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 extra cellular matrix-mediated signal transduction events, We showed previou sly that the c-Src nonreceptor PTK and the Grb2 SH2/SH3 adaptor protei n bound directly to FAK after fibronectin stimulation (D. D. Schlaepfe r, S, K, Hanks, T, Hunter, and P, van der Geer, Nature [London] 372:78 6-791, 1994). Here, we present evidence that c-Src association with FA K 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 3T 3 cells and was constitutively phosphorylated in v-Src-transformed NIH 3T3 cells, In vitro, c-Src phosphorylated FAK Tyr-925 in a glutathion e S-transferase-FAK C-terminal domain fusion protein, whereas FAK did not, Using epitope-tagged FAK constructs, transiently expressed in hum an 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 b inding, whereas mutation of the c-Src binding site on FAK (Tyr-397) di srupted both c-Src and Grb2 binding to FAK in vivo. These results supp ort a model whereby Src-family PTKs are recruited to FAK and focal adh esions 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 acti vation of the Ras signal transduction pathway, In Src-transformed cell s, this pathway may be constitutively activated as a result of FAK Tyr -925 phosphorylation in the absence of integrin stimulation.