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Syndecan-4 Modulates Focal Adhesion Kinase Phosphorylation*

  • Sarah A. Wilcox-Adelman
    Footnotes
    Affiliations
    Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129
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  • Fabienne Denhez
    Affiliations
    Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129
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  • Paul F. Goetinck
    Correspondence
    To whom correspondence should be addressed: Cutaneous Biology Research Center, MGH-East, Bldg. 149, 13th St., Charlestown, MA 02129. Tel.: 617-726-4183; Fax: 617-726-4189
    Affiliations
    Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129
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  • Author Footnotes
    * This work was supported in part by National Institute of Health, NICHD Grant HD-37490 and grants from the Cutaneous Biology Research Center through the MGH/Shiseido Company (to P. F. G.) and the Dermatology Foundation Research (to F. D.)The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.This paper is dedicated to the memory of Merton Bernfield who pioneered the field of syndecan biology.
    ‡ Supported by National Institutes of Health Postdoctoral Fellowship F32 HD41235.
Open AccessPublished:June 26, 2002DOI:https://doi.org/10.1074/jbc.M201283200
      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.
      PKC
      protein kinase C
      FAK
      focal adhesion kinase
      FN
      fibronectin
      CBD
      cell-binding domain
      LPA
      lysophosphatidic acid
      WT
      wild type
      PBS
      phosphate-buffered saline
      PMA
      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.
      • Woods A.
      ,
      • Woods A., Oh, E.-S.
      • Couchman J.R.
      ,
      • Carey D.J.
      ,
      • Bernfield M.
      • Gotte M.
      • Park P.W.
      • Reizes O.
      • Fitzgerald M.L.
      • Lincecum J.
      • Zako M.
      ). Although all members of the syndecan family arose from a single ancestral gene, their expression patterns in tissues and during development are highly regulated (
      • Carey D.J.
      ,
      • Bernfield M.
      • Gotte M.
      • Park P.W.
      • Reizes O.
      • Fitzgerald M.L.
      • Lincecum J.
      • Zako M.
      ,
      • Elenius K.
      • Jalkanen M.
      ). 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 (
      • Ethell I.M.
      • Hagihara K.
      • Miura Y.
      • Irie F.
      • Yamaguchi Y.
      ,
      • Hsueh Y.-P.
      • Sheng M.
      ,
      • Gao Y., Li, M.
      • Chen W.
      • Simons M.
      ,
      • Grootjans J.J.
      • Zimmermann P.
      • Reekmans G.
      • Smets A.
      • Degeest G.
      • Durr J.
      • David G.
      ,
      • Cohen D.J.
      • Woods D.F.
      • Marfatia S.M.
      • Walther Z.
      • Chishti A.H.
      • Anderson J.M.
      • Wood D.F.
      ). Unlike other family members, syndecan-4 binds protein kinase C-α (PKC-α)1 through the intermediary phosphatidylinositol bisphosphate (
      • Horowitz A.
      • Murakami M.
      • Gao Y.
      • Simons M.
      ,
      • Oh E.-S.
      • Woods A.
      • Couchman J.R.
      ) at the variable region and the cytoplasmic protein syndesmos through both the variable and the membrane-proximal constant regions (
      • Baciu P.C.
      • Saoncella S.
      • Lee S.H.
      • Denhez F.
      • Leuthardt D.
      • Goetinck P.F.
      ). Syndecans-1, -2, and -4 have been shown to bind extracellular matrix proteins (
      • Lebakken C.S.
      • McQuade K.J.
      • Rapraeger A.C.
      ,
      • Utani A.
      • Nomizu M.
      • Matsuura H.
      • Kato K.
      • Kobayashi T.
      • Takeda U.
      • Aota S.
      • Nielsen P.K.
      • Shinkai H.
      ,
      • Woods A.
      • Longley R.L.
      • Tumova S.
      • Couchman J.R.
      ). However, syndecan-4 is the only family member to localize to sites of cell-matrix adhesions (
      • Woods A.
      • Couchman J.R.
      ). 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 (
      • Baciu P.C.
      • Goetinck P.F.
      ).
      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.
      • Burridge K.
      • Chrzanowska-Wodnicka M.
      ,
      • Zamir E.
      • Geiger B.
      ,
      • Petit V.
      • Thiery J.P.
      ). 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 (
      • Woods A.
      • Couchman J.R.
      • Johansson S.
      • Hook M.
      ,
      • Bloom L.
      • Ingham K.C.
      • Hynes R.O.
      ,
      • Saoncella S.
      • Echtermeyer F.
      • Denhez F.
      • Nowlen J.K.
      • Mosher D.F.
      • Robinson S.D.
      • Hynes R.O.
      • Goetinck P.F.
      ). 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 (
      • Saoncella S.
      • Echtermeyer F.
      • Denhez F.
      • Nowlen J.K.
      • Mosher D.F.
      • Robinson S.D.
      • Hynes R.O.
      • Goetinck P.F.
      ). The syndecan-4 signal can be bypassed in CBD-adherent fibroblasts by directly stimulating the small GTPase Rho with lysophosphatidic acid (LPA) (
      • Saoncella S.
      • Echtermeyer F.
      • Denhez F.
      • Nowlen J.K.
      • Mosher D.F.
      • Robinson S.D.
      • Hynes R.O.
      • Goetinck P.F.
      ). 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 (
      • Saoncella S.
      • Echtermeyer F.
      • Denhez F.
      • Nowlen J.K.
      • Mosher D.F.
      • Robinson S.D.
      • Hynes R.O.
      • Goetinck P.F.
      ).
      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 (
      • Ishiguro K.
      • Kadomatsu K.
      • Kojima T.
      • Muramatsu H.
      • Tsuzuki S.
      • Nakamura E.
      • Kusugami K.
      • Saito H.
