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-2) plays a critical role in vasculogenesis and angiogenesis. However, the mechanism by which VEGFR-2 activation elicits these cellular events is not fully understood. We recently constructed a chimeric receptor containing the extracellular domain of human CSF-1R/c-fms, fused with the entire transmembrane and cytoplasmic domains of murine VEGFR-2 (Rahimi, N., Dayanir, V., and Lashkari, K. (2000) J. Biol. Chem. 275, 16986-16992). In this study we used VEGFR-2 chimera (herein named CKR) to elucidate the signal transduction relay of 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. Further analysis revealed that tyrosines 799 and 1173 are docking sites for p85 of phosphatidylinositol 3-kinase (PI3K). Pretreatment of cells with wortmannin, an inhibitor of PI3K, and rapamycin, a potent inhibitor of S6 kinase, abrogated CKR-mediated cell growth. However, expression of a dominant negative form of ras (N(17)ras) and inhibition of the mitogen-activated protein kinase (MAPK) pathway by PD98059 did not attenuate CKR-stimulated cell growth. Altogether, these results demonstrate that activation of VEGFR-2 results in activation of PI3K and that activation of PI3K/S6kinase pathway, but not Ras/MAPK, is responsible for VEGFR-2-mediated cell growth.
Department of Ophthalmology and Biochemistry, School of Medicine, Boston University, Boston, Massachusetts 02118, USA.
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Dayanir
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Meyer
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Lashkari
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Rahimi
D000818:Animals; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004789:Enzyme Activation; D016297:Mutagenesis, Site-Directed; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D017978:Receptors, Growth Factor / Q000235:genetics / Q000378:metabolism*; D040262:Receptors, Vascular Endothelial Growth Factor; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014443:Tyrosine
D017978:Receptors, Growth Factor; D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor