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The Proto-oncogene Product p120cbl Links c-Src and Phosphatidylinositol 3′-Kinase to the Integrin Signaling Pathway*

  • Marja Ojaniemi
    Affiliations
    La Jolla Cancer Research Center, The Burnham Institute, La Jolla, California 92037 and the
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  • Stuart S. Martin
    Affiliations
    § Division of Endocrinology and Metabolism, Department of Medicine, University of California at San Diego, La Jolla, California 92093
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  • Fabrizio Dolfi
    Footnotes
    Affiliations
    La Jolla Cancer Research Center, The Burnham Institute, La Jolla, California 92037 and the
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  • Jerrold M. Olefsky
    Affiliations
    § Division of Endocrinology and Metabolism, Department of Medicine, University of California at San Diego, La Jolla, California 92093
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  • Kristiina Vuori
    Correspondence
    To whom correspondence should be addressed
    Affiliations
    La Jolla Cancer Research Center, The Burnham Institute, La Jolla, California 92037 and the
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  • Author Footnotes
    * This work was supported in part by the Finnish Academy of Sciences and by National Institutes of Health Grants CA71560 (to K. V.) and DK33651 (to J. M. O). 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.
    Recipient of a postdoctoral fellowship from the Associazione Italiana per la Ricerca sul Cancro.
Open AccessPublished:February 07, 1997DOI:https://doi.org/10.1074/jbc.272.6.3780
      Integrin-mediated cell adhesion triggers intracellular signaling cascades, including tyrosine phosphorylation of intracellular proteins. We show in this report that p120cbl (Cbl), the 120-kDa c-cbl proto-oncogene product, becomes tyrosine-phosphorylated during integrin-mediated macrophage cell adhesion to extracellular matrix substrata and anti-integrin antibodies. This tyrosine phosphorylation does not occur when cells attach to polylysine, to which cells adhere in a nonspecific fashion. It also does not take place when adhesion-induced reorganization of the cytoskeleton is inhibited with cytochalasin D. In contrast to the rapid and transient tyrosine phosphorylation of Cbl by CSF-1 stimulation, tyrosine phosphorylation of Cbl by cell attachment was gradual and persistent. Tyrosine-phosphorylated Cbl was found to form complexes with the SH2 domain-containing signaling proteins Src and phosphatidylinositol 3-kinase; in vitro kinase assays demonstrated that these kinases were active in the Cbl complexes following integrin ligand binding. Furthermore, Cbl was found to translocate to the plasma membrane in response to cell adhesion to fibronectin. These observations suggest that Cbl serves as a docking protein and may transduce signals to downstream signaling pathways following integrin-mediated cell adhesion in macrophages.

      INTRODUCTION

      Cell-extracellular matrix (ECM)
      The abbreviations used are: ECM
      extracellular matrix
      FAK
      focal adhesion kinase
      PI
      phosphatidylinositol
      RIPA
      radioimmune precipitation assay
      GST
      glutathione S-transferase
      Pipes
      1,4-piperazinediethanesulfonic acid
      PAGE
      polyacrylamide gel electrophoresis.
      interactions play an important role in a variety of biological processes, including cell growth, differentiation, and migration. Integrins compose the major class of receptors used by cells to interact with ECM proteins (,
      • Ruoslahti E.
      ). The integrin family currently consists of over 20 distinct α/β heterodimeric transmembrane receptors, with the combination of a particular α and β subunit determining the ligand specificity (
      • Schwartz M.A.
      • Schaller M.D.
      • Ginsberg M.H.
      ). Upon ligand binding, integrins form clusters on the cell surface; this clustering takes place at cellular sites termed focal adhesions and leads to the assembly of intracellular multiprotein complexes associated with the actin cytoskeleton (
      • Burridge K.
      • Fath K.
      • Kelly T.
      • Nuckolls G.
      • Turner C.
      ). Focal adhesions are thought to act not only as structural links between the ECM and the actin cytoskeleton, but also as sites of signal transduction from the ECM; engagement of cell-surface integrins is associated with a rapid tyrosine phosphorylation of several focal adhesion proteins. In fibroblasts, these proteins include the focal adhesion kinase (FAK) (for a review, see Ref.
      • Clark E.A.
