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J. Biol. Chem., Vol. 282, Issue 48, 34787-34800, November 30, 2007

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Regulation of Nox1 Activity via Protein Kinase A-mediated Phosphorylation of NoxA1 and 14-3-3 Binding^*

Jun-Sub Kim, Becky A. Diebold¹, Bernard M. Babior^¶, Ulla G. Knaus, and Gary M. Bokoch^||2

From the Departments of Immunology, ^||Cell Biology, and ^¶Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037 and Department of Pathology, Emory University, Atlanta, Georgia 30322

Nox activator 1 (NoxA1) is a homologue of p67^phox that acts in conjunction with Nox organizer 1 (NoxO1) to regulate reactive oxygen species (ROS) production by the NADPH oxidase Nox1. The phosphorylation of cytosolic regulatory components by multiple kinases plays important roles in assembly and activity of the phagocyte NADPH oxidase (Nox2) system, but little is known about regulation by phosphorylation in the Nox1 system. Here we identify Ser¹⁷² and Ser⁴⁶¹ of NoxA1 as phosphorylation sites for protein kinase A (PKA). A consequence of this phosphorylation was the enhancement of NoxA1 complex formation with 14-3-3 proteins. Using both a transfected human embryonic kidney 293 cell Nox1 model system and endogenous Nox1 in colon cell lines, we showed that the elevation of cAMP inhibits, whereas the inhibition of PKA enhances, Nox1-dependent ROS production through effects on NoxA1. Inhibition of Nox1 activity was intensified by the availability of 14-3-3 protein, and this regulatory interaction was dependent on PKA-phosphorylatable sites at Ser¹⁷² and Ser⁴⁶¹ in NoxA1. We showed that phosphorylation and 14-3-3 binding induce the dissociation of NoxA1 from the Nox1 complex at the plasma membrane, suggesting a mechanism for the inhibitory effect on Nox1 activity. Our data establish that PKA-phosphorylated NoxA1 is a new binding partner of 14-3-3 protein(s) and that this forms the basis of a novel mechanism regulating the formation of ROS by Nox1 and, potentially, other NoxA1-regulated Nox family members.

Received for publication, June 11, 2007 , and in revised form, September 28, 2007.

^* This work was supported by Centers for Disease Control Grant CI000095 (to G. M. B. and U. G. K.), National Institutes of Health Grant CA068376 (to B. M. B.), and American Heart Association Award 0535081N (to B. A. D.). 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.

We dedicate this work to Bernie Babior, who helped in its initiation.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4.

¹ Senior co-author. To whom correspondence may be addressed: Emory University, Whitehead Bldg., Rm. 165, 615 Michael St., Atlanta, GA 30322. Tel.: 404-727-5880; Fax: 404-727-8538; E-mail: bdiebol{at}emory.edu. ² Senior co-author. To whom correspondence may be addressed: The Scripps Research Inst., IMM 14, 10550 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-8217; Fax: 858-784-8218; E-mail: bokoch{at}scripps.edu.

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