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-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. 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. Vascular endothelial growth factor receptor-2 (VEGFR 3The abbreviations used are: VEGFR, vascular endothelial growth factor receptor; BAEC, bovine aortic endothelial cell(s); eNOS, endothelial nitricoxide synthase; HA, hemagglutinin; myr-Akt, myristoylated-Akt; NO, nitric oxide; PI3K, phosphatidylinositol 3-kinase; PLC-γ, phospholipase C-γ; VEGF, vascular endothelial growth factor; WT, wild type.-2/Flk-1/KDR) is mainly responsible for the biological effects of VEGF in endothelial cells (1Takahashi H. Shibuya M. Clin. Sci. (Lond.). 2005; 109: 227-241Crossref PubMed Scopus (730) Google Scholar, 2Munoz-Chapuli R. Quesada A.R. Angel M.M. Cell Mol. Life Sci. 2004; 61: 2224-2243Crossref PubMed Scopus (269) Google Scholar, 3Ferrara N. Endocr. Rev. 2004; 25: 581-611Crossref PubMed Scopus (3009) Google Scholar, 4Zachary I. Gliki G. Cardiovasc. Res. 2001; 49: 568-581Crossref PubMed Scopus (577) Google Scholar). Other transmembrane tyrosine kinase receptors, such as VEGFR-1 and -3, have also been shown to transduce the intracellular signals of VEGF (1Takahashi H. Shibuya M. Clin. Sci. (Lond.). 2005; 109: 227-241Crossref PubMed Scopus (730) Google Scholar, 5Shibuya M. Claesson-Welsh L. Exp. Cell Res. 2006; 312: 549-560Crossref PubMed Scopus (861) Google Scholar). The role of VEGFR-1 appears, however, to be mostly evident during embryonic angiogenesis, and VEGFR-3 signaling is restricted to lymphatic endothelial cells (5Shibuya M. Claesson-Welsh L. Exp. Cell Res. 2006; 312: 549-560Crossref PubMed Scopus (861) Google Scholar). Gene inactivation studies in mice have revealed an essential role for VEGFR-2 in vasculo-genesis, since VEGFR-2-/- animals die between embryonic day 8.5 and 9.5 due to a lack of vascular development similar to the phenotype observed in VEGF null mice (6Shalaby F. Rossant J. Yamaguchi T.P. Gertsenstein M. Wu X.F. Breitman M.L. Schuh A.C. Nature. 1995; 376: 62-66Crossref PubMed Scopus (3371) Google Scholar, 7Carmeliet P. Ferreira V. Breier G. Pollefeyt S. Kieckens L. Gertsenstein M. Fahrig M. Vandenhoeck A. Harpal K. Eberhardt C. Declercq C. Pawling J. Moons L. Collen D. Risau W. Nagy A. Nature. 1996; 380: 435-439Crossref PubMed Scopus (3475) Google Scholar). Signal transduction of the VEGFR-2 in endothelial cells leads to the endothelial responses that are characteristic of proangiogenic factors. VEGF-dependent DNA synthesis and proliferation of endothelial cells are strongly dependent on the activation of extracellular signal-regulated kinases 1 and 2 by VEGFR-2 (2Munoz-Chapuli R. Quesada A.R. Angel M.M. Cell Mol. Life Sci. 2004; 61: 2224-2243Crossref PubMed Scopus (269) Google Scholar, 8Takahashi T. Yamaguchi S. Chida K. Shibuya M. EMBO J. 2001; 20: 2768-2778Crossref PubMed Scopus (610) Google Scholar, 9Ng Y.S. Krilleke D. Shima D.T. Exp. Cell Res. 2006; 312: 527-537Crossref PubMed Scopus (117) Google Scholar, 10Takahashi T. Shibuya M. Oncogene. 1997; 14: 2079-2089Crossref PubMed Scopus (273) Google Scholar). Endothelial cell migration stimulated by VEGF involves both the phosphatidylinositol 3-kinase and the kinase M. D. G. K. Res. PubMed Scopus Google Scholar, S. F. J. J. Oncogene. 1997; PubMed Scopus Google Scholar, S. F. H. L. J. J. J. J. PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar). of endothelial cell or signaling by VEGF in the of and in the of their VEGF endothelial cell via the A. J. M. V. N. J. PubMed Scopus Google Scholar). VEGF-stimulated nitric oxide (NO) release from endothelial cells also on the activation of NO is an endothelial that of and in vascular M. D. G. K. Res. PubMed Scopus Google Scholar, S. R. Res. PubMed Scopus Google Scholar, A. L. F. M. J. PubMed Scopus Google Scholar, L. F. M. J. 1996; PubMed Google Scholar, A. G. J. Clin. 1997; PubMed Scopus Google Scholar, T. M. D. A. PubMed Scopus Google Scholar). mice responses to VEGF both in of and in vascular D. T. A. J. Sci. S. A. 2001; PubMed Scopus Google Scholar). VEGF stimulation of endothelial