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Insulin-like Growth Factor I Stimulates Tyrosine Phosphorylation of p130Cas, Focal Adhesion Kinase, and Paxillin

ROLE OF PHOSPHATIDYLINOSITOL 3′-KINASE AND FORMATION OF A p130Cas·Crk COMPLEX*
  • Adele Casamassima
    Footnotes
    Affiliations
    Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, United Kingdom and Department of Medicine, School of Medicine and Molecular Biology Institute, University of California, Los Angeles, California 90095-1786
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  • Enrique Rozengurt
    Correspondence
    To whom correspondence should be addressed: 900 Veteran Ave., Warren Hall, Rm. 11-124, Dept. of Medicine, School of Medicine, University of California, Los Angeles, CA 90095-1786. Tel.: 310-794-6610; Fax: 310-267-2399
    Affiliations
    Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, United Kingdom and Department of Medicine, School of Medicine and Molecular Biology Institute, University of California, Los Angeles, California 90095-1786
    Search for articles by this author
  • Author Footnotes
    * 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.
    ‡ Present address: Dipartimento di Biologia e Patologia Cellulare e Molecolare, Universitá Federico II, via Pansini 5, 80131 Naples, Italy.
Open AccessPublished:October 02, 1998DOI:https://doi.org/10.1074/jbc.273.40.26149
      Addition of insulin growth factor-I (IGF-I) to quiescent Swiss 3T3 cells rapidly induced tyrosine phosphorylation of the p130Crk-associated substrate (p130Cas), a novel adaptor protein localized at focal adhesions. Half-maximal effect was obtained at 0.6 nm. IGF-I also promoted the formation of a complex between p130Cas and c-Crk and elicited a parallel increase in the tyrosine phosphorylation of p125Fak and paxillin. IGF-I-induced p130Cas, p125Fak, and paxillin tyrosine phosphorylation could be dissociated from mitogen-activated protein kinase kinase, p70S6K, and protein kinase C activation. In contrast, the structurally unrelated phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 markedly attenuated the increase in tyrosine phosphorylation of p130Cas, p125Fak, and paxillin induced by IGF-I. Cytochalasin D, which disrupts the network of actin microfilaments, completely prevented tyrosine phosphorylation of p130Cas, p125Fak, and paxillin and the formation of a p130Cas·Crk complex in response to IGF-I. Thus, our results identified a phosphatidylinositol 3-kinase-dependent pathway that requires the integrity of the actin cytoskeleton to induce tyrosine phosphorylation of p130Cas, p125Fak, and paxillin in response to IGF-I and suggest that tyrosine phosphorylation of these focal adhesion proteins, together with the recruitment of c-Crk into a complex with p130Cas, may play a novel role in IGF-I signal transduction.
      IGF-I
      insulin-like growth factor I
      anti-Tyr(P)
      anti-phosphotyrosine
      DMEM
      Dulbecco's modified Eagle's medium
      mAb
      monoclonal antibody
      p125Fak
      p125 focal adhesion kinase
      p130Cas
      p130 Crk-associated substrate
      PAGE
      polyacrylamide gel electrophoresis
      PDBu
      phorbol 12,13-dibutyrate
      PI 3-kinase
      phosphatidylinositol 3-kinase
      PKC
      protein kinase C
      SH2 and SH3
      Src homology domain 2 and 3, respectively
      PDGF
      platelet-derived growth factor.
      The insulin-like growth factor-1 (IGF-I)1 is implicated in many normal and abnormal processes including development, cell growth, and malignant transformation (
      • Baserga R.
      ,
      • Blakesley V.A.
      • Kalebic T.
      • Helman L.J.
      • Stannard B.
      • Faria T.N.
      • Roberts Jr., C.T.
      • LeRoith D.
      ,
      • Kulik G.
      • Klippel A.
      • Weber M.J.
      ,
      • Baserga R.
      • Resnicoff M.
      • D'Ambrosio C.
      • Valentinis B.
      ). The initial event in IGF-I signaling involves its binding to the IGF-I receptor, an α2 β2 heterotetramer with ligand-dependent tyrosine kinase activity (
      • LeRoith D.
      ). The insulin receptor substrates 1 and 2 are rapidly phosphorylated on multiple tyrosine residues in response to IGF-I (
      • Myers Jr., M.G.
      • Grammer T.C.
      • Wang L.M.
      • Sun X.J.
      • Pierce J.H.
      • Blenis J.
      • White M.F.
      ,
      • Patti M.-E.
      • Sun X.-J.
