Blanes Mariela Garcia, Oubaha Malika, Rautureau Yohann, Gratton Jean-Philippe
Laboratory of Endothelial Cell Biology, Institut de Recherches Cliniques de Montréal, Université de Montréal, Montreal, Quebec H2W 1R7, Canada.
J Biol Chem. 2007 Apr 6;282(14):10660-9. doi: 10.1074/jbc.M609048200. Epub 2007 Feb 15.
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.
血管内皮生长因子（VEGF）刺激内皮细胞释放一氧化氮（NO）是通过激活VEGF受体2（VEGFR-2）介导的。在此，我们试图确定VEGFR-2上的哪个自磷酸化酪氨酸残基对于VEGF介导的内皮型一氧化氮合酶（eNOS）激活以及内皮细胞产生NO至关重要。将VEGFR-2的酪氨酸残基801、1175和1214突变为苯丙氨酸，并分析突变受体刺激NO产生的能力。我们发现在与VEGFR-2突变体和eNOS共转染的COS-7细胞以及牛主动脉内皮细胞中，与野生型、Y1175F-VEGFR-2和Y1214F-VEGFR-2相比，Y801F-VEGFR-2突变体无法刺激NO合成和eNOS激活。然而，与Y1175F-VEGFR-2相比，Y801F突变体保留了激活磷脂酶C-γ的能力。有趣的是，与野生型受体相比，Y801F-VEGFR-2在受体激活时不能完全激活磷脂酰肌醇3-激酶或募集p85亚基。这导致Y801F-VEGFR-2完全无法刺激内皮细胞中Akt激活和eNOS在丝氨酸1179处的磷酸化。此外，Akt的组成性激活或eNOS的磷酸模拟突变体（S1179D）完全挽救了Y801F-VEGFR-2诱导NO释放的无能。最后，我们生成了一种特异性识别VEGFR-2酪氨酸801磷酸化形式的抗体，并证明该残基在VEGF刺激内皮细胞时被积极磷酸化。因此，我们得出结论，VEGFR-2酪氨酸残基801的自磷酸化对于VEGF刺激内皮细胞产生NO至关重要，这主要是通过激活磷脂酰肌醇3-激酶和Akt信号传导至eNOS来实现的。
