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Mol Cell Biol. Mar 1997; 17(3): 1702–1713.
PMCID: PMC231895

Fibronectin-stimulated signaling from a focal adhesion kinase-c-Src complex: involvement of the Grb2, p130cas, and Nck adaptor proteins.

Abstract

The focal adhesion kinase (FAK), a protein-tyrosine kinase (PTK), associates with integrin receptors and is activated by cell binding to extracellular matrix proteins, such as fibronectin (FN). FAK autophosphorylation at Tyr-397 promotes Src homology 2 (SH2) domain binding of Src family PTKs, and c-Src phosphorylation of FAK at Tyr-925 creates an SH2 binding site for the Grb2 SH2-SH3 adaptor protein. FN-stimulated Grb2 binding to FAK may facilitate intracellular signaling to targets such as ERK2-mitogen-activated protein kinase. We examined FN-stimulated signaling to ERK2 and found that ERK2 activation was reduced 10-fold in Src- fibroblasts, compared to that of Src- fibroblasts stably reexpressing wild-type c-Src. FN-stimulated FAK phosphotyrosine (P.Tyr) and Grb2 binding to FAK were reduced, whereas the tyrosine phosphorylation of another signaling protein, p130cas, was not detected in the Src- cells. Stable expression of residues 1 to 298 of Src (Src 1-298, which encompass the SH3 and SH2 domains of c-Src) in the Src- cells blocked Grb2 binding to FAK; but surprisingly, Src 1-298 expression also resulted in elevated p130cas P.Tyr levels and a two- to threefold increase in FN-stimulated ERK2 activity compared to levels in Src- cells. Src 1-298 bound to both FAK and p130cas and promoted FAK association with p130cas in vivo. FAK was observed to phosphorylate p130cas in vitro and could thus phosphorylate p130cas upon FN stimulation of the Src 1-298-expressing cells. FAK-induced phosphorylation of p130cas in the Src 1-298 cells promoted the SH2 domain-dependent binding of the Nck adaptor protein to p130cas, which may facilitate signaling to ERK2. These results show that there are additional FN-stimulated pathways to ERK2 that do not involve Grb2 binding to FAK.

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Selected References

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  • Akiyama SK, Yamada SS, Yamada KM, LaFlamme SE. Transmembrane signal transduction by integrin cytoplasmic domains expressed in single-subunit chimeras. J Biol Chem. 1994 Jun 10;269(23):15961–15964. [PubMed]
  • Avraham S, London R, Fu Y, Ota S, Hiregowdara D, Li J, Jiang S, Pasztor LM, White RA, Groopman JE, et al. Identification and characterization of a novel related adhesion focal tyrosine kinase (RAFTK) from megakaryocytes and brain. J Biol Chem. 1995 Nov 17;270(46):27742–27751. [PubMed]
  • Bockholt SM, Burridge K. An examination of focal adhesion formation and tyrosine phosphorylation in fibroblasts isolated from src-, fyn-, and yes- mice. Cell Adhes Commun. 1995 May;3(2):91–100. [PubMed]
  • Böhmer RM, Scharf E, Assoian RK. Cytoskeletal integrity is required throughout the mitogen stimulation phase of the cell cycle and mediates the anchorage-dependent expression of cyclin D1. Mol Biol Cell. 1996 Jan;7(1):101–111. [PMC free article] [PubMed]
  • Bokoch GM, Wang Y, Bohl BP, Sells MA, Quilliam LA, Knaus UG. Interaction of the Nck adapter protein with p21-activated kinase (PAK1). J Biol Chem. 1996 Oct 18;271(42):25746–25749. [PubMed]
