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 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.