Synergistic activation of the Arabidopsis NADPH oxidase AtrbohD by Ca2+ and phosphorylation.
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.
Ogasawara
Yoko
Y
Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba, Japan.
Kaya
Hidetaka
H
Hiraoka
Goro
G
Yumoto
Fumiaki
F
Kimura
Sachie
S
Kadota
Yasuhiro
Y
Hishinuma
Haruka
H
Senzaki
Eriko
E
Yamagoe
Satoshi
S
Nagata
Koji
K
Nara
Masayuki
M
Suzuki
Kazuo
K
Tanokura
Masaru
M
Kuchitsu
Kazuyuki
K
D020816:Amino Acid Motifs / Q000502:physiology; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000096:biosynthesis* / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology*; D018506:Gene Expression Regulation, Plant / Q000187:drug effects / Q000502:physiology*; D057927:Hydrophobic and Hydrophilic Interactions; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008387:Marine Toxins; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000235:genetics; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics; D010080:Oxazoles / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000502:physiology; D017382:Reactive Oxygen Species / Q000378:metabolism*
D029681:Arabidopsis Proteins; D004791:Enzyme Inhibitors; D007476:Ionophores; D008387:Marine Toxins; D009097:Multienzyme Complexes; D010080:Oxazoles; D017382:Reactive Oxygen Species; D015759:Ionomycin; C059041:calyculin A; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D002118:Calcium