  • Boudreau N, Sympson CJ, Werb Z, Bissell MJ. Suppression of ICE and apoptosis in mammary epithelial cells by extracellular matrix. Science. 1995 Feb 10;267(5199):891–893. [PMC free article] [PubMed]
  • Boyle WJ, van der Geer P, Hunter T. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 1991;201:110–149. [PubMed]
  • Broome MA, Hunter T. The PDGF receptor phosphorylates Tyr 138 in the c-Src SH3 domain in vivo reducing peptide ligand binding. Oncogene. 1997 Jan 9;14(1):17–34. [PubMed]
  • Broome MA, Hunter T. Requirement for c-Src catalytic activity and the SH3 domain in platelet-derived growth factor BB and epidermal growth factor mitogenic signaling. J Biol Chem. 1996 Jul 12;271(28):16798–16806. [PubMed]
  • Calalb MB, Polte TR, Hanks SK. Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity: a role for Src family kinases. Mol Cell Biol. 1995 Feb;15(2):954–963. [PMC free article] [PubMed]
  • Cary LA, Chang JF, Guan JL. Stimulation of cell migration by overexpression of focal adhesion kinase and its association with Src and Fyn. J Cell Sci. 1996 Jul;109(Pt 7):1787–1794. [PubMed]
  • Chan PY, Kanner SB, Whitney G, Aruffo A. A transmembrane-anchored chimeric focal adhesion kinase is constitutively activated and phosphorylated at tyrosine residues identical to pp125FAK. J Biol Chem. 1994 Aug 12;269(32):20567–20574. [PubMed]
  • White MF, Kahn CR. The insulin signaling system. J Biol Chem. 1994 Jan 7;269(1):1–4. [PubMed]
  • Chen Q, Lin TH, Der CJ, Juliano RL. Integrin-mediated activation of MEK and mitogen-activated protein kinase is independent of Ras [corrected]. J Biol Chem. 1996 Jul 26;271(30):18122–18127. [PubMed]
  • Clark EA, Brugge JS. Integrins and signal transduction pathways: the road taken. Science. 1995 Apr 14;268(5208):233–239. [PubMed]
  • Clark EA, Hynes RO. Ras activation is necessary for integrin-mediated activation of extracellular signal-regulated kinase 2 and cytosolic phospholipase A2 but not for cytoskeletal organization. J Biol Chem. 1996 Jun 21;271(25):14814–14818. [PubMed]
  • Cobb BS, Schaller MD, Leu TH, Parsons JT. Stable association of pp60src and pp59fyn with the focal adhesion-associated protein tyrosine kinase, pp125FAK. Mol Cell Biol. 1994 Jan;14(1):147–155. [PMC free article] [PubMed]
  • Fang F, Orend G, Watanabe N, Hunter T, Ruoslahti E. Dependence of cyclin E-CDK2 kinase activity on cell anchorage. Science. 1996 Jan 26;271(5248):499–502. [PubMed]
  • Filardo EJ, Brooks PC, Deming SL, Damsky C, Cheresh DA. Requirement of the NPXY motif in the integrin beta 3 subunit cytoplasmic tail for melanoma cell migration in vitro and in vivo. J Cell Biol. 1995 Jul;130(2):441–450. [PMC free article] [PubMed]
  • Galisteo ML, Chernoff J, Su YC, Skolnik EY, Schlessinger J. The adaptor protein Nck links receptor tyrosine kinases with the serine-threonine kinase Pak1. J Biol Chem. 1996 Aug 30;271(35):20997–21000. [PubMed]
  • George EL, Georges-Labouesse EN, Patel-King RS, Rayburn H, Hynes RO. Defects in mesoderm, neural tube and vascular development in mouse embryos lacking fibronectin. Development. 1993 Dec;119(4):1079–1091. [PubMed]
  • Grant SG, Karl KA, Kiebler MA, Kandel ER. Focal adhesion kinase in the brain: novel subcellular localization and specific regulation by Fyn tyrosine kinase in mutant mice. Genes Dev. 1995 Aug 1;9(15):1909–1921. [PubMed]
  • Guan JL, Shalloway D. Regulation of focal adhesion-associated protein tyrosine kinase by both cellular adhesion and oncogenic transformation. Nature. 1992 Aug 20;358(6388):690–692. [PubMed]