cells signaling that NO production. VEGF intracellular in endothelial cells through the activation of phospholipase C-γ H. J. PubMed Scopus Google Scholar). the activation of Akt by VEGFR-2 is responsible for eNOS phosphorylation on serine 1179 in which leads to eNOS and NO release S. I. C. R. Nature. PubMed Scopus Google Scholar, D. J. K. A. Nature. PubMed Scopus Google Scholar). Interestingly, the of in NO release not been for receptor tyrosine phosphorylation of tyrosine residues in the intracellular of the VEGFR-2 been shown to be essential for the and activation of in intracellular of autophosphorylated have been through of receptors or have been to be phosphorylated on their essential role in the activation of the signaling by of the phosphorylated residues are 801, 1175, and 1214 T. Yamaguchi S. Chida K. Shibuya M. EMBO J. 2001; 20: 2768-2778Crossref PubMed Scopus (610) Google Scholar, T. S. J. T. A. P. L. C. C. Wu J. S. H. D. A. Claesson-Welsh L. EMBO J. 2005; PubMed Scopus Google Scholar, M. P. Res. PubMed Scopus Google Scholar, Res. 1997; PubMed Scopus Google Scholar, V. K. N. J. 2001; PubMed Scopus Google Scholar). are in the on the of residues in VEGFR-2 a on the role of Tyrosine residues and in the kinase to be for VEGFR-2 kinase M. Oncogene. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). Other phosphorylated residues have been to the and activation of as is the for and the and for and T. S. J. T. A. P. L. C. C. Wu J. S. H. D. A. Claesson-Welsh L. EMBO J. 2005; PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. J. PubMed Scopus Google Scholar, Oncogene. PubMed Scopus Google Scholar, K. C. R. N. T. Claesson-Welsh L. M. J. 2004; PubMed Scopus Google Scholar). of been to cell and to in mice T. Yamaguchi S. Chida K. Shibuya M. EMBO J. 2001; 20: 2768-2778Crossref PubMed Scopus (610) Google Scholar, Res. 1997; PubMed Scopus Google Scholar, A. Oncogene. PubMed Scopus Google Scholar, K. N. Shibuya M. Sci. S. A. 2005; PubMed Scopus Google Scholar). of is for and migration L. F. G. J. Oncogene. 2004; PubMed Scopus Google Scholar). Finally, phosphorylation of been to to the VEGFR-2 and activation V. K. N. J. 2001; PubMed Scopus Google Scholar). Herein, we have attempted to determine which phosphorylated tyrosine on the VEGFR-2 is essential for VEGF-mediated NO production from endothelial cells. eNOS activation is the of intracellular signals and we also which of of eNOS, are essential for activation. results show, both in a COS-7 cell and in bovine aortic endothelial cells that NO synthesis by the mutant and is similar to that of the wild type In of tyrosine 801 results in a complete of NO the Y801F-VEGFR-2, in contrast to the wild type receptor, activate the signaling and induce eNOS phosphorylation on serine However, the Y801F mutant activate in contrast to the Y1175F-VEGFR-2. Finally, we a that tyrosine 801 of the VEGFR-2 is actively phosphorylated in response to VEGF stimulation of endothelial cells. We thus conclude that autophosphorylation of the 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 and Akt signaling to eNOS. Cell cells were in with bovine and aortic endothelial cells were in with bovine and Endothelial cells were used or VEGF cells were or in with and VEGF from the of the and used for cell stimulation this and in from tyrosine to for residues and and to for residue were the on VEGFR-2 The used were and were by DNA for bovine eNOS, and were D. J. K. A. Nature. PubMed Scopus Google Scholar, M. S. C. J. PubMed Scopus Google Scholar, J. D. N. T. Res. PubMed Scopus Google Scholar). bovine eNOS and and were by C. of and COS-7 and were in or in or and were to the the of NO from cells, of were and for the of the of NO in by a NO as G. J. A. J. S. J. 1995; PubMed Scopus Google Scholar). the of NO production in COS-7 and BAEC, cell and to of VEGF-stimulated NO production from on cells. The for the of a VEGF antibody from and and were from Cell antibody from antibody from antibody from and antibody from The antibody a and the the The through a to the and the of this through a to the phosphorylated the of the the phosphorylated