Identification of VEGF receptor-2 tyrosine phosphorylation sites involved in VEGF-mediated endothelial platelet-activating factor synthesis.
Vascular endothelial growth factor (VEGF)-mediated inflammation requires the synthesis of acute platelet-activating factor (PAF) by endothelial cells (ECs). We previously reported that VEGF-mediated PAF synthesis involves the activation of the homodimeric tyrosine kinase receptor VEGFR-2/R-2, leading to the recruitment of p38 and p42/p44 mitogen-activated protein kinases (MAPKs) and activation of secreted group V phospholipase A₂ (sPLA₂-V). We have also reported that VEGF-A₁₆₅-mediated prostacyclin (PGI₂) synthesis requires VEGFR-1/R-2 heterodimeric receptor activation. Selective activation of VEGF receptors can coordinate the synthesis of pro-PAF and anti-PGI₂ inflammatory factors. It is unknown which VEGFR-2 tyrosine phosphorylation site(s) contribute(s) to PAF synthesis. Bovine aortic endothelial cells (BAECs) were transfected with pcDNA vectors encoding for native VEGF receptor-2 (VEGFR-2) cDNA or VEGFR-2 cDNA containing tyrosine phosphorylation sites mutated into phenylalanine residues (Y801F, Y1059F, Y1175F, Y1214F); an empty pcDNA vector was used as a negative control. Treatment of pcDNA-transfected BAECs with VEGF (10⁻⁹ mol/L) for 15 min increased PAF synthesis by 180%. In BAECs transfected with pcDNA vectors encoding mutated Y801F, Y1059F, Y1175F, or Y1214F VEGFR-2 cDNA, we observed a marked reduction of VEGF-mediated PAF synthesis by 38%, 46%, 69%, and 31%, respectively, compared with BAECs transfected with pcDNA vector encoding VEGFR-2 cDNA. Our data provide a novel insight as to the mechanisms by which VEGF promotes PAF synthesis.
Rechka
Abdennebi
A
Research Centre, Montreal Heart Institute, QC, Canada.
Neagoe
Paul-Eduard
PE
Gratton
Jean-Philippe
JP
Sirois
Martin G
MG
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010972:Platelet Activating Factor / Q000096:biosynthesis*; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism*
D010972:Platelet Activating Factor; D042461:Vascular Endothelial Growth Factor A; D014443:Tyrosine; D040301:Vascular Endothelial Growth Factor Receptor-2