Autophosphorylation of the Focal Adhesion Kinase, ppl25^FAK, Directs SH2-Dependent Binding of pp60<i>^src</i>

TLDR

Phosphorylation of protein tyrosine kinases on tyrosine residues regulates activity and mediates SH2 domain interactions, and the focal adhesion kinase pp125FAK is phosphorylated in an integrin‑dependent manner at a novel site, Tyr‑397, that enables binding to Src family kinases. The study aimed to determine whether Tyr‑397 is the principal autophosphorylation site on pp125FAK and to assess its role in Src association. Tyr‑397 is the major autophosphorylation site on pp125FAK; mutation to phenylalanine abolishes phosphorylation and disrupts stable complex formation with pp60src, establishing Tyr‑397 as a high‑affinity SH2 binding site that influences FAK‑Src signaling and integrin‑mediated pathways.

Abstract

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, pp125FAK, exhibits extracellular matrix-dependent phosphorylation on tyrosine and physically associates with two nonreceptor PTKs, pp60src and pp59fyn, via their SH2 domains. Herein, we identify Tyr-397 as the major site of tyrosine phosphorylation on pp125FAK both in vivo and in vitro. Tyrosine 397 is located at the juncture of the N-terminal and catalytic domains, a novel site for PTK autophosphorylation. Mutation of Tyr-397 to a nonphosphorylatable residue dramatically impairs the phosphorylation of pp125FAK on tyrosine in vivo and in vitro. The mutation of Tyr-397 to Phe also inhibits the formation of stable complexes with pp60src in cells expressing Src and FAK397F, suggesting that autophosphorylation of pp125FAK may regulate the association of pp125FAK 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 pp125FAK, the regulation of the Src family of PTKs, and signal transduction through the integrins.