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The APC/C maintains the spindle assembly checkpoint by targeting Cdc20 for destruction

Nature Cell Biology volume 10, pages 1411–1420 (2008)Cite this article

Abstract

The spindle assembly checkpoint (SAC) is required to block sister chromatid separation until all chromosomes are properly attached to the mitotic apparatus. The SAC prevents cells from entering anaphase by inhibiting the ubiquitylation of cyclin B1 and securin by the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase. The target of the SAC is the essential APC/C activator Cdc20. It is unclear how the SAC inactivates Cdc20 but most current models suggest that Cdc20 forms a stable complex with the Mad2 checkpoint protein. Here we show that most Cdc20 is not in a complex with Mad2; instead Mad2 is required for Cdc20 to form a complex with another checkpoint protein, BubR1. We further show that during the SAC, the APC/C ubiquitylates Cdc20 to target it for degradation. Thus, ubiquitylation of human Cdc20 is not required to release it from the checkpoint complex, but to degrade it to maintain mitotic arrest.

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Figure 1: Cdc20 is degraded in checkpoint-arrested cells.
Figure 2: Cdc20 degradation is dependent on APC/C activity and a functional SAC.
Figure 3: Cdc20 requires both APC/C- and Mad2-binding motifs to be degraded in the SAC.
Figure 4: Analysis of checkpoint complexes by gel-filtration chromatography.
Figure 5: A Cdc20 mutant without lysines overrides the SAC.
Figure 6: Dissociation of checkpoint proteins from Cdc20 does not require ubiquitylation of Cdc20.
Figure 7: Spindle checkpoint model.

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Acknowledgements

We thank Daisuke Izawa for pioneering APC3 and APC11 siRNA depletion, Paola Marco for help with the BubR1 constructs, Stephen Taylor, Gowei Fang and Andrea Musacchio for reagents, Athanassios Giannis for dimethylanastron, Mercedes Pardo and Lu Yu of the Mass Spectroscopy group at the Sanger Centre for mass spectroscopy, and Andrea Musacchio and members of our laboratory for helpful discussions. M.Y. is particularly grateful to Takahiro Matsusaka for help with microscopy and microinjection. J.N. was supported by fellowships from the Carlsberg Foundation and the Danish Cancer Society, M.Y. was supported by a Yousef Jameel scholarship. This work was supported by a Program Grant from Cancer Research UK to J.P. (C29/A3211).

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  1. Jakob Nilsson and Mona Yekezare: These authors contributed equally to the work

Authors and Affiliations

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

    Jakob Nilsson, Mona Yekezare & Jonathon Pines

  2. DNA 2.0 Inc, 1430 O'Brien Drive, Menlo Park, CA 94025, USA.,

    Jeremy Minshull

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  1. Jakob Nilsson
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Contributions

J.N. performed the biochemical experiments; M.Y. performed the imaging experiments; J.M. constructed the Lys-less Cdc20 and control Cdc20 constructs; J.N., M.Y. and J.P. designed the experiments; J.P. wrote the paper with help from J.N. and M.Y.

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Correspondence to Jonathon Pines.

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Nilsson, J., Yekezare, M., Minshull, J. et al. The APC/C maintains the spindle assembly checkpoint by targeting Cdc20 for destruction. Nat Cell Biol 10, 1411–1420 (2008). https://doi.org/10.1038/ncb1799

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