      • Muramatsu T.
      ,
      • Echtermeyer F.
      • Streit M.
      • Wilcox-Adelman S.
      • Saoncella S.
      • Denhez F.
      • Detmar M.
      • Goetinck P.
      ). 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 (
      • Ishiguro K.
      • Kadomatsu K.
      • Kojima T.
      • Muramatsu H.
      • Tsuzuki S.
      • Nakamura E.
      • Kusugami K.
      • Saito H.
      • Muramatsu T.
      ). 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” (
      • Echtermeyer F.
      • Streit M.
      • Wilcox-Adelman S.
      • Saoncella S.
      • Denhez F.
      • Detmar M.
      • Goetinck P.
      ,
      • Ishiguro 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.
      ,
      • Ishiguro K.
      • Kadomatsu K.
      • Kojima T.
      • Muramatsu H.
      • Matsuo S.
      • Kusugami K.
      • Saito H.
      • Muramatsu T.
      ). 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 (
      • Echtermeyer F.
      • Streit M.
      • Wilcox-Adelman S.
      • Saoncella S.
      • Denhez F.
      • Detmar M.
      • Goetinck P.
      ). 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 (
      • Palecek S.P.
      • Loftus J.C.
      • Ginsberg M.H.
      • Lauffenburger D.A.
      • Horwitz A.F.
      ). 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 (
      • Ilic D.
      • Furuta Y.
      • Kanazawa S.
      • Takeda N.
      • Sobue K.
      • Nakatsuji N.
      • Nomura S.
      • Fujimoto J.
      • Okada M.
      • Yamamoto T.
      • Aizawa S.
      ,
      • Ren X.D.
      • Kiosses W.B.
      • Sieg D.J.
      • Otey C.A.
      • Schlaepfer D.D.
      • Schwartz M.A.
      ). Loss of FAK is associated with decreased cell migration and increased focal adhesion size (
      • Ilic D.
      • Furuta Y.
      • Kanazawa S.
      • Takeda N.
      • Sobue K.
      • Nakatsuji N.
      • Nomura S.
      • Fujimoto J.
      • Okada M.
      • Yamamoto T.
      • Aizawa S.
      ,
      • Ilic D.
      • Kanazawa S.
      • Furuta Y.
      • Yamamoto T.
      • Aizawa S.
      ,
      • Sieg D.J.
      • Ilic D.
      • Jones K.C.
      • Damsky C.H.
      • Hunter T.
      • Schlaepfer D.D.
      ,
      • Owen J.D.
      • Ruest P.J.
      • Fry D.W.
      • Hanks S.K.
      ), whereas overexpression of FAK increases cell migration (
      • Owen J.D.
      • Ruest P.J.
      • Fry D.W.
      • Hanks S.K.
      ,
      • Cary L.A.
      • Chang J.F.
      • Guan J.-L.
      ,
      • Sieg D.J.
      • Hauck C.R.
      • Schlaepfer D.D.
      ).
      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 (
      • Zachary I.
      ). 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.
      • Schaller M.D.
      • Hildebrand J.D.
      • Shannon J.D.
      • Fox J.W.
      • Vines R.R.
      • Parsons J.T.
      and
      • Miyamoto S.
      • Teramoto H.
      • Coso O.A.
      • Gutkind J.S.
      • Burbelo P.D.
      • Akiyama S.K.
      • Yamada K.M.
      and reviewed in Refs.
      • Otey C.A.
      and
      • Schlaepfer D.D.
      • Hauck C.R.
      • Sieg D.J.
      ). FAK-mediated cell migration is dependent on phosphorylation of FAK Tyr397 (
      • Cary L.A.
      • Chang J.F.
      • Guan J.-L.
      ). The phosphorylated form of Tyr397 binds Src (
      • Schaller M.D.
      • Hildebrand J.D.
      • Shannon J.D.
      • Fox J.W.
      • Vines R.R.
      • Parsons J.T.
      ), the p85 subunit of phosphatidylinositol 3-kinase (
      • Chen H.-C.
      • Appeddu P.A.
      • Isoda H.
      • Guan J.-L.
      ), phospholipase Cγ1 (
      • Zhang X.
      • Chattopadhyay A., Ji, Q.-S.
      • Owen J.D.
      • Ruest P.J.
      • Carpenter G.
      • Hanks S.K.
      ), the adaptor proteins Grb7 (
      • Han D.C.
      • Guan J.-L.
      ) and Shc (
      • Schlaepfer D.D.
      • Jones K.C.
      • Hunter T.
      ), and the phosphatase PTEN (
      • Tamura M., Gu, J.
      • Danen E.H.
      • Takino T.
      • Miyamoto S.
      • Yamada K.M.
      ). The unlikely possibility that all of these proteins bind FAK simultaneously suggests that distinct FAK-containing signaling complexes probably exist within the focal adhesion (
      • Schaller M.D.
      ). The phosphorylation of the remaining tyrosine residues (407, 576, 577, 861, and 925) occurs in a Src-dependent manner (
      • Calalb M.B.
      • Polte T.R.
      • Hanks S.K.
      ,
      • Calalb M.B.
      • Zhang X.
      • Polte T.R.
      • Hanks S.K.
      ,
      • Schlaepfer D.D.
      • Hanks S.K.
      • Hunter T.
      • van der Geer P.
      ,
      • Schlaepfer D.D.
      • Hunter T.
      ). Phosphotyrosines 576 and 577 are required for maximal FAK kinase activity (
      • Calalb M.B.
      • Polte T.R.
      • Hanks S.K.
      ) and phosphotyrosine 925 acts as a binding site for the Grb2 adaptor protein leading to activation of the Ras pathway (
      • Schlaepfer D.D.
      • Hanks S.K.
      • Hunter T.
      • van der Geer P.
      ).
