A single autophosphorylation site on KDR/Flk-1 is essential for VEGF-A-dependent activation of PLC-gamma and DNA synthesis in vascular endothelial cells

KDR/Flk-1 tyrosine kinase, one of the two vascular endothelial growth facto r (VEGF) receptors, induces mitogenesis and differentiation of vascular end othelial cells. To understand the mechanisms underlying the VEGF-A-induced growth signaling pathway, we constructed a series of human KDR mutants and examined their biological properties. An in vitro kinase assay and subseque nt tryptic peptide mapping revealed that Y1175 and Y1214 are the two major VEGF-A-dependent autophosphorylation sites. Using an antibody highly specif ic to the phosphoY1175 region, we demonstrated that Y1175 is phosphorylated rapidly in vivo in primary endothelial cells. When the mutated KDRs were i ntroduced into the endothelial cell lines by adenoviral vectors, only the Y 1175F KDR, Tyr1175 to phenylalanine mutant, Lost the ability to tyrosine ph osphorylate phospholipase C-gamma and, significantly, reduced MAP kinase ph osphorylation and DNA synthesis in response to VEGF-A. Furthermore, primary endothelial cells microinjected with anti-phosphoY1175 antibody clearly de creased DNA synthesis compared with control cells. These findings strongly suggest that autophosphorylation of Y1175 on KDR is crucial for endothelial cell proliferation, and that this region is a new target for anti-angiogen ic reagents.