Spatial regulation of greatwall by Cdk1 and PP2A-Tws in the cell cycle

Entry into mitosis requires the phosphorylation of multiple substrates by cyclin B-Cdk1, while exit from mitosis requires their dephosphorylation, which depends largely on the phosphatase PP2A in complex with its B55 regulatory subunit (Tws in Drosophila). At mitotic entry, cyclin B-Cdk1 activates the Greatwall kinase, which phosphorylates Endosulfine proteins, thereby activating their ability to inhibit PP2A-B55 competitively. The inhibition of PP2A-B55 at mitotic entry facilitates the accumulation of phosphorylated Cdk1 substrates. The coordination of these enzymes involves major changes in their localization. In interphase, Gwl is nuclear while PP2A-B55 is cytoplasmic. We recently showed that Gwl suddenly relocalizes from the nucleus to the cytoplasm in prophase, before nuclear envelope breakdown and that this controlled localization of Gwl is required for its function. We and others have shown that phosphorylation of Gwl by cyclin B-Cdk1 at multiple sites is required for its nuclear exclusion, but the precise mechanisms remained unclear. In addition, how Gwl returns to its nuclear localization was not explored. Here we show that cyclin B-Cdk1 directly inactivates a Nuclear Localization Signal in the central region of Gwl. This phosphorylation facilitates the cytoplasmic retention of Gwl, which is exported to the cytoplasm in a Crm1-dependent manner. In addition, we show that PP2A-Tws promotes the return of Gwl to its nuclear localization during cytokinesis. Our results indicate that the cyclic changes in Gwl localization at mitotic entry and exit are directly regulated by the antagonistic cyclin B-Cdk1 and PP2A-Tws enzymes.

有丝分裂启动需要细胞周期蛋白B-Cdk1（cyclin B-Cdk1）对多种底物进行磷酸化；而有丝分裂退出则需要这些底物发生去磷酸化，该过程主要依赖于与其B55调节亚基结合的磷酸酶PP2A（phosphatase PP2A），在果蝇中该调节亚基被称为Tws。在有丝分裂启动阶段，细胞周期蛋白B-Cdk1会激活大壁激酶（Greatwall kinase，下文简称Gwl），后者可磷酸化内硫蛋白（Endosulfine proteins），从而激活其竞争性抑制PP2A-B55的能力。有丝分裂启动阶段对PP2A-B55的抑制，会促进Cdk1底物的磷酸化积累。这些酶的功能协调依赖于其定位的显著改变。在分裂间期，Gwl定位于细胞核内，而PP2A-B55则分布于细胞质中。我们近期的研究发现，Gwl会在核膜破裂前的前期突然从细胞核转位至细胞质，且Gwl的这种受控定位对于其功能发挥是必需的。我们及其他研究团队均已证实，细胞周期蛋白B-Cdk1通过对Gwl的多个位点进行磷酸化，可使其被排出细胞核，但具体的分子机制仍不明确。此外，Gwl如何恢复其细胞核定位的机制尚未被探究。本研究证实，细胞周期蛋白B-Cdk1可直接使Gwl中央区域的核定位信号（Nuclear Localization Signal）失活。该磷酸化修饰可促进Gwl在细胞质中的滞留，而Gwl向细胞质的输出依赖于Crm1蛋白。此外，我们还发现PP2A-Tws可在胞质分裂阶段促进Gwl恢复其细胞核定位。本研究结果表明，在有丝分裂启动与退出过程中Gwl定位的周期性变化，直接由相互拮抗的细胞周期蛋白B-Cdk1与PP2A-Tws酶复合物所调控。