      Fibroblasts treated with LPA, which directly stimulates the small GTPase Rho, show increases in FAK phosphorylation and its subsequent localization to focal adhesions (
      • Kumagai N.
      • Morii N.
      • Fujisawa K.
      • Yoshimasa T.
      • Nakao K.
      • Narumiya S.
      ,
      • Chrzanowska-Wodnicka M.
      • Burridge K.
      ,
      • Ridley A.J.
      • Hall A.
      ,
      • Barry S.T.
      • Critchley D.R.
      ,
      • Flinn H.M.
      • Ridley A.J.
      ,
      • Rodriguez-Fernandez J.L.
      • Rozengurt E.
      ). Closer examination of early adhesion events documented initial FAK phosphorylation occurring in a Rho-independent manner followed by Rho-mediated FAK phosphorylation (
      • Clark E.A.
      • King W.G.
      • Brugge J.S.
      • Symons M.
      • Hynes R.O.
      ). Recently, Ren et al. (
      • Ren X.D.
      • Kiosses W.B.
      • Sieg D.J.
      • Otey C.A.
      • Schlaepfer D.D.
      • Schwartz M.A.
      ) 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 (
      • Ren X.D.
      • Kiosses W.B.
      • Sieg D.J.
      • Otey C.A.
      • Schlaepfer D.D.
      • Schwartz M.A.
      ). 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 (
      • Burridge K.
      • Turner C.E.
      • Romer L.H.
      ) but maximal phosphorylation requires both the integrin-binding and heparin-binding domains of FN (
      • Jeong J.
      • Han I.
      • Lim Y.
      • Kim J.
      • Park I.
      • Woods A.
      • Couchman J.R.
      • Oh E.S.
      ). As syndecan-4 binds the heparin-binding domain of FN (
      • Tumova S.
      • Woods A.
      • Couchman J.R.
      ) and has been shown to act in a Rho-dependent manner to influence focal adhesions and actin stress fibers (
      • Saoncella S.
      • Echtermeyer F.
      • Denhez F.
      • Nowlen J.K.
      • Mosher D.F.
      • Robinson S.D.
      • Hynes R.O.
      • Goetinck P.F.
      ), 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.

      DISCUSSION

      The heparan sulfate proteoglycan syndecan-4 has two main cellular functions. It acts as a co-receptor for heparin-binding growth factors (such as the family of fibroblast growth factors and heparin-binding vascular endothelial growth factor isoforms) regulating the ligand-dependent activation of the primary receptor (
      • Carey D.J.
      ). Syndecan-4 also functions, in a Rho-dependent manner, with the α5β1 integrin to promote the adhesion-dependent formation of actin stress fibers and focal adhesions (
      • Saoncella S.
      • Echtermeyer F.
      • Denhez F.
      • Nowlen J.K.
      • Mosher D.F.
      • Robinson S.D.
      • Hynes R.O.
      • Goetinck P.F.
      ). Syndecan-4-null fibroblasts will form focal adhesions and actin stress fibers when plated on a FN substrate (
      • Ishiguro K.
      • Kadomatsu K.
      • Kojima T.
      • Muramatsu H.
      • Tsuzuki S.
      • Nakamura E.
      • Kusugami K.
      • Saito H.
      • Muramatsu T.
      ,
      • Echtermeyer F.
      • Streit M.
      • Wilcox-Adelman S.
      • Saoncella S.
      • Denhez F.
      • Detmar M.
      • Goetinck P.
      ). However, the cells are unable to generate focal adhesions when seeded on the CBD of FN and incubated with either syndecan-4 antibodies or the heparin-binding domain of FN (
      • Ishiguro K.
      • Kadomatsu K.
      • Kojima T.
      • Muramatsu H.
      • Tsuzuki S.
      • Nakamura E.
      • Kusugami K.
      • Saito H.
      • Muramatsu T.
      ). Therefore, although a compensatory cellular mechanism exists to bypass syndecan-4 deficiency, it is possible to solely evaluate the syndecan-4 signaling pathway.
      Transfection of full-length syndecan-4 core protein enhances cell spreading and focal adhesion formation and decreases cell migration (
      • Longley R.L.
      • Woods A.
      • Fleetwood A.
      • Cowling G.J.
      • Gallagher J.T.
      • Couchman J.R.
      ,
      • Echtermeyer F.
      • Baciu P.C.
      • Saoncella S., Ge, Y.
      • Goetinck P.F.
      ). Interestingly, transfection of cells with syndecan-4 constructs that lack cytoplasmic domains also exhibit decreased cell migration although they do not form stress fibers or extensive focal adhesions when seeded on a FN or vitronectin substrate (
      • Longley R.L.
      • Woods A.
      • Fleetwood A.
      • Cowling G.J.
      • Gallagher J.T.
      • Couchman J.R.
      ). Fibroblasts generated from mice lacking syndecan-4 also show migration delays compared with wild type controls (
      • Echtermeyer F.
      • Streit M.
      • Wilcox-Adelman S.
      • Saoncella S.
      • Denhez F.
      • Detmar M.
      • Goetinck P.
      ). The impaired migration in syndecan-4-null cells may result from the inability of the adhesions to generate enough tension required for migration or from the inability to disengage established contacts so that new adhesions may form (
      • Palecek S.P.
      • Loftus J.C.
      • Ginsberg M.H.
      • Lauffenburger D.A.
      • Horwitz A.F.
      ). Alternatively, although not mutually exclusive from the former, syndecan-4 may regulate components of a signaling pathway involved in cell migration and the absence of syndecan-4 disrupts the efficiency with which the pathway acts.
      It has previously been shown that integrin ligation to the CBD of FN does not induce tyrosine phosphorylation of FAK to the same level as cells that are ligated to full-length FN (
      • Jeong J.
      • Han I.
      • Lim Y.