      • Brugge J.S.
      ), paxillin (
      • Burridge K.
      • Turner C.E.
      • Romer L.H.
      ), tensin (
      • Bockholt S.M.
      • Burridge K.
      ), and p130cas (
      • Petch L.A.
      • Bockholt S.M.
      • Bouton A.
      • Parsons J.T.
      • Burridge K.
      ,
      • Nojima Y.
      • Morino N.
      • Mimura T.
      • Hamasaki K.
      • Furuya H.
      • Sakai R.
      • Sato T.
      • Tachibana K.
      • Morimoto C.
      • Yazaki Y.
      • Hirai H.
      ,
      • Vuori K.
      • Ruoslahti E.
      ,
      • Vuori K.
      • Hirai H.
      • Aizawa S.
      • Ruoslahti E.
      ). Following tyrosine phosphorylation, these proteins engage in multiple protein-protein interactions by binding to signaling proteins containing SH2 domains, including Src family kinases, Csk, and the adaptor proteins Grb2 and Crk (
      • Vuori K.
      • Hirai H.
      • Aizawa S.
      • Ruoslahti E.
      ,
      • Cobb B.S.
      • Schaller M.D.
      • Leu T.H.
      • Parsons J.T.
      ,
      • Eide B.L.
      • Turck C.W.
      • Escobedo J.A.
      ,
      • Schaller M.D.
      • Hildebrand J.D.
      • Shannon J.D.
      • Fox J.W.
      • Vines R.R.
      • Parsons J.T.
      ,
      • Xing Z.
      • Chen H.C.
      • Nowlen J.K.
      • Taylor S.J.
      • Shalloway D.
      • Guan J.L.
      ,
      • Calalb M.B.
      • Polte T.R.
      • Hanks S.K.
      ,
      • Schlaepfer D.D.
      • Hanks S.K.
      • Hunter T.
      • van der Geer P.
      ,
      • Sabe H.
      • Hata A.
      • Okada M.
      • Nakagawa H.
      • Hanafusa H.
      ,
      • Schaller M.D.
      • Parsons J.T.
      ,
      • Birge R.B.
      • Fajardo J.E.
      • Reichman C.
      • Shoelson S.E.
      • Songyang Z.
      • Cantley L.C.
      • Hanafusa H.
      ,
      • Birge R.B.
      • Fajardo J.E.
      • Mayer B.J.
      • Hanafusa H.
      ,
      • Kanner S.B.
      • Reynolds A.B.
      • Wang H.C.
      • Vines R.R.
      • Parsons J.T.
      ,
      • Petruzzelli L.
      • Takami M.
      • Herrera R.
      ,
      • Nakamoto T.
      • Sakai R.
      • Ozawa K.
      • Yazaki Y.
      • Hirai H.
      ,
      • Bergman M.
      • Joukov V.
      • Virtanen I.
      • Alitalo K.
      ). Collectively, these observations indicate that ligand binding by integrins regulates the functions of multiple docking proteins that may transmit signals to downstream pathways.
      In this report, we have studied the tyrosine phosphorylation events taking place during integrin-mediated cell adhesion in macrophages. In myeloid cells, adherence to ECM components or ligation of integrins with antibodies results in a rapid induction of multiple inflammatory mediator genes, cytokines, and collagenases and in a modulation of the proliferative capacity and the phagocytic activity of these cells (for reviews, see Refs.
      • Mondal K.
      • Lofquist A.K.
      • Watson J.M.
      • Morris J.S.
      • Price L.K.
      • Haskill J.S.
      ,
      • Brown E.J.
      ,
      • Levesque J.P.
      • Hatzfeld A.
      • Hatzfeld J.
      ). In parallel to these events, monocyte/macrophage cell adhesion to ECM proteins is accompanied by a rapid increase in protein tyrosine phosphorylation (
      • Lin T.H.
      • Yurochko A.
      • Kornberg L.
      • Morris J.
      • Walker J.J.
      • Haskill S.
      • Juliano R.L.
      ); the protein-tyrosine kinase Syk (
      • Lin T.H.
      • Rosales C.
      • Mondal K.
      • Bolen J.B.
      • Haskill S.
      • Juliano R.L.
      ) and paxillin (
      • De Nichilo M.O.
