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.

Keywords: Cdk1; Cell cycle; Greatwall; Mitosis; PP2A.

Figure 1.

The nuclear exclusion of Gwl in prophase depends on active export. (A) The nuclear exclusion of Gwl in prophase is inhibited by leptomycin B. Cells were treated with 50 μM leptopmycin B or ethanol alone and filmed immediately. (B) Identification of an NES motif in Gwl (residues 553–558). This motif was predicted as a highly probable NES using ValidNESs. (C) Mutation of the NES abolished the nuclear exclusion of Gwl in prophase. NESm = L556A, I558A. Time zero (T₀) was defined as the time when an increase in cytoplasmic Gwl is first noticed. Scale bars: 5 μm.

Figure 2.

Gwl shuttles between the nucleus and the cytoplasm. (A) Mutation of the NES motif partially rescues the localization defects due to mutations of NLS motifs in Gwl. The relative fluorescence intensities of the cytoplasm and nucleus were measured for the indicated versions of Gwl-GFP. Scale bar: 5 μm. (B) Fluorescence loss in photobleaching (FLIP). Irradiation of a small portion of the cytoplasm (A1) for repeated cycles (30 total) induces the bleaching of Gwl-GFP in the nucleus (measured in A2). A region A3 in a neighboring cell was measured to subtract the bleaching induced by the image acquisition (see Materials & Methods). Calculations were done for the indicated time points. Numbers are averages of multiple cells ± SEM. Note that mutation of the NES in Gwl slows down the bleaching.

Figure 3.

Phosphorylation near an NLS is required for the nuclear exclusion of Gwl in prophase. (A) Thr562 (red) is a phosphorylation site immediately adjacent to NLS2 (green) in Gwl. To be compared with Gwl-WT-GFP in Fig. 1A. (B) Alanine mutation of Thr562 abolishes the nuclear exclusion of Gwl in prophase. (C) Phosphomimetic mutations of Thr562 inactivate NLS2 and promote the cytoplasmic retention of Gwl. Scale bars: 5 μm. The relative fluorescence intensities of the cytoplasm and nucleus were measured for the indicated versions of Gwl-GFP. * p = 0.02, *** p < 1 × 10⁻¹³.

Figure 4.

Gwl associates with PP2A-Tws in the cytoplasm during late mitotic exit. (A) D-Mel cells were transfected with plasmids for the expression of the indicated proteins. Cells were used in Protein A-affinity purifications followed by Western blots. Tws-Myc was co-purified specifically with Gwl-PrA. Mutation of the 2 NLSs enhanced this association. (B) D-Mel cells expressing Tws-Myc and Gwl-PrA were submitted to Proximal Ligation Assay (PLA). PLA foci were visualized on a confocal microscope and quantified. PLA foci were detected mostly in telophase and during cytokinesis, in the cytoplasm and not in the nuclei. (C) Tws-Flag/Gwl-Myc PLA signals are enhanced when the 2 NLSs in Gwl are mutated. Numbers = average ± SEM

Figure 5.

PP2A-Tws promotes the nuclear localization of Gwl in vivo. (A) Gwl is hyperphosphorylated in embryos from mothers heterozygous for mutations in mts and tws, as detected by slower-migrating forms of Gwl by Western blot. (B) Gwl is more cytoplasmic in embryos from mothers heterozygous for a mutation in tws. Left: immunofluorescence in fixed syncytial embryos. Center: quantification of the nuclear vs cytoplasmic fluorescence intensities of Gwl. Right: Western blot showing the lower levels of Tws in embryos from tws^P/+ mothers. (C) Gwl is more cytoplasmic in tws^P/tws^P larval neuroblasts. Left: immunofluorescence in fixed larval neuroblasts. Dashed lines indicate the approximated limits of nuclear areas. Center: quantification of the nuclear vs cytoplasmic fluorescence intensities of Gwl. Right: Western blot showing the lower levels of Tws in tws^P/tws^P whole larvae.

Figure 6.

PP2A-Tws is required for timely return of Gwl to the nuclei during late mitotic exit. (A) Stable cell lines allowing the inducible expression of RFP-Lamin B and Gwl-GFP or Gwl-T562A-GFP were transfected with dsRNA against tws or the bacterial KAN gene. Expression was induced 3 days later and cells were filmed on day 4. The time between the initial enrichment of RFP-Lamin B at the nuclear periphery (T₀) and the observation of a clearly higher concentration of Gwl-GFP in daughter nuclei compared to the cytoplasm (red squares) was measured. Scale bars: 5 μm. (B) Quantification of measurements done as in (A). The delay in Gwl import caused by the depletion of Tws depends on Thr562. Error bars: SEM. (C) Western blots for the indicated proteins under the conditions used.