Journal of Biological Chemistry · 2002 · 68 citations · 21 references
Life SciencesCell BiologyMolecular BiologyCell AdhesionCell SignalingMolecular Cell BiologyAdhesion MoleculesCell Adhesion ReceptorsCell-matrix AdhesionCytoskeletal SignalingFocal AdhesionsIntegrin SignalingAdaptor Protein InteractionsAdaptor Protein RecruitmentAdhesion Complex AssemblyAdhesion ReceptorsFocal Adhesion AssemblyFocal Adhesion ComplexesFocal Adhesion OrganizationIntegrin Binding
Syndecan-4 and integrins are the primary transmembrane receptors of focal adhesions in cells adherent to extracellular matrix molecules. Syndesmos is a cytoplasmic protein that interacts specifically with the cytoplasmic domain of syndecan-4, and it co-localizes with syndecan-4 in focal contacts. In the present study we sought possible interactors with syndesmos. We find that syndesmos interacts with the focal adhesion adaptor protein paxillin. The binding of syndesmos to paxillin is direct, and these interactions are triggered by the activation of protein kinase C. Syndesmos also binds the paxillin homolog, Hic-5. The connection of syndecan-4 with paxillin through syndesmos parallels the connection between paxillin and integrins and may thus reflect the cooperative signaling of these two receptors in the assembly of focal adhesions and actin stress fibers. Syndecan-4 and integrins are the primary transmembrane receptors of focal adhesions in cells adherent to extracellular matrix molecules. Syndesmos is a cytoplasmic protein that interacts specifically with the cytoplasmic domain of syndecan-4, and it co-localizes with syndecan-4 in focal contacts. In the present study we sought possible interactors with syndesmos. We find that syndesmos interacts with the focal adhesion adaptor protein paxillin. The binding of syndesmos to paxillin is direct, and these interactions are triggered by the activation of protein kinase C. Syndesmos also binds the paxillin homolog, Hic-5. The connection of syndecan-4 with paxillin through syndesmos parallels the connection between paxillin and integrins and may thus reflect the cooperative signaling of these two receptors in the assembly of focal adhesions and actin stress fibers. Cell adhesion is important for such biological functions as cell growth, proliferation, survival, and migration. Fibroblasts adherent to extracellular matrix (ECM) 1The abbreviations used are: ECMextracellular matrixCBDcell-binding domainFAKfocal adhesion kinaseMAPKmitogen-activated protein kinasePKCprotein kinase CPIP2phosphatidylinositol 4,5-bisphosphateGSTglutathione S-transferaseTPA12-O-tetradecanoylphorbol-13-acetateCEFchick embryo fibroblastsDTSP3,3′-dithiobispropionic acid N-hydroxysuccinimide ester 1The abbreviations used are: ECMextracellular matrixCBDcell-binding domainFAKfocal adhesion kinaseMAPKmitogen-activated protein kinasePKCprotein kinase CPIP2phosphatidylinositol 4,5-bisphosphateGSTglutathione S-transferaseTPA12-O-tetradecanoylphorbol-13-acetateCEFchick embryo fibroblastsDTSP3,3′-dithiobispropionic acid N-hydroxysuccinimide esterproteins (i.e. fibronectin, vitronectin, collagen) in vitro, cluster transmembrane adhesion receptors and intracellular cytoplasmic adaptor and signaling molecules into macromolecular complexes termed focal adhesions (1.Burridge K. Chrzanowska-Wodnicka M. Annu. Rev. Cell Dev. Biol. 1996; 12: 463-518Crossref PubMed Scopus (1639) Google Scholar). The primary transmembrane receptors associated with the focal adhesion are members of the integrin superfamily and the heparan-sulfate proteoglycan (HSPG) syndecan-4 (2.Baciu P.C. Goetinck P.F. Mol. Biol. Cell. 1995; 6: 1503-1513Crossref PubMed Scopus (120) Google Scholar, 3.Bernfield M. Gotte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 69: 729-777Crossref Scopus (2281) Google Scholar, 4.Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3757) Google Scholar, 5.Woods A. Couchman J.R. Mol. Biol. Cell. 1994; 5: 183-192Crossref PubMed Scopus (276) Google Scholar). Many integrins (such as the α5β1 and αvβ3) bind to an Arg-Gly-Asp (RGD) sequence found within their ECM ligands. Previous studies have demonstrated that fibroblasts seeded onto an RGD-containing cell-binding domain fragment (CBD) of fibronectin will attach but not spread or form focal adhesions and actin stress fibers (6.Saoncella 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 (328) Google Scholar, 7.Woods A. Couchman J.R. Johansson S. Hook M. EMBO J. 1986; 5: 665-670Crossref PubMed Scopus (322) Google Scholar). This laboratory demonstrated that syndecan-4, working in cooperation with the α5β1 integrin, can stimulate a Rho-dependent pathway that will elicit focal adhesion and stress fiber formation in CBD-adherent cells (6.Saoncella 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 (328) Google Scholar). extracellular matrix cell-binding domain focal adhesion kinase mitogen-activated protein kinase protein kinase C phosphatidylinositol 4,5-bisphosphate glutathione S-transferase 12-O-tetradecanoylphorbol-13-acetate chick embryo fibroblasts 3,3′-dithiobispropionic acid N-hydroxysuccinimide ester extracellular matrix cell-binding domain focal adhesion kinase mitogen-activated protein kinase protein kinase C phosphatidylinositol 4,5-bisphosphate glutathione S-transferase 12-O-tetradecanoylphorbol-13-acetate chick embryo fibroblasts 3,3′-dithiobispropionic acid N-hydroxysuccinimide ester The cytoplasmic face of the focal adhesion serves two purposes, physical organization of the adhesion complex and a convergence point for various signaling pathways (1.Burridge K. Chrzanowska-Wodnicka M. Annu. Rev. Cell Dev. Biol. 1996; 12: 463-518Crossref PubMed Scopus (1639) Google Scholar, 4.Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3757) Google Scholar, 8.Clark E.A. Brugge J.S. Science. 1995; 268: 233-239Crossref PubMed Scopus (2802) Google Scholar, 9.Critchley D.R. Curr. Opin. Cell Biol. 2000; 12: 133-139Crossref PubMed Scopus (490) Google Scholar, 10.Schoenwaelder S.M. Burridge K. Curr. Opin. Cell Biol. 1999; 11: 274-286Crossref PubMed Scopus (647) Google Scholar). Structural proteins (such as actin, α-actinin, talin, tensin, vinculin, zyxin, actopaxin, paxillin, and Hic-5) (11.Couchman J.R. Woods A. J. Cell Sci. 1999; 112: 3415-3420Crossref PubMed Google Scholar, 12.Nikolopoulos S.N. Turner C.E. J. Cell Biol. 2000; 151: 1435-1448Crossref PubMed Scopus (169) Google Scholar, 13.Turner C.E. Nat. Cell Biol. 2000; 2: E231-E236Crossref PubMed Scopus (643) Google Scholar) serve to stabilize the focal adhesion complex, couple cell surface receptors to the actomyosin contractile apparatus (9.Critchley D.R. Curr. Opin. Cell Biol. 2000; 12: 133-139Crossref PubMed Scopus (490) Google Scholar), and act as a scaffold to which other proteins can bind. Paxillin, in particular, is a multifaceted scaffolding protein containing two main structural domains: an SH2- and SH3-containing N terminus and a C-terminal region composed of four sequential LIM (lin-11, isl-1, mec-3) domains (14.Turner C.E. J. Cell Sci. 2000; 113: 4139-4140Crossref PubMed Google Scholar). Between these two domains paxillin is capable of binding both structural (α-tubulin and actopaxin) as well as signaling (focal adhesion kinase (FAK), Csk, and c-Abl) molecules, allowing it to play an intimate role in the dynamics of the focal adhesion (12.Nikolopoulos S.N. Turner C.E. J. Cell Biol. 2000; 151: 1435-1448Crossref PubMed Scopus (169) Google Scholar, 13.Turner C.E. Nat. Cell Biol. 2000; 2: E231-E236Crossref PubMed Scopus (643) Google Scholar, 14.Turner C.E. J. Cell Sci. 2000; 113: 4139-4140Crossref PubMed Google Scholar, 15.Schaller M.D. Parsons J.T. Mol. Cell. Biol. 1995; 15: 2635-2645Crossref PubMed Scopus (498) Google Scholar). Signaling molecules act as conduits between the extracellular environment and the cell, influencing focal adhesion turnover, nuclear transcription, cell morphology, and cell survival (4.Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3757) Google Scholar, 8.Clark E.A. Brugge J.S. Science. 1995; 268: 233-239Crossref PubMed Scopus (2802) Google Scholar). Tyrosine kinases (FAK, Src, Csk, Fyn, c-Abl, and Pyk2) are activated upon integrin engagement and subsequently trigger such cellular events as mitosis, cytoskeletal rearrangement (4.Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3757) Google Scholar, 16.Avraham H. Park S.Y. Schinkmann K. Avraham S. Cell Signal. 2000; 12: 123-133Crossref PubMed Scopus (406) Google Scholar, 17.Salgia R. Brunkhorst B. Pisick E. Li J.L. Lo S.H. Chen L.B. Griffin J.D. Oncogene. 1995; 11: 1149-1155PubMed Google Scholar) and activation of the Ras-extracellular signal-regulated kinase (ERK) MAPK cascade (4.Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3757) Google Scholar). In addition, enhanced tyrosine phosphorylation is associated with increased focal adhesion and stress fiber formation (10.Schoenwaelder S.M. Burridge K. Curr. Opin. Cell Biol. 1999; 11: 274-286Crossref PubMed Scopus (647) Google Scholar). The serine/threonine kinase protein kinase C (PKC) is also associated with focal adhesions. Cells adherent to the CBD of fibronectin and treated with phorbol esters to directly stimulate PKC generate focal adhesions and actin stress fibers (18.Woods A. Couchman J.R. J. Cell Sci. 1992; 101: 277-290Crossref PubMed Google Scholar), indicating that this kinase is involved in the focal adhesion assembly process. PKC activation directs the localization of syndecan-4 to focal adhesion sites (2.Baciu P.C. Goetinck P.F. Mol. Biol. Cell. 1995; 6: 1503-1513Crossref PubMed Scopus (120) Google Scholar), and in turn, PKC activation is potentiated following oligomerization of syndecan-4 core proteins (19.Oh E.S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 8133-8136Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 20.Oh E.S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 11805-11811Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 21.Oh E.S. Woods A. Lim S.T. Theibert A.W. Couchman J.R. J. Biol. Chem. 1998; 273: 10624-10629Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar) and the binding of phosphatidylinositol 4,5-bisphosphate (PIP2) to syndecan-4 (22.Horowitz A. Murakami M. Gao Y. Simons M. Biochemistry. 1999; 38: 15871-15877Crossref PubMed Scopus (77) Google Scholar). Interestingly, PKC stimulation also enhances the shedding of syndecan-4 from the plasma membrane, thereby modifying its function from a cell surface receptor to an ECM-associated molecule (3.Bernfield M. Gotte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 69: 729-777Crossref Scopus (2281) Google Scholar, 23.Subramanian S.V. Fitzgerald M.L. Bernfield M. J. Biol. Chem. 1997; 272: 14713-14720Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). Syndecan-4 is a member of a family of transmembrane HSPG (syndecans 1–4) that are characterized by divergent extracellular domains and cytoplasmic domains that contain two constant regions separated by a variable region that is unique to each family member (3.Bernfield M. Gotte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 69: 729-777Crossref Scopus (2281) Google Scholar, 24.Zimmermann P. David G. FASEB J. 1999; 13: S91-S100Crossref PubMed Google Scholar). Recently, several proteins have been identified that bind to the cytoplasmic domain of the syndecan family members. These proteins (syntenin, synectin, synbindin, and CASK) contain PDZ domains that ligate the invariant C-terminal 4 amino acids (EFYA) present in all 4 syndecan molecules (25.Cohen A.R. 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 (318) Google Scholar, 26.Ethell I.M. Hagihara K. Miura Y. Irie F. Yamaguchi Y. J. Cell Biol. 2000; 151: 53-68Crossref PubMed Scopus (103) Google Scholar, 27.Gao Y. Li M. Chen W. Simons M. J. Cell. Physiol. 2000; 184: 373-379Crossref PubMed Scopus (151) Google Scholar, 28.Grootjans 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 (337) Google Scholar, 29.Hsueh Y.P. Yang F.C. Kharazia V. Naisbitt S. Cohen A.R. Weinberg R.J. Sheng M. J. Cell Biol. 1998; 142: 139-151Crossref PubMed Scopus (283) Google Scholar). Other molecules ligate the unique variable region and, therefore, bind selectively to one family member. PIP2binds the variable region of syndecan-4 (22.Horowitz A. Murakami M. Gao Y. Simons M. Biochemistry. 