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APC15 drives the turnover of MCC-CDC20 to make the spindle assembly checkpoint responsive to kinetochore attachment

Nature Cell Biology volume 13, pages 1234–1243 (2011)Cite this article

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Abstract

Faithful chromosome segregation during mitosis depends on the spindle assembly checkpoint (SAC), which monitors kinetochore attachment to the mitotic spindle. Unattached kinetochores generate mitotic checkpoint proteins complexes (MCCs) that bind and inhibit the anaphase-promoting complex, or cyclosome (APC/C). How the SAC proficiently inhibits the APC/C but still allows its rapid activation when the last kinetochore attaches to the spindle is important for the understanding of how cells maintain genomic stability. We show that the APC/C subunit APC15 is required for the turnover of the APC/C co-activator CDC20 and release of MCCs during SAC signalling but not for APC/C activity per se. In the absence of APC15, MCCs and ubiquitylated CDC20 remain ‘locked’ onto the APC/C, which prevents the ubiquitylation and degradation of cyclin B1 when the SAC is satisfied. We conclude that APC15 mediates the constant turnover of CDC20 and MCCs on the APC/C to allow the SAC to respond to the attachment state of kinetochores.

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Figure 1: APC15 is a subunit of the human APC/C.
Figure 2: APC15 is an APC/C subunit required for timely entry into anaphase.
Figure 3: APC15 depletion causes a SAC-dependent delay in mitosis but does not affect APC/C activity.
Figure 4: APC15 is required for the turnover of MAD2, BUBR1, BUB3 and CDC20 on the APC/C during prometaphase.
Figure 5: APC15 is required for the release of MAD2, BUBR1 and BUB3 from the APC/C after the SAC had been satisfied.
Figure 6: Ubiquitylation of CDC20 is not required to release MCCs from the APC/C.
Figure 7: Ubiquitylation contributes to the release of MCCs from the APC/C.

References

  1. Sudakin, V., Chan, G.K. & Yen, T.J. Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2. J. Cell Biol. 154, 925–936 (2001).

    Article  CAS  Google Scholar 

  2. Zeng, X. et al. Pharmacologic inhibition of the anaphase-promoting complex induces a spindle checkpoint-dependent mitotic arrest in the absence of spindle damage. Cancer Cell 18, 382–395 (2010).

    Article  CAS  Google Scholar 

  3. Visconti, R., Palazzo, L. & Grieco, D. Requirement for proteolysis in spindle assembly checkpoint silencing. Cell Cycle 9, 564–569 (2010).

    Article  CAS  Google Scholar 

  4. Reddy, S. K., Rape, M., Margansky, W. A. & Kirschner, M. W. Ubiquitination by the anaphase-promoting complex drives spindle checkpoint inactivation. Nature 446, 921–925 (2007).

    Article  CAS  Google Scholar 

  5. Nilsson, J., Yekezare, M., Minshull, J. & Pines, J. The APC/C maintains the spindle assembly checkpoint by targeting Cdc20 for destruction. Nat. Cell Biol. 10, 1411–1420 (2008).

    Article  CAS  Google Scholar 

  6. Yoon, H. J. et al. Proteomics analysis identifies new components of the fission and budding yeast anaphase-promoting complexes. Curr. Biol. 12, 2048–2054 (2002).

    Article  CAS  Google Scholar 

  7. Hall, M. C., Torres, M. P., Schroeder, G. K. & Borchers, C. H. Mnd2 and Swm1 are core subunits of the Saccharomyces cerevisiae anaphase-promoting complex. J. Biol. Chem. 278, 16698–16705 (2003).

    Article  CAS  Google Scholar 

  8. Schreiber, A. et al. Structural basis for the subunit assembly of the anaphase-promoting complex. Nature 470, 227–232 (2011).

    Article  CAS  Google Scholar 

  9. Penkner, A. M., Prinz, S., Ferscha, S. & Klein, F. Mnd2, an essential antagonist of the anaphase-promoting complex during meiotic prophase. Cell 120, 789–801 (2005).

    Article  CAS  Google Scholar 

  10. Hubner, N. C. et al. Quantitative proteomics combined with BAC TransgeneOmics reveals in vivo protein interactions. J. Cell Biol. 189, 739–754 (2010).

    Article  CAS  Google Scholar 

  11. Clute, P. & Pines, J. Temporal and spatial control of cyclin B1 destruction in metaphase. Nat. Cell Biol. 1, 82–87 (1999).

    Article  CAS  Google Scholar 

  12. Thornton, B. R. et al. An architectural map of the anaphase-promoting complex. Genes Dev. 20, 449–460 (2006).

    Article  CAS  Google Scholar 

  13. Izawa, D. & Pines, J. How APC/C-Cdc20 changes its substrate specificity in mitosis. Nat. Cell Biol. 13, 223–233 (2011).