      • Yamada K.M.
      ) have recently been identified to be among these proteins. FAK, although important in integrin signaling in many other cell types, including fibroblasts, may not play a crucial role in macrophages, as it is absent or expressed at low levels in these cells (
      • Lin T.H.
      • Yurochko A.
      • Kornberg L.
      • Morris J.
      • Walker J.J.
      • Haskill S.
      • Juliano R.L.
      ,
      • De Nichilo M.O.
      • Yamada K.M.
      ,
      • Choi K.
      • Kennedy M.
      • Keller G.
      ). Consistent with these reports, we found in this study that integrin ligand binding does not induce tyrosine phosphorylation of FAK in mouse macrophages. A profound increase in tyrosine phosphorylation of proteins with apparent molecular masses of 120-130 kDa, however, accompanied integrin-mediated cell adhesion. We sought to determine whether p120cbl (Cbl) might be among the 120-130-kDa tyrosine-phosphorylated proteins. Cbl, which was originally identified as the cellular homolog of the Cas NS-1 murine leukemia retroviral oncogene v-cbl, is a novel signaling molecule primarily expressed in hematopoietic cells. Cbl lacks any obvious catalytic domains, but it possesses multiple potential tyrosine phosphorylation sites and proline-rich motifs, which could mediate concurrent association with SH2 and SH3 domain-containing polypeptides, respectively. Cbl also has a carboxyl-terminal leucine zipper, a motif known to promote homo- and heterodimerization of other proteins (for a review, see Ref.
      • Langdon W.Y.
      ). Thus, Cbl is well suited for a potential role in assembling intracellular signaling complexes. More important, Cbl has been shown to be abundant in macrophages (
      • Rivero-Lezcano O.M.
      • Sameshima J.H.
      • Marcilla A.
      • Robbins K.C.
      ,
      • Tanaka S.
      • Neff L.
      • Baron R.
      • Levy J.B.
      ) and was recently identified as a major tyrosine kinase substrate following CSF-1 stimulation or engagement of Fcγ receptors in these cells (
      • Tanaka S.
      • Neff L.
      • Baron R.
      • Levy J.B.
      • Marcilla A.
      • Rivero-Lezcano O.M.
      • Agarwal A.
      • Robbins K.C.
      ). We report here that Cbl is a predominant phosphorylated component in macrophages upon cell adhesion and forms complexes with SH2 domain-containing signaling molecules, such as Src and PI 3-kinase. Thus, protein-protein interactions mediated by Cbl may connect integrin signaling to downstream signaling pathways in macrophages.

      DISCUSSION

      In macrophages as well as in other cell systems, a limited number of proteins have been shown to undergo tyrosine phosphorylation in response to cell adhesion and spreading on extracellular matrix substrata. We show here that p120cbl (Cbl) is a predominant phosphorylated component in macrophages upon integrin-mediated cell adhesion. Upon cell adhesion, Cbl was found to form complexes with SH2 domain-containing signaling molecules, such as Src and PI 3-kinase, and may thus connect integrins to these downstream signaling pathways.
      Cbl tyrosine phosphorylation has been reported to occur in response to a number of stimuli, including activation of the T-cell antigen receptor and ligand-induced stimulation of the granulocyte-macrophage colony-stimulating factor and erythropoietin receptors, the Fcγ receptor, the epidermal growth factor receptor, the CSF-1 receptor, and the B-cell antigen receptor. In addition, Cbl is heavily tyrosine-phosphorylated in v-abl- and bcr-abl-transformed cells (see Refs.
      • Langdon W.Y.
      ,
      • Donovan J.A.
      • Wange R.L.
      • Langdon W.Y.
      • Samelson L.E.
      ,
      • Fukazawa T.
      • Reedquist K.A.
      • Trub T.
      • Soltoff S.
      • Panchamoorthy G.
      • Druker B.
      • Cantley L.
      • Shoelson S.E.
      • Band H.
      , and
      • Fukazawa T.
      • Miyake S.
      • Band V.
      • Band H.
      • Sattler M.
      • Salgia R.
      • Okuda K.
      • Uemura N.
      • Durstin M.A.
      • Pisick E.
      • Xu G.
      • Li J.-L.
