Induced focal adhesion kinase (FAK) expression in FAK-null cells enhances cell spreading and migration requiring both auto- and activation loop phosphorylation sites and inhibits adhesion-dependent tyrosine phosphorylation of Pyk2

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase involv ed in integrin-mediated control of cell behavior. Following cell adhesion t o components of the extracellular matrix, FAK becomes phosphorylated at mul tiple sites, including tyrosines 397, 576, and 577. Tyr-397 is an autophosp horylation site that promotes interaction with c-Src or Fyn. Tyr-576 and Ty r-577 lie in the putative activation loop of the kinase domain, and FAK cat alytic activity may be elevated through phosphorylation of these residues b y associated Src family kinase. Recent studies have implicated FAK as a pos itive regulator of cell spreading and migration. To further study the mecha nism of adhesion-induced FAK activation and the possible role and signaling requirements for FAK in cell spreading and migration, we utilized the tetr acycline repression system to achieve inducible expression of either wild-t ype FAK or phosphorylation site mutants in fibroblasts derived from FAK-nul l mouse embryos. Using these Tet-FAK cells, we demonstrated that both the F AK autophosphorylation and activation loop sites are critical for maximum a dhesion-induced FAK activation and FAK-enhanced cell spreading and migratio n responses. Negative effects on cell spreading and migration, as well as d ecreased phosphorylation of the substrate p130(Cas), were observed upon ind uced expression of the FAK autophosphorylation site mutant. These negative effects appear to result from an inhibition of integrin-mediated signaling by the FAK-related kinase Pyk2/CAK beta/RAFTK/CadTK.