and and were with a 1 1 and were by and were in by and a were with the antibody for 2 of a and for an 1 The were by with in by and to antibody by a or by a or were on cells were stimulated for with VEGF were for in were with and with in for cells were with bovine and for 1 with the antibody in bovine in antibody 1 of antibody were observed a were analyzed by of by as VEGFR-2 Tyrosine NO release from endothelial cells the autophosphorylation on tyrosine residues of the In to the phosphorylated tyrosine residues of the essential for the activation of intracellular signaling to eNOS activation and NO we generated on the Tyrosine residues 801, 1175, and in the as autophosphorylation of the VEGFR-2 receptor, were to T. Yamaguchi S. Chida K. Shibuya M. EMBO J. 2001; 20: 2768-2778Crossref PubMed Scopus (610) Google Scholar, Res. 1997; PubMed Scopus Google Scholar, V. K. N. J. 2001; PubMed Scopus Google Scholar, A. Oncogene. PubMed Scopus Google Scholar, K. N. Shibuya M. Sci. S. A. 2005; PubMed Scopus Google Scholar, L. F. G. J. Oncogene. 2004; PubMed Scopus Google Scholar, V. F. N. J. PubMed Scopus Google Scholar). In addition, a receptor that a to residue in the of the tyrosine kinase generated as a that the not the kinase ability of the receptor, the of tyrosine phosphorylation of mutants wild type receptors Cell from COS-7 cells the wild type or mutant receptors were an and of tyrosine phosphorylation were by with a antibody VEGFR-2 were and autophosphorylation on tyrosine for of the tyrosine mutants and wild type receptors in contrast to the tyrosine receptor This that the tyrosine kinase of the receptor not by the Tyrosine 801 of the VEGFR-2 for of NO to the tyrosine residues of the VEGFR-2 phosphorylation is essential for the activation of eNOS. we used a COS-7 cells to the capacity of the VEGFR-2 mutants or to stimulate NO release. a of from COS-7 cells, with eNOS or eNOS in with the wild type or the mutated receptors, were for the of are the nitric oxide in Sci. S. A. PubMed Scopus Google Scholar). of were in the of COS-7 cells eNOS in the of cells 1 and the of wild type VEGFR-2 receptor with eNOS a in the production of NO with the production of cells the 2 and In of the receptor not this results are with the that the VEGFR-2 receptor the capacity of NO production J. J. Res. PubMed Scopus Google and that this COS-7 is for of eNOS activation by the Interestingly, of the VEGFR-2 tyrosine 801 to in NO production with the wild type The of NO synthesis by the Y801F mutant similar to the NO from cells with the receptor or eNOS that the Y801F the NO production. In of and 1214 not the of NO by the cells with the from the wild type cells. results an essential role for tyrosine 801 of the VEGFR-2 in the stimulation of NO release. The not in NO release by the and 1214 mutants with wild type receptor a role for residues in eNOS in contrast to which is shown to be In addition, we cotransfected with eNOS a mutant that been shown to have tyrosine kinase not M. Oncogene. PubMed Scopus Google Scholar, J. PubMed Scopus Google and the of NO from cells were similar to the and Y801F mutants not the of in transduction to eNOS we the of this on NO in an endothelial cell were with an wild type, or Y801F or mutated were for and stimulated for with VEGF and were for the of the of 2 the VEGF-stimulated in NO from the stimulation of the VEGFR-2 receptor in cells in NO production. of the VEGFR-2 a in VEGFR-2 as revealed by and observed on eNOS The of the VEGFR-2 in a VEGF in the of in the with cells the of tyrosine not the VEGF-stimulated in NO production with cells the wild type receptor 2 and In VEGF stimulation of the mutant Y801F receptor to a in NO production with cells the wild type and receptors Interestingly, NO from cells the Y801F mutant from cells VEGFR-2 1 and on this that the mutant Y801F receptor the ability to as a on the VEGFR-2 receptor and thus eNOS activation by results to the tyrosine 801 residue of the VEGFR-2 as essential for VEGF-dependent eNOS activation and NO synthesis from endothelial cells. of by the VEGFR-2 the activation of eNOS by the VEGFR-2 