Phosphorylation of Tyr1214 within VEGFR-2 triggers the recruitment of Nck and activation of Fyn leading to SAPK2/p38 activation and endothelial cell migration in response to VEGF.
VEGFR-2 is the major receptor that regulates the different functions of VEGF in adults. We have previously reported that following VEGF treatment of endothelial cells, VEGFR-2 is phosphorylated on Tyr1214 upstream of the Cdc42-SAPK2/p38-MAPKAP K2 pathway. However, little is known of the earliest molecular events that compose the SAPK2/p38 pathway following VEGFR-2 activation. In this study, we address this question using HA-tagged constructs of either wild-type VEGFR-2 or Y1214F VEGFR-2 mutant in immunoprecipitation assays. We show that the Src family kinase member Fyn, but not c-Src itself, is recruited to VEGFR-2 and is activated in a p-Tyr1214-dependent manner. We also report that the SH2 domain-containing adapter molecule Nck, but not Grb2, is recruited to VEGFR-2 in a p-Tyr1214-dependent manner and that it associates with Fyn. Moreover, PAK-2 is phosphorylated in a Fyn-dependent manner. Using chemical and genetic inhibitors, we show that Fyn activity is required for SAPK2/p38 but not for FAK activation in response to VEGF. In contrast, c-Src permits activation of FAK, but not that of SAPK2/p38. In addition, Fyn is required for stress fiber formation and endothelial cell migration. We propose a model in which Fyn forms a molecular complex with Nck and PAK-2 and suggest that this complex assembles in a p-Tyr1214-dependent manner within VEGFR-2 following VEGF treatment. In turn, this triggers the activation of the SAPK2/p38 MAP kinase module, and promotes stress fiber formation and endothelial cell migration.
Lamalice
Laurent
L
Centre de Recherche en Cancérologie de l'Université Laval, 9 rue McMahon, Québec G1R 2J6, Canada.
Houle
François
F
Huot
Jacques
J
D000199:Actins / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002465:Cell Movement; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation; D005455:Fluorescent Antibody Technique; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D048290:Mitogen-Activated Protein Kinase 11 / Q000378:metabolism*; D041681:NIH 3T3 Cells; D015513:Oncogene Proteins / Q000378:metabolism*; D010766:Phosphorylation; D051076:Proto-Oncogene Proteins c-fyn / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*; D014471:Umbilical Veins; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism*; D019061:src-Family Kinases
D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; C078332:Nck protein; D015513:Oncogene Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D014443:Tyrosine; D040301:Vascular Endothelial Growth Factor Receptor-2; C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn; D019061:src-Family Kinases; D048290:Mitogen-Activated Protein Kinase 11