UBE2S elongates ubiquitin chains on APC/C substrates to promote mitotic exit

Abstract

The anaphase-promoting complex (APC/C), a ubiquitin ligase, is the target of the spindle-assembly checkpoint (SAC), and it ubiquitylates protein substrates whose degradation regulates progress through mitosis. The identity of the ubiquitin-conjugating (E2) enzymes that work with the APC/C is unclear. In an RNA interference (RNAi) screen for factors that modify release from drug-induced SAC activation, we identified the E2 enzyme UBE2S as an APC/C auxiliary factor that promotes mitotic exit. UBE2S is dispensable in a normal mitosis, but its depletion prolongs drug-induced mitotic arrest and suppresses mitotic slippage. In vitro, UBE2S elongates ubiquitin chains initiated by the E2 enzymes UBCH10 and UBCH5, enhancing the degradation of APC/C substrates by the proteasome. Indeed, following release from SAC-induced mitotic arrest, UBE2S-depleted cells neither degrade crucial APC/C substrates, nor silence this checkpoint, whereas bypassing the SAC through BUBR1 depletion or Aurora-B inhibition negates the requirement for UBE2S. Thus, UBE2S functions with the APC/C in a two-step mechanism to control substrate ubiquitylation that is essential for mitotic exit after prolonged SAC activation, providing a new model for APC/C function in human cells.

Figure 1. siRNA screen identifies UBE2S as a modifier of release from arrest at the SAC

(a) Schematic of screen design using reverse transfection in 96-well plates. (b) Representative fluorescent microscope images from screen data. Cal51 cells were transfected with control or UBE2S siRNA and treated with the Eg5 inhibitor Monastrol as described in (a). Cells are stained for DNA and the mitotic marker phospho-Ser10 Histone H3 (pSer10-H3). DNA staining is used for cell identification. pSer10-H3 staining intensity was measured for >5,000 cells per well and the frequency of pSer10-H3 positive cells reported as a percentage. (c) A graph of ranked ΔMI standard scores (Z-score) for all 535 siRNA included in the screen. The standard score for a non-targeting control siRNA is also presented (coloured blue) and putative hits that give a standard score >2 (dashed red line) and p-value <0.01 are coloured red. (d) Western blots demonstrating efficiency of knockdown using 4 different UBE2S siRNA oligos. β-actin is shown as a loading control. (e) Validation of UBE2S as a screen hit using 4 different siRNA oligos. The ΔMI was determined as described in (a) and in the text.

Figure 2. UBE2S regulates the outcome of drug-induced mitotic arrest

(a) Schematic of experimental design for a-e. (b) UBE2S prevents release from mitotic arrest. Cells were treated with control or UBE2S siRNA and the mitotic index was measured following arrest with Monastrol. UBE2S-depleted cells were treated with Monastrol for 20 hours (t=20 arrest) and then either released from inhibitor (t=40 release) or left in drug for additional 20 hours (t=40 constant). A DMSO control at t=40 is also shown and the results for 4 different siRNA targeting UBE2S are presented. (c) UBE2S prevents release from mitotic arrest induced by different anti-mitotics. The MI was determined following treatment with DMSO, Monastrol (Mona), S-trityl-l-cysteine (S-Cys), Dimethylenastron (DMA), Nocodazole (Noc) or taxol. (d and e) UBE2S regulates release from mitotic arrest in (d) HeLa and (e) RPE cells. All data are averages of 3 replicates with error bars representing standard deviations.