      • Bruening J.C.
      • Araki E.
      • Lipes M.A.
      • White M.F.
      • Kahn C.R.
      ) and act as docking proteins that bind, through their phosphorylated residues, SH2 domain-containing proteins which propagate the IGF-I signal (
      • Brüning J.C.
      • Winnay J.
      • Cheatham B.
      • Kahn C.R.
      ). These include the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) and the adaptor proteins Grb-2 and Shc which lead to activation of the Ras/Raf/ERK pathway (
      • LeRoith D.
      ,
      • Kasus-Jacobi A.
      • Perdereau D.
      • Tartare-Deckert S.
      • Van Obberghen E.
      • Girard J.
      • Burnol A.-F.
      ). In addition, IGF-I also promotes cell migration of normal and tumor cells and induces the recruitment of actin into membrane ruffles (
      • Bornfeldt K.E.
      • Raines E.W.
      • Nakano T.
      • Graves L.M.
      • Krebs E.G.
      • Ross R.
      ,
      • Kotani K.
      • Yonezawa K.
      • Hara K.
      • Ueda H.
      • Kitamura Y.
      • Sakaue H.
      • Ando A.
      • Chavanieu A.
      • Calas B.
      • Grigorescu F.
      • Nishiyama M.
      • Waterfield M.D.
      • Kasuga M.
      ,
      • Leventhal P.S.
      • Shelden E.A.
      • Kim B.
      • Feldman E.L.
      ,
      • Brooks P.C.
      • Klemke R.L.
      • Schon S.
      • Lewis J.M.
      • Schwartz M.A.
      • Cheresh D.A.
      ).
      Changes in the organization of the actin cytoskeleton induced by many extracellular factors are accompanied by striking changes in the tyrosine phosphorylation of several signaling proteins localized at the focal adhesion plaques (
      • Rozengurt E.
      ,
      • Rozengurt E.
      • Rodriguez-Fernandez J.L.
      ), the distinct sites of the plasma membrane that are in close contact with the extracellular matrix proteins (
      • Burridge K.
      • Chrzanowska-Wodnicka M.
      ). Specifically, the tyrosine phosphorylation of the non-receptor tyrosine kinase p125Fak (
      • Hanks S.K.
      • Calalb M.B.
      • Harper M.C.
      • Patel S.K.
      ,
      • Schaller M.D.
      • Borgman C.A.
      • Cobb B.S.
      • Vines R.R.
      • Reynolds A.B.
      • Parsons J.T.
      ) and of the adaptor proteins p130Cas (
      • Sakai R.
      • Iwamatsu A.
      • Hirano N.
      • Ogawa S.
      • Tanaka T.
      • Mano H.
      • Yazaki Y.
      • Hirai H.
      ) and paxillin (
      • Salgia R.
      • Li J.-L.
      • Lo S.H.
      • Brunkhorst B.
      • Kansas G.S.
      • Sobhany E.S.
      • Sun Y.
      • Pisick E.
      • Hallek M.
      • Ernst T.
      • Tantravahi R.
      • Chen L.B.
      • Griffin J.D.
      ,
      • Turner C.E.
      • Miller J.T.
      ) is increased by diverse signaling molecules that regulate cell growth, differentiation, and migration including neuropeptide agonists (
      • Zachary I.
      • Sinnett-Smith J.
      • Rozengurt E.
      ,
      • Zachary I.
      • Sinnett-Smith J.
      • Turner C.E.
      • Rozengurt E.
      ,
      • Sinnett-Smith J.
      • Zachary I.
      • Valverde A.M.
      • Rozengurt E.
      ,
      • Seufferlein T.
      • Withers D.J.
      • Broad S.
      • Herget T.
      • Walsh J.H.
      • Rozengurt E.
      ,
      • Casamassima A.
      • Rozengurt E.
      ), bioactive lipids (
      • Casamassima A.
      • Rozengurt E.
      ,
      • Seufferlein T.
      • Rozengurt E.
      ,
      • Seufferlein T.
      • Rozengurt E.
      ,
      • Seufferlein T.
      • Rozengurt E.
      ), and growth factors (
      • Casamassima A.
      • Rozengurt E.
      ,
      • Rankin S.
      • Rozengurt E.
      ,
      • Rankin S.
      • Hooshmand-Rad R.
      • Claesson-Welsh L.
      • Rozengurt E.
      ,
      • Ojaniemi M.
      • Vuori K.
