Biochemical characterization of in vitro phosphorylation and dephosphorylation of the plasma membrane H+-ATPase.
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 Mg(2+) but not Ca(2+), 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 Mg(2+)-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.
Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, 464-8602 Japan.
Yuki
Y
Hayashi
Suguru
S
Nakamura
Atsushi
A
Takemiya
Yohei
Y
Takahashi
Ken-Ichiro
K
Shimazaki
Toshinori
T
Kinoshita
D017360:Arabidopsis / Q000201:enzymology; D002227:Carbazoles / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D026121:Indole Alkaloids / Q000494:pharmacology; D008027:Light; D008274:Magnesium / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology; D017830:Octoxynol / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D054046:Plant Stomata / Q000201:enzymology; D011494:Protein Kinases / Q000378:metabolism*; D000071636:Protein Phosphatase 2C; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011523:Protoplasts / Q000201:enzymology; D013912:Threonine / Q000378:metabolism; D031307:Vicia faba / Q000201:enzymology*
D002227:Carbazoles; D026121:Indole Alkaloids; D013912:Threonine; D017830:Octoxynol; C049985:staurosporine aglycone; D004492:Edetic Acid; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases; D000071636:Protein Phosphatase 2C; D006180:Proton-Translocating ATPases; D008274:Magnesium