TYROSINE PHOSPHORYLATION OF FOCAL ADHESION KINASE AT SITES IN THE CATALYTIC DOMAIN REGULATES KINASE-ACTIVITY - A ROLE FOR SRC FAMILY KINASES

Focal adhesion kinase (FAK) is a widely expressed nonreceptor protein- tyrosine kinase implicated in integrin-mediated signal transduction pa thways and in the process of oncogenic transformation by v-Src. Elevat ion of FAK's phosphotyrosine content, following both cell adhesion to extracellular matrix substrata and cell transformation by Rous sarcoma virus, correlates directly with an increased kinase activity. To help elucidate the role of FAR phosphorylation in signal transduction even ts, we used a tryptic phosphopeptide mapping approach to identify tyro sine sites of phosphorylation responsive to both cell adhesion and Src transformation. We have identified four tyrosines, 397, 407, 576, and 577, which are phosphorylated in mouse BALB/3T3 fibroblasts in an adh esion dependent manner. Tyrosine 397 has been previously recognized as the major site of FAK autophosphorylation. Phosphorylation of tyrosin es 407, 576, and 577, which are previously unrecognized sites, is sign ificantly elevated in the presence of c-Src in vitro and v-Src in vivo . Tyrosines 576 and 577 lie within catalytic subdomain VIII-a region r ecognized as a target for phosphorylation-mediated regulation of prote in kinase activity. We found that maximal kinase activity of FAK immun e complexes requires phosphorylation of both tyrosines 576 and 577. Ou r results indicate that phosphorylation of FAK by Src (or other Src fa mily kinases) is an important step in the formation of an active signa ling complex.