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

Nature Cell Biology volume 11, pages 1363–1369 (2009)Cite this article

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 mitosis1,2,3. 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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Figure 1: RNAi screen identifies UBE2S as a modifier of release from arrest at the SAC.
Figure 2: UBE2S regulates the outcome of drug-induced mitotic arrest.
Figure 3: UBE2S is necessary for mitotic release and slippage.
Figure 4: UBE2S elongates pre-initiated ubiquitin chains on cyclin B1.
Figure 5: UBE2S is necessary for the degradation of APC/C substrates and to antagonise the SAC.

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Acknowledgements

We thank P. Lehner (Cambridge Institute of Medical Research) for providing the ubiquitin-proteasome siRNA library, the Newton Trust for funding its purchase, and L. Passmore for helpful discussions. M.J.G. was supported by a Canadian Institute of Health Research fellowship, J.M., by a FEBS fellowship and C.G., by a Churchill Foundation Scholarship. M.J.G., C.G. and P.R. were also supported by a UK Medical Research Council (MRC) grant to A.R.V., and J.W. and by a Wellcome Trust grant to A.R.V. Work in J.P.'s laboratory is supported by Cancer Research UK, and in A.R.V.'s, by the MRC.

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Author notes

  1. Mathew J. Garnett, Jörg Mansfeld and Ashok R. Venkitaraman: These authors contributed equally to this work.

Authors and Affiliations

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

    Mathew J. Garnett, Colin Godwin, Jiahua Wu, Paul Russell & Ashok R. Venkitaraman

  2. Wellcome/Cancer Research UK Gurdon Institute and Department of Zoology, Tennis Court Road, Cambridge, 1QN, CB2, UK

    Jörg Mansfeld, Takahiro Matsusaka & Jonathon Pines

  3. Correspondence should be addressed to M.J.G or A.R.V.arv22@cam.ac.uk,

    Mathew J. Garnett & Ashok R. Venkitaraman

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Contributions

M.J.G., A.R.V. and P.R. designed the siRNA screen; M.J.G. and C.G. performed the screen; M.J.G, C.G., P.R. and J.W. analysed the screen data. M.J.G. performed the analysis of UBE2S cellular function. J.M. performed the APC/C in vitro activity assays, and T.M., the cyclin-B1 degradation assays and microinjection studies. M.J.G. and T.M. performed the time-lapse studies, which J.W. helped quantify. All authors analysed and interpreted the data. The manuscript was written by M.J.G. and A.R.V.

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Garnett, M., Mansfeld, J., Godwin, C. et al. UBE2S elongates ubiquitin chains on APC/C substrates to promote mitotic exit. Nat Cell Biol 11, 1363–1369 (2009). https://doi.org/10.1038/ncb1983

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