AUTOPHOSPHORYLATION OF THE FOCAL ADHESION KINASE, PP125(FAK), DIRECTS SH2 DEPENDENT BINDING OF PP60(SRC)

The phosphorylation of protein tyrosine kinases (PTKs) on tyrosine residues is a critical regulatory event that modulates catalytic activity and triggers the physical association of PTKs with Src homology 2 (SH2)-containing proteins. The integrin-linked focal adhesion kinase, ppl25(FAK), exhibits extracellular matrix-dependent phosphorylation on tyrosine and physically associates with two nonreceptor PTKs, pp6O(src) and pp59(fyn), via their SH2 domains. Herein, we identify Tyr-397 as the major site of tyrosine phosphorylation on ppl25(FAK) both in vivo and in vitro. Tyrosine 397 is located at the juncture of the N-terminaI and catalytic domains, a novel site for PTK autophosphorylation. Mutation of Tyr-397 to a nonphosphorylatable residue dramatically impairs the phosphorylation of ppl25(FAK) on tyrosine in vivo and in vitro. The mutation of Tyr-397 to Phe also inhibits the formation of stable complexes with pp60(src) in cells expressing Src and FAK(397F) suggesting that autophosphorylation of ppl25(FAK) may regulate the association of pp125(FAK) with Src family kinases in vivo. The identification of Tyr-397 as a major site for FAK autophosphorylation provides one of the first examples of a cellular protein containing a high-affinity binding site for a Src family kinase SH2 domain. This finding has implications for models describing the mechanisms of action of pp125(FAK), the regulation of the Src family of PTKs, and signal transduction through the integrins.