Figure 3. UBE2S is necessary for mitotic release and slippage

(a) FACS analysis of cells exiting mitosis following release from arrest. Cells were transfected with control or UBE2S siRNAs (2 different oligos), arrested with Monastrol for 20 hours, collected by mitotic shake-off and analysed following release by FACS staining for the mitotic marker MPM2. (b) Cumulative frequency of cell exiting mitosis determined using DIC time-lapse imaging. Cells were transfected with control or UBE2S siRNA as indicated, arrested for 20 hours before imaging. The time taken to exit mitosis following release was measured based on cellular morphology. (c) A time-course of mitotic index (measured as pSer10-H3 positive cells) following treatment with taxol. Data are the average of 3 replicates with error bars representing standard deviations. Some error bars are too small to be seen. (d) Cumulative frequency graph representing the duration of mitosis following Monastrol treatment as measured by time-lapse imaging. Mitotic arrest induced by Mona is incomplete under the conditions used here, and ~25% of siCTRL and siUBE2S cells complete mitosis within the first ~100 minutes. siUBE2S cells that arrest during mitosis undergo a prolonged arrest compared to siCTRL cells. (e) A box-and-whisker plot showing the duration of an unperturbed mitosis. The duration from NEBD (nuclear envelope breakdown) to anaphase in HeLa cells was measured following depletion of UBE2S (left two plots) or microinjection of cells with a plasmid encoding an untagged version of UBE2S or catalytically inactive UBE2S (C95S). These results are representative of 2 independent experiments.

Figure 4. UBE2S elongates pre-initiated ubiquitin chains on Cyclin B1

(a) Autoradiograph of an in vitro APC/C ubiquitylation assay. Recombinant UBCH10, UBCH5, UBE2S, or the indicated combinations (at 1:1 molar ratio) were used for APC/C in vitro activity assays with ³³P-Cyclin B1 (aa1-86) as substrate. Reactions were performed for the indicated time before separation by SDS-PAGE and analysis using a phosphoimager. (b) Same as experiment in (a) but using methyl-ubiquitin to prevent the elongation but not initiation of ubiquitin chains. (c) Quantification of Cyclin B1 ubiquitylation. In vitro ubiquitylation reactions were performed as in (a) but with shorter reaction times to prevent limiting amounts of unmodified substrate (Supplemental Information, Fig. S3). Cyclin B1-ubiquitin conjugates with 1-4, 5-9 or >9 ubiquitin molecules (as indicated in Supplemental Information, Fig. S3) from three independent experiments were quantified. Data are normalised to the total amount of ubiquitylated Cyclin B1 and plotted as stacked area charts. Error bars show the SEM.

Figure 5. UBE2S is necessary for degradation of APC/C substrates and to antagonise the SAC

(a) Western blots for APC substrates following release from a mitotic arrest in control or UBE2S-depleted cells. Cells were arrested for 20 hours in Monastrol, collected by mitotic shake-off, washed 3 times and released into media for 3 and 9 hours. A hyper-phosphorylated form of BUBR1 indicative of checkpoint activation is indicated with an asterisk. Accumulation of Cyclin A is observed 9 hours following release in control cells as cells enter the next cell-cycle. (b) Rate of Cyclin B1 degradation during mitotic arrest. HeLa cells were injected during G2-phase with a plasmid encoding Cyclin B1-Venus and Cyclin B1 degradation analysed by time-lapse fluorescence microscopy in the presence of 100 nM taxol. The fluorescence intensity for each cell is normalised to when Cyclin B1 degradation began. Data are the mean from 6 UBE2S-depleted cells and 14 control cells with error bars representing 95% confidence intervals. These data are representative of 2 independent experiments. (c) Dose-response curve to titration of Monastrol concentrations. Cells were treated with increasing concentrations of Monastrol and the MI determined at t=20 arrest, t=40 release and t=40 constant. Values are the average of 3 replicates and error bars represent standard deviations. In some instances the error bars are too small be seen. (d) Depletion of UBE2S stabilizes SAC activation. The amount of BUBR1 immunoprecipitating with CDC20 was determined. The hyperphosphorylated form of BUBR1 is not observed with the % SDS-PAGE used in this experiment. (e) Inactivation of the SAC bypasses the requirement for UBE2S. The mitotic index in cells co-depleted of UBE2S and BUBR1, and treated with DMSO, Monastrol or taxol. Values are the average of 3 replicates and error bars represent standard deviations. (f and g) Acute inactivation of the checkpoint in arrested cells overcomes the requirement for UBE2S. Monastrol arrested mitotic cells were treated with the Aurora inhibitor ZM 447439 (ZM) to inactivate the checkpoint and (f) the MI was measured by FACS and (g) Cyclin B1 degradation examined by Western blotting. β-actin blots are shown as a control for loading.