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

Abstract

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.

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

“…Interestingly, both Cdc25 and Greatwall depend on Polo and Cdk1 for their phosphorylation and nuclear exclusion, suggesting that they may be regulated by similar mechanisms 43,49 .…”

Greatwall-Endosulfine: A Molecular Switch that Regulates PP2A/B55 Protein Phosphatase Activity in Dividing and Quiescent Cells

“…This phosphorylation triggers Greatwall autophosphorylation at its Cterminal domain (S875 in humans, or S883 in Xenopus), resulting in full activation of the kinase [22,23] ( Figure 2). In Drosophila, Cdk1/CyclinB phosphorylation also mediates Greatwall relocalization from the nucleus to the cytosol by its association to exportin-1 (CRM1) [55]. Greatwall is phosphorylated by Polo kinase, which results in binding to 14-3-3Ɛ proteins and cytosolic retention [56].…”

“…Dephosphorylation and reactivation of PP1 partially inactivates Greatwall by promoting its dephosphorylation of the S875 residue (in Mastl), which in turn partially releases ENSA and ARPP-19 inhibition of PP2A/B55 [60][61][62]. This is sufficient to promote Greatwall dephosphorylation on T194 and S875 residues by PP2A/B55 phosphatase activity [60][61][62], resulting in full inhibition of Greatwall kinase activity and in its translocation to the nucleus [55]. Recent studies have also described an additional layer of phosphatase regulation of Greatwall-Endosulfine because the RNA polymerase II C-terminal domain phosphatase (Fcp1) dephosphorylates Greatwall, ENSA, and ARPP-19 [63,64].…”

Cell Cycle Regulators in Female Meiosis of Drosophila melanogaster

“…The activity of Gwl itself is subject to regulation via phosphorylation via both Cdk1 and Polo kinases [21,22]. Both kinases phosphorylate Gwl in the central region of the protein, disrupting the function of two nuclear localization sequences, thus promoting the cytoplasmic accumulation of Gwl.…”

“…Both kinases phosphorylate Gwl in the central region of the protein, disrupting the function of two nuclear localization sequences, thus promoting the cytoplasmic accumulation of Gwl. This may allow it to efficiently inhibit PP2A, which is predominantly cytoplasmic [21,22].…”