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

Garnett MJ, Mansfeld J, Godwin C, Matsusaka T, Wu J, Russell P, Pines J, Venkitaraman AR - Nat. Cell Biol. (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: University of Cambridge, Department of Oncology and The Medical Research Council Cancer Cell Unit, Hutchison/MRC Research Centre, Hills Road, Cambridge, CB2 OXZ, UK.

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.

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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.
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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.

Mentions: UBE2S depletion significantly impaired the ability to resume mitotic progression after exposure to S-trityl-l-cysteine and Dimethylenastron (which, like Mona, inhibit the mitotic kinesin Eg5 19-22), or mitotic inhibitors with different modes of action such as taxol and nocadazole, which respectively stabilize or suppress microtubule assembly (Figure 2c). UBE2S depletion had the largest effect after taxol exposure, and the smallest after Mona, consistent with the magnitude of the initial mitotic arrest induced by these compounds (see Figure 5c). These effects were manifest in several cell types, including the cervical cancer cell line, HeLa, as well as immortalized Retinal Pigmented Epithelial (RPE) cells (Figures 2d and e).


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

Garnett MJ, Mansfeld J, Godwin C, Matsusaka T, Wu J, Russell P, Pines J, Venkitaraman AR - Nat. Cell Biol. (2009)

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.
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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.
Mentions: UBE2S depletion significantly impaired the ability to resume mitotic progression after exposure to S-trityl-l-cysteine and Dimethylenastron (which, like Mona, inhibit the mitotic kinesin Eg5 19-22), or mitotic inhibitors with different modes of action such as taxol and nocadazole, which respectively stabilize or suppress microtubule assembly (Figure 2c). UBE2S depletion had the largest effect after taxol exposure, and the smallest after Mona, consistent with the magnitude of the initial mitotic arrest induced by these compounds (see Figure 5c). These effects were manifest in several cell types, including the cervical cancer cell line, HeLa, as well as immortalized Retinal Pigmented Epithelial (RPE) cells (Figures 2d and e).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: University of Cambridge, Department of Oncology and The Medical Research Council Cancer Cell Unit, Hutchison/MRC Research Centre, Hills Road, Cambridge, CB2 OXZ, UK.

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.

Show MeSH
Related in: MedlinePlus