VEGF-induced Rac1 activation in endothelial cells is regulated by the guanine nucleotide exchange factor Vav2.
Vascular endothelial growth factor (VEGF) signaling is critical for both normal and disease-associated vascular development. Dysregulated VEGF signaling has been implicated in ischemic stroke, tumor angiogenesis, and many other vascular diseases. VEGF signals through several effectors, including the Rho family of small GTPases. As a member of this family, Rac1 promotes VEGF-induced endothelial cell migration by stimulating the formation of lamellipodia and membrane ruffles. To form these membrane protrusions, Rac1 is activated by guanine nucleotide exchange factors (GEFs) that catalyze the exchange of GDP for GTP. The goal of this study was to identify the GEF responsible for activating Rac1 in response to VEGF stimulation. We have found that VEGF stimulates biphasic activation of Rac1 and for these studies we focused on the peak of activation that occurs at 30 min. Inhibition of VEGFR-2 signaling blocks VEGF-induced Rac1 activation. Using a Rac1 nucleotide-free mutant (G15ARac1), which has a high affinity for binding activated GEFs, we show that the Rac GEF Vav2 associates with G15ARac1 after VEGF stimulation. Additionally, we show that depleting endothelial cells of endogenous Vav2 with siRNA prevents VEGF-induced Rac1 activation. Moreover, Vav2 is tyrosine phosphorylated upon VEGF treatment, which temporally correlates with Rac1 activation and requires VEGFR-2 signaling and Src kinase activity. Finally, we show that depressing Vav2 expression by siRNA impairs VEGF-induced endothelial cell migration. Taken together, our results provide evidence that Vav2 acts downstream of VEGF to activate Rac1.
Department of Cell and Developmental Biology, University of North Carolina-Chapel Hill, Lineberger Comprehensive Cancer Center, Campus Box # 7295, Chapel Hill, NC 27599, USA.
Tiana A
TA
Garrett
Jaap D
JD
Van Buul
Keith
K
Burridge
D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D002633:Chemotaxis; D002522:Chlorocebus aethiops; D042783:Endothelial Cells / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D006801:Humans; D051567:Proto-Oncogene Proteins c-vav / Q000378:metabolism*; D015398:Signal Transduction; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology* / Q000502:physiology; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
D051567:Proto-Oncogene Proteins c-vav; C093651:VAV2 protein, human; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D040301:Vascular Endothelial Growth Factor Receptor-2; D020830:rac1 GTP-Binding Protein