Journal Article

Biochemical Evidence for the Requirement of 14-3-3 Protein Binding in Activation of the Guard-cell Plasma Membrane H<sup>+</sup>-ATPase by Blue Light

Toshinori Kinoshita and Ken-ichiro Shimazaki

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 43, issue 11, pages 1359-1365
Published in print November 2002 | ISSN: 0032-0781
Published online November 2002 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pcf167
Biochemical Evidence for the Requirement of 14-3-3 Protein Binding in Activation of the Guard-cell Plasma Membrane H+-ATPase by Blue Light

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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-T950V 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 (Thr950) 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.

Keywords: Keywords: Blue light — 14-3-3 protein — Fusicoccin — Guard cells — H+-ATPase (EC 3.6.1.35) — Phosphothreonine residue — Stomata.; Abbreviations: BL, blue light; CNBr, cyanogen bromide; FC, fusicoccin; GCPs, guard-cell protoplasts; GST, glutathione-S-transferase; RL, red light, VHA; Vicia plasma membrane H+-ATPase.

Journal Article.  5610 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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