Vascular endothelial growth factor causes translocation of p47phox to membrane ruffles through WAVE1.
Growth factors initiate cytoskeletal rearrangements tightly coordinated with nuclear signaling events. We hypothesized that the angiogenic growth factor, vascular endothelial growth factor (VEGF), may utilize oxidants that are site-directed to a complex critical to both cytoskeletal and mitogenic signaling. We identified the WASP-family verprolin homologous protein-1 (WAVE1) as a binding partner for the NADPH oxidase adapter p47phox within membrane ruffles of VEGF-stimulated cells. Within 15 min of VEGF stimulation, p47phox coprecipitated with WAVE1, with the ruffle and oxidase agonist Rac1, and with the Rac1 effector PAK1. VEGF also increased p47phox phosphorylation, oxidant production, and ruffle formation, all of which were dependent upon PAK1 kinase activity. The antioxidant Mn (III) tetrakis(4-benzoic acid) porphyrin and ectopic expression of either the p47-binding WAVE1 domain or the WAVE1-binding p47phox domain decreased VEGF-induced ruffling, whereas the active mutant p4-(S303D, S304D,S328D) stimulated oxidant production and formation of circular dorsal ruffles. Both kinase-dead PAK1-(K298A) and Mn (III) tetrakis(4-benzoic acid) porphyrin decreased c-Jun N-terminal kinase (JNK) activation by VEGF, whereas dominant-negative JNK did not block ruffle formation, suggesting a bifurcation of mitogenic and cytoskeletal signaling events at or distal to the oxidase but proximal to JNK. Thus, WAVE1 may act as a scaffold to recruit the NADPH oxidase to a complex involved with both cytoskeletal regulation and downstream JNK activation.
Wu
Ru Feng
RF
University of Texas Southwestern and The Dallas Veterans Medical Center, Dallas, Texas 75216, USA.
Gu
Ying
Y
Xu
You Cheng
YC
Nwariaku
Fiemu E
FE
Terada
Lance S
LS
D000975:Antioxidants / Q000494:pharmacology; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology; D004789:Enzyme Activation; D016166:Free Radical Scavengers / Q000494:pharmacology; D015723:Gene Library; D005822:Genetic Vectors; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D008164:Luminescent Proteins / Q000378:metabolism; D008665:Metalloporphyrins / Q000494:pharmacology; D008840:Microfilament Proteins / Q000737:chemistry* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism; D016877:Oxidants / Q000737:chemistry / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism; D014471:Umbilical Veins / Q000166:cytology; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D051300:Wiskott-Aldrich Syndrome Protein Family
D000975:Antioxidants; D016166:Free Radical Scavengers; D008164:Luminescent Proteins; D008665:Metalloporphyrins; D008840:Microfilament Proteins; D016877:Oxidants; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D042461:Vascular Endothelial Growth Factor A; D051300:Wiskott-Aldrich Syndrome Protein Family; C097284:manganese(III)-tetrakis(4-benzoic acid)porphyrin; D049452:Green Fluorescent Proteins; D014443:Tyrosine; D000242:Cyclic AMP; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D005982:Glutathione Transferase; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D005978:Glutathione