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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Abstract
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.