1999; 38: 15871-15877Crossref PubMed Scopus (77) Google Scholar), as does the cytoplasmic protein syndesmos (30.Baciu P.C. Saoncella S. Lee S.H. Denhez F. Leuthardt D. Goetinck P.F. J. Cell Sci. 2000; 113: 315-324Crossref PubMed Google Scholar). Syndesmos was isolated from a yeast two-hybrid screen in which the cytoplasmic domain of syndecan-4 was used as bait and was found to co-localize with syndecan-4 in the focal contacts of ventral plasma membranes of fibronectin adherent cells. Syndesmos can be myristylated suggesting that it may interact directly with the plasma membrane in addition to its association with syndecan-4. When overexpressed in NIH3T3 cells, syndesmos accelerates cell spreading and the assembly of actin stress fibers (30.Baciu P.C. Saoncella S. Lee S.H. Denhez F. Leuthardt D. Goetinck P.F. J. Cell Sci. 2000; 113: 315-324Crossref PubMed Google Scholar). Due to the impact syndesmos has on cell morphology, we deduced that it might interact with other focal adhesion-associated proteins and sought to identify other potential cytoplasmic interactors. Here we report that syndesmos binds the adaptor protein paxillin. The connection of syndecan-4 with paxillin and possible downstream signaling events through syndesmos parallels the connection between paxillin and integrins and may thus reflect the cooperativity between these two receptors in the assembly of focal adhesions and actin stress fibers. Primary chick embryo fibroblasts (CEFs) were obtained and cultured as previously described (2.Baciu P.C. Goetinck P.F. Mol. Biol. Cell. 1995; 6: 1503-1513Crossref PubMed Scopus (120) Google Scholar). All experiments were done on cultures that were 85% confluent. Syndesmos was immunoprecipitated from cell lysates with anti-syndesmos polyclonal antibodies (30.Baciu P.C. Saoncella S. Lee S.H. Denhez F. Leuthardt D. Goetinck P.F. J. Cell Sci. 2000; 113: 315-324Crossref PubMed Google Scholar). The immunoprecipitates were washed four times in lysis buffer (phosphate-buffered saline/Triton X-100 (0.1%), protease inhibitors (Complete, protease inhibitor mixture, Roche Molecular Biochemicals) and 1 mm sodium orthovanadate), solubilized in SDS-PAGE buffer, electrophoresed, transferred to an immobilon-P membrane, and probed for tyrosine-phosphorylated proteins (PY 99, sc-720, Santa-Cruz Biotechnology, Inc.) or paxillin (P13520-Transduction Laboratories, Lexington, KY). The membranes were then incubated with a horseradish peroxidase-conjugated anti-mouse secondary antibody (170-6516, Bio-Rad), and visualization of immunoreactive bands was performed with enhanced chemiluminescence following the manufacturer's instructions (Pierce, Rockford, IL). GST-paxillin, GST-Hic-5, GST-syndesmos, or GST alone were expressed in Escherichia coli (BL21) and purified on glutathione-agarose beads according to the manufacturer's directions (Amersham Biosciences, Inc.). The construction of GST-syndesmos has been previously described (30.Baciu P.C. Saoncella S. Lee S.H. Denhez F. Leuthardt D. Goetinck P.F. J. Cell Sci. 2000; 113: 315-324Crossref PubMed Google Scholar). The GST-paxillin and GST-Hic-5 constructs were a gift from Dr. Sheila Thomas (Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA). Cell lysates from CEFs were incubated overnight at 4 °C with the glutathione-agarose bead-bound GST constructs or GST alone. The beads were then washed six times in the lysis buffer, solubilized in SDS-PAGE buffer, electrophoresed, transferred to an immobilon-P membrane, and analyzed for bound syndesmos using the anti-syndesmos polyclonal antibody (30.Baciu P.C. Saoncella S. Lee S.H. Denhez F. Leuthardt D. Goetinck P.F. J. Cell Sci. 2000; 113: 315-324Crossref PubMed Google Scholar) as described above for the immunoprecipitations. In vitro transcription-translation was carried out using the TNT in vitro transcription-translation kit from Promega (Madison, WI). Reactions were carried out as recommended by the manufacturer. The template used for these experiments was syndesmos cDNA that was cloned in pBluescript (Stratagene, La Jolla, CA). In vivo cell labeling grade l-[35S]methionine (SJ 1015; >1000 Ci/mmol) was purchased from Amersham Biosciences, Inc. CEFs were treated for 30 min with the