Millar J B, McGowan C H, Lenaers G, Jones R, Russell P
Department of Molecular Biology, Scripps Research Institute, La Jolla, CA 92037.
EMBO J. 1991 Dec;10(13):4301-9. doi: 10.1002/j.1460-2075.1991.tb05008.x.
We have investigated the mechanism by which fission yeast p80cdc25 induces mitosis. The in vivo active domain was localized to the C-terminal 23 kDa of p80cdc25. This domain produced as a bacterial fusion protein (GST-cdc25) caused tyrosyl dephosphorylation and activation of immunoprecipitated p34cdc2. Furthermore, GST-cdc25 dephosphorylated both para-nitrophenyl-phosphate (pNPP) and casein phosphorylated on serine in vitro. Reaction requirements and inhibitor sensitivities were the same as those of phosphotyrosine phosphatases (PTPases). Analysis of cdc25 C-terminal domains from a variety of species revealed a conserved motif having critical residues present at the active site of PTPases. Mutation of the cdc25 Cys480 codon, corresponding to an essential cysteine in the active site of PTPases, abolished the phosphatase activity of GST-cdc25. These data indicate that cdc25 proteins define a novel subclass of eukaryotic PTPases, and strongly argue that cdc25 proteins directly dephosphorylate and activate p34cdc2 kinase to induce M-phase.
我们研究了裂殖酵母p80cdc25诱导有丝分裂的机制。体内活性结构域定位于p80cdc25的C端23 kDa区域。作为细菌融合蛋白（GST-cdc25）产生的该结构域导致免疫沉淀的p34cdc2的酪氨酸去磷酸化并激活。此外，GST-cdc25在体外使对硝基苯磷酸酯（pNPP）和丝氨酸磷酸化的酪蛋白去磷酸化。反应要求和抑制剂敏感性与磷酸酪氨酸磷酸酶（PTPases）相同。对来自多种物种的cdc25 C端结构域的分析揭示了一个保守基序，在PTPases的活性位点存在关键残基。与PTPases活性位点的必需半胱氨酸相对应的cdc25 Cys480密码子的突变消除了GST-cdc25的磷酸酶活性。这些数据表明cdc25蛋白定义了真核PTPases的一个新亚类，并有力地证明cdc25蛋白直接使p34cdc2激酶去磷酸化并激活以诱导M期。
