AUTOPHOSPHORYLATION OF THE FOCAL ADHESION KINASE, PP125(FAK), DIRECTSSH2 DEPENDENT BINDING OF PP60(SRC)

The phosphorylation of protein tyrosine kinases (PTKs) on tyrosine res idues is a critical regulatory event that modulates catalytic activity and triggers the physical association of PTKs with Src homology 2 (SH 2)-containing proteins. The integrin-linked focal adhesion kinase, ppl 25(FAK), exhibits extracellular matrix-dependent phosphorylation on ty rosine and physically associates with two nonreceptor PTKs, pp6O(src) and pp59(fyn), via their SH2 domains. Herein, we identify Tyr-397 as t he major site of tyrosine phosphorylation on ppl25(FAK) both in vivo a nd in vitro. Tyrosine 397 is located at the juncture of the N-terminaI and catalytic domains, a novel site for PTK autophosphorylation. Muta tion of Tyr-397 to a nonphosphorylatable residue dramatically impairs the phosphorylation of ppl25(FAK) on tyrosine in vivo and in vitro. Th e mutation of Tyr-397 to Phe also inhibits the formation of stable com plexes with pp60(src) in cells expressing Src and FAK(397F) suggesting that autophosphorylation of ppl25(FAK) may regulate the association o f pp125(FAK) with Src family kinases in vivo. The identification of Ty r-397 as a major site for FAK autophosphorylation provides one of the first examples of a cellular protein containing a high-affinity bindin g site for a Src family kinase SH2 domain. This finding has implicatio ns for models describing the mechanisms of action of pp125(FAK), the r egulation of the Src family of PTKs, and signal transduction through t he integrins.