Journal Article

Biochemical Characterization of In Vitro Phosphorylation and Dephosphorylation of the Plasma Membrane H<sup>+</sup>-ATPase

Yuki Hayashi, Suguru Nakamura, Atsushi Takemiya, Yohei Takahashi, Ken-ichiro Shimazaki and Toshinori Kinoshita

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 51, issue 7, pages 1186-1196
Published in print July 2010 | ISSN: 0032-0781
Published online June 2010 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pcq078
Biochemical Characterization of In Vitro Phosphorylation and Dephosphorylation of the Plasma Membrane H+-ATPase

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Stomatal opening, which is mediated by blue light receptor phototropins, is driven by activation of the plasma membrane H+-ATPase via phosphorylation of the penultimate threonine in the C-terminus and subsequent binding of a 14-3-3 protein. However, the biochemical properties of the protein kinase and protein phosphatase for H+-ATPase are largely unknown. We therefore investigated in vitro phosphorylation and dephosphorylation of H+-ATPase. H+-ATPase was phosphorylated in vitro on the penultimate threonine in the C-terminus in isolated microsomes from guard cell protoplasts of Vicia faba. Phosphorylated H+-ATPase was dephosphorylated in vitro, and the dephosphorylation was inhibited by EDTA, a divalent cation chelator, but not by calyculin A, an inhibitor of type 1 and 2A protein phosphatases. Essentially the same results were obtained in purified plasma membranes from etiolated Arabidopsis seedlings, indicating that a similar protein kinase and phosphatase are involved in plant cells. Further analyses revealed that phosphorylation of the H+-ATPase is insensitive to K-252a, a potent inhibitor of protein kinase, and is hypersensitive to Triton X-100, a non-ionic detergent. Moreover, dephosphorylation required Mg2+ but not Ca2+, and protein phosphatase was localized in the 1% Triton X-100-insoluble fraction. These results demonstrate that a protein kinase–phosphatase pair, K-252a-insensitive protein kinase and Mg2+-dependent type 2C protein phosphatase, co-localizes at least in part with the H+-ATPase in the plasma membrane and regulates the phosphorylation status of the penultimate threonine of the H+-ATPase.

Keywords: 14-3-3 protein; Arabidopsis thaliana; Guard cells; Phosphothreonine residue; Plasma membrane H+-ATPase; Vicia faba

Journal Article.  6933 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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