Evidence for in vivo phosphorylation of the Grb2 SH2-domain binding site on focal adhesion kinase by Src-family protein-tyrosine kinases.
Focal adhesion kinase (FAK) is a nonreceptor protein-tyrosine kinase (PTK) that associates with integrin receptors and participates in extracellular matrix-mediated signal transduction events. We showed previously that the c-Src nonreceptor PTK and the Grb2 SH2/SH3 adaptor protein bound directly to FAK after fibronectin stimulation (D. D. Schlaepfer, S.K. Hanks, T. ... Hunter, and P. van der Geer, Nature [London] 372:786-791, 1994). Here, we present evidence that c-Src association with FAK is required for Grb2 binding to FAK. Using a tryptic phosphopeptide mapping approach, the in vivo phosphorylation of the Grb2 binding site on FAK (Tyr-925) was detected after fibronectin stimulation of NIH 3T3 cells and was constitutively phosphorylated in v-Src-transformed NIH 3T3 cells. In vitro, c-Src phosphorylated FAK Tyr-925 in a glutathione S-transferase-FAK C-terminal domain fusion protein, whereas FAK did not. Using epitope-tagged FAK constructs, transiently expressed in human 293 cells, we determined the effect of site-directed mutations on c-Src and Grb2 binding to FAK. Mutation of FAK Tyr-925 disrupted Grb2 binding, whereas mutation of the c-Src binding site on FAK (Tyr-397) disrupted both c-Src and Grb2 binding to FAK in vivo. These results support a model whereby Src-family PTKs are recruited to FAK and focal adhesions following integrin-induced autophosphorylation and exposure of FAK Tyr-397. Src-family binding and phosphorylation of FAK at Tyr-925 creates a Grb2 SH2-domain binding site and provides a link to the activation of the Ras signal transduction pathway. In Src-transformed cells, this pathway may be constitutively activated as a result of FAK Tyr-925 phosphorylation in the absence of integrin stimulation.
Mesh Terms:
3T3 Cells, Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Animals, Binding Sites, Cell Adhesion, Cell Adhesion Molecules, Cell Line, ErbB Receptors, Focal Adhesion Kinase 1, Focal Adhesion Protein-Tyrosine Kinases, GRB2 Adaptor Protein, Glutathione Transferase, Humans, Integrins, Mice, Models, Biological, Mutagenesis, Site-Directed, Phosphopeptides, Phosphorylation, Polylysine, Polymerase Chain Reaction, Protein-Tyrosine Kinases, Proteins, Recombinant Fusion Proteins, Tyrosine, src Homology Domains, src-Family Kinases
3T3 Cells, Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Animals, Binding Sites, Cell Adhesion, Cell Adhesion Molecules, Cell Line, ErbB Receptors, Focal Adhesion Kinase 1, Focal Adhesion Protein-Tyrosine Kinases, GRB2 Adaptor Protein, Glutathione Transferase, Humans, Integrins, Mice, Models, Biological, Mutagenesis, Site-Directed, Phosphopeptides, Phosphorylation, Polylysine, Polymerase Chain Reaction, Protein-Tyrosine Kinases, Proteins, Recombinant Fusion Proteins, Tyrosine, src Homology Domains, src-Family Kinases
Mol. Cell. Biol.
Date: Oct. 01, 1996
PubMed ID: 8816475
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