Stimulus-dependent regulation of the phagocyte NADPH oxidase by a VAV1, Rac1, and PAK1 signaling axis.
The p21-activated kinase-1 (PAK1) is best known for its role in the regulation of cytoskeletal and transcriptional signaling pathways. We show here in the microglia cell line Ra2 that PAK1 regulates NADPH oxidase (NOX-2) activity in a stimulus-specific manner. Thus, conditional expression of PAK1 dominant-positive mutants enhanced, whereas dominant-negative mutants inhibited, NADPH oxidase-mediated superoxide generation following formyl-methionyl-leucylphenylalanine or phorbol 12-myristate 13-acetate stimulation. Both Rac1 and the GTP exchange factor VAV1 were required as upstream signaling proteins in the formyl-methionyl-leucyl-phenylalanine-induced activation of endogenous PAK1. In contrast, PAK1 mutants had no effect on superoxide generation downstream of FcgammaR signaling during phagocytosis of IgG-immune complexes. We further present evidence that the effect of PAK1 on the respiratory burst is mediated through phosphorylation of p47(Phox), and we show that expression of a p47(Phox) (S303D/S304D/S320D) mutant, which mimics phosphorylation by PAK1, induced basal superoxide generation in vivo. In contrast PAK1 substrates LIMK-1 or RhoGDI are not likely to contribute to the PAK1 effect on NADPH oxidase activation. Collectively, our findings define a VAV1-Rac1-PAK1 signaling axis in mononuclear phagocytes regulating superoxide production in a stimulus-dependent manner.
Roepstorff
Kirstine
K
Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, 2200N Copenhagen, Denmark.
Rasmussen
Izabela
I
Sawada
Makoto
M
Cudre-Maroux
Cristophe
C
Salmon
Patrick
P
Bokoch
Gary
G
van Deurs
Bo
B
Vilhardt
Frederik
F
D019943:Amino Acid Substitution; D000818:Animals; D000936:Antigen-Antibody Complex / Q000378:metabolism / Q000494:pharmacology; D002273:Carcinogens / Q000494:pharmacology; D002460:Cell Line; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D054461:Lim Kinases / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D017628:Microglia / Q000166:cytology / Q000378:metabolism; D020125:Mutation, Missense; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism*; D010586:Phagocytes / Q000378:metabolism; D051567:Proto-Oncogene Proteins c-vav / Q000235:genetics / Q000378:metabolism*; D017452:Receptors, IgG / Q000235:genetics / Q000378:metabolism; D016897:Respiratory Burst; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013481:Superoxides / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D054462:p21-Activated Kinases / Q000235:genetics / Q000378:metabolism*; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*; D062948:rho Guanine Nucleotide Dissociation Inhibitor alpha; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors
C569638:ARHGDIA protein, human; D000936:Antigen-Antibody Complex; D002273:Carcinogens; D020730:Guanine Nucleotide Dissociation Inhibitors; D008562:Membrane Glycoproteins; D009479:Neuropeptides; D051567:Proto-Oncogene Proteins c-vav; C107953:RAC1 protein, human; C484602:Rac1 protein, mouse; D017452:Receptors, IgG; C496618:VAV1 protein, human; C496619:Vav1 protein, mouse; D062948:rho Guanine Nucleotide Dissociation Inhibitor alpha; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D013481:Superoxides; D009240:N-Formylmethionine Leucyl-Phenylalanine; C056558:CYBB protein, human; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; C515892:LIMK1 protein, human; D054461:Lim Kinases; C515893:Limk1 protein, mouse; C515903:PAK1 protein, human; C515905:Pak1 protein, mouse; D054462:p21-Activated Kinases; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D013755:Tetradecanoylphorbol Acetate