Binding of 14-3-3 beta to the carboxyl terminus of Wee1 increases Wee1 stability, kinase activity, and G(2)-M cell population

Wee1 protein kinase plays an important regulatory role in cell cycle progre ssion. It inhibits Cdc-5 activity by phosphorylating Tyr15 and arrests cell s at G(2)-M phase. In an attempt to understand Wee1 regulation during cell cycle, yeast two-hybrid screening was used to identify Wee1-binding protein (s). Five of the eight positive clones identified encode 14-3-3 beta. In vi vo binding assay in 293 cells showed that both full-length and NH2-terminal truncated Wee1 bind with 14-3-3 beta, The 14-3-3 beta binding site was map ped to a COOH-terminal consensus motif, RSVSLT (codons 639 to 646). Binding with 14-3-3 beta increases the protein level of full-length Wee1 but not o f the truncated Wee1, Accompanying the protein level increases, the kinase activity of Wee1 also increases when coexpressed with 14-3-3 beta, Increase d Wee1 protein level/enzymatic activity is accountable, at least in part, t o an increased Wee1 protein half-life when coexpressed with 14-3-3 beta, Th e protein half-life of the NH2-terminal truncated Wee1 is much longer than that of the full-length protein and is not affected by 14-3-3 beta cotransf ection, Biologically, 14-3-3 beta/Wee1 coexpression increases the cell popu lation at G(2)-M phase. Thus, Wee1 binding with 14-3-3 beta increases its b iochemical activity as well as its biological function. The finding reveals a novel mechanism by which 14-3-3 regulates G(2)-M arrest and suggests tha t the NH2-terminal domain of Wee1 contains a negative regulatory sequence t hat determines Wee1 stability.