PP2A/B55 and Fcp1 Regulate Greatwall and Ensa Dephosphorylation during Mitotic Exit

Entry into mitosis is triggered by activation of Cdk1 and inactivation of its counteracting phosphatase PP2A/B55. Greatwall kinase inactivates PP2A/B55 via its substrates Ensa and ARPP19. Both Greatwall and Ensa/ARPP19 are regulated by phosphorylation, but the dynamic regulation of Greatwall activity and the phosphatases that control Greatwall kinase and its substrates are poorly understood. To address these questions we applied a combination of mathematical modelling and experiments using phospho-specific antibodies to monitor Greatwall, Ensa/ARPP19 and Cdk substrate phosphorylation during mitotic entry and exit. We demonstrate that PP2A/B55 is required for Gwl dephosphorylation at the essential Cdk site Thr194. Ensa/ARPP19 dephosphorylation is mediated by the RNA Polymerase II carboxy terminal domain phosphatase Fcp1. Surprisingly, inhibition or depletion of neither Fcp1 nor PP2A appears to block dephosphorylation of the bulk of mitotic Cdk1 substrates during mitotic exit. Taken together our results suggest a hierarchy of phosphatases coordinating Greatwall, Ensa/ARPP19 and Cdk substrate dephosphorylation during mitotic exit.

有丝分裂进入这一过程，由细胞周期蛋白依赖性激酶1（Cdk1）的激活，与其拮抗磷酸酶蛋白磷酸酶2A/B55（PP2A/B55）的失活共同触发。长城激酶（Greatwall kinase）可通过其底物Ensa与ARPP19，对PP2A/B55进行失活调控。长城激酶与Ensa/ARPP19均受磷酸化修饰调控，但目前学界对长城激酶活性的动态调控机制，以及调控长城激酶及其底物的磷酸酶种类，仍缺乏深入认知。为解答上述科学问题，我们结合数学建模与实验方法，利用磷酸化特异性抗体监测了有丝分裂进入与退出过程中，长城激酶、Ensa/ARPP19及Cdk底物的磷酸化状态。实验证实，PP2A/B55是长城激酶在其关键Cdk位点苏氨酸194（Thr194）处发生去磷酸化的必需因子。Ensa/ARPP19的去磷酸化过程，则由RNA聚合酶II羧基末端结构域磷酸酶Fcp1介导。令人意外的是，无论是抑制还是耗竭Fcp1与PP2A/B55，似乎均无法阻断有丝分裂退出过程中大部分有丝分裂Cdk1底物的去磷酸化。综上，本研究结果揭示了一套磷酸酶层级调控机制，可在有丝分裂退出过程中协同调控长城激酶、Ensa/ARPP19与Cdk1底物的去磷酸化过程。