      ). The increase in the tyrosine phosphorylation of focal adhesion proteins induced by these agents is critically dependent on the integrity of the actin cytoskeleton (
      • Sinnett-Smith J.
      • Zachary I.
      • Valverde A.M.
      • Rozengurt E.
      ,
      • Seufferlein T.
      • Rozengurt E.
      ,
      • Seufferlein T.
      • Rozengurt E.
      ,
      • Rankin S.
      • Rozengurt E.
      ,
      • Seufferlein T.
      • Withers D.J.
      • Mann D.
      • Rozengurt E.
      ) and is mediated by PI 3-kinase- and PKC-dependent and -independent pathways (
      • Sinnett-Smith J.
      • Zachary I.
      • Valverde A.M.
      • Rozengurt E.
      ,
      • Seufferlein T.
      • Rozengurt E.
      ,
      • Rankin S.
      • Hooshmand-Rad R.
      • Claesson-Welsh L.
      • Rozengurt E.
      ). Tyrosine phosphorylation of p125Fak, p130Cas, and paxillin is also induced by extracellular matrix proteins (
      • Hanks S.K.
      • Calalb M.B.
      • Harper M.C.
      • Patel S.K.
      ,
      • Burridge K.
      • Turner C.E.
      • Romer L.H.
      ,
      • Lipfert L.
      • Haimovich B.
      • Schaller M.D.
      • Cobb B.S.
      • Parsons J.T.
      • Brugge J.S.
      ,
      • Vuori K.
      • Ruoslahti E.
      ) and transforming variants of p60src (
      • Guan J.L.
      • Shalloway D.
      ). Thus, the phosphorylation of these focal adhesion proteins represents a point of convergence in the action of growth factors, integrins, and oncogenes (
      • Rozengurt E.
      ,
      • Zachary I.
      • Rozengurt E.
      ).
      In contrast to other growth factors, insulin has been shown to decrease the level of tyrosine phosphorylation of p125Fak and paxillin in a variety of cell types (
      • Knight J.B.
      • Yamauchi K.
      • Pessin J.E.
      ,
      • Pillay T.S.
      • Sasaoka T.
      • Olefsky J.M.
      ,
      • Tobe K.
      • Sabe H.
      • Yamamoto T.
      • Yamauchi T.
      • Asai S.
      • Kaburagi Y.
      • Tamemoto H.
      • Ueki K.
      • Kimura H.
      • Akanuma Y.
      • Yazaki Y.
      • Hanafusa H.
      • Kadowaki T.
      ,
      • Konstantopoulos N.
      • Clark S.
      ,
      • Ouwens D.M.
      • Mikkers H.M.
      • van der Zon G.C.
      • Stein-Gerlach M.
      • Ullrich A.
      • Maassen J.A.
      ) through pathways involving Csk (
      • Tobe K.
      • Sabe H.
      • Yamamoto T.
      • Yamauchi T.
      • Asai S.
      • Kaburagi Y.
      • Tamemoto H.
      • Ueki K.
      • Kimura H.
      • Akanuma Y.
      • Yazaki Y.
      • Hanafusa H.
      • Kadowaki T.
      ), phosphotyrosine phosphatase 1D (
      • Ouwens D.M.
      • Mikkers H.M.
      • van der Zon G.C.
      • Stein-Gerlach M.
      • Ullrich A.
      • Maassen J.A.
      ), and integrins (
      • Baron V.
      • Calleja V.
      • Ferrari P.
      • Alengrin F.
      • Van Obberghen E.
      ). The influence of IGF-I on the tyrosine phosphorylation of p125Fak and paxillin has been less studied and is the subject of conflicting reports (
      • Leventhal P.S.
      • Shelden E.A.
      • Kim B.
      • Feldman E.L.
      ,
      • Baron V.
      • Calleja V.
      • Ferrari P.
      • Alengrin F.
      • Van Obberghen E.
      ,
      • Konstantopoulos N.
      • Clark S.
      ). The effect of IGF-I on the tyrosine phosphorylation of p130Cas, a novel adaptor protein implicated in transformation (
      • Sakai R.
      • Iwamatsu A.
      • Hirano N.
      • Ogawa S.
      • Tanaka T.
      • Mano H.
      • Yazaki Y.
      • Hirai H.
      ) and recently identified as a critical mediator of cell migration (
      • Cary L.A.
      • Han D.C.
      • Polte T.R.
      • Hanks S.K.
      • Guan J.L.
      ,
      • Klemke R.L.
      • Leng J.
      • Molander R.
      • Brooks P.C.
      • Vuori K.
      • Cheresh D.A.
