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PP2A-twins is antagonized by greatwall and collaborates with polo for cell cycle progression and centrosome attachment to nuclei in drosophila embryos.

ABSTRACT: Cell division and development are regulated by networks of kinases and phosphatases. In early Drosophila embryogenesis, 13 rapid nuclear divisions take place in a syncytium, requiring fine coordination between cell cycle regulators. The Polo kinase is a conserved, crucial regulator of M-phase. We have recently reported an antagonism between Polo and Greatwall (Gwl), another mitotic kinase, in Drosophila embryos. However, the nature of the pathways linking them remained elusive. We have conducted a comprehensive screen for additional genes functioning with polo and gwl. We uncovered a strong interdependence between Polo and Protein Phosphatase 2A (PP2A) with its B-type subunit Twins (Tws). Reducing the maternal contribution of Polo and PP2A-Tws together is embryonic lethal. We found that Polo and PP2A-Tws collaborate to ensure centrosome attachment to nuclei. While a reduction in Polo activity leads to centrosome detachments observable mostly around prophase, a reduction in PP2A-Tws activity leads to centrosome detachments at mitotic exit, and a reduction in both Polo and PP2A-Tws enhances the frequency of detachments at all stages. Moreover, we show that Gwl antagonizes PP2A-Tws function in both meiosis and mitosis. Our study highlights how proper coordination of mitotic entry and exit is required during embryonic cell cycles and defines important roles for Polo and the Gwl-PP2A-Tws pathway in this process.

SUBMITTER: Wang P

PROVIDER: S-EPMC3154958 | biostudies-literature |

REPOSITORIES: biostudies-literature

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PP2A-B55/SUR-6 collaborates with the nuclear lamina for centrosome separation during mitotic entry.

Project description:Across most sexually reproducing animals, centrosomes are provided to the oocyte through fertilization and must be positioned properly to establish the zygotic mitotic spindle. How centrosomes are positioned in space and time through the concerted action of key mitotic entry biochemical regulators, including protein phosphatase 2A (PP2A-B55/SUR-6), biophysical regulators, including dynein, and the nuclear lamina is unclear. Here, we uncover a role for PP2A-B55/SUR-6 in regulating centrosome separation. Mechanistically, PP2A-B55/SUR-6 regulates nuclear size before mitotic entry, in turn affecting nuclear envelope-based dynein density and motor capacity. Computational simulations predicted the requirement of PP2A-B55/SUR-6 regulation of nuclear size and nuclear-envelope dynein density for proper centrosome separation. Conversely, compromising nuclear lamina integrity led to centrosome detachment from the nuclear envelope and migration defects. Removal of PP2A-B55/SUR-6 and the nuclear lamina simultaneously further disrupted centrosome separation, leading to unseparated centrosome pairs dissociated from the nuclear envelope. Taking these combined results into consideration, we propose a model in which centrosomes migrate and are positioned through the concerted action of PP2A-B55/SUR-6-regulated nuclear envelope-based dynein pulling forces and centrosome-nuclear envelope tethering. Our results add critical precision to models of centrosome separation relative to the nucleus during spindle formation in cell division.

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Greatwall maintains mitosis through regulation of PP2A.

Project description:Greatwall (GW) is a new kinase that has an important function in the activation and the maintenance of cyclin B-Cdc2 activity. Although the mechanism by which it induces this effect is unknown, it has been suggested that GW could maintain cyclin B-Cdc2 activity by regulating its activation loop. Using Xenopus egg extracts, we show that GW depletion promotes mitotic exit, even in the presence of a high cyclin B-Cdc2 activity by inducing dephosphorylation of mitotic substrates. These results indicate that GW does not maintain the mitotic state by regulating the cyclin B-Cdc2 activation loop but by regulating a phosphatase. This phosphatase is PP2A; we show that (1) PP2A binds GW, (2) the inhibition or the specific depletion of this phosphatase from mitotic extracts rescues the phenotype induced by GW inactivation and (3) the PP2A-dependent dephosphorylation of cyclin B-Cdc2 substrates is increased in GW-depleted Xenopus egg extracts. These results suggest that mitotic entry and maintenance is not only mediated by the activation of cyclin B-Cdc2 but also by the regulation of PP2A by GW.

