with a polyclonal Rho antibody (Upstate Biotechnology Inc.) overnight at 4 °C. A membrane containing identical amounts of protein used in the Rho activation assay was incubated with polyclonal antibodies to Rho (Upstate Biotechnology Inc.) and actin (Sigma) to serve as loading controls. The blots were washed, incubated with the appropriate secondary antibody, and exposed to film following treatment with the West Pico enhanced chemiluminescent reagent (Pierce). Localization of syndecan-4 to focal adhesion sites" @default.
• W2029206521 created "2016-06-24" @default.
• W2029206521 creator A5065610431 @default.
• W2029206521 creator A5089442069 @default.
• W2029206521 creator A5089902491 @default.
• W2029206521 date "2002-09-01" @default.
• W2029206521 modified "2023-10-18" @default.
• W2029206521 title "Syndecan-4 Modulates Focal Adhesion Kinase Phosphorylation" @default.
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