Phosphorylation of tyrosine 1214 on VEGFR2 is required for VEGF-induced activation of Cdc42 upstream of SAPK2/p38.
Activation of the tyrosine kinase receptor vascular endothelial growth factor receptor 2 (VEGFR2) by VEGF leads to the activation of stress-activated protein kinase (SAPK)2/p38 and then to actin polymerization and reorganization into stress fibers in endothelial cells. In turn, this triggers endothelial cell migration. Yet, nothing is known about the molecular mechanisms that couple VEGFR2 to SAPK2/p38. Here, we found that VEGF increased by twofold the activity of the small GTPase Cdc42 and that the expression of two different constitutively active forms of Cdc42 (Cdc42 V12 and Cdc42 L61) led to a marked increase in the formation of stress fibers that was sensitive to SAPK2/p38 inhibition by SB203580. Moreover, the expression of a dominant-negative form of Cdc42 (Cdc42 N17) inhibited the activation of SAPK2/p38 and of its direct target MAP kinase-activated protein kinase 2. These results indicate that Cdc42 is upstream of SAPK2/p38 in response to the activation of VEGFR2 by VEGF. In contrast, we found that neither RhoA nor Rac was involved in the SAPK2/p38-mediated actin reorganization induced by VEGF. Using a site-specific mutant of the major autophosphorylation site Y1214 on VEGFR2, we found that the mutant Y1214F inhibited the activation of both Cdc42 and SAPK2/p38 in response to VEGF. We conclude that phosphorylation of Y1214 on VEGFR2 is required to trigger the sequential activation of Cdc42 and SAPK2/p38 and to drive the SAPK2/p38-mediated actin remodeling in stress fibers in endothelial cells exposed to VEGF.
Lamalice
Laurent
L
Le Centre de recherche en cancérologie de l'Université Laval, L'Hôtel-Dieu de Québec, 9 rue McMahon, Québec, Canada G1R 2J6.
Houle
François
F
Jourdan
Guillaume
G
Huot
Jacques
J
D000199:Actins / Q000378:metabolism; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008954:Models, Biological; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D022502:Stress Fibers / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism*; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D000199:Actins; D042461:Vascular Endothelial Growth Factor A; D019000:Phosphotyrosine; D040301:Vascular Endothelial Growth Factor Receptor-2; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein