Synergistic activation of the Arabidopsis NADPH oxidase AtrbohD by Ca2+ and phosphorylation |
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Authors: | Ogasawara Yoko Kaya Hidetaka Hiraoka Goro Yumoto Fumiaki Kimura Sachie Kadota Yasuhiro Hishinuma Haruka Senzaki Eriko Yamagoe Satoshi Nagata Koji Nara Masayuki Suzuki Kazuo Tanokura Masaru Kuchitsu Kazuyuki |
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Institution: | Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba, Japan. |
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Abstract: | 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 Ca(2+). However, the significance of Ca(2+) 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 Ca(2+) ionophore that induces Ca(2+) influx into the cell. This activation required a conformational change in the EF-hand region, as a result of Ca(2+) 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 Ca(2+) binding and phosphorylation synergistically activate the ROS-producing enzyme activity of AtrbohD. |
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