is mainly by intracellular signaling the and the in intracellular by the activation of We analyzed the activation of by the VEGFR-2 by the phosphorylation of by COS-7 cell the wild type or mutant or of a wild type VEGFR-2 receptor in COS-7 cells results in phosphorylation In the of the receptor not induce of the mutant Y801F and receptors tyrosine phosphorylation to similar to by the wild type the the since COS-7 cells this mutant of phosphorylation similar to by the kinase mutant and that were from the wild type results were by the of the phosphorylation of in endothelial cells. the VEGFR-2 receptor or the mutants Y801F and were stimulated with VEGF and activation via the of tyrosine that VEGF stimulation of BAEC, the wild type VEGFR-2 receptor, in a in for the COS-7 cells the Y801F mutant to a VEGF-dependent in phosphorylation and of stimulation to the by the wild type In similar in the phosphorylation of in response to VEGF stimulation were not observed in the Y1175F-VEGFR-2. The phosphorylation in from the wild type since this phosphorylation of is to the observed in VEGF-stimulated results the the essential role of tyrosine phosphorylation in the activation of by VEGF T. Yamaguchi S. Chida K. Shibuya M. EMBO J. 2001; 20: 2768-2778Crossref PubMed Scopus (610) Google Scholar, Res. 1997; PubMed Scopus Google Scholar, A. Oncogene. PubMed Scopus Google Scholar). In addition, results also demonstrate that phosphorylation on tyrosine 801 of the VEGFR-2 is for the phosphorylation of Tyrosine 801 a for PI3K, and eNOS we the Y801F mutant induce tyrosine phosphorylation of the subunit of PI3K, COS-7 cells were cotransfected with p85 in the or in of or Cell were for the and tyrosine phosphorylation of p85 by that of the VEGFR-2 a in the tyrosine phosphorylation of p85 with cells In the Y801F mutant of the phosphorylation of p85 with the VEGFR-2 that tyrosine 801 is for the activation of by the We also the between p85 and the thus from of COS-7 cells the WT, or that the VEGFR-2 in the cotransfected with the receptor, a between the 1 and Interestingly, with the receptor, the Y801F-VEGFR-2 a with p85 2 and between the receptor, and p85 results are in with the of tyrosine 801 phosphorylation in in the activation and of p85 to the VEGFR-2 V. K. N. J. 2001; PubMed Scopus Google however, that tyrosine residues also in the activation of by the The VEGF-dependent activation of Akt in the and Y801F-VEGFR-2 were in BAEC, and the activation of VEGF via serine phosphorylation VEGF stimulation of the VEGFR-2 receptor in cells in Akt This phosphorylation in the wild type VEGFR-2 However, the of VEGF-dependent Akt phosphorylation in the Y801F mutant were similar to observed in cells the receptor and with cells the This that the Y801F mutant is of Akt since does not in an in Akt activation the observed in cells. We by the Akt phosphorylation on eNOS, serine in response to VEGF stimulation of BAEC, with an or the wild type and the mutant Y801F-VEGFR-2, were stimulated with VEGF of as in eNOS phosphorylation on and this phosphorylation in cells the the Y801F mutant eNOS phosphorylation in response to VEGF that were to in cells and from observed in VEGFR-2 We thus conclude as for Akt the Y801F mutant is of eNOS phosphorylation on since does not in eNOS phosphorylation to in cells the of the VEGFR-2 on tyrosine 801 is essential for the VEGF-dependent of Akt and eNOS in endothelial cells. eNOS by Akt NO demonstrate that the inability of the Y801F mutant to stimulate NO release is due to incapacity to induce eNOS phosphorylation by we activation of Akt and eNOS the phenotype by the of tyrosine 801 of the that the of eNOS and the wild type VEGFR-2 in COS-7 cells in NO production with eNOS the Y801F-VEGFR-2 mutant not NO production. of a form of with eNOS NO production eNOS of the VEGFR-2 with eNOS and NO release with the of NO in and cells. Interestingly, the Y801F mutant not NO release from cells, of this mutant with eNOS and NO production to in cells the wild type eNOS, and This that the activation of Akt signaling the capacity of the Y801F mutant to induce eNOS activation. In