title: Analysis of the phosphorylation level in guard-cell plasma membrane H+-ATPase in response to fusicoccin.
abstract: A fungal phytotoxin fusicoccin (FC) causes irreversible opening of stomata by activation of the plasma membrane H+-ATPase in guard cells. However, the mechanism by which FC activates the H+-ATPase is not fully understood with respect to the event of phosphorylation. In this study, we provide quantitative evidence that FC-dependent activation of H+-ATPase requires the phosphorylation of the C-terminus, and that FC maintains the activated state by preventing the dephosphorylation. The plasma membrane H+-ATPase in guard cells was phosphorylated on serine and threonine residues in the C-termini of both VHA1 and VHA2 by FC, and the phosphorylation level paralleled the rates of H+-pumping and ATP hydrolysis. An endogenous 14-3-3 protein was co-precipitated with the H+-ATPase, and the amount of 14-3-3 protein was proportional to the phosphorylation level of H+-ATPASE: The recombinant 14-3-3 protein bound to the C-terminus only when it was phosphorylated, even in the presence of FC. The phosphorylated C-terminus was dephosphorylated by alkaline phosphatase, and the dephosphorylation was completely prevented when the C-terminus had been incubated with both FC and 14-3-3 protein. The results suggest that FC activates the H+-ATPase by accumulating the complex of phosphorylated H+-ATPase and 14-3-3 protein through inhibition of the dephosphorylation in guard cells.
authors.affiliation: Department of Biology, Faculty of Sciences, Kyushu University, Ropponmatsu, Fukuoka, 810-8560 Japan.
authors.forename: T
authors.initials: T
authors.lastname: Kinoshita
authors.forename: K
authors.initials: K
authors.lastname: Shimazaki
mesh_terms: D048948:14-3-3 Proteins; D000255:Adenosine Triphosphate / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D003488:Cyanogen Bromide; D004789:Enzyme Activation; D007887:Fabaceae / Q000201:enzymology*; D006027:Glycosides / Q000494:pharmacology*; D007700:Kinetics; D008027:Light; D008274:Magnesium / Q000494:pharmacology; D009183:Mycotoxins / Q000494:pharmacology*; D010766:Phosphorylation; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*; D011523:Protoplasts / Q000201:enzymology; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
chemical_list: D048948:14-3-3 Proteins; D006027:Glycosides; D009183:Mycotoxins; C007808:fusicoccin; D000255:Adenosine Triphosphate; D014446:Tyrosine 3-Monooxygenase; D006180:Proton-Translocating ATPases; D008274:Magnesium; D003488:Cyanogen Bromide