    Article  CAS  Google Scholar 

  14. Di Fiore, B. & Pines, J. How cyclin A destruction escapes the spindle assembly checkpoint. J. Cell Biol. 190, 501–509 (2010).

    Article  CAS  Google Scholar 

  15. Ge, S., Skaar, J. R. & Pagano, M. APC/C- and Mad2-mediated degradation of Cdc20 during spindle checkpoint activation. Cell Cycle 8, 167–171 (2009).

    Article  CAS  Google Scholar 

  16. Pan, J. & Chen, R. H. Spindle checkpoint regulates Cdc20p stability in Saccharomyces cerevisiae. Genes Dev. 18, 1439–1451 (2004).

    Article  CAS  Google Scholar 

  17. Kulukian, A., Han, J. S. & Cleveland, D. W. Unattached kinetochores catalyze production of an anaphase inhibitor that requires a Mad2 template to prime Cdc20 for BubR1 binding. Dev. Cell 16, 105–117 (2009).

    Article  CAS  Google Scholar 

  18. Meraldi, P., Draviam, V. M. & Sorger, P. K. Timing and checkpoints in the regulation of mitotic progression. Dev. Cell 7, 45–60 (2004).

    Article  CAS  Google Scholar 

  19. Dobles, M., Liberal, V., Scott, M. L., Benezra, R. & Sorger, P. K. Chromosome missegregation and apoptosis in mice lacking the mitotic checkpoint protein Mad2. Cell 101, 635–645 (2000).

    Article  CAS  Google Scholar 

  20. Santaguida, S., Tighe, A., D’Alise, A. M., Taylor, S. S. & Musacchio, A. Dissecting the role of MPS1 in chromosome biorientation and the spindle checkpoint through the small molecule inhibitor reversine. J. Cell Biol. 190, 73–87 (2010).

    Article  CAS  Google Scholar 

  21. Carroll, C. W. & Morgan, D. O. The Doc1 subunit is a processivity factor for the anaphase-promoting complex. Nat. Cell Biol. 4, 880–887 (2002).

    Article  CAS  Google Scholar 

  22. Garnett, M. J. et al. UBE2S elongates ubiquitin chains on APC/C substrates to promote mitotic exit. Nat. Cell Biol. 11, 1363–1369 (2009).

    Article  CAS  Google Scholar 

  23. Yang, M. et al. p31 (comet) blocks mad2 activation through structural mimicry. Cell 131, 744–755 (2007).

    Article  CAS  Google Scholar 

  24. Xia, G. et al. Conformation-specific binding of p31 (comet) antagonizes the function of Mad2 in the spindle checkpoint. EMBO J. 23, 3133–3143 (2004).

    Article  CAS  Google Scholar 

  25. Mapelli, M. et al. Determinants of conformational dimerization of Mad2 and its inhibition by p31 comet. EMBO J. 25, 1273–1284 (2006).

    Article  CAS  Google Scholar 

  26. Habu, T., Kim, S. H., Weinstein, J. & Matsumoto, T. Identification of a MAD2-binding protein, CMT2, and its role in mitosis. EMBO J. 21, 6419–6428 (2002).

    Article  CAS  Google Scholar 

  27. Herzog, F. et al. Structure of the anaphase-promoting complex/cyclosome interacting with a mitotic checkpoint complex. Science 323, 1477–1481 (2009).

    Article  CAS  Google Scholar 

  28. Gascoigne, K. E. & Taylor, S. S. Cancer cells display profound intra- and interline variation following prolonged exposure to antimitotic drugs. Cancer Cell 14, 111–122 (2008).

    Article  CAS  Google Scholar 

  29. Huang, H. C., Shi, J., Orth, J. D. & Mitchison, T. J. Evidence that mitotic exit is a better cancer therapeutic target than spindle assembly. Cancer Cell 16, 347–358 (2009).

    Article  CAS  Google Scholar 

  30. Brito, D. A. & Rieder, C. L. Mitotic checkpoint slippage in humans occurs via cyclin B destruction in the presence of an active checkpoint. Curr. Biol. 16, 1194–1200 (2006).

    Article  CAS  Google Scholar 

  31. Ditchfield, C. et al. Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores. J. Cell Biol. 161, 267–280 (2003).

    Article  CAS  Google Scholar 

  32. Nezi, L. & Musacchio, A. Sister chromatid tension and the spindle assembly checkpoint. Curr. Opin. Cell Biol. 21, 785–795 (2009).

    Article  CAS  Google Scholar 

  33. Miniowitz-Shemtov, S., Teichner, A., Sitry-Shevah, D. & Hershko, A. ATP is required for the release of the anaphase-promoting complex/cyclosome from inhibition by the mitotic checkpoint. Proc. Natl Acad. Sci. USA 107, 5351–5356 (2010).