      • Kim J.
      • Park I.
      • Woods A.
      • Couchman J.R.
      • Oh E.S.
      ). We analyzed the autophosphorylated form of FAK and now demonstrate that FAK Tyr397 phosphorylation is lower in the absence of the heparin-binding domain of FN. Incubation of CBD-adherent FN-null cells with the tyrosine phosphatase inhibitor sodium vanadate augmented the phosphorylation levels of FAK Tyr397 to that of vanadate-treated cells seeded on full-length FN suggesting that syndecan-4 may influence FAK Tyr397 phosphorylation by regulating the activity of a cellular tyrosine phosphatase. Candidates for such tyrosine phosphatases are: PTEN (
      • Tamura M., Gu, J.
      • Matsumoto K.
      • Aota S.-I.
      • Parsons R.
      • Yamada K.M.
      ), PTP-PEST (
      • Angers-Loustau A.
      • Cote J.-F.
      • Charest A.
      • Dowbenko D.
      • Spencer S.
      • Lasky L.A.
      • Trembley M.L.
      ), and Shp-2 (
      • Manes S.
      • Mira E.
      • Gomez-Mouton C.
      • Zhao Z.J.
      • Lacalle R.A.
      • Martinez A.C.
      ), which have been shown to dephosphorylate FAK in vitro. Incubation of our cells with the Shp-2 inhibitor calpeptin did not enhance phosphorylation of FAK Tyr397 (data not shown) suggesting that under our conditions Shp-2 is not involved in modulating the phosphorylation state of FAK Tyr397.
      Syndecan-4-regulated focal adhesion formation is primarily associated with two signaling molecules: the serine/threonine kinase PKC and the small GTPase Rho. Fibroblasts seeded on CBD can be stimulated to generate actin stress fibers and focal adhesions by incubating the cells with either PMA (to directly activate PKC) (
      • Woods A.
      • Courchman J.R.
      ) or LPA (to directly stimulate Rho) (
      • Saoncella S.
      • Echtermeyer F.
      • Denhez F.
      • Nowlen J.K.
      • Mosher D.F.
      • Robinson S.D.
      • Hynes R.O.
      • Goetinck P.F.
      ). It is unclear whether these signaling molecules collaborate in one signaling pathway or if they work in parallel but separate pathways. Our study shows that increased phosphorylation of FAK Tyr397 is associated with the activation of the Rho pathway and not the PKC pathway, as only LPA and not PMA increased Tyr397 phosphorylation in our conditions. Similarly, LPA, but not PMA, treatment also induced the localization of FAK phosphorylated on Tyr397 to vinculin-containing focal adhesions in syndecan-4-null cells plated on FN. Therefore, our data indicate that syndecan-4-mediated FAK phosphorylation occurs only through a Rho-mediated process and not through a collaboration of both Rho and PKC stimulation.
      PKC activation augmented general tyrosine phosphorylation in CBD-adherent cells but this stimulation did not translate into increased phosphorylation of FAK Tyr397. The lack of effect with PMA treatment does not imply that a syndecan-4-PKC pathway does not exist. Indeed, syndecan-4 and PKC have a close functional association. PKC recruits syndecan-4 to focal adhesion sites (
      • Baciu P.C.
      • Goetinck P.F.
      ) and, conversely, syndecan-4 binds PKC through the intermediary phosphatidylinositol bisphosphate (
      • Horowitz A.
      • Murakami M.
      • Gao Y.
      • Simons M.
      ,
      • Oh E.S.
      • Woods A.
      • Lim S.T.
      • Theibert A.W.
      • Couchman J.R.
      ) and potentiates its activity (
      • Oh E.-S.
      • Woods A.
      • Couchman J.R.
      ,
      • Oh E.-S.
      • Woods A.
      • Couchman J.R.
      ).
      It is possible that PKC promotes focal adhesion formation through an alternative cell-surface heparan sulfate proteoglycan. Syndecan-4-null cells will generate actin stress fibers and focal adhesions when seeded on a combination substrate of CBD and heparin-binding domain FN fragments, and this effect can be inhibited with the addition of heparin to the cell medium (
      • Ishiguro K.
      • Kadomatsu K.
      • Kojima T.
      • Muramatsu H.
      • Tsuzuki S.
      • Nakamura E.
      • Kusugami K.
      • Saito H.
      • Muramatsu T.
      ). These data indicate that another cell-surface heparan sulfate proteoglycan can compensate for the lack of syndecan-4 to promote focal adhesion and stress fiber formation.
      Alternatively, the PKC pathway may be activated following the association of syndecan-4 with a heparin-binding growth factor receptor. Growth factor ligation (such as fibroblast growth factor-2) may promote the association of syndecan-4 with PKC, leading to its activation and the subsequent formation of focal adhesions and actin stress fibers. Syndecan-4 acts as a co-receptor for several heparin-binding growth factors, regulating the ligand-dependent activation of the primary receptors (
      • Carey D.J.
      ). The cytoplasmic domain of syndecan-4 has been implicated in promoting fibroblast growth factor 2-dependent cell proliferation and migration (
      • Volk R.
      • Schwartz J.J., Li, J.
      • Rosenberg R.D.
      • Simons M.
      ) and fibroblast growth factor-2 regulates the syndecan-4 dependent activation of PKC (
      • Horowitz A.
      • Murakami M.
      • Gao Y.
      • Simons M.
      ,
      • Horowitz A.
      • Simons M.
      ).
      To demonstrate conclusively that the signaling mechanism affecting the phosphorylation of FAK Tyr397 acts specifically through syndecan-4, syndecan-4-null fibroblasts were used. The cells were seeded on a FN substrate as they have been shown to develop focal adhesions under these conditions. The lack of syndecan-4 expression in these cells dictates that adhesion to a FN substrate will not stimulate a syndecan-4 signaling pathway but will activate the unknown complementary pathway, if it is involved (
      • Ishiguro K.