  • Hanks SK, Calalb MB, Harper MC, Patel SK. Focal adhesion protein-tyrosine kinase phosphorylated in response to cell attachment to fibronectin. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8487–8491. [PMC free article] [PubMed]
  • Harte MT, Hildebrand JD, Burnham MR, Bouton AH, Parsons JT. p130Cas, a substrate associated with v-Src and v-Crk, localizes to focal adhesions and binds to focal adhesion kinase. J Biol Chem. 1996 Jun 7;271(23):13649–13655. [PubMed]
  • Hildebrand JD, Schaller MD, Parsons JT. Identification of sequences required for the efficient localization of the focal adhesion kinase, pp125FAK, to cellular focal adhesions. J Cell Biol. 1993 Nov;123(4):993–1005. [PMC free article] [PubMed]
  • Hildebrand JD, Taylor JM, Parsons JT. An SH3 domain-containing GTPase-activating protein for Rho and Cdc42 associates with focal adhesion kinase. Mol Cell Biol. 1996 Jun;16(6):3169–3178. [PMC free article] [PubMed]
  • Hirsch E, Iglesias A, Potocnik AJ, Hartmann U, Fässler R. Impaired migration but not differentiation of haematopoietic stem cells in the absence of beta1 integrins. Nature. 1996 Mar 14;380(6570):171–175. [PubMed]
  • Howell BW, Cooper JA. Csk suppression of Src involves movement of Csk to sites of Src activity. Mol Cell Biol. 1994 Aug;14(8):5402–5411. [PMC free article] [PubMed]
  • Hu Q, Milfay D, Williams LT. Binding of NCK to SOS and activation of ras-dependent gene expression. Mol Cell Biol. 1995 Mar;15(3):1169–1174. [PMC free article] [PubMed]
  • Huhtala P, Humphries MJ, McCarthy JB, Tremble PM, Werb Z, Damsky CH. Cooperative signaling by alpha 5 beta 1 and alpha 4 beta 1 integrins regulates metalloproteinase gene expression in fibroblasts adhering to fibronectin. J Cell Biol. 1995 May;129(3):867–879. [PMC free article] [PubMed]
  • Ilić D, Furuta Y, Kanazawa S, Takeda N, Sobue K, Nakatsuji N, Nomura S, Fujimoto J, Okada M, Yamamoto T. Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice. Nature. 1995 Oct 12;377(6549):539–544. [PubMed]
  • Jockusch BM, Bubeck P, Giehl K, Kroemker M, Moschner J, Rothkegel M, Rüdiger M, Schlüter K, Stanke G, Winkler J. The molecular architecture of focal adhesions. Annu Rev Cell Dev Biol. 1995;11:379–416. [PubMed]
  • Kaplan KB, Bibbins KB, Swedlow JR, Arnaud M, Morgan DO, Varmus HE. Association of the amino-terminal half of c-Src with focal adhesions alters their properties and is regulated by phosphorylation of tyrosine 527. EMBO J. 1994 Oct 17;13(20):4745–4756. [PMC free article] [PubMed]
  • Kaplan KB, Swedlow JR, Morgan DO, Varmus HE. c-Src enhances the spreading of src-/- fibroblasts on fibronectin by a kinase-independent mechanism. Genes Dev. 1995 Jun 15;9(12):1505–1517. [PubMed]
  • Law SF, Estojak J, Wang B, Mysliwiec T, Kruh G, Golemis EA. Human enhancer of filamentation 1, a novel p130cas-like docking protein, associates with focal adhesion kinase and induces pseudohyphal growth in Saccharomyces cerevisiae. Mol Cell Biol. 1996 Jul;16(7):3327–3337. [PMC free article] [PubMed]
  • Lehmann JM, Riethmüller G, Johnson JP. Nck, a melanoma cDNA encoding a cytoplasmic protein consisting of the src homology units SH2 and SH3. Nucleic Acids Res. 1990 Feb 25;18(4):1048–1048. [PMC free article] [PubMed]
