Endothelial cell migration and survival are essential for angiogenesis. Focal adhesions are multicomponent juxtamembrane structures that are thought to play critical roles in the adhesion, survival and migration of endothelial and other adherent cell types. Focal Adhesion Kinase (FAK) 1 is an important component of endothelial focal adhesion complexes that is essential for embryogenesis and is strongly implicated in cell migration, adhesion, survival and cell cycle control. Tyrosine phosphorylation of FAK plays a central role in focal adhesion function by transducing signals from integrins and receptors for diverse extracellular factors including the polypeptide growth factor ligands for receptor tyrosine kinases. Specific phosphorylated tyrosine residues in FAK are thought to mediate complex formation between FAK and other signalling molecules. Phosphorylation of FAK at Y397, the major FAK autophosphorylation site, creates a high-affinity binding site for the SH2 domains of pp60src and pp59fyn, while phosphorylation at tyrosines 576 and 577 in the activation loop region of the catalytic domain enhances FAK kinase activity. FAK is also phosphorylated by Src in vitro at tyrosine residues 407 and 861, but the function of these sites is unclear. The non-catalytic NH2-terminal region of FAK can associate with peptides corresponding to the cytoplasmic domains of integrins, but otherwise little is known about the function of this domain.
Previous findings have suggested that FAK proteolytic cleavage mediated by caspases plays a central role in inhibiting FAK function, leading to the loss of focal adhesion architecture, cell retraction and detachment, and subsequent apoptotic cell death. Our studies in HUVECs and porcine aortic endothelial cells (PAECs) show that dephosphorylations at Y397 and Y861 are very early events preceding the commitment to programmed cell death, whereas FAK proteolysis is a late event during endothelial cell apoptosis which occurs after focal adhesion disassembly, annexin V staining, DNA fragmentation and cell detachment.
The angiogenic cytokine, vascular endothelial growth factor (VEGF), is a potent survival and chemotactic factor for human umbilical vein endothelial cells (HUVECs). We previously reported that VEGF stimulates FAK tyrosine phosphorylation and increases focal adhesion formation in HUVECs. VEGF increased focal adhesion localization of FAK phosphorylated at Y397 and Y861, but stimulated a marked increase in phosphorylation at Y861 without significantly affecting the total level of phosphoY397FAK. Inhibition of Src using the specific inhibitor PP2 completely blocked VEGF-induced Y861 phosphorylation without reducing the level of phosphoY397FAK. The role of Src in mediating endothelial functions of VEGF linked to FAK was also examined. PP2 markedly inhibited VEGF-induced chemotaxis and wound healing cell migration. The Src inhibitor also reduced the anti-apoptotic effect of VEGF determined by surface annexin V staining but did not increase FAK proteolysis or prevent the VEGF-dependent inhibition of FAK proteolysis. In contrast, the specific phosphatidylinositol 3′-kinase inhibitor LY294002 induced apoptosis and markedly decreased p125FAK expression and increased FAK proteolysis, but had little effect on Y861 phosphorylation.
These findings identify Y861 as a major FAK phosphorylation site in endothelial cells and identify Src as the key mediator of phosphorylation at this site. Whereas negative regulation of FAK phosphorylation is a very early event in endothelial cell apoptosis, VEGF induced Src-dependent Y861 phosphorylation. Since inhibition of Src blocked VEGF-induced endothelial cell migration and anti-apoptosis, these results suggest that Src-dependent FAK phosphorylation may be an important pathway integrating the mechanisms mediating these two VEGF-regulated endothelial cell functions.