Stimulus-dependent Regulation of the Phagocyte NADPH Oxidase by a VAV1, Rac1, and PAK1 Signaling Axis

doi:10.1074/jbc.M708281200

Stimulus-dependent Regulation of the Phagocyte NADPH Oxidase by a VAV1, Rac1, and PAK1 Signaling Axis^*

^{^‡}Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, 2200N Copenhagen, Denmark, the ^{^¶}Department of Microbiology, Université de Genève, 1211 Genève, Switzerland, the ^{^∥}Departments of Immunology and Cell Biology, Scripps Research Institute, La Jolla, California 92037, and the ^{^§}Department of Brain Function, Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-8601, Japan

1 To whom correspondence should be addressed: Dept. of Cellular and Molecular Medicine, Bldg. 18.4, The Panum Institute, Copenhagen University, Blegdamsvej 3, 2200N Copenhagen, Denmark. Tel.: 45-35327120; Fax: 45-35327285; E-mail: f.vilhardt{at}mai.ku.dk.

Abstract

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 FcγR 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.

Footnotes

↵² The abbreviations used are: cyt b₅₅₈, flavocytochrome b₅₅₈; AA, arachidonic acid; BIM-1, bisindolylmaleimide-1; GEF, GTP exchange factor; LIMK, LIM kinase; NOS, NO synthetase; PAK, p21-activated kinase; PBD, p21-binding domain; fMLP, formyl-methionyl-leucyl-phenylalanine; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; mAb, monoclonal antibody; HA, hemagglutinin; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GFP, green fluorescent protein; WT, wild type; PVDF, polyvinylidene difluoride; DPI, diphenyliodonium; HBSS, Hanks' balanced salt solution; FACS, fluorescence-activated cell sorter; MOPS, 4-morpholinepropanesulfonic acid; luminol E-CL, luminol-enhanced chemiluminescence; DHCF, 5,7-dichlorodihydrofluorescein.
↵³ I. Rasmussen, K. Roepstorff, M. Sawada, K. Suzuki, and F. Vilhardt, manuscript in preparation.
↵^* This work was supported by Danish Research Council Grant 22-04-0336, grants from Scleroseforeningen and the Lundbeck Foundation (to F. V.), and National Institutes of Health Grant HL48008 (to G. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵ The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1-4.
- Received October 4, 2007.
- Revision received December 17, 2007.
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