PP2A-B55 promotes nuclear envelope reformation after mitosis in Drosophila.

As a dividing cell exits mitosis and daughter cells enter interphase, many proteins must be dephosphorylated. The protein phosphatase 2A (PP2A) with its B55 regulatory subunit plays a crucial role in this transition, but the identity of its substrates and how their dephosphorylation promotes mitotic exit are largely unknown. We conducted a maternal-effect screen in Drosophila melanogaster to identify genes that function with PP2A-B55/Tws in the cell cycle. We found that eggs that receive reduced levels of Tws and of components of the nuclear envelope (NE) often fail development, concomitant with NE defects following meiosis and in syncytial mitoses. Our mechanistic studies using Drosophila cells indicate that PP2A-Tws promotes nuclear envelope reformation (NER) during mitotic exit by dephosphorylating BAF and suggests that PP2A-Tws targets additional NE components, including Lamin and Nup107. This work establishes Drosophila as a powerful model to further dissect the molecular mechanisms of NER and suggests additional roles of PP2A-Tws in the completion of meiosis and mitosis.

Coupling of Polo kinase activation to nuclear localization by a bifunctional NLS is required during mitotic entry.

The Polo kinase is a master regulator of mitosis and cytokinesis conserved from yeasts to humans. Polo is composed of an N-term kinase domain (KD) and a C-term polo-box domain (PBD), which regulates its subcellular localizations. The PBD and KD can interact and inhibit each other, and this reciprocal inhibition is relieved when Polo is phosphorylated at its activation loop. How Polo activation and localization are coupled during mitotic entry is unknown. Here we report that PBD binding to the KD masks a nuclear localization signal (NLS). Activating phosphorylation of the KD leads to exposure of the NLS and entry of Polo into the nucleus before nuclear envelope breakdown. Failures of this mechanism result in misregulation of the Cdk1-activating Cdc25 phosphatase and lead to mitotic and developmental defects in Drosophila. These results uncover spatiotemporal mechanisms linking master regulatory enzymes during mitotic entry.

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.