      • Kadomatsu K.
      • Kojima T.
      • Muramatsu H.
      • Tsuzuki S.
      • Nakamura E.
      • Kusugami K.
      • Saito H.
      • Muramatsu T.
      ). Our experiments reveal that there is less FAK phosphorylated on Tyr397 in syndecan-4-null fibroblasts than wild type fibroblasts under basal conditions and this can be rescued through re-expression of syndecan-4, demonstrating that syndecan-4 is directly involved in influencing FAK Tyr397 phosphorylation. The lack of FAK phosphorylated on Tyr397 in the syndecan-4-null cells was not because of an inability of Tyr397 to be phosphorylated, as treatment with LPA or sodium vanadate (data not shown) increased Tyr397phosphorylation to syndecan-4-WT control levels, but was attributable to a decrease in the levels of active Rho in the cells. Correspondingly, direct inactivation of Rho using C3 exotransferase resulted in the loss of FAK phosphorylated at Tyr397 in the focal adhesions of syndecan-4-WT cells. The syndecan-4-null fibroblasts still generate vinculin-containing focal complexes so the limited level of GTP-bound Rho present in the null cells is sufficient to generate stress fibers and focal adhesions. Incubation of syndecan-4-null fibroblasts with LPA augmented the level of active Rho, indicating that the molecule is capable of functioning appropriately but is either not activated to the same degree as in wild type cells or is unable to maintain the active state for the “normal” length of time. Rho activation has been shown to increase FAK phosphorylation (
      • Clark E.A.
      • King W.G.
      • Brugge J.S.
      • Symons M.
      • Hynes R.O.
      ) and cell motility (
      • Matsumoto Y.
      • Tanaka K.
      • Harimaya K.
      • Nakatani F.
      • Matsuda S.
      • Iwamoto Y.
      ), whereas inhibition of Rho attenuates both (
      • Bobak D.
      • Moorman J.
      • Guanzon A.
      • Gilmer L.
      • Hahn C.
      ,
      • Imamura F.
      • Mukai M.
      • Ayaki M.
      • Akedo H.
      ). Although studies have demonstrated that FAK can inhibit Rho activity, this attenuation occurs during early cell spreading (within 40 min of cell plating) (
      • Ren X.D.
      • Kiosses W.B.
      • Sieg D.J.
      • Otey C.A.
      • Schlaepfer D.D.
      • Schwartz M.A.
      ). The cells in our experiments were adherent for 4 h during which Rho activity is higher (
      • Ren X.-D.
      • Kiosses W.B.
      • Schwartz M.A.
      ). This is the first description, to our knowledge, that the heparan sulfate proteoglycan syndecan-4 influences the level of active Rho in cells.
      Rho cycles between an active GTP-bound state and an inactive GDP-bound state. While active it can bind to its effector molecules: Dia, ROCKs (Rho kinases), and phosphatidylinositol-4-phosphate 5-kinase to induce actin polymerization, cell body contraction, and stress fiber and focal adhesion formation (
      • Ridley A.J.
      ,
      • Geiger B.
      • Bershadsky A.
      ). Rho also stimulates cell motility (
      • Yoshioka K.
      • Matsumura F.
      • Akedo H.
      • Itoh K.
      ,
      • Takaishi K.
      • Kikuchi A.
      • Kuroda S.
      • Kotani K.
      • Sasaki T.
      • Takai Y.
      ,
      • Stasia M.J.
      • Jouan A.
      • Bourmeyster N.
      • Boquet P.
      • Vignais P.V.
      ) and this may be a function separate to that of forming focal adhesions and stress fibers (
      • Nabi I.R.
      ,
      • Machesky L.M.
      • Hall A.
      ). Phosphorylation of FAK at Tyr397 is necessary for optimal cell migration (
      • Owen J.D.
      • Ruest P.J.
      • Fry D.W.
      • Hanks S.K.
      ,
      • Sieg D.J.
      • Hauck C.R.
      • Schlaepfer D.D.
      ,
      • Wang H.B.
      • Dembo M.
      • Hanks S.K.
      • Wang Y.
      ). Phosphorylation of FAK on Tyr397 generates a binding site for SH2-containing proteins. Some of these binding partners (phosphatidylinositol 3-kinase, Grb7, and Src) increase cell migration (
      • Cary L.A.
      • Chang J.F.
      • Guan J.-L.
      ,
      • Chen H.-C.
      • Appeddu P.A.
      • Isoda H.
      • Guan J.-L.
      ,
      • Han D.C.
      • Guan J.-L.
      ,
      • Reiske H.R.
      • Kao S.C.
      • Cary L.A.
      • Guan J.L.
      • Lai J.F.
      • Chen H.C.
      ,
      • Shen T.L.
      • Guan J.L.
      ) and it may be that FAK serves to enhance the association of these proteins with their downstream effectors by binding them within the focal adhesion (
      • Shen T.L.
      • Guan J.L.
      ). Indeed, recent studies have linked Rho activity with the targeting of Src to focal adhesion sites (
      • Timpson P.
      • Jones G.E.
      • Frame M.C.
      • Brunton V.G.
      ).
      As syndecan-4-null cells are deficient in active Rho and phosphorylated FAK Tyr397 and show impaired cell migration (
      • Echtermeyer F.
      • Streit M.
      • Wilcox-Adelman S.
      • Saoncella S.
      • Denhez F.
      • Detmar M.
      • Goetinck P.
      ) it seems likely that syndecan-4 signaling is associated with cell migration. Decreased cell migration is associated with either lack of syndecan-4 expression or by overexpressing full-length syndecan-4 or syndecan-4 containing cytoplasmic deletion mutants (
      • Echtermeyer F.
      • Streit M.