      • Prasad K.V.
      • Griffin J.D.
      ). In these systems, tyrosine-phosphorylated Cbl has been shown to interact with the SH2 domains of Src family kinases (
      • Reedquist K.A.
      • Fukazawa T.
      • Druker B.
      • Panchamoorthy G.
      • Shoelson S.E.
      • Band H.
      ,
      • Donovan J.A.
      • Wange R.L.
      • Langdon W.Y.
      • Samelson L.E.
      ), PI 3-kinase (
      • Fukazawa T.
      • Reedquist K.A.
      • Trub T.
      • Soltoff S.
      • Panchamoorthy G.
      • Druker B.
      • Cantley L.
      • Shoelson S.E.
      • Band H.
      ,
      • Soltoff S.P.
      • Cantley L.C.
      ,
      • Panchamoorthy G.
      • Fukazawa T.
      • Miyake S.
      • Soltoff S.
      • Reedquist K.
      • Druker B.
      • Shoelson S.
      • Cantley L.
      • Band H.
      ,
      • Meisner H.
      • Conway B.R.
      • Hartley D.
      • Czech M.P.
      • Hartley D.
      • Meisner H.
      • Corvera S.
      ), Crk (
      • Sattler M.
      • Salgia R.
      • Okuda K.
      • Uemura N.
      • Durstin M.A.
      • Pisick E.
      • Xu G.
      • Li J.-L.
      • Prasad K.V.
      • Griffin J.D.
      ,
      • Reedquist K.A.
      • Fukazawa T.
      • Panchamoorthy G.
      • Langdon W.Y.
      • Shoelson S.E.
      • Druker B.J.
      • Band H.
      ,
      • Buday L.
      • Khwaja A.
      • Sipeki S.
      • Farago A.
      • Downward J.
      ,
      • Ribon V.
      • Hubbell S.
      • Herrera R.
      • Saltiel A.R.
      ), CRKL (
      • Andoniou C.E.
      • Thien C.B.F.
      • Langdon W.Y.
      ), and Abl (
      • Sattler M.
      • Salgia R.
      • Okuda K.
      • Uemura N.
      • Durstin M.A.
      • Pisick E.
      • Xu G.
      • Li J.-L.
      • Prasad K.V.
      • Griffin J.D.
      ). Cbl has also been shown to bind SH3 domain-containing signaling molecules, such as the adaptor proteins Grb2 and Nck (
      • Rivero-Lezcano O.M.
      • Sameshima J.H.
      • Marcilla A.
      • Robbins K.C.
      ,
      • Donovan J.A.
      • Wange R.L.
      • Langdon W.Y.
      • Samelson L.E.
      ,
      • Fukazawa T.
      • Reedquist K.A.
      • Trub T.
      • Soltoff S.
      • Panchamoorthy G.
      • Druker B.
      • Cantley L.
      • Shoelson S.E.
      • Band H.
      ,
      • Panchamoorthy G.
      • Fukazawa T.
      • Miyake S.
      • Soltoff S.
      • Reedquist K.
      • Druker B.
      • Shoelson S.
      • Cantley L.
      • Band H.
      ,
      • Meisner H.
      • Conway B.R.
      • Hartley D.
      • Czech M.P.
      ,
      • Odai H.
      • Sasaki K.
      • Iwamatsu A.
      • Hanazono Y.
      • Tanaka T.
      • Mitani K.
      • Yazaki Y.
      • Hirai H.
      ), members of the Src family kinases (
      • Marcilla A.
      • Rivero-Lezcano O.M.
      • Agarwal A.
      • Robbins K.C.
      ,
      • Reedquist K.A.
      • Fukazawa T.
      • Druker B.
      • Panchamoorthy G.
      • Shoelson S.E.
      • Band H.
      • Tanaka S.
      • Morishita T.
      • Hashimoto Y.
      • Hattori S.
      • Nakamura S.
      • Shibuya M.
      • Matuoka K.
      • Takenawa T.
      • Kurata T.
      • Nagashima K.
      • Matsuda M.
      ), and PI 3-kinase (
      • Fukazawa T.
      • Reedquist K.A.
      • Trub T.
      • Soltoff S.
      • Panchamoorthy G.
      • Druker B.
