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 endoth...

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Published in:The Journal of biological chemistry 2007-04, Vol.282 (14), p.10660-10669
Main Authors: Garcia Blanes, Mariela, Oubaha, Malika, Rautureau, Yohann, Gratton, Jean-Philippe
Format: Article
Language:English
Subjects:
Amino Acid Substitution
Animals
Aorta - cytology
Aorta - enzymology
Cattle
Chlorocebus aethiops
COS Cells
Endothelial Cells - cytology
Endothelial Cells - enzymology
Enzyme Activation - genetics
Mutation, Missense
Nitric Oxide - biosynthesis
Nitric Oxide Synthase Type III - genetics
Nitric Oxide Synthase Type III - metabolism
Phosphatidylinositol 3-Kinases - metabolism
Phospholipase C gamma - metabolism
Phosphorylation
Protein Processing, Post-Translational - genetics
Proto-Oncogene Proteins c-akt - metabolism
Signal Transduction - physiology
Tyrosine - genetics
Tyrosine - metabolism
Vascular Endothelial Growth Factor Receptor-2 - genetics
Vascular Endothelial Growth Factor Receptor-2 - metabolism
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Summary: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-γ 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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M609048200