      • Wilcox-Adelman S.
      • Saoncella S.
      • Denhez F.
      • Detmar M.
      • Goetinck P.
      ,
      • Longley R.L.
      • Woods A.
      • Fleetwood A.
      • Cowling G.J.
      • Gallagher J.T.
      • Couchman J.R.
      ). That both ends of the spectrum (overexpression of syndecan-4 and lack of syndecan-4) inhibit cell migration suggests that a homeostasis is generated through syndecan-4 signaling and a balance may be required for optimal cell migration. We hypothesize that under conditions in which syndecan-4 is not ligated (FN-null cells seeded on the CBD or syndecan-4-null cells seeded on full-length FN) integrin ligation induces the phosphorylation of FAK Tyr397. Tyrosine phosphatase activity leads to the subsequent dephosphorylation of this tyrosine residue. The levels of GTP-bound Rho are also low. This situation is most likely not a static event but probably encourages the cycling of FAK between a phosphorylated and nonphosphorylated state on Tyr397. Ligation of syndecan-4 (whether in FN-null or syndecan-4-WT cells plated on full-length FN) results in the increased activation of Rho. This attenuates the tyrosine phosphatase activity causing FAK Tyr397 to remain phosphorylated longer. The tyrosine phosphatase activity is not completely inhibited, therefore, cycling between phosphorylation and dephosphorylation of Tyr397probably occurs but under a different kinetic. This is pictured diagrammatically in Fig. 6.
      Figure thumbnail gr6
      Figure 6Model of syndecan-4 signaling influencing FAK phosphorylation of Tyr397.A, ligation of both integrin and syndecan-4 (syndecan-4-WT cells on FN and FN-null cells on FN) lead to increased levels of active Rho and diminished activity of a tyrosine phosphatase. This shifts the equilibrium of FAK Tyr397 to the phosphorylated state. B, in the absence of syndecan-4 ligation (syndecan-4-null cells on FN) Rho is not activated. FAK is phosphorylated on Tyr397 but is subsequently dephosphorylated through the action of a tyrosine phosphatase. Under these conditions FAK potentially would be cycling between the phosphorylated and dephosphorylated state more rapidly than in the situation described in A. This scenario is also valid for FN-null cells seeded on CBD.

      Acknowledgments

      We thank Hui Su for confocal assistance and Stefania Saoncella, Tokuro Iwabuchi, and Enzo Calautti for helpful discussions.

      REFERENCES

        • Woods A.
        J. Clin. Invest. 2001; 107: 935-941
        • Woods A., Oh, E.-S.
        • Couchman J.R.
        Matrix Biol. 1998; 17: 477-483
        • Carey D.J.
        Biochem. J. 1997; 327: 1-16
        • Bernfield M.
        • Gotte M.
        • Park P.W.
        • Reizes O.
        • Fitzgerald M.L.
        • Lincecum J.
        • Zako M.
        Annu. Rev. Biochem. 1999; 68: 729-777
        • Elenius K.
        • Jalkanen M.
        J. Cell Sci. 1994; 107: 2975-2982
        • Ethell I.M.
        • Hagihara K.
        • Miura Y.
        • Irie F.
        • Yamaguchi Y.
        J. Cell Biol. 2000; 151: 53-68
        • Hsueh Y.-P.
        • Sheng M.
        J. Neurosci. 1999; 19: 7415-7425
        • Gao Y., Li, M.
        • Chen W.
        • Simons M.
        J. Cell. Physiol. 2000; 184: 373-379
        • Grootjans J.J.
        • Zimmermann P.
        • Reekmans G.
        • Smets A.
        • Degeest G.
        • Durr J.
        • David G.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13683-13688
        • Cohen D.J.
        • Woods D.F.
        • Marfatia S.M.
        • Walther Z.
        • Chishti A.H.
        • Anderson J.M.
        • Wood D.F.
        J. Cell Biol. 1998; 142: 129-138
        • Horowitz A.
        • Murakami M.
        • Gao Y.
        • Simons M.
        Biochemistry. 1999; 38: 15871-15877
        • Oh E.-S.
        • Woods A.
        • Couchman J.R.
        J. Biol. Chem. 1997; 272: 8133-8136
        • Baciu P.C.
        • Saoncella S.
        • Lee S.H.
        • Denhez F.
        • Leuthardt D.
        • Goetinck P.F.
        J. Cell Sci. 2000; 113: 315-324
        • Lebakken C.S.
        • McQuade K.J.
        • Rapraeger A.C.
        Exp. Cell Res. 2000; 259: 315-325
        • Utani A.
        • Nomizu M.
        • Matsuura H.
        • Kato K.
        • Kobayashi T.
        • Takeda U.
        • Aota S.
        • Nielsen P.K.
        • Shinkai H.
        J. Biol. Chem. 2001; 276: 28779-28788
        • Woods A.
        • Longley R.L.
        • Tumova S.
        • Couchman J.R.
        Arch. Biochem. Biophys. 2000; 374: 66-72
        • Woods A.
        • Couchman J.R.
        Mol. Biol. Cell. 1994; 5: 183-192
        • Baciu P.C.
        • Goetinck P.F.
        Mol. Biol. Cell. 1995; 6: 1503-1513
        • Burridge K.
        • Chrzanowska-Wodnicka M.
        Annu. Rev. Cell Dev. Biol. 1996; 12: 463-519
        • Zamir E.
        • Geiger B.
        J. Cell Sci. 2001; 114: 3583-3590
        • Petit V.
        • Thiery J.P.
        Biol. Cell. 2000; 92: 477-494
        • Woods A.
        • Couchman J.R.
        • Johansson S.
        • Hook M.
        EMBO J. 1986; 5: 665-670
        • Bloom L.
        • Ingham K.C.
        • Hynes R.O.
