Biochemical evidence for the requirement of 14-3-3 protein binding in activation of the guard-cell plasma membrane H+-ATPase by blue light.
Blue light (BL) activates the plasma membrane H(+)-ATPase via phosphorylation of the C-terminus with concomitant binding of 14-3-3 protein to the terminus in stomatal guard cells. However, the binding site and role of 14-3-3 protein in this physiological response have not been elucidated. We investigated the above using synthetic phosphopeptides designed from the C-terminus of Vicia H(+)-ATPase (isoform 1; VHA1). The presence of KGLDIDTIQQHYphospho-T(950)V peptide (P-950) prevented binding of 14-3-3 protein to the phosphorylated H(+)-ATPase. Dephosphorylated P-950 and other phosphopeptides, including typical phosphorylation sites in the C-terminus, had no effect on the binding. Incubation of BL-activated plasma membrane H(+)-ATPase with P-950 dissociated the 14-3-3 protein from the H(+)-ATPase without affecting phosphorylation levels and decreased the H(+)-ATPase activity. By contrast, incubation of P-950 with the activated H(+)-ATPase from fusicoccin-treated guard-cell protoplasts neither dissociated the 14-3-3 protein nor decreased the H(+)-ATPase activity. These results indicate that BL induces phosphorylation on threonine residue (Thr(950)) in the C-terminus of H(+)-ATPase, and that the binding of 14-3-3 to this site is required for the activation of H(+)-ATPase in stomatal guard cells.
Kinoshita
Toshinori
T
Department of Biology, Faculty of Sciences, Kyushu University, Ropponmatsu, Fukuoka, 810-8560 Japan.
Shimazaki
Ken-ichiro
K
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D006027:Glycosides / Q000494:pharmacology; D008027:Light; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010860:Pigments, Biological / Q000494:pharmacology; D019441:Plant Epidermis / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D011485:Protein Binding / Q000187:drug effects / Q000528:radiation effects; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013912:Threonine / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D031306:Vicia / Q000502:physiology* / Q000528:radiation effects
D048948:14-3-3 Proteins; D006027:Glycosides; D010860:Pigments, Biological; C007808:fusicoccin; D013912:Threonine; D014446:Tyrosine 3-Monooxygenase; D006180:Proton-Translocating ATPases