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J. Biol. Chem., Vol. 283, Issue 14, 8885-8892, April 4, 2008

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Synergistic Activation of the Arabidopsis NADPH Oxidase AtrbohD by Ca²⁺ and Phosphorylation^*

Yoko Ogasawara¹, Hidetaka Kaya¹, Goro Hiraoka, Fumiaki Yumoto^¶||, Sachie Kimura, Yasuhiro Kadota, Haruka Hishinuma, Eriko Senzaki, Satoshi Yamagoe^**, Koji Nagata^¶, Masayuki Nara, Kazuo Suzuki^**, Masaru Tanokura^¶, and Kazuyuki Kuchitsu²

From the Department of Applied Biological Science and the Genome & Drug Research Center, Tokyo University of Science, Noda, Chiba 278-8510, Japan, the ^¶Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan, the ^||Department of Physiology II, Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan, the ^**Department of Bioactive Molecules, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo 162-8640, Japan, and the Laboratory of Chemistry, College of Liberal Arts and Sciences, Tokyo Medical and Dental University, Ichikawa, Chiba 272-0827, Japan

Plant respiratory burst oxidase homolog (rboh) proteins, which are homologous to the mammalian 91-kDa glycoprotein subunit of the phagocyte oxidase (gp91^phox) or NADPH oxidase 2 (NOX2), have been implicated in the production of reactive oxygen species (ROS) both in stress responses and during development. Unlike mammalian gp91^phox/NOX2 protein, plant rboh proteins have hydrophilic N-terminal regions containing two EF-hand motifs, suggesting that their activation is dependent on Ca²⁺. However, the significance of Ca²⁺ binding to the EF-hand motifs on ROS production has been unclear. By employing a heterologous expression system, we showed that ROS production by Arabidopsis thaliana rbohD (AtrbohD) was induced by ionomycin, which is a Ca²⁺ ionophore that induces Ca²⁺ influx into the cell. This activation required a conformational change in the EF-hand region, as a result of Ca²⁺ binding to the EF-hand motifs. We also showed that AtrbohD was directly phosphorylated in vivo, and that this was enhanced by the protein phosphatase inhibitor calyculin A (CA). Moreover, CA itself induced ROS production and dramatically enhanced the ionomycin-induced ROS production of AtrbohD. Our results suggest that Ca²⁺ binding and phosphorylation synergistically activate the ROS-producing enzyme activity of AtrbohD.

Received for publication, September 28, 2007 , and in revised form, December 26, 2007.

^* This work was supported by a Grant-in-Aid for Young Scientists (B) (19770035) from Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (to H. K.), by a Grant-in-Aid for Plant Graduate Student from Nara Institute of Science and Technology, supported by MEXT of Japan (to S. K.), in part by a Grant-in-Aid for Japanese Society for the Promotion of Science Fellows (18-6801, to Y. K.), and in part by Grants-in-Aid for Scientific Research (B) (19370023) from MEXT of Japan (to K. K.). 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 These authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan. Tel.: 81-4-7122-9404; Fax: 81-4-7123-9767; E-mail: kuchitsu{at}rs.noda.tus.ac.jp.

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