Biochemical characterization of in vitro phosphorylation and dephosphorylation of the plasma membrane H+-ATPase

Plant Cell Physiol. 2010 Jul;51(7):1186-96. doi: 10.1093/pcp/pcq078. Epub 2010 Jun 1.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arabidopsis / enzymology
  • Carbazoles / pharmacology
  • Edetic Acid / pharmacology
  • Indole Alkaloids / pharmacology
  • Light
  • Magnesium / metabolism
  • Microsomes / enzymology
  • Octoxynol / pharmacology
  • Phosphoprotein Phosphatases / metabolism*
  • Phosphorylation
  • Plant Stomata / enzymology
  • Protein Kinases / metabolism*
  • Protein Phosphatase 2C
  • Proton-Translocating ATPases / metabolism*
  • Protoplasts / enzymology
  • Threonine / metabolism
  • Vicia faba / enzymology*

Substances

  • Carbazoles
  • Indole Alkaloids
  • Threonine
  • Octoxynol
  • staurosporine aglycone
  • Edetic Acid
  • Protein Kinases
  • Phosphoprotein Phosphatases
  • Protein Phosphatase 2C
  • Proton-Translocating ATPases
  • Magnesium