The v-src SH3 domain facilitates a cell adhesion-independent association with focal adhesion kinase

Integrins facilitate cell attachment to the extracellular matrix, and these interactions generate cell survival, proliferation, and motility signals. Integrin signals are relayed in part by focal adhesion kinase (FAK) activat ion and the formation of a transient signaling complex initiated by Src hom ology 2 (SH2)-dependent binding of Src family protein-tyrosine kinases to t he FAK Tyr-397 autophosphorylation site. Here we show that in viral Src (v- Src)-transformed NIH3T3 fibroblasts, an adhesion-independent FAK-Src signal ing complex occurs. Co-expression studies in human 293T cells showed that v -Src could associate with and phosphorylate a Phe-397 FAK mutant at Tyr-925 promoting Grb2 binding to FAK in suspended cells. In vitro, glutathione S- transferase fusion proteins of the v-Src SH3 but not c-Src SH3 domain bound to FAK in lysates of NIH3T3 fibroblasts, The v-Src SH3-binding sites were mapped to known proline-X-X-proline (PXXP) SH3-binding motifs in the FAK N- (residues 371-377) and C-terminal domains (residues 712-718 and 871-882) b y in vitro pull-down assays, and these sites are composed of a PXXPXX Phi ( where Phi, is a hydrophobic residue) v-Src SH3 binding consensus. Sequence comparisons show that residues in the RT loop region of the c-Src and v-Src SH3 domains differ. Substitution of c-Src RT loop residues (Arg-97 and Thr -98) for those found in the v-Src SH3 domain (Trp-97 and Ile-98) enhanced t he binding of distinct NIH3T3 cellular proteins to a glutathione S-transfer ase fusion protein of the c-Src (Trp-97 + Ile-98) SH3 domain, FAK was ident ified as a c-Src (Trp-97 + Ile-98) SH3 domain target in fibroblasts, and co -expression studies in 293T cells showed that full-length c-Src (Trp-97 + I le-98) could associate in vivo with Phe-397 FAK in an SH2-independent manne r. These studies establish a functional role for the v-Src SH3 domain in st abilizing an adhesion-independent signaling complex with FAK.