      • Cantley L.
      • Shoelson S.E.
      • Band H.
      ,
      • Soltoff S.P.
      • Cantley L.C.
      ). Therefore, Cbl appears to act as a docking protein with the potential of regulating downstream signaling pathways through protein-protein interactions. Our finding that Cbl is part of the integrin signaling cascade indicates that Cbl is a point of convergence in the actions of a variety of factors known to influence cell morphology, locomotion, growth, and differentiation.
      Our observation that Cbl becomes tyrosine-phosphorylated following cell adhesion to ECM substrates, but not to polylysine, is consistent with the tyrosine phosphorylation being mediated by integrins. This is further supported by the observation that cell adhesion to different anti-integrin antibodies also results in elevated tyrosine phosphorylation of Cbl. It is not clear how integrin ligand binding initiates activation of intracellular tyrosine kinases; integrins themselves lack any known enzymatic activity, and no direct in vivo association between integrins and tyrosine kinases has been observed (
      • Schwartz M.A.
      • Schaller M.D.
      • Ginsberg M.H.
      ). Tyrosine phosphorylation of Cbl requires the presence of intact cytoskeleton since the adhesion-induced tyrosine phosphorylation of Cbl can be prevented by cytochalasin D treatment. A similar situation has been observed in other cells with FAK, tensin, and p130cas (
      • Bockholt S.M.
      • Burridge K.
      ,
      • Nojima Y.
      • Morino N.
      • Mimura T.
      • Hamasaki K.
      • Furuya H.
      • Sakai R.
      • Sato T.
      • Tachibana K.
      • Morimoto C.
      • Yazaki Y.
      • Hirai H.
      ,
      • Vuori K.
      • Ruoslahti E.
      ). It is therefore possible that kinases associate with integrins through interactions with cytoskeletal complexes induced by cross-linking of integrins, and intact functional cytoskeleton may be required to bring together the various components of this signaling complex.
      The structure of Cbl suggests that it is a signal assembly protein; consistent with earlier findings, we found that Cbl can bind in vitro to SH2 and SH3 domains of various signaling molecules. SH3 domains of Src and PI 3-kinase bound Cbl in an adhesion-independent manner, whereas the interactions between Cbl and the SH2 domains of Crk, Src, and PI 3-kinase required integrin-mediated cell adhesion. Coprecipitation of Cbl with Src and PI 3-kinase was observed only in adherent cells, suggesting that a stable complex formation between these proteins may require the SH2 domain binding to phosphorylated Cbl. No coprecipitation between Cbl and Crk was observed under our experimental conditions.
      Our finding that Src coprecipitates with Cbl in a kinase-active form suggests that Src might be responsible for the tyrosine phosphorylation of Cbl during integrin-mediated ligand binding. In our model, the binding of Cbl through the Src SH3 domain would have a role in the initial substrate recognition before tyrosine phosphorylation. The Src SH2 domain would reinforce the binding after tyrosine phosphorylation of Cbl, and the tight SH2 domain-mediated association would cause the effective hyperphosphorylation of Cbl by Src tyrosine kinase during integrin-mediated ligand binding. The molecular events leading to the initial step of the tyrosine phosphorylation of Cbl are currently unknown. The SH2 domain-mediated interaction may enzymatically activate Src by releasing the autoinhibition imposed by the interaction between its SH2 domain and a tyrosine-phosphorylated residue near its C-terminal tail; similar activation of Src kinases has been proposed to take place during FAK-Src interaction upon integrin ligand binding (
      • Schaller M.D.
      • Hildebrand J.D.
      • Shannon J.D.
      • Fox J.W.
      • Vines R.R.
      • Parsons J.T.
      ,
      • Calalb M.B.
      • Polte T.R.
      • Hanks S.K.
      ). Tyrosine phosphorylation of Cbl in turn should allow the recruitment of SH2 domain-containing signaling molecules such as PI 3-kinase and Crk and their associated proteins. This might enable these proteins to be tyrosine-phosphorylated by Src or to interact with the molecules associated with the other domains of Cbl. Thus, Cbl may serve as a docking protein linking Src to downstream signaling molecules in the integrin signaling pathway.
