Phosphorylation of CDC25A on SER283 in late S/G2 by CDK/cyclin complexes accelerates mitotic entry

The Cdc25A phosphatase is an essential activator of CDK-cyclin complexes at all steps of the eukaryotic cell cycle. The activity of Cdc25A is itself regulated in part by positive and negative feedback regulatory loops performed by its CDK-cyclin substrates that occur in G1 as well as during the G1/S and G2/M transitions. However, the regulation of Cdc25A during G2 phase progression before mitotic entry has not been intensively characterized. Here, we identify by mass spectrometry analysis a new phosphorylation event of Cdc25A on Serine283. Phospho-specific antibodies revealed that the phosphorylation of this residue appears in late S/G2 phase of an unperturbed cell cycle and is performed by CDK-cyclin complexes. Overexpression studies of wild-type and non-phosphorylatable mutant forms of Cdc25A indicated that Ser283 phosphorylation increases the G2/M-promoting activity of the phosphatase without impacting its stability or subcellular localization. Our results therefore identify a new positive regulatory loop between Cdc25A and its CDK-cyclin substrates which contributes to accelerate entry into mitosis through the regulation of Cdc25A activity in G2.

Cdc25A磷酸酶（Cdc25A phosphatase）是真核细胞周期各阶段中CDK-细胞周期蛋白复合物（CDK-cyclin complexes）的必需激活因子。Cdc25A的活性本身部分由其CDK-细胞周期蛋白底物介导的正负反馈调控环路所调控，此类调控环路存在于G1期以及G1/S和G2/M转换过程中。然而，针对有丝分裂进入前G2期进程中Cdc25A的调控机制，目前尚未得到深入解析。本研究通过质谱分析鉴定出Cdc25A蛋白丝氨酸283（Serine283）位点的一处新型磷酸化修饰。磷酸化特异性抗体实验显示，该残基的磷酸化出现于未受扰动的细胞周期的晚S/G2期，且由CDK-细胞周期蛋白复合物催化完成。对Cdc25A野生型与非磷酸化突变体的过表达研究表明，丝氨酸283的磷酸化可增强该磷酸酶的G2/M期促进活性，但不会影响其稳定性或亚细胞定位。综上，本研究鉴定出Cdc25A与其CDK-细胞周期蛋白底物之间的新型正反馈调控环路，该环路通过调控G2期内Cdc25A的活性，加速细胞进入有丝分裂的进程。