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J. Biol. Chem., Vol. 281, Issue 31, 21857-21868, August 4, 2006

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Direct Involvement of the Small GTPase Rac in Activation of the Superoxide-producing NADPH Oxidase Nox1^*

Kei Miyano, Noriko Ueno, Ryu Takeya, and Hideki Sumimoto¹

From the Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, and CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan

Activation of the non-phagocytic superoxide-producing NADPH oxidase Nox1, complexed with p22^phox at the membrane, requires its regulatory soluble proteins Noxo1 and Noxa1. However, the role of the small GTPase Rac remained to be clarified. Here we show that Rac directly participates in Nox1 activation via interacting with Noxa1. Electropermeabilized HeLa cells, ectopically expressing Nox1, Noxo1, and Noxa1, produce superoxide in a GTP-dependent manner, which is abrogated by expression of a mutant Noxa1(R103E), defective in Rac binding. Superoxide production in Nox1-expressing HeLa and Caco-2 cells is decreased by depletion or sequestration of Rac; on the other hand, it is enhanced by expression of the constitutively active Rac1(Q61L), but not by that of a mutant Rac1 with the A27K substitution, deficient in binding to Noxa1. We also demonstrate that Nox1 activation requires membrane recruitment of Noxa1, which is normally mediated via Noxa1 binding to Noxo1, a protein tethered to the Nox1 partner p22^phox: the Noxa1-Noxo1 and Noxo1-p22^phox interactions are both essential for Nox1 activity. Rac likely facilitates the membrane localization of Noxa1: although Noxa1(W436R), defective in Noxo1 binding, neither associates with the membrane nor activates Nox1, the effects of the W436R substitution are restored by expression of Rac1(Q61L). The Rac-Noxa1 interaction also serves at a step different from the Noxa1 localization, because the binding-defective Noxa1(R103E), albeit targeted to the membrane, does not support superoxide production by Nox1. Furthermore, a mutant Noxa1 carrying the substitution of Ala for Val-205 in the activation domain, which is expected to undergo a conformational change upon Rac binding, fully localizes to the membrane but fails to activate Nox1.

Received for publication, December 22, 2005 , and in revised form, June 5, 2006.

^* This work was supported in part by Grant-in-aid for Scientific Research and National Project on Protein Structural and Functional Analyses from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and CREST and BIRD projects of JST (Japan Science and Technology Agency). 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.

¹ To whom correspondence should be addressed. Tel.: 81-92-642-6806; Fax: 81-92-642-6807; E-mail: hsumi{at}bioreg.kyushu-u.ac.jp.

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