Phosphorylation of tyrosine 801 of vascular endothelial growth factor receptor-2 is necessary for Akt-dependent endothelial nitric-oxide synthase activation and nitric oxide release from endothelial cells.
Vascular endothelial growth factor (VEGF)-stimulated nitric oxide (NO) release from endothelial cells is mediated through the activation of VEGF receptor-2 (VEGFR-2). Herein, we have attempted to determine which autophosphorylated tyrosine residue on the VEGFR-2 is essential for VEGF-mediated endothelial nitric-oxide synthase (eNOS) activation and NO production from endothelial cells. Tyrosine residues 801, 1175, and 1214 of the VEGFR-2 were mutated to phenylalanine, and the mutated receptors were analyzed for their ability to stimulate NO production. We show, both in COS-7 cells cotransfected with the VEGFR-2 mutants and eNOS and in bovine aortic endothelial cells, that the Y801F-VEGFR-2 mutant is unable to stimulate NO synthesis and eNOS activation in contrast to the wild type, Y1175F-VEGFR-2, and Y1214F-VEGFR-2. However, the Y801F mutant retains the capacity to activate phospholipase C-gamma in contrast to the Y1175F-VEGFR-2. Interestingly, the Y801F-VEGFR-2, in contrast to the wild type receptor, does not fully activate phosphatidylinositol 3-kinase or recruit the p85 subunit upon receptor activation. This results in a complete incapacity of the Y801F-VEGFR-2 to stimulate Akt activation and eNOS phosphorylation on serine 1179 in endothelial cells. In addition, constitutive activation of Akt or a phosphomimetic mutant of eNOS (S1179D) fully rescues the inability of the Y801F-VEGFR-2 to induce NO release. Finally, we generated an antibody that specifically recognizes the phosphorylated form of tyrosine 801 of the VEGFR-2 and demonstrate that this residue is actively phosphorylated in response to VEGF stimulation of endothelial cells. We thus conclude that autophosphorylation of tyrosine residue 801 of the VEGFR-2 is essential for VEGF-stimulated NO production from endothelial cells, and this is primarily accomplished via the activation of phosphatidylinositol 3-kinase and Akt signaling to eNOS.
Blanes
Mariela Garcia
MG
Laboratory of Endothelial Cell Biology, Institut de Recherches Cliniques de Montréal, Université de Montréal, Montreal, Quebec H2W 1R7, Canada.
Oubaha
Malika
M
Rautureau
Yohann
Y
Gratton
Jean-Philippe
JP
D019943:Amino Acid Substitution; D000818:Animals; D001011:Aorta / Q000166:cytology / Q000201:enzymology; D019556:COS Cells; D002417:Cattle; D002522:Chlorocebus aethiops; D042783:Endothelial Cells / Q000166:cytology / Q000201:enzymology*; D004789:Enzyme Activation / Q000235:genetics; D020125:Mutation, Missense; D009569:Nitric Oxide / Q000096:biosynthesis*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051966:Phospholipase C gamma / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000235:genetics; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism*
D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D051966:Phospholipase C gamma; D051057:Proto-Oncogene Proteins c-akt; D014443:Tyrosine; D040301:Vascular Endothelial Growth Factor Receptor-2