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（CDK-cyclin）复合物的必需激活因子。Cdc25A的活性本身部分受到其CDK-细胞周期蛋白底物所介导的正负反馈调控环路的调节，这类调控事件既发生于G1期，也存在于G1/S与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（mass spectrometry analysis），鉴定出Cdc25A上一个全新的磷酸化位点——丝氨酸283（Serine283）。磷酸化特异性抗体实验显示，该位点的磷酸化信号出现于未受干扰的细胞周期的晚S/G2期，且由CDK-细胞周期蛋白复合物催化完成。针对野生型与非磷酸化突变型Cdc25A的过表达研究表明，Ser283位点的磷酸化可增强该磷酸酶的G2/M促进活性，而不会对其稳定性或亚细胞定位造成影响。综上，本研究揭示了Cdc25A与其CDK-细胞周期蛋白底物之间的全新正反馈调控环路，该环路通过调控G2期内的Cdc25A活性，进而加速细胞进入有丝分裂的进程。