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Spatiotemporal coordination of Greatwall-Endos-PP2A promotes mitotic progression.

Project description:Mitotic entry involves inhibition of protein phosphatase 2A bound to its B55/Tws regulatory subunit (PP2A-B55/Tws), which dephosphorylates substrates of mitotic kinases. This inhibition is induced when Greatwall phosphorylates Endos, turning it into an inhibitor of PP2A-Tws. How this mechanism operates spatiotemporally in the cell is incompletely understood. We previously reported that the nuclear export of Greatwall in prophase promotes mitotic progression. Here, we examine the importance of the localized activities of PP2A-Tws and Endos for mitotic regulation. We find that Tws shuttles through the nucleus via a conserved nuclear localization signal (NLS), but expression of Tws in the cytoplasm and not in the nucleus rescues the development of tws mutants. Moreover, we show that Endos must be in the cytoplasm before nuclear envelope breakdown (NEBD) to be efficiently phosphorylated by Greatwall and to bind and inhibit PP2A-Tws. Disrupting the cytoplasmic function of Endos before NEBD results in subsequent mitotic defects. Evidence suggests that this spatiotemporal regulation is conserved in humans.

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Greatwall and Polo-like kinase 1 coordinate to promote checkpoint recovery.

Project description:Checkpoint recovery upon completion of DNA repair allows the cell to return to normal cell cycle progression and is thus a crucial process that determines cell fate after DNA damage. We previously studied this process in Xenopus egg extracts and established Greatwall (Gwl) as an important regulator. Here we show that preactivated Gwl kinase can promote checkpoint recovery independently of cyclin-dependent kinase 1 (Cdk1) or Plx1 (Xenopus polo-like kinase 1), whereas depletion of Gwl from extracts exhibits no synergy with that of Plx1 in delaying checkpoint recovery, suggesting a distinct but related relationship between Gwl and Plx1. In further revealing their functional relationship, we found mutual dependence for activation of Gwl and Plx1 during checkpoint recovery, as well as their direct association. We characterized the protein association in detail and recapitulated it in vitro with purified proteins, which suggests direct interaction. Interestingly, Gwl interaction with Plx1 and its phosphorylation by Plx1 both increase at the stage of checkpoint recovery. More importantly, Plx1-mediated phosphorylation renders Gwl more efficient in promoting checkpoint recovery, suggesting a functional involvement of such regulation in the recovery process. Finally, we report an indirect regulatory mechanism involving Aurora A that may account for Gwl-dependent regulation of Plx1 during checkpoint recovery. Our results thus reveal novel mechanisms underlying the involvement of Gwl in checkpoint recovery, in particular, its functional relationship with Plx1, a well characterized regulator of checkpoint recovery. Coordinated interplays between Plx1 and Gwl are required for reactivation of these kinases from the G(2)/M DNA damage checkpoint and efficient checkpoint recovery.

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The BEG (PP2A-B55/ENSA/Greatwall) pathway ensures cytokinesis follows chromosome separation.

Project description:Cytokinesis follows separase activation and chromosome segregation. This order is ensured in budding yeast by the mitotic exit network (MEN), where Cdc14p dephosphorylates key conserved Cdk1-substrates exemplified by the anaphase spindle-elongation protein Ase1p. However, in metazoans, MEN and Cdc14 function is not conserved. Instead, the PP2A-B55?/ENSA/Greatwall (BEG) pathway controls the human Ase1p ortholog PRC1. In this pathway, PP2A-B55 inhibition is coupled to Cdk1-cyclin B activity, whereas separase inhibition is maintained by cyclin B concentration. This creates two cyclin B thresholds during mitotic exit. Simulation and experiments using PRC1 as a model substrate show that the first threshold permits separase activation and chromosome segregation, and the second permits PP2A-B55 activation and initiation of cytokinesis. Removal of the ENSA/Greatwall (EG) timer module eliminates this second threshold, as well as associated delay in PRC1 dephosphorylation and initiation of cytokinesis, by uncoupling PP2A-B55 from Cdk1-cyclin B activity. Therefore, temporal order during mitotic exit is promoted by the metazoan BEG pathway.