membrane-permeable homobifunctional cross-linking reagent DTSP (3,3′-dithiobispropionic acid N-hydroxysuccinimide ester, C14H16O4S2, Sigma), which contains a cleavable disulfide linkage. Cells were then rinsed with phosphate-buffered saline and blocked with 0.2 m glycine in phosphate-buffered saline for 10 min. After two washes with phosphate-buffered saline, the cells were lysed in radioimmune precipitation buffer, and the lysates were incubated with either anti-syndesmos serum or preimmune serum coupled to protein-G beads. The immunoprecipitates were washed four times with radioimmune precipitation buffer, resuspended in electrophoresis buffer containing 20% β-mercaptoethanol, boiled for 5 min to cleave the disulfide bond, and then analyzed by SDS-PAGE under reducing conditions and Western blotted using anti-paxillin antibodies. After starvation in Dulbecco's modified Eagle's medium without fetal bovine serum, CEFs were treated for the indicated times with TPA (Sigma) at a of Cell lysates and immunoprecipitates were obtained as described identify possible syndesmos CEFs were treated with for in the of fetal bovine serum and immunoprecipitated from the cell with antibodies syndesmos were and analyzed by Western using an tyrosine-phosphorylated protein that with an of was on its we that this protein be paxillin, one of the tyrosine-phosphorylated focal adhesion-associated This was proteins from cell lysates were immunoprecipitated with antibodies and blotted with an antibody paxillin. immunoreactive bands and be with this antibody as previously R. Li J.L. Lo S.H. Brunkhorst B. E.S. Y. Pisick E. M. R. Chen L.B. Griffin J.D. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). the paxillin antibody used also the paxillin (12.Nikolopoulos S.N. Turner C.E. J. Cell Biol. 2000; 151: 1435-1448Crossref PubMed Scopus (169) Google Scholar), the is not but a fragment of paxillin, we also this in in vitro transcription-translation using a paxillin template not of syndesmos with paxillin also be demonstrated in GST GST-syndesmos, but not GST is to two paxillin immunoreactive bands from lysates and GST-paxillin, but not GST syndesmos from lysates In addition, GST-Hic-5 also syndesmos from lysates suggesting that the syndesmos binding is present in both paxillin and its Hic-5. complexes CEFs were treated with the membrane and immunoprecipitates were obtained from cell lysates with either anti-syndesmos serum or preimmune The of these immunoprecipitates by SDS-PAGE and Western with antibodies paxillin that paxillin was to syndesmos is thus that a intracellular between the two that the interactions between syndesmos and paxillin or in the and GST in the above experiments are direct, we performed GST experiments with in vitro syndesmos to the potential of other associated GST-paxillin and GST-Hic-5, but not GST bound in vitro syndesmos these a between paxillin Hic-5) and syndesmos. PKC is associated with focal adhesion and actin stress fiber formation and also with increased tyrosine phosphorylation in cells. PKC activation is involved in the of syndesmos and paxillin, CEFs were incubated with which directly for various of was with syndesmos upon 1 min of TPA with the tyrosine inhibitor the for at min indicating that these are also on tyrosine phosphorylation The cross-linking done in the of and also the in vitro experiments and that tyrosine phosphorylation is not for the association between syndesmos and paxillin. the described in 4 not for the tyrosine phosphorylation has a on the by the of the paxillin binding region of syndesmos. The region on the syndesmos is in a region in the syndesmos TPA has been to trigger focal adhesion formation and to the of syndecan-4 in these focal adhesions (18.Woods A. Couchman J.R. J. Cell Sci. 1992; 101: 277-290Crossref PubMed Google P.C. Goetinck P.F. Mol. Biol. Cell. 1995; 6: 1503-1513Crossref PubMed Scopus (120) Google Scholar). we are that TPA also the association of syndesmos with paxillin. is to that this is and that the of the laboratory has that the of syndecan-4 in focal adhesions be triggered by TPA we that the in association is involved