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J. Biol. Chem., Vol. 283, Issue 19, 12736-12746, May 9, 2008

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Identification of a Conserved Rac-binding Site on NADPH Oxidases Supports a Direct GTPase Regulatory Mechanism^*

Yu-Ya Kao, Davide Gianni, Benjamin Bohl, Ross M. Taylor, and Gary M. Bokoch¹

From the Departments of Immunology and Cell Biology, The Scripps Research Institute, La Jolla, California 92037 and the Departments of Microbiology and Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717

The NADPH oxidases (Noxs) are a family of superoxide-generating enzymes implicated in a variety of biological processes. Full activity of Nox1, -2, and -3 requires the action of a Rac GTPase. A direct regulatory interaction of Rac with Nox2 has been proposed as part of a two-step mechanism for regulating electron transfer during superoxide formation. Using truncation analysis of Rac binding to the cytoplasmic tail of Nox2, along with peptides derived from this region in cell-free assays, we identify a Rac interaction site within amino acids 419–430 of Nox2. This region is required for binding Rac2 but not p47^phox or p67^phox cytosolic regulatory factors. A cell-permeant version of the peptide encompassing amino acids 419–430 specifically inhibits NADPH oxidase activation in intact human neutrophils. Mutational analysis of the putative Rac-binding site revealed specific residues, particularly Lys-421, Tyr-425, and Lys-426, individually required for Rac-dependent NADPH oxidase activity that are conserved in the Rac-regulated Nox1, Nox2, and Nox3 enzymes but not in Nox4 or Nox5. Mutation of the conserved residues in the Rac-binding site of Nox1 also result in the loss of Rac-dependent activity. Our data identify a functional Rac interaction site conserved in Rac-dependent Noxs and support a direct regulatory interaction of Rac GTPases to promote activation of these NADPH oxidases.

Received for publication, February 7, 2008

^* This work was supported, in whole or in part, by National Institutes of Health Grant HL48008 (to G. M. B.). This work was also supported by a grant from the Chronic Granulomatous Disorder Research Trust (to Y.-Y. K. and G. M. B.). This is Manuscript 19141 from the Department of Immunology, The Scripps Research Institute. 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. S1–S3.

¹ To whom correspondence should be addressed. Tel.: 858-784-8217; Fax: 858-784-8218; E-mail: Bokoch{at}scripps.edu.

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