    Article  Google Scholar 

  34. Ma, H. T. & Poon, R. Y. Orderly inactivation of the key checkpoint protein mitotic arrest deficient 2 (MAD2) during mitotic progression. J. Biol. Chem. 15, 13052–13059 (2011).

    Article  CAS  Google Scholar 

  35. Danielsen, J. M. et al. Mass spectrometric analysis of lysine ubiquitylation reveals promiscuity at site level. Mol. Cell Proteomics 10, M110.003590 (2011).

    Article  CAS  Google Scholar 

  36. Gmachl, M., Gieffers, C., Podtelejnikov, A. V., Mann, M. & Peters, J. M. The RING-H2 finger protein APC11 and the E2 enzyme UBC4 are sufficient to ubiquitinate substrates of the anaphase-promoting complex. Proc. Natl Acad. Sci. USA 97, 8973–8978 (2000).

    Article  CAS  Google Scholar 

  37. Leverson, J. D. et al. The APC11 RING-H2 finger mediates E2-dependent ubiquitination. Mol. Biol. Cell 11, 2315–2325 (2000).

    Article  CAS  Google Scholar 

  38. Williamson, A. et al. Identification of a physiological E2 module for the human anaphase-promoting complex. Proc. Natl Acad. Sci. USA 106, 18213–18218 (2009).

    Article  Google Scholar 

  39. Walker, A., Acquaviva, C., Matsusaka, T., Koop, L. & Pines, J. UbcH10 has a rate-limiting role in G1 phase but might not act in the spindle checkpoint or as part of an autonomous oscillator. J. Cell Sci. 121, 2319–2326 (2008).

    Article  CAS  Google Scholar 

  40. Stegmeier, F. et al. Anaphase initiation is regulated by antagonistic ubiquitination and deubiquitination activities. Nature 446, 876–881 (2007).

    Article  CAS  Google Scholar 

  41. Choi, E. et al. BubR1 acetylation at prometaphase is required for modulating APC/C activity and timing of mitosis. EMBO J. 28, 2077–2089 (2009).

    Article  CAS  Google Scholar 

  42. Oelschlaegel, T. et al. The yeast APC/C subunit Mnd2 prevents premature sister chromatid separation triggered by the meiosis-specific APC/C-Ama1. Cell 120, 773–788 (2005).

    Article  CAS  Google Scholar 

  43. Maciejowski, J. et al. Mps1 directs the assembly of Cdc20 inhibitory complexes during interphase and mitosis to control M phase timing and spindle checkpoint signaling. J. Cell Biol. 190, 89–100 (2010).

    Article  CAS  Google Scholar 

  44. Berdougo, E., Terret, M. E. & Jallepalli, P. V. Functional dissection of mitotic regulators through gene targeting in human somatic cells. Methods Mol. Biol. 545, 21–37 (2009).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank S. Taylor for the HeLa FRT cell line, A. Musacchio for the anti-MAD2 and anti-p31comet antibodies and for communicating results before publication, T. Hunt for the anti-APC3 antibody, members of our laboratory for discussions and O. Nashchekina, F. Walton, E. Russe and S. Wieser for help with somatic cell knock-ins, mass spectrometry, statistical and flow cytometry analyses, respectively. J.M. was supported by FEBS and EMBO fellowships. This work was supported by a project grant from the BBSRC and by a Programme grant from Cancer Research UK to J.P. and core grant support to the Sanger Institute (M.O.C. and J.S.C.) from The Wellcome Trust (grant number 079643/Z/06/Z).

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  1. Philippe Collin and Mark O. Collins: These authors contributed equally

Authors and Affiliations

  1. The Gurdon Institute and Department of Zoology, Tennis Court Road, Cambridge CB2 1QN, UK

    Jörg Mansfeld, Philippe Collin & Jonathon Pines

  2. Proteomic Mass Spectrometry Laboratory, The Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK

    Mark O. Collins & Jyoti S. Choudhary

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  1. Jörg Mansfeld
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Contributions

J.M. carried out all of the experiments, P.C. generated the RPE1 cyclin-A2– and B1–Venus knock-in cell lines, M.O.C. and J.S.C. carried out the mass spectrometry that identified APC15 and the CDC20 ubiquitylation site. J.M. and J.P. designed the experiments and wrote the paper. All authors contributed to the interpretation of the results.

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Correspondence to Jonathon Pines.

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Mansfeld, J., Collin, P., Collins, M. et al. APC15 drives the turnover of MCC-CDC20 to make the spindle assembly checkpoint responsive to kinetochore attachment. Nat Cell Biol 13, 1234–1243 (2011). https://doi.org/10.1038/ncb2347

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