        Mol. Biol. Cell. 1999; 10: 1521-1536
        • 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: 2805-2810
        • Ishiguro K.
        • Kadomatsu K.
        • Kojima T.
        • Muramatsu H.
        • Tsuzuki S.
        • Nakamura E.
        • Kusugami K.
        • Saito H.
        • Muramatsu T.
        J. Biol. Chem. 2000; 275: 5249-5252
        • Echtermeyer F.
        • Streit M.
        • Wilcox-Adelman S.
        • Saoncella S.
        • Denhez F.
        • Detmar M.
        • Goetinck P.
        J. Clin. Invest. 2001; 107: R9-R14
        • Ishiguro 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: 47483-47488
        • Ishiguro K.
        • Kadomatsu K.
        • Kojima T.
        • Muramatsu H.
        • Matsuo S.
        • Kusugami K.
        • Saito H.
        • Muramatsu T.
        Lab. Invest. 2001; 81: 509-516
        • Palecek S.P.
        • Loftus J.C.
        • Ginsberg M.H.
        • Lauffenburger D.A.
        • Horwitz A.F.
        Nature. 1997; 385: 537-540
        • Ilic D.
        • Furuta Y.
        • Kanazawa S.
        • Takeda N.
        • Sobue K.
        • Nakatsuji N.
        • Nomura S.
        • Fujimoto J.
        • Okada M.
        • Yamamoto T.
        • Aizawa S.
        Nature. 1995; 377: 539-544
        • Ren X.D.
        • Kiosses W.B.
        • Sieg D.J.
        • Otey C.A.
        • Schlaepfer D.D.
        • Schwartz M.A.
        J. Cell Sci. 2000; 113: 3673-3678
        • Ilic D.
        • Kanazawa S.
        • Furuta Y.
        • Yamamoto T.
        • Aizawa S.
        Exp. Cell Res. 1996; 222: 298-303
        • Sieg D.J.
        • Ilic D.
        • Jones K.C.
        • Damsky C.H.
        • Hunter T.
        • Schlaepfer D.D.
        EMBO J. 1998; 17: 5933-5947
        • Owen J.D.
        • Ruest P.J.
        • Fry D.W.
        • Hanks S.K.
        Mol. Cell. Biol. 1999; 19: 4806-4818
        • Cary L.A.
        • Chang J.F.
        • Guan J.-L.
        J. Cell Sci. 1996; 109: 1787-1794
        • Sieg D.J.
        • Hauck C.R.
        • Schlaepfer D.D.
        J. Cell Sci. 1999; 112: 2677-2691
        • Zachary I.
        Int. J. Biochem. Cell Biol. 1997; 29: 929-934
        • Schaller M.D.
        • Hildebrand J.D.
        • Shannon J.D.
        • Fox J.W.
        • Vines R.R.
        • Parsons J.T.
        Mol. Cell. Biol. 1994; 14: 1680-1688
        • Miyamoto S.
        • Teramoto H.
        • Coso O.A.
        • Gutkind J.S.
        • Burbelo P.D.
        • Akiyama S.K.
        • Yamada K.M.
        J. Cell Biol. 1995; 131: 791-805
        • Otey C.A.
        Int. Rev. Cytol. 1996; 167: 161-183
        • Schlaepfer D.D.
        • Hauck C.R.
        • Sieg D.J.
        Prog. Biophys. Mol. Biol. 1999; 71: 435-478
        • Chen H.-C.
        • Appeddu P.A.
        • Isoda H.
        • Guan J.-L.
        J. Biol. Chem. 1996; 271: 26329-26334
        • Zhang 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: 9021-9026
        • Han D.C.
        • Guan J.-L.
        J. Biol. Chem. 1999; 274: 24425-24430
        • Schlaepfer D.D.
        • Jones K.C.
        • Hunter T.
        Mol. Cell. Biol. 1998; 18: 2571-2585
        • Tamura M., Gu, J.
        • Danen E.H.
        • Takino T.
        • Miyamoto S.
        • Yamada K.M.
        J. Biol. Chem. 1999; 274: 20693-20703
        • Schaller M.D.
        Biochim. Biophys. Acta. 2001; 1540: 1-21
        • Calalb M.B.
        • Polte T.R.
        • Hanks S.K.
        Mol. Cell. Biol. 1995; 15: 954-963
        • Calalb M.B.
        • Zhang X.
        • Polte T.R.
        • Hanks S.K.
        Biochem. Biophys. Res. Commun. 1996; 228: 662-668
        • Schlaepfer D.D.
        • Hanks S.K.
        • Hunter T.
        • van der Geer P.
        Nature. 1994; 372: 786-791
        • Schlaepfer D.D.
        • Hunter T.
        Mol. Cell. Biol. 1996; 16: 5623-5633
        • Kumagai N.
        • Morii N.
        • Fujisawa K.
        • Yoshimasa T.
        • Nakao K.
        • Narumiya S.
        FEBS Lett. 1993; 329: 273-276
        • Chrzanowska-Wodnicka M.
        • Burridge K.
        J. Cell Sci. 1994; 107: 3643-3654
        • Ridley A.J.
        • Hall A.
        EMBO J. 1994; 13: 2600-2610
        • Barry S.T.
        • Critchley D.R.
        J. Cell Sci. 1994; 107: 2033-2045
        • Flinn H.M.
        • Ridley A.J.
        J. Cell Sci. 1996; 109: 1133-1141
        • Rodriguez-Fernandez J.L.
        • Rozengurt E.
        J. Biol. Chem. 1998; 273: 19321-19328
        • Clark E.A.
        • King W.G.
        • Brugge J.S.
        • Symons M.
        • Hynes R.O.
        J. Cell Biol. 1998; 142: 573-586
        • Burridge K.
        • Turner C.E.
        • Romer L.H.