a similar we eNOS, an residue which as a the Y801F-VEGFR-2 NO release S. I. C. R. Nature. PubMed Scopus Google Scholar, D. J. K. A. Nature. PubMed Scopus Google Scholar, D. J. PubMed Scopus Google Scholar). of COS-7 cells with wild type eNOS and the wild type or mutant or VEGFR-2 similar results as the wild type and both an in NO production with cells eNOS the Y801F this in NO synthesis However, the of the receptor and were in with the mutant of eNOS, the in NO synthesis by the VEGFR-2 were not of the Akt phosphorylation on eNOS the ability to the stimulation of NO production S. I. C. R. Nature. PubMed Scopus Google Scholar, D. J. K. A. Nature. PubMed Scopus Google Scholar). the COS-7 cells, the of NO as that by eNOS S. I. C. R. Nature. PubMed Scopus Google Scholar, D. J. K. A. Nature. PubMed Scopus Google Scholar, D. J. PubMed Scopus Google Scholar). of with the VEGFR-2 in a in NO that signaling serine 1179 phosphorylation on eNOS to NO release. of the with similar of NO with the Interestingly, the Y801F-VEGFR-2 to stimulate NO production from to to of the wild type This that of the phosphorylation of eNOS on the Akt for a complete of the capacity of the Y801F-VEGFR-2 to stimulate NO release. This also that the incapacity of the Y801F mutant to stimulate eNOS activation is due to inability to induce eNOS phosphorylation on Tyrosine 801 in to VEGF to demonstrate that tyrosine 801 of the VEGFR-2 is phosphorylated activation of the receptor by VEGF and that a role in VEGF we have generated an antibody that recognizes the phosphorylated form of the receptor tyrosine Tyrosine residues 1175, and 1214 of the VEGFR-2 have been as autophosphorylation VEGF stimulation T. Yamaguchi S. Chida K. Shibuya M. EMBO J. 2001; 20: 2768-2778Crossref PubMed Scopus (610) Google Scholar, T. S. J. T. A. P. L. C. C. Wu J. S. H. D. A. Claesson-Welsh L. EMBO J. 2005; PubMed Scopus Google Scholar). tyrosine 801 been as a VEGFR-2 activation for the signaling V. K. N. J. 2001; PubMed Scopus Google and eNOS of phosphorylation been the of antibody for the phosphorylation of we the phosphorylated and The the phosphorylated of the with the we COS-7 cells with the wild type and mutant VEGFR-2 receptors Cell were for the VEGFR-2 and with the tyrosine The antibody specifically phosphorylation in the wild type and the mutant to the receptor and the Y801F-VEGFR-2 In addition, we for the of tyrosine phosphorylation the antibody and as shown in that the phosphorylation of the mutant receptor Y801F similar to that of the mutant and wild type receptors The phosphorylation by this antibody in the Y801F mutant is due to of phosphorylated tyrosine residues on however, that this antibody a for the phosphorylated form of demonstrate that this residue on the VEGFR-2 is phosphorylated in endothelial cells in response to VEGF were stimulated with VEGF for or and the receptor and to a the an in phosphorylation in response to VEGF in endothelial cells that phosphorylation of stimulation The of tyrosine 801 phosphorylation the autophosphorylation of the VEGFR-2 as revealed by the with the antibody VEGF stimulation of endothelial cells the of the as revealed by of the with an antibody M. S. C. J. PubMed Scopus Google Scholar). Finally, we to determine this antibody phosphorylation in in endothelial cells. we studies on VEGF-stimulated and of of the VEGFR-2 by in BAEC, the of phosphorylation are since the is the in cells with the antibody This is with the results in and phosphorylation of the VEGFR-2 is in cells. VEGF stimulation of for a in the a of the This intracellular phosphorylation of tyrosine 801 of the VEGFR-2 is in with the of an of the intracellular in VEGFR-2 signaling A. L. V. H. 2006; PubMed Scopus Google Scholar, F. C. J. Cell 2006; PubMed Scopus Google Scholar). The phosphorylation is similar to phosphorylation by F. C. J. Cell 2006; PubMed Scopus Google Scholar). This antibody to demonstrate that the tyrosine residue 801 of the VEGFR-2 is an of this receptor VEGF stimulation in the to eNOS activation and NO release from endothelial cells. The results that tyrosine 801 of the VEGFR-2 is for