in the focal adhesion assembly as The a of that of a cDNA with a syndesmos cDNA a of that a protein of amino This sequence is that the amino acids of syndesmos (30.Baciu P.C. Saoncella S. Lee S.H. Denhez F. Leuthardt D. Goetinck P.F. J. Cell Sci. 2000; 113: 315-324Crossref PubMed Google Scholar). of the sequence has an to a region of the sequence The sequence does not either the four potential or an acid C-terminal found in syndesmos. the binding in syndesmos that interacts with paxillin we performed GST experiments with a of GST-syndesmos constructs of or The of these that syndesmos binds to paxillin through the amino acid sequence between amino acids and of the sequence This sequence is in the region of with the sequence that also binds paxillin. adhesions are macromolecular complexes that are cells are on extracellular matrix are of the transmembrane molecules, integrins and syndecan-4, and a of cytoplasmic signaling and adaptor is an important cytoplasmic adaptor protein that binds several other focal adhesion-associated proteins such as vinculin, actopaxin, Src, and M.D. Parsons J.T. Mol. Cell. Biol. 1995; 15: 2635-2645Crossref PubMed Scopus (498) Google Scholar, R. Li J.L. Lo S.H. Brunkhorst B. E.S. Y. Pisick E. M. R. Chen L.B. Griffin J.D. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, Z. Turner C.E. Brugge J.S. C. J. Biol. Chem. 268: Full Text PDF PubMed Google Scholar). connection between paxillin and integrins can be a binds both paxillin and talin, and the binds the cytoplasmic domain of the of In the present we report on a connection between paxillin and syndecan-4, the transmembrane receptor of focal adhesions. We that paxillin binds directly to a cytoplasmic protein that binds specifically to the cytoplasmic domain of syndecan-4 (30.Baciu P.C. Saoncella S. Lee S.H. Denhez F. Leuthardt D. Goetinck P.F. J. Cell Sci. 2000; 113: 315-324Crossref PubMed Google Scholar). paxillin, as an important focal adhesion complex adaptor can between the intracellular focal adhesion complex and integrins and syndecan-4, the two transmembrane proteins of that The connection of paxillin with integrins is through a and that with syndecan-4 is through syndesmos. is that the interactions between paxillin and syndesmos can be triggered by the activation of The variable region of the cytoplasmic domain of syndecan-4 binds and thus and PKC activation Y. Li M. Chen W. Simons M. J. Cell. Physiol. 2000; 184: 373-379Crossref PubMed Scopus (151) Google Scholar). PKC activation also to the assembly of focal adhesions and actin stress fibers in cells on the cell binding domain of fibronectin, a in which syndecan-4 is not (18.Woods A. Couchman J.R. J. Cell Sci. 1992; 101: 277-290Crossref PubMed Google Scholar). cells on the cell binding domain of fibronectin can the focal adhesion complex treated with antibodies syndecan-4 (6.Saoncella 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 (328) Google Scholar). syndecan-4 and integrins in the assembly of the focal adhesion We have previously that of syndesmos enhances cell spreading (30.Baciu P.C. Saoncella S. Lee S.H. Denhez F. Leuthardt D. Goetinck P.F. J. Cell Sci. 2000; 113: 315-324Crossref PubMed Google Scholar). The of syndesmos with paxillin is enhanced following PKC syndesmos is in the it may with paxillin. PKC activation may then the localization of syndesmos to focal adhesions. The connection of paxillin with the cytoplasmic domains of both syndecan-4 and integrins one of the cooperativity between these receptors in which be to intracellular signaling pathways through the binding of paxillin to These signaling pathways the binding of to an SH3-containing protein for or the phosphorylation of tyrosine on which an with and in the for into the the interactions of phosphorylation of paxillin a binding for the adaptor protein which in binds the to the signaling pathway E.A. Brugge J.S. Science. 1995; 268: 233-239Crossref PubMed Scopus (2802) Google Scholar). These possible interactions for syndecan-4 are in We Dr. Sheila Thomas for the GST-paxillin and We also and for
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