      ), is unknown.
      In the present study we report, for the first time, that IGF-I induces a rapid and transient increase in the tyrosine phosphorylation of p130Cas in Swiss 3T3 cells, a useful model system to elucidate signal transduction pathways in the action of growth factors (
      • Rozengurt E.
      ). Our results also show that IGF-I promotes the formation of a complex between p130Cas and c-Crk and elicits a coordinate increase in the tyrosine phosphorylation of p125Fak and paxillin. IGF-I stimulates tyrosine phosphorylation of these focal adhesion proteins through a PI 3-kinase-dependent pathway that requires the integrity of the actin cytoskeleton.

      DISCUSSION

      There is increasing evidence indicating that IGF-I signaling is involved in multiple biological processes including cell migration, proliferation, and transformation, but the downstream targets that mediate these effects have not been fully identified. The findings presented here demonstrate that IGF-I induces tyrosine phosphorylation of the adaptor molecule p130Cas in Swiss 3T3 cells. The rapidity and the low, physiological concentrations of IGF-I inducing this effect suggest that this event may be functionally important in the biological action of IGF-I. Our results also demonstrate that IGF-I induces tyrosine phosphorylation of p125Fak and paxillin with kinetics that parallel those of p130Cas tyrosine phosphorylation.
      The signal transduction events initiated by the IGF-IR have been extensively investigated, but the pathway(s) leading to tyrosine phosphorylation of the focal adhesion proteins p130Cas, p125Fak and paxillin by IGF-I was unknown. Given that the mitogen-activated protein kinase kinase-1/ERK and PI 3-kinase pathways have emerged as major mediators of the biological effects promoted by IGF-I, we examined their role in IGF-mediated tyrosine phosphorylation of focal adhesion proteins. We found that IGF-I stimulated tyrosine phosphorylation of p130Cas, p125Fak, and paxillin independently of ERK activation. In contrast, the structurally unrelated PI 3-kinase inhibitors wortmannin and LY294002 markedly attenuated the increase in the tyrosine phosphorylation of p130Cas, p125Fak, and paxillin induced by IGF-I. Thus, our results identify a PI 3-kinase-dependent pathway in the tyrosine phosphorylation of these focal adhesion proteins in response to IGF-I stimulation.
      PI 3-kinase activity is required for the formation of membrane ruffles by several motility-inducing growth factors (
      • Kotani K.
      • Yonezawa K.
      • Hara K.
      • Ueda H.
      • Kitamura Y.
      • Sakaue H.
      • Ando A.
      • Chavanieu A.
      • Calas B.
      • Grigorescu F.
      • Nishiyama M.
      • Waterfield M.D.
      • Kasuga M.
      ,
      • Wennström S.
      • Siegbahn A.
      • Yokote K.
      • Arvidsson A.K.
      • Heldin C.H.
      • Mori S.
      • Claesson-Welsh L.
      ), and the lipid products of this enzyme have been shown to induce actin reorganization and to increase cell migration (
      • Derman M.P.
      • Toker A.
      • Hartwig J.H.
      • Spokes K.
      • Falck J.R.
      • Chen C.-S.
      • Cantley L.C.
      • Cantley L.G.
      ). The small GTP-binding protein Rac is known to mediate the recruitment of actin into membrane ruffles induced by extracellular stimuli (
      • Hall A.
      ,
      • Han J.
      • Luby-Phelps K.
      • Das B.
      • Shu X.
      • Xia Y.
      • Mosteller R.D.
      • Krishna U.M.
      • Falck J.R.
      • White M.A.
      • Broek D.
      ). Previous studies have demonstrated the existence of a signal transduction pathway in the action of PDGF and epidermal growth factor involving PI 3-kinase, Rac, and actin cytoskeletal reorganization that leads to the tyrosine phosphorylation of p130Cas, p125Fak, and paxillin (
      • Casamassima A.
      • Rozengurt E.
      ,
      • Rankin S.
      • Hooshmand-Rad R.
      • Claesson-Welsh L.
      • Rozengurt E.
      ,
      • Ojaniemi M.
      • Vuori K.
      ). The results presented here show that treatment of Swiss 3T3 cells with either cytochalasin D or high concentrations of PDGF, at concentrations known to disrupt the organization of the actin cytoskeleton (
      • Sinnett-Smith J.
      • Zachary I.
      • Valverde A.M.
      • Rozengurt E.
      ,
      • Rankin S.
      • Rozengurt E.