  • Lev S, Moreno H, Martinez R, Canoll P, Peles E, Musacchio JM, Plowman GD, Rudy B, Schlessinger J. Protein tyrosine kinase PYK2 involved in Ca(2+)-induced regulation of ion channel and MAP kinase functions. Nature. 1995 Aug 31;376(6543):737–745. [PubMed]
  • Li J, Avraham H, Rogers RA, Raja S, Avraham S. Characterization of RAFTK, a novel focal adhesion kinase, and its integrin-dependent phosphorylation and activation in megakaryocytes. Blood. 1996 Jul 15;88(2):417–428. [PubMed]
  • Lukashev ME, Sheppard D, Pytela R. Disruption of integrin function and induction of tyrosine phosphorylation by the autonomously expressed beta 1 integrin cytoplasmic domain. J Biol Chem. 1994 Jul 15;269(28):18311–18314. [PubMed]
  • Marshall CJ. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell. 1995 Jan 27;80(2):179–185. [PubMed]
  • Mayer BJ, Hirai H, Sakai R. Evidence that SH2 domains promote processive phosphorylation by protein-tyrosine kinases. Curr Biol. 1995 Mar 1;5(3):296–305. [PubMed]
  • Miyamoto S, Teramoto H, Coso OA, Gutkind JS, Burbelo PD, Akiyama SK, Yamada KM. Integrin function: molecular hierarchies of cytoskeletal and signaling molecules. J Cell Biol. 1995 Nov;131(3):791–805. [PMC free article] [PubMed]
  • Montgomery AM, Reisfeld RA, Cheresh DA. Integrin alpha v beta 3 rescues melanoma cells from apoptosis in three-dimensional dermal collagen. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):8856–8860. [PMC free article] [PubMed]
  • Nakamoto T, Sakai R, Ozawa K, Yazaki Y, Hirai H. Direct binding of C-terminal region of p130Cas to SH2 and SH3 domains of Src kinase. J Biol Chem. 1996 Apr 12;271(15):8959–8965. [PubMed]
  • Nojima Y, Morino N, Mimura T, Hamasaki K, Furuya H, Sakai R, Sato T, Tachibana K, Morimoto C, Yazaki Y, et al. Integrin-mediated cell adhesion promotes tyrosine phosphorylation of p130Cas, a Src homology 3-containing molecule having multiple Src homology 2-binding motifs. J Biol Chem. 1995 Jun 23;270(25):15398–15402. [PubMed]
  • Petch LA, Bockholt SM, Bouton A, Parsons JT, Burridge K. Adhesion-induced tyrosine phosphorylation of the p130 src substrate. J Cell Sci. 1995 Apr;108(Pt 4):1371–1379. [PubMed]
  • Polte TR, Hanks SK. Interaction between focal adhesion kinase and Crk-associated tyrosine kinase substrate p130Cas. Proc Natl Acad Sci U S A. 1995 Nov 7;92(23):10678–10682. [PMC free article] [PubMed]
  • Quilliam LA, Lambert QT, Mickelson-Young LA, Westwick JK, Sparks AB, Kay BK, Jenkins NA, Gilbert DJ, Copeland NG, Der CJ. Isolation of a NCK-associated kinase, PRK2, an SH3-binding protein and potential effector of Rho protein signaling. J Biol Chem. 1996 Nov 15;271(46):28772–28776. [PubMed]
  • Rusanescu G, Qi H, Thomas SM, Brugge JS, Halegoua S. Calcium influx induces neurite growth through a Src-Ras signaling cassette. Neuron. 1995 Dec;15(6):1415–1425. [PubMed]
  • Sakai R, Iwamatsu A, Hirano N, Ogawa S, Tanaka T, Mano H, Yazaki Y, Hirai H. A novel signaling molecule, p130, forms stable complexes in vivo with v-Crk and v-Src in a tyrosine phosphorylation-dependent manner. EMBO J. 1994 Aug 15;13(16):3748–3756. [PMC free article] [PubMed]
  • Sasaki H, Nagura K, Ishino M, Tobioka H, Kotani K, Sasaki T. Cloning and characterization of cell adhesion kinase beta, a novel protein-tyrosine kinase of the focal adhesion kinase subfamily. J Biol Chem. 1995 Sep 8;270(36):21206–21219. [PubMed]
