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.

有丝分裂进入需要细胞周期蛋白B-Cdk1（cyclin B-Cdk1）对多种底物进行磷酸化修饰，而有丝分裂退出则依赖于对这些底物的去磷酸化，这一过程主要由与其B55调节亚基（B55 regulatory subunit，果蝇（Drosophila）中称为Tws）结合的蛋白磷酸酶2A（PP2A）复合物介导。在有丝分裂进入阶段，细胞周期蛋白B-Cdk1会激活Greatwall激酶（Greatwall kinase，Gwl），后者可磷酸化Endosulfine蛋白（Endosulfine proteins），进而激活其竞争性抑制PP2A-B55的能力。有丝分裂进入时对PP2A-B55的抑制作用，能够促进Cdk1底物磷酸化水平的积累。这些酶的功能协调伴随着其细胞定位的显著改变：在间期（interphase），Gwl定位于细胞核内，而PP2A-B55则分布于细胞质中。我们近期的研究证实，Gwl会在核膜破裂（nuclear envelope breakdown）前的早前期突然从细胞核转移至细胞质，且这种受控的定位变化对其功能不可或缺。我们与其他研究团队均发现，细胞周期蛋白B-Cdk1对Gwl的多位点磷酸化是其核输出所必需的，但具体的分子机制仍未阐明。此外，Gwl如何返回细胞核定位的过程也尚未被探究。本研究中，我们发现细胞周期蛋白B-Cdk1可直接使Gwl中央区域的核定位信号（Nuclear Localization Signal）失活，该磷酸化修饰会促进Gwl在细胞质中的滞留，且Gwl以依赖于Crm1的方式被转运至细胞质。除此之外，我们还证实，在胞质分裂（cytokinesis）阶段，PP2A-Tws复合物会促进Gwl返回细胞核定位。我们的研究结果表明，有丝分裂进入与退出阶段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酶复合物调控。