      ), abrogated the increase in the tyrosine phosphorylation of these focal adhesion proteins induced by IGF-I. Our results reveal a novel cross-talk between IGF-I and PDGF and indicate that an intact actin cytoskeleton is essential for IGF-I-induced tyrosine phosphorylation of p130Cas, p125Fak, and paxillin via the PI 3-kinase pathway. The integrity of the actin cytoskeleton is likely to be required for promoting the assembly of the focal contacts (
      • Burridge K.
      • Chrzanowska-Wodnicka M.
      ), the distinct sites of the plasma membrane where p130Cas, p125Fak, and paxillin are recruited and become tyrosine-phosphorylated.
      The signaling pathways that lie downstream of PI 3-kinase also include the serine/threonine kinases p70S6K (
      • Grammer T.C.
      • Cheatham L.
      • Chou M.M.
      • Blenis J.
      ) and the novel and atypical isoforms of PKC (

      Toker, A., and Cantley, L. C. (1997) Nature387,

      ). Our results demonstrate that inhibition of either p70S6K or PKC activation, with the immunosuppressant rapamycin or the specific PKC antagonist GF 109203X, respectively, did not prevent IGF-I-stimulated tyrosine phosphorylation of p130Cas, p125Fak, and paxillin. Thus, IGF-I-mediated tyrosine phosphorylation of focal adhesion proteins is PI 3-kinase-dependent but can be dissociated from activation of either p70S6K or PKC.
      The results presented here also demonstrate that the tyrosine phosphorylation of p130Cas in response to bombesin is not prevented by either wortmannin or LY294002, at concentrations that virtually abolished the tyrosine phosphorylation of p130Casinduced by IGF-I. These results imply that there is a PI 3′-kinase-dependent and PI 3′-kinase-independent signal transduction pathway stimulating the tyrosine phosphorylation of focal adhesion proteins in the same cells.
      The molecular cloning of p130Cas revealed an adaptor protein that contains an SH3 domain, proline-rich regions, and a cluster of 15 putative SH2-binding motifs (
      • Sakai R.
      • Iwamatsu A.
      • Hirano N.
      • Ogawa S.
      • Tanaka T.
      • Mano H.
      • Yazaki Y.
      • Hirai H.
      ). This suggests that tyrosine-phosphorylated p130Cas may serve to promote the assembly of multiple SH2-containing molecules. In fact, p130Cas forms stable complexes with other signaling proteins, including c-Crk and c-Src, in an SH2-dependent manner (
      • Sakai R.
      • Iwamatsu A.
      • Hirano N.
      • Ogawa S.
      • Tanaka T.
      • Mano H.
      • Yazaki Y.
      • Hirai H.
      ,
      • Burnham M.R.
      • Harte M.T.
      • Richardson A.
      • Parsons J.T.
      • Bouton A.H.
      ,
      • Harte M.T.
      • Hildebrand J.D.
      • Burnham M.R.
      • Bouton A.H.
      • Parsons J.T.
      ,
      • Nakamoto T.
      • Sakai R.
      • Ozawa K.
      • Yazaki Y.
      • Hirai H.
      ) and associates with p125Fak, through its SH3 domain (
      • Polte T.R.
      • Hanks S.K.
      ). Recent reports have shown that integrin-dependent cell adhesion, neuropeptide agonists, and low concentrations of PDGF and epidermal growth factor stimulate association of p130Cas with c-Crk, an SH2 and SH3 domain-containing adaptor protein (
      • Casamassima A.
      • Rozengurt E.
      ,
      • Ojaniemi M.
      • Vuori K.
      ,
      • Vuori K.
      • Ruoslahti E.
      ,
      • Nojima Y.
      • Morino N.
      • Mimura T.
      • Hamasaki K.
      • Furuya H.
      • Sakai R.
      • Sato T.
      • Tachibana K.
      • Morimoto C.
      • Yazaki Y.
      • Hirai H.
      ).
      The results presented here demonstrate, for the first time, that IGF-I rapidly induces the formation of a p130Cas·c-Crk complex that is dependent on the integrity of the actin cytoskeleton. Interestingly, c-Crk has been implicated as a positive effector of IGF-I-mediated mitogenic signaling (
      • Beitner-Johnson D.
      • LeRoith D.
      ), but the mechanisms involved remain unclear. IGF-I has been shown to increase c-Crk dissociation from insulin receptor substrate-1 (
      • Beitner-Johnson D.
      • Blakesley V.A.
      • Shen-Orr Z.
      • Jimenez M.
      • Stannard B.
