Letter | Published:

The mitotic checkpoint complex binds a second CDC20 to inhibit active APC/C

Nature volume 517, pages 631634 (29 January 2015) | Download Citation



The spindle assembly checkpoint (SAC) maintains genomic stability by delaying chromosome segregation until the last chromosome has attached to the mitotic spindle. The SAC prevents the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase from recognizing cyclin B and securin by catalysing the incorporation of the APC/C co-activator, CDC20, into a complex called the mitotic checkpoint complex (MCC). The SAC works through unattached kinetochores generating a diffusible ‘wait anaphase’ signal1,2 that inhibits the APC/C in the cytoplasm, but the nature of this signal remains a key unsolved problem. Moreover, the SAC and the APC/C are highly responsive to each other: the APC/C quickly targets cyclin B and securin once all the chromosomes attach in metaphase, but is rapidly inhibited should kinetochore attachment be perturbed3,4. How this is achieved is also unknown. Here, we show that the MCC can inhibit a second CDC20 that has already bound and activated the APC/C. We show how the MCC inhibits active APC/C and that this is essential for the SAC. Moreover, this mechanism can prevent anaphase in the absence of kinetochore signalling. Thus, we propose that the diffusible ‘wait anaphase’ signal could be the MCC itself, and explain how reactivating the SAC can rapidly inhibit active APC/C.

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  1. 1.

    , , & The checkpoint delaying anaphase in response to chromosome monoorientation is mediated by an inhibitory signal produced by unattached kinetochores. J. Cell Biol. 130, 941–948 (1995)

  2. 2.

    et al. Mitosis in vertebrate somatic cells with two spindles: implications for the metaphase/anaphase transition checkpoint and cleavage. Proc. Natl Acad. Sci. USA 94, 5107–5112 (1997)

  3. 3.

    & Temporal and spatial control of cyclin B1 destruction in metaphase. Nature Cell Biol. 1, 82–87 (1999)

  4. 4.

    & Kinetic framework of spindle assembly checkpoint signalling. Nature Cell Biol. 15, 1370–1377 (2013)

  5. 5.

    , & 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)

  6. 6.

    , , , & Structure of the mitotic checkpoint complex. Nature 484, 208–213 (2012)

  7. 7.

    et al. Catalytic assembly of the mitotic checkpoint inhibitor BubR1-Cdc20 by a Mad2-induced functional switch in Cdc20. Mol. Cell 51, 92–104 (2013)

  8. 8.

    & Mad2 and the APC/C compete for the same site on Cdc20 to ensure proper chromosome segregation. J. Cell Biol. 199, 27–37 (2012)

  9. 9.

    & Panta rhei: the APC/C at steady state. J. Cell Biol. 201, 177–189 (2013)

  10. 10.

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

  11. 11.

    , , & Mad2-independent inhibition of APCCdc20 by the mitotic checkpoint protein BubR1. Dev. Cell 1, 227–237 (2001)

  12. 12.

    & Mad3p, a pseudosubstrate inhibitor of APCCdc20 in the spindle assembly checkpoint. Genes Dev. 21, 655–667 (2007)

  13. 13.

    , & Mad3 KEN boxes mediate both Cdc20 and Mad3 turnover, and are critical for the spindle checkpoint. PLoS ONE 2, e342 (2007)

  14. 14.

    et al. Structural analysis of human Cdc20 supports multisite degron recognition by APC/C. Proc. Natl Acad. Sci. USA 109, 18419–18424 (2012)

  15. 15.

    et al. Uncoupling of the spindle-checkpoint and chromosome-congression functions of BubR1. J. Cell Sci. 123, 84–94 (2010)

  16. 16.

    , , , & BubR1 blocks substrate recruitment to the APC/C in a KEN-box-dependent manner. J. Cell Sci. 124, 4332–4345 (2011)

  17. 17.

    , , & The spindle assembly checkpoint works like a rheostat rather than a toggle switch. Nature Cell Biol. 15, 1378–1385 (2013)

  18. 18.

    et al. A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins. Mol. Cell. Proteomics 7, 282–289 (2008)

  19. 19.

    & Mad2 and Mad3 cooperate to arrest budding yeast in mitosis. Curr. Biol. 22, 180–190 (2012)

  20. 20.

    , , , & 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)

  21. 21.

    , & Human blinkin/AF15q14 is required for chromosome alignment and the mitotic checkpoint through direct interaction with Bub1 and BubR1. Dev. Cell 13, 663–676 (2007)

  22. 22.

    et al. The Mad1/Mad2 complex as a template for Mad2 activation in the spindle assembly checkpoint. Curr. Biol. 15, 214–225 (2005)

  23. 23.

    et al. Role of the Mad2 dimerization interface in the spindle assembly checkpoint independent of kinetochores. Curr. Biol. (2012)

  24. 24.

    , , , & Phosphorylation of Cdc20 by Bub1 provides a catalytic mechanism for APC/C inhibition by the spindle checkpoint. Mol. Cell 16, 387–397 (2004)

  25. 25.

    , , & p31comet-mediated extraction of Mad2 from the MCC promotes efficient mitotic exit. J. Cell Sci. 124, 3905–3916 (2011)

  26. 26.

    , , , & Homeostatic control of mitotic arrest. Mol. Cell 44, 710–720 (2011)

  27. 27.

    , , , & APC15 drives the turnover of MCC-Cdc20 to make the spindle assembly checkpoint responsive to kinetochore attachment. Nature Cell Biol. 13, 1234–1243 (2011)

  28. 28.

    , , & The APC/C maintains the spindle assembly checkpoint by targeting Cdc20 for destruction. Nature Cell Biol. 10, 1411–1420 (2008)

  29. 29.

    & Analysis of activator-binding sites on the APC/C supports a cooperative substrate-binding mechanism. Mol. Cell 34, 68–80 (2009)

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We are grateful to T. Matsusaka for developing infrared-dye-conjugated ubiquitylation substrates, to A. Musacchio, W. Earnshaw, T. Kiyomitsu and M. Yanagida for reagents, and to A. Musacchio and members of our laboratory for critical discussions. This work was supported by a project and a programme grant from Cancer Research UK to J.P. J.P. acknowledges core funding to the Gurdon Institute from the Wellcome Trust and CR UK.

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

    • Daisuke Izawa
    •  & Jonathon Pines


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Experiments were designed by D.I. and J.P., carried out by D.I., and analysed by D.I. and J.P.; D.I. and J.P. wrote the paper.

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The authors declare no competing financial interests.

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Correspondence to Jonathon Pines.

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