Binding of 14-3-3beta to the carboxyl terminus of Wee1 increases Wee1 stability, kinase activity, and G2-M cell population.
Wee1 protein kinase plays an important regulatory role in cell cycle progression. It inhibits Cdc-2 activity by phosphorylating Tyr15 and arrests cells at G2-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 14-3-3beta. In vivo binding assay in 293 cells showed that both full-length and NH2-terminal truncated Wee1 bind with 14-3-3beta. The 14-3-3beta binding site was mapped to a COOH-terminal consensus motif, RSVSLT (codons 639 to 646). Binding with 14-3-3beta increases the protein level of full-length Wee1 but not of the truncated Wee1. Accompanying the protein level increases, the kinase activity of Wee1 also increases when coexpressed with 14-3-3beta. Increased Wee1 protein level/enzymatic activity is accountable, at least in part, to an increased Wee1 protein half-life when coexpressed with 14-3-3beta. The protein half-life of the NH2-terminal truncated Wee1 is much longer than that of the full-length protein and is not affected by 14-3-3beta cotransfection. Biologically, 14-3-3beta/Wee1 coexpression increases the cell population at G2-M phase. Thus, Wee1 binding with 14-3-3beta increases its biochemical activity as well as its biological function. The finding reveals a novel mechanism by which 14-3-3 regulates G2-M arrest and suggests that the NH2-terminal domain of Wee1 contains a negative regulatory sequence that determines Wee1 stability.
Wang
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Y
Department of Molecular Biology, Parke-Davis Pharmaceutical Research, Ann Arbor, Michigan, USA.
Jacobs
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C
Hook
K E
KE
Duan
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H
Booher
R N
RN
Sun
Y
Y
D048948:14-3-3 Proteins; D001665:Binding Sites; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins; D002460:Cell Line; D004274:DNA, Recombinant / Q000235:genetics; D016195:G2 Phase; D006801:Humans; D008938:Mitosis; D009687:Nuclear Proteins; D010957:Plasmids; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins; D014162:Transfection; D020798:Two-Hybrid System Techniques; D014446:Tyrosine 3-Monooxygenase
D048948:14-3-3 Proteins; D018797:Cell Cycle Proteins; D004274:DNA, Recombinant; D009687:Nuclear Proteins; D011506:Proteins; D029702:Schizosaccharomyces pombe Proteins; C482982:YWHAB protein, human; D014446:Tyrosine 3-Monooxygenase; C068689:wee1 protein, S pombe; D011505:Protein-Tyrosine Kinases; C499349:WEE1 protein, human