Src mediates stimulation by vascular endothelial growth factor of the phosphorylation of focal adhesion kinase at tyrosine 861, and migration and anti-apoptosis in endothelial cells

Vascular endothelial growth factor (VEGF) stimulates the tyrosine phosphory lation of focal adhesion kinase (FAK), increases focal adhesion formation a nd is chemotactic for human umbilical-vein endothelial cells (HUVECs). In t he present study we identified the major sites of VEGF-induced FAK tyrosine phosphorylation and investigated the mechanism mediating this pathway in t he action of VEGF. VEGF increased the focal adhesion localization of FAK ph osphorylated at Tyr-397 (Y397) and Y861 but stimulated a marked increase in phosphorylation at Y861 without significantly affecting the total level of phospho-Y397 FAK. Inhibition of Src with the specific inhibitor 4-amino-5- (4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) completely bloc ked VEGF-induced Y861 phosphorylation without decreasing the level of phosp ho-Y397 FAK. We also examined the role of Src in mediating endothelial func tions of VEGF in which FAK has been implicated as having a role. PP2 marked ly inhibited V-EGF-induced chemotaxis and wound-healing cell migration. The Src inhibitor also decreased the anti-apoptotic effect of VEGF determined by surface staining of annexin V but did not increase FAK proteolysis or pr event the VEGF-dependent inhibition of FAK proteolysis. In contrast, the sp ecific PtdIns 3-kinase inhibitor LY294002 induced apoptosis and markedly de creased p125(FAK) expression and increased FAK proteolysis but had little e ffect on Y861 phosphorylation. These findings identify Src-dependent FAK ph osphorylation at Y861 as a novel VEGF-induced signalling pathway in endothe lial cells and suggest that this pathway might be involved in the mechanism s mediating VEGF-induced endothelial cell migration and anti-apoptosis.