      • Wang L.M.
      • Pierce J.
      • LeRoith D.
      ), resulting in a pool of c-Crk available for new molecular interactions. The assembly of a p130Cas·c-Crk complex, as shown by our results, is one of the new interactions of c-Crk triggered by IGF-I. The complex between p130Cas and c-Crk may be important in regulating the subcellular distribution of c-Crk and/or the activity of new downstream effectors in IGF-I signal transduction pathways.
      The findings presented here assume an added interest in view of the increasing evidence implicating p130Cas and p125Fak in cell migration, proliferation, and transformation. The adaptor protein p130Cas also been implicated in agonist-stimulated mitogenesis (
      • Casamassima A.
      • Rozengurt E.
      ) and in cell transformation (
      • Sakai R.
      • Iwamatsu A.
      • Hirano N.
      • Ogawa S.
      • Tanaka T.
      • Mano H.
      • Yazaki Y.
      • Hirai H.
      ,
      • Auvinen M.
      • Paasinen-Sohns A.
      • Hirai H.
      • Andersson L.C.
      • Hölttä E.
      ), and it has recently been identified as a mediator of p125Fak-mediated cell migration (
      • Cary L.A.
      • Han D.C.
      • Polte T.R.
      • Hanks S.K.
      • Guan J.L.
      ). In this context, it is interesting that the formation of a p130Cas·c-Crk complex is emerging as a critical switch in the promotion of cell migration (
      • Klemke R.L.
      • Leng J.
      • Molander R.
      • Brooks P.C.
      • Vuori K.
      • Cheresh D.A.
      ). In addition, c-Crk binds to a number of signaling proteins through its SH3 terminal domain including C3G (
      • Gotoh T.
      • Hattori S.
      • Nakamura S.
      • Kitayama H.
      • Noda M.
      • Takai Y.
      • Kaibuchi K.
      • Matsui H.
      • Hatase O.
      • Takahashi H.
      • Kurata T.
      • Matsuda M.
      ), a guanine nucleotide exchange factor for Rap-1, which is a small GTP-binding protein that induces mitogenesis in Swiss 3T3 cells (
      • Yoshida Y.
      • Kawata M.
      • Miura Y.
      • Musha T.
      • Sasaki T.
      • Kikuchi A.
      • Takai Y.
      ). Thus, the increase in the tyrosine phosphorylation of p130Cas and the induction of complex formation between p130Cas and c-Crk may be important novel early events in IGF-I signal transduction leading to cell migration and mitogenesis.
      Gene disruption experiments have demonstrated a critical role of p125Fak in embryonic development, cell migration, and turnover of focal adhesions (
      • Illc D.
      • Furuta Y.
      • Kanazawa S.
      • Takeda N.
      • Sobue K.
      • Nakatsuji N.
      • Nomura S.
      • Fujimoto J.
      • Okada M.
      • Yamamoto T.
      • Aizawa S.
      ,
      • Cary L.A.
      • Chang J.F.
      • Guan J.L.
      ,
      • Ilic D.
      • Damsky C.H.
      • Yamamoto T.
      ). Furthermore, microinjection of dominant-negative fragments of p125Fak which displaces endogenous p125Fak from focal adhesions and prevents its activation inhibited cell motility and serum stimulation of DNA synthesis (
      • Gilmore A.P.
      • Romer L.H.
      ). In addition, there is increasing evidence linking overexpression of p125Fak to cell migration (
      • Cary L.A.
      • Chang J.F.
      • Guan J.L.
      ) and to the invasive properties of cancer cells (
      • Owens L.V.
      • Xu L.
      • Craven R.J.
      • Dent G.A.
      • Weiner T.M.
      • Kornberg L.
      • Liu E.T.
      • Cance W.G.
      ). IGF-I signaling is also of critical importance in cell proliferation, migration, and transformation (
      • Baserga R.
      • Hongo A.
      • Rubini M.
      • Prisco M.
      • Valentinis B.
      ). For example, IGF-I has been shown to stimulate tumor cell metastasis, a process that requires cell migration (
      • Brooks P.C.
      • Klemke R.L.
      • Schon S.
      • Lewis J.M.
      • Schwartz M.A.
      • Cheresh D.A.
      ). Consequently, our results showing that IGF induces a coordinate increase in the level of tyrosine phosphorylation of p130Cas, p125Fak, and paxillin and the formation of a complex between p130Cas and c-Crk through a PI 3-kinase-dependent signaling pathway suggest novel mechanisms of action for this important growth promoting factor.

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