        J. Cell Biol. 1992; 119: 893-903
        • Jeong J.
        • Han I.
        • Lim Y.
        • Kim J.
        • Park I.
        • Woods A.
        • Couchman J.R.
        • Oh E.S.
        Biochem. J. 2001; 356: 531-537
        • Tumova S.
        • Woods A.
        • Couchman J.R.
        J. Biol. Chem. 2000; 275: 9410-9417
        • Rapraeger A.C.
        • Ott V.L.
        J. Biol. Chem. 1998; 273: 35291-35298
        • Xu F.
        • Zhao Z.J.
        Exp. Cell Res. 2001; 262: 49-58
        • Small J.V.
        • Celis J.E.
        J. Cell Sci. 1978; 31: 393-409
        • Reid T.
        • Furuyashiki T.
        • Ishizaki T.
        • Watanabe G.
        • Watanabe N.
        • Fujisawa K.
        • Morii N.
        • Madaule P.
        • Narumiya S.
        J. Biol. Chem. 1996; 271: 13556-13560
        • Vuori K.
        • Ruoslahti E.
        J. Biol. Chem. 1993; 268: 21459-21462
        • Brown P.J.
        Biochem. Biophys. Res. Commun. 1988; 155: 603-607
        • Woods A.
        • Courchman J.R.
        J. Cell Sci. 1992; 101: 277-290
        • Bruce-Staskal P.J.
        • Bouton A.H.
        Exp. Cell Res. 2001; 264: 296-306
        • Ridley A.J.
        • Hall A.
        Cell. 1992; 70: 389-399
        • Longley R.L.
        • Woods A.
        • Fleetwood A.
        • Cowling G.J.
        • Gallagher J.T.
        • Couchman J.R.
        J. Cell Sci. 1999; 112: 3421-3431
        • Zohn I.M.
        • Campbell S.L.
        • Khosravi-Far R.
        • Rossman K.L.
        • Der C.J.
        Oncogene. 1998; 17: 1415-1438
        • Ridley A.J.
        J. Cell Sci. 2001; 114: 2713-2722
        • Mackay D.J.
        • Hall A.
        J. Biol. Chem. 1998; 273: 20685-20688
        • Ren X.-D.
        • Kiosses W.B.
        • Schwartz M.A.
        EMBO J. 1999; 18: 578-585
        • Echtermeyer F.
        • Baciu P.C.
        • Saoncella S., Ge, Y.
        • Goetinck P.F.
        J. Cell Sci. 1999; 112: 3433-3441
        • Tamura M., Gu, J.
        • Matsumoto K.
        • Aota S.-I.
        • Parsons R.
        • Yamada K.M.
        Science. 1998; 280: 1614-1617
        • Angers-Loustau A.
        • Cote J.-F.
        • Charest A.
        • Dowbenko D.
        • Spencer S.
        • Lasky L.A.
        • Trembley M.L.
        J. Cell Biol. 1999; 144: 1019-1031
        • Manes S.
        • Mira E.
        • Gomez-Mouton C.
        • Zhao Z.J.
        • Lacalle R.A.
        • Martinez A.C.
        Mol. Cell. Biol. 1999; 19: 3125-3135
        • Oh E.S.
        • Woods A.
        • Lim S.T.
        • Theibert A.W.
        • Couchman J.R.
        J. Biol. Chem. 1998; 273: 10624-10629
        • Oh E.-S.
        • Woods A.
        • Couchman J.R.
        J. Biol. Chem. 1997; 272: 11805-11811
        • Volk R.
        • Schwartz J.J., Li, J.
        • Rosenberg R.D.
        • Simons M.
        J. Biol. Chem. 1999; 274: 24417-24424
        • Horowitz A.
        • Simons M.
        J. Biol. Chem. 1998; 273: 10914-10918
        • Matsumoto Y.
        • Tanaka K.
        • Harimaya K.
        • Nakatani F.
        • Matsuda S.
        • Iwamoto Y.
        Jpn. J. Cancer Res. 2001; 92: 429-438
        • Bobak D.
        • Moorman J.
        • Guanzon A.
        • Gilmer L.
        • Hahn C.
        Oncogene. 1997; 15: 2179-2189
        • Imamura F.
        • Mukai M.
        • Ayaki M.
        • Akedo H.
        Jpn. J. Cancer Res. 2000; 91: 811-816
        • Geiger B.
        • Bershadsky A.
        Curr. Opin. Cell Biol. 2001; 13: 584-592
        • Yoshioka K.
        • Matsumura F.
        • Akedo H.
        • Itoh K.
        J. Biol. Chem. 1998; 273: 5146-5154
        • Takaishi K.
        • Kikuchi A.
        • Kuroda S.
        • Kotani K.
        • Sasaki T.
        • Takai Y.
        Mol. Cell. Biol. 1993; 13: 72-79
        • Stasia M.J.
        • Jouan A.
        • Bourmeyster N.
        • Boquet P.
        • Vignais P.V.
        Biochem. Biophys. Res. Commun. 1991; 180: 615-622
        • Nabi I.R.
        J. Cell Sci. 1999; 112: 1803-1811
        • Machesky L.M.
        • Hall A.
        J. Cell Biol. 1997; 138: 913-926
        • Wang H.B.
        • Dembo M.
        • Hanks S.K.
        • Wang Y.
        Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11295-11300
        • Reiske H.R.
        • Kao S.C.
        • Cary L.A.
        • Guan J.L.
        • Lai J.F.
        • Chen H.C.
        J. Biol. Chem. 1999; 274: 12361-12366
        • Shen T.L.
        • Guan J.L.
        FEBS Lett. 2001; 499: 176-181
        • Timpson P.
        • Jones G.E.
        • Frame M.C.
        • Brunton V.G.
        Curr. Biol. 2001; 11: 1836-1846