      We used enolase as a substrate in the in vitro tyrosine kinase assays in order to detect Src kinase activity in the Cbl immunoprecipitates. Since enolase can be phosphorylated not only by Src, but also by other members of the Src family, it is possible that some of the other family members may contribute to the tyrosine kinase activity observed in Cbl complexes following integrin ligand binding. These kinases include the Src family members Hck, Fgr, and Lyn, which are known to be coexpressed in myeloid cells (
      • Lowell C.A.
      • Soriano P.
      ). Indeed, recent biochemical evidence has implicated some of these kinases in integrin-mediated signaling in myeloid cells; both Fgr and Lyn have been shown to be activated in a β2 integrin-dependent manner in human neutrophils (
      • Berton G.
      • Fumagalli L.
      • Laudanna C.
      • Sorio C.
      ,
      • Yan S.R.
      • Fumagalli L.
      • Berton G.
      ). Furthermore, examination of bone marrow-derived neutrophils from hckfgr double mutants has revealed a severe defect in neutrophil function elicited by plating cells on ECM protein-coated surfaces or by directly cross-linking cell-surface integrins (
      • Lowell C.A.
      • Fumagalli L.
      • Berton G.
      ). Similar results have recently been seen with monocytes and macrophages derived from hckfgr double mutant mice (cited in Ref.
      • Lowell C.A.
      • Soriano P.
      ). Our preliminary results suggest that, in addition to Src, Lyn and Fgr may also become associated with Cbl in an integrin-dependent fashion, but a lack of suitable reagents has prevented us from studying these putative interactions in more detail. Together, these results suggest that Src family kinases are involved in integrin signaling in myeloid cells and that Cbl is a candidate molecule in connecting Src kinases to integrin signaling complexes in these cells.
      The findings reported here show an in vivo association between Cbl and PI 3-kinase upon integrin ligand binding. In addition, we have demonstrated that a substantial level of PI 3-kinase activity associates with Cbl in an adhesion-dependent manner. PI 3-kinase is known to be activated when one or both of the SH2 domains in the p85 subunit of this enzyme bind to tyrosine-phosphorylated proteins that contain YXXM motifs (
      • Songyang Z.
      • Shoelson S.E.
      • Chaudhuri M.
      • Gish G.
      • Pawson T.
      • Haser W.G.
      • King F.
      • Roberts T.
      • Ratnofsky S.
      • Lechleider R.J.
      • Neel B.G.
      • Birge R.B.
      • Fajardo J.E.
      • Chou M.M.
      • Hanafusa H.
      • Schaffhausen B.
      • Cantley L.C.
      ). We found that the N-terminal SH2 domain of p85 PI 3-kinase bound to Cbl in an adhesion-dependent manner; Cbl has two YXXM motifs (
      • Langdon W.Y.
      ) that can serve as binding sites for the SH2 domains of PI 3-kinase. Based on these observations, we postulate that one functional consequence of Cbl tyrosine phosphorylation upon integrin ligand binding is the catalytic activation of PI 3-kinase. Thus, Cbl may play a prominent role in macrophages in coupling the PI 3-kinase enzyme with the integrin signaling machinery. In other systems, activation of PI 3-kinase has been connected to a number of biological effects, such as the mitogenic effects of certain growth factors, changes in actin rearrangement, and growth factor-mediated membrane ruffling and chemotactic migration (for a review, see Ref.
      • Kapeller R.
      • Cantley L.C.
      ). Similarly, Cbl-activated PI 3-kinase may be involved in integrin-mediated chemotaxis and phagocytosis in macrophages.
      In summary, the ligand binding of integrins seems to control the tyrosine phosphorylation status of a number of intracellular proteins that can function as docking proteins connecting multiple downstream signaling pathways via SH2 and SH3 domain interactions; Cbl appears to mediate integrin signaling through direct recruitment and activation of Src and PI 3-kinase in macrophages. Further studies are needed to define the cause-effect relationships between the tyrosine-phosphorylated proteins, integrins, and actin cytoskeleton and to reveal intermediate steps between integrins and the kinases.

      Acknowledgments

      We thank Drs. Joan Brugge, Hisamaru Hirai, Rich Maki, Michiyuki Matsuda, Erkki Ruoslahti, and Stephen Taylor for providing reagents used in this study.

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