VEGF-stimulated NO is for phosphorylation and of the p85 subunit of to the both Akt and eNOS VEGFR-2 the incapacity of the Y801F-VEGFR-2 to stimulate NO release be by the phosphorylation of eNOS on the Akt and tyrosine 801 on the VEGFR-2 is phosphorylated in endothelial cells in response to VEGF we demonstrate that phosphorylation of eNOS on VEGFR-2 activation is an essential for NO and this is dependent on the phosphorylation of tyrosine 801 of the results eNOS phosphorylation on serine 1179 by Akt as an for eNOS activation and NO production in response to VEGF and that VEGF signaling such as are not for eNOS activation. of to NO both in a COS-7 cell and in endothelial cells. we that the Y801F mutant is of Akt phosphorylation on and eNOS phosphorylation on that the incapacity of the mutated receptor to induce NO release is due to signaling Interestingly, the Y801F VEGFR-2 mutant is fully of phosphorylation in contrast to the mutant that eNOS phosphorylation by Akt is a for NO release. Other signaling are in VEGF-stimulated NO by the that the VEGFR-2 is of NO release from the in intracellular and activation have been in VEGF-stimulated NO release H. J. PubMed Scopus Google Scholar). However, results to and demonstrate in the of an PLC-γ, NO release does not phosphorylation of eNOS on by Akt phosphorylation of the receptor is not that in the of an of the intracellular of by PLC-γ, phosphorylation of of eNOS is to NO release to results also that the of eNOS activation by VEGF in contrast to the in the of NO by endothelial cells VEGF stimulation A. P. C. J. 2001; PubMed Scopus Google Scholar, S. J. PubMed Scopus Google Scholar). this in an by S. P. M. T. F. L. D. A. Res. 2006; PubMed Scopus Google that of tyrosine of the VEGFR-2 results in stimulated NO release. the receptor, demonstrate that phosphorylation of tyrosine of the VEGFR-2 is in eNOS through activation of Akt by This results in a by of the NO release. of this of the stimulated NO the conclude that tyrosine residues are in eNOS activation by the that phosphorylation of is a for this signaling via Akt since results in a complete of eNOS phosphorylation and activation. Interestingly, the by S. P. M. T. F. L. D. A. Res. 2006; PubMed Scopus Google that the tyrosine residue of the VEGFR-1 is essential for eNOS activation by this receptor, which demonstrate for the of NO release by demonstrate that of the VEGFR-1 eNOS activation through Akt This is tyrosine of the VEGFR-1 is to tyrosine 801 of the to tyrosine 801 of the VEGFR-2 not been for role in eNOS activation. studies demonstrate that both VEGF receptors a signaling to stimulate NO production from endothelial cells and that eNOS phosphorylation by Akt is a and for NO release. of tyrosine 801 of the VEGFR-2 been as in activation V. K. N. J. 2001; PubMed Scopus Google Scholar). We that the of to of the VEGFR-2 p85 phosphorylation and however, both are not This that the phosphorylation of tyrosine residues does in the and activation of p85 to the receptor through such as to with phosphorylated or the T. S. J. T. A. P. L. C. C. Wu J. S. H. D. A. Claesson-Welsh L. EMBO J. 2005; PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, M. A. A. P. J. 2006; PubMed Scopus Google Scholar). results that a in the activation of the p85 subunit of by the VEGFR-2 leads to a complete of the signaling Akt and eNOS, in the of NO release. of tyrosine of the VEGFR-2 been shown to be essential for embryonic in mice a of this residue die in of a of endothelial cell proliferation and to the VEGFR-2 null mice K. N. Shibuya M. Sci. S. A. 2005; PubMed Scopus Google Scholar). that phosphorylation of this residue is not essential for NO which is in with a role for NO in and in that the phosphorylation of the residues and in of VEGF-mediated In we have phosphorylation of tyrosine 801 of the VEGFR-2 as the intracellular essential for VEGF transduction to eNOS via We also that eNOS phosphorylation on is a that is essential for VEGF-stimulated release of an of VEGF effects on endothelial cells, nitric We for of the and for

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