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PP2A/B55 and Fcp1 regulate Greatwall and Ensa dephosphorylation during mitotic exit.

Project description: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.

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

Project description: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.

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Therapeutic relevance of the PP2A-B55 inhibitory kinase MASTL/Greatwall in breast cancer.

Project description:PP2A is a major tumor suppressor whose inactivation is frequently found in a wide spectrum of human tumors. In particular, deletion or epigenetic silencing of genes encoding the B55 family of PP2A regulatory subunits is a common feature of breast cancer cells. A key player in the regulation of PP2A/B55 phosphatase complexes is the cell cycle kinase MASTL (also known as Greatwall). During cell division, inhibition of PP2A-B55 by MASTL is required to maintain the mitotic state, whereas inactivation of MASTL and PP2A reactivation is required for mitotic exit. Despite its critical role in cell cycle progression in multiple organisms, its relevance as a therapeutic target in human cancer and its dependence of PP2A activity is mostly unknown. Here we show that MASTL overexpression predicts poor survival and shows prognostic value in breast cancer patients. MASTL knockdown or knockout using RNA interference or CRISPR/Cas9 systems impairs proliferation of a subset of breast cancer cells. The proliferative function of MASTL in these tumor cells requires its kinase activity and the presence of PP2A-B55 complexes. By using a new inducible CRISPR/Cas9 system in breast cancer cells, we show that genetic ablation of MASTL displays a significant therapeutic effect in vivo. All together, these data suggest that the PP2A inhibitory kinase MASTL may have both prognostic and therapeutic value in human breast cancer.

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Tgfb3 collaborates with PP2A and notch signaling pathways to inhibit retina regeneration.

Project description:Neuronal degeneration in the zebrafish retina stimulates Müller glia (MG) to proliferate and generate multipotent progenitors for retinal repair. Controlling this proliferation is critical to successful regeneration. Previous studies reported that retinal injury stimulates pSmad3 signaling in injury-responsive MG. Contrary to these findings, we report pSmad3 expression is restricted to quiescent MG and suppressed in injury-responsive MG. Our data indicates that Tgfb3 is the ligand responsible for regulating pSmad3 expression. Remarkably, although overexpression of either Tgfb1b or Tgfb3 can stimulate pSmad3 expression in the injured retina, only Tgfb3 inhibits injury-dependent MG proliferation; suggesting the involvement of a non-canonical Tgfb signaling pathway. Furthermore, inhibition of Alk5, PP2A or Notch signaling rescues MG proliferation in Tgfb3 overexpressing zebrafish. Finally, we report that this Tgfb3 signaling pathway is active in zebrafish MG, but not those in mice, which may contribute to the different regenerative capabilities of MG from fish and mammals.

| S-EPMC7250569 | biostudies-literature

Cyclin B-Cdk1 inhibits protein phosphatase PP2A-B55 via a Greatwall kinase-independent mechanism.

Project description:Entry into M phase is governed by cyclin B-Cdk1, which undergoes both an initial activation and subsequent autoregulatory activation. A key part of the autoregulatory activation is the cyclin B-Cdk1-dependent inhibition of the protein phosphatase 2A (PP2A)-B55, which antagonizes cyclin B-Cdk1. Greatwall kinase (Gwl) is believed to be essential for the autoregulatory activation because Gwl is activated downstream of cyclin B-Cdk1 to phosphorylate and activate ?-endosulfine (Ensa)/Arpp19, an inhibitor of PP2A-B55. However, cyclin B-Cdk1 becomes fully activated in some conditions lacking Gwl, yet how this is accomplished remains unclear. We show here that cyclin B-Cdk1 can directly phosphorylate Arpp19 on a different conserved site, resulting in inhibition of PP2A-B55. Importantly, this novel bypass is sufficient for cyclin B-Cdk1 autoregulatory activation. Gwl-dependent phosphorylation of Arpp19 is nonetheless necessary for downstream mitotic progression because chromosomes fail to segregate properly in the absence of Gwl. Such a biphasic regulation of Arpp19 results in different levels of PP2A-B55 inhibition and hence might govern its different cellular roles.

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