  • Schaller MD, Borgman CA, Cobb BS, Vines RR, Reynolds AB, Parsons JT. pp125FAK a structurally distinctive protein-tyrosine kinase associated with focal adhesions. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5192–5196. [PMC free article] [PubMed]
  • Schaller MD, Hildebrand JD, Shannon JD, Fox JW, Vines RR, Parsons JT. Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src. Mol Cell Biol. 1994 Mar;14(3):1680–1688. [PMC free article] [PubMed]
  • Schlaepfer DD, Hanks SK, Hunter T, van der Geer P. Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase. Nature. 1994 Dec 22;372(6508):786–791. [PubMed]
  • Schlaepfer DD, Hunter T. Evidence for in vivo phosphorylation of the Grb2 SH2-domain binding site on focal adhesion kinase by Src-family protein-tyrosine kinases. Mol Cell Biol. 1996 Oct;16(10):5623–5633. [PMC free article] [PubMed]
  • Schwartz MA, Schaller MD, Ginsberg MH. Integrins: emerging paradigms of signal transduction. Annu Rev Cell Dev Biol. 1995;11:549–599. [PubMed]
  • Songyang Z, Shoelson SE, Chaudhuri M, Gish G, Pawson T, Haser WG, King F, Roberts T, Ratnofsky S, Lechleider RJ, et al. SH2 domains recognize specific phosphopeptide sequences. Cell. 1993 Mar 12;72(5):767–778. [PubMed]
  • Soriano P, Montgomery C, Geske R, Bradley A. Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Cell. 1991 Feb 22;64(4):693–702. [PubMed]
  • Tanaka M, Gupta R, Mayer BJ. Differential inhibition of signaling pathways by dominant-negative SH2/SH3 adapter proteins. Mol Cell Biol. 1995 Dec;15(12):6829–6837. [PMC free article] [PubMed]
  • Thomas JE, Soriano P, Brugge JS. Phosphorylation of c-Src on tyrosine 527 by another protein tyrosine kinase. Science. 1991 Oct 25;254(5031):568–571. [PubMed]
  • Thomas SM, Soriano P, Imamoto A. Specific and redundant roles of Src and Fyn in organizing the cytoskeleton. Nature. 1995 Jul 20;376(6537):267–271. [PubMed]
  • Vuori K, Hirai H, Aizawa S, Ruoslahti E. Introduction of p130cas signaling complex formation upon integrin-mediated cell adhesion: a role for Src family kinases. Mol Cell Biol. 1996 Jun;16(6):2606–2613. [PMC free article] [PubMed]
  • Vuori K, Ruoslahti E. Association of insulin receptor substrate-1 with integrins. Science. 1994 Dec 2;266(5190):1576–1578. [PubMed]
  • Vuori K, Ruoslahti E. Tyrosine phosphorylation of p130Cas and cortactin accompanies integrin-mediated cell adhesion to extracellular matrix. J Biol Chem. 1995 Sep 22;270(38):22259–22262. [PubMed]
  • Wary KK, Mainiero F, Isakoff SJ, Marcantonio EE, Giancotti FG. The adaptor protein Shc couples a class of integrins to the control of cell cycle progression. Cell. 1996 Nov 15;87(4):733–743. [PubMed]
  • Xing Z, Chen HC, Nowlen JK, Taylor SJ, Shalloway D, Guan JL. Direct interaction of v-Src with the focal adhesion kinase mediated by the Src SH2 domain. Mol Biol Cell. 1994 Apr;5(4):413–421. [PMC free article] [PubMed]
  • Yamada KM, Miyamoto S. Integrin transmembrane signaling and cytoskeletal control. Curr Opin Cell Biol. 1995 Oct;7(5):681–689. [PubMed]
  • Zhang Z, Vuori K, Reed JC, Ruoslahti E. The alpha 5 beta 1 integrin supports survival of cells on fibronectin and up-regulates Bcl-2 expression. Proc Natl Acad Sci U S A. 1995 Jun 20;92(13):6161–6165. [PMC free article] [PubMed]
  • Zhu X, Assoian RK. Integrin-dependent activation of MAP kinase: a link to shape-dependent cell proliferation. Mol Biol Cell. 1995 Mar;6(3):273–282. [PMC free article] [PubMed]

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