A TRANSMEMBRANE-ANCHORED CHIMERIC FOCAL ADHESION KINASE IS CONSTITUTIVELY ACTIVATED AND PHOSPHORYLATED AT TYROSINE RESIDUES IDENTICAL TO PP125(FAK)

Focal adhesion kinase, pp125(FAK), is a nonmyristylated cytosolic tyro sine kinase unrelated to protein-tyrosine kinase families categorized to date. The kinase activity and tyrosine phosphorylation of pp125(FAK ) induced by beta(1) and beta(3) integrin-mediated cell adherence or a ggregation. pp125(FAK) is also a tyrosine phosphorylation substrate in v-src-transformed cells and is localized to focal adhesion contacts o f adherent fibroblasts and carcinoma cells. In this report, we have tr ansiently expressed in COS cells a transmembrane-anchored chimeric rec eptor kinase, CD2FAK, consisting of CD2 and pp125(FAK). We analyzed it s kinase activity and tyrosine phosphorylation and compared to those o f pp125(FAK). We found that CD2FAK exhibited constitutive kinase activ ity and a high basal tyrosine phosphorylation level when COS transfect ants were suspended in serum-free media. The kinase activity of CD2FAK was similarly up-regulated upon beta(1) integrin mediated cell adhere nce as the endogenous pp125(FAK). Both CD2FAK and pp125(FAK) appeared to be active as autophosphorylating kinases as shown by mutation of th e ATP binding site. We determined the major tyrosine phosphorylation s ite, Tyr(397), identical for both the constitutively activated CD2FAK and pp125(FAK) in response to beta(1) integrin-mediated cell adherence by site-directed mutagenesis. Deletions of the NH2- or the COOH-termi nal noncatalytic domain of FAK, including Tyr(397) did not lead to abo lition of the kinase activity of pp125(FAK) or CD2FAK. Taken together, CD2FAK exhibits properties of an activated pp125(FAK) and the kinase activity does not appear to require tyrosine phosphorylation in vitro or in vivo.