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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 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.
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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.

Mentions: We examined more closely the kinetics of checkpoint arrest and mitotic slippage after UBE2S depletion, by analysing Mona-arrested cells released into drug-free media. Approximately 35% of control-treated cells exit mitosis in the first 3 hours, and the majority (85%) by 12 hours (Figure 3a). UBE2S depletion significantly delayed mitotic exit; even by 12 hours, only about 30% of cells had exited. A similar delay occurs in individual cells examined by time-lapse imaging (Figure 3b). The average time to exit mitosis after release from drug-induced arrest was 255 +/− 169 minutes (n=49 cells) in controls versus 668 +/− 412 minutes (n=48) after UBE2S depletion. Similar effects occurred after release from a short (6 hour) arrest in synchronised cells; thus, the duration of arrest did not alter the requirement for UBE2S (Supplemental Information, Fig. S1a).


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 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.
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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.
Mentions: We examined more closely the kinetics of checkpoint arrest and mitotic slippage after UBE2S depletion, by analysing Mona-arrested cells released into drug-free media. Approximately 35% of control-treated cells exit mitosis in the first 3 hours, and the majority (85%) by 12 hours (Figure 3a). UBE2S depletion significantly delayed mitotic exit; even by 12 hours, only about 30% of cells had exited. A similar delay occurs in individual cells examined by time-lapse imaging (Figure 3b). The average time to exit mitosis after release from drug-induced arrest was 255 +/− 169 minutes (n=49 cells) in controls versus 668 +/− 412 minutes (n=48) after UBE2S depletion. Similar effects occurred after release from a short (6 hour) arrest in synchronised cells; thus, the duration of arrest did not alter the requirement for UBE2S (Supplemental Information, Fig. S1a).

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