Identification of tyrosine residues in vascular endothelial growth factor receptor-2/FLK-1 involved in activation of phosphatidylinositol 3-kinase and cell proliferation

Activation of vascular endothelial growth factor receptor-2 (VEGFR-8) plays a critical role in vasculogenesis and angiogenesis, However, the mechanism by which VEGFR-2 activation elicits these cellular events is not fully und erstood. We recently constructed a chimeric receptor containing the extrace llular domain of human CSF-1R/c-fms, fused with the entire transmembrane an d cytoplasmic domains of murine VEGFR-2 (Rahimi, N., Dayanir, V., and Lashk ari, K, (2000) J. Biol, Chem, 275, 16986-16992), In this study we used VEGF R-2 chimera (herein named CKR) to elucidate the signal transduction relay o f VEGFR-2 in porcine aortic endothelial (PAE) cells. Mutation of tyrosines 799 and 1173 individually on CKR resulted in partial loss of CKR's ability to stimulate cell growth. Double mutation of these sites caused total loss of CKR's ability to stimulate cell growth. Interestingly, mutation of these sites had no effect on the ability of CKR to stimulate cell migration. Fur ther analysis revealed that tyrosines 799 and 1173 are docking sites for p8 5 of phosphatidylinositol 3-kinase (PI3K), Pretreatment of cells with wortm annin, an inhibitor of PI3K, and rapamycin, a potent inhibitor of S6 kinase , abrogated CKR-mediated cell growth. However, expression of a dominant neg ative form of res (N(17)ras) and inhibition of the mitogen-activated protei n kinase (MAPK) pathway by PD98059 did not attenuate CKR-stimulated cell gr owth. Altogether, these results demonstrate that activation of VEGFR-2 resu lts in activation of PI3K and that activation of PI3K/S6kinase pathway, but not Ras/MAPK, is responsible for VEGFR-2-mediated cell growth.