Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Anaphase initiation is regulated by antagonistic ubiquitination and deubiquitination activities


The spindle checkpoint prevents chromosome mis-segregation by delaying sister chromatid separation until all chromosomes have achieved bipolar attachment to the mitotic spindle. Its operation is essential for accurate chromosome segregation, whereas its dysregulation can contribute to birth defects and tumorigenesis. The target of the spindle checkpoint is the anaphase-promoting complex (APC), a ubiquitin ligase that promotes sister chromatid separation and progression to anaphase. Using a short hairpin RNA screen targeting components of the ubiquitin-proteasome pathway in human cells, we identified the deubiquitinating enzyme USP44 (ubiquitin-specific protease 44) as a critical regulator of the spindle checkpoint. USP44 is not required for the initial recognition of unattached kinetochores and the subsequent recruitment of checkpoint components. Instead, it prevents the premature activation of the APC by stabilizing the APC-inhibitory Mad2–Cdc20 complex. USP44 deubiquitinates the APC coactivator Cdc20 both in vitro and in vivo, and thereby directly counteracts the APC-driven disassembly of Mad2–Cdc20 complexes (discussed in an accompanying paper). Our findings suggest that a dynamic balance of ubiquitination by the APC and deubiquitination by USP44 contributes to the generation of the switch-like transition controlling anaphase entry, analogous to the way that phosphorylation and dephosphorylation of Cdk1 by Wee1 and Cdc25 controls entry into mitosis.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type



Prices may be subject to local taxes which are calculated during checkout

Figure 1: Validation of candidate genes from Taxol screen.
Figure 2: USP44 activity is cell-cycle regulated and is required for proper spindle checkpoint function and anaphase timing.
Figure 3: USP44 regulates APC Cdc20 downstream of Mad2 recruitment to kinetochores.
Figure 4: USP44 inhibits APCCdc20 activation in vitro.
Figure 5: USP44 deubiquitinates Cdc20 in vitro and in vivo.


  1. Lengauer, C., Kinzler, K. W. & Vogelstein, B. Genetic instabilities in human cancers. Nature 396, 643–649 (1998)

    Article  ADS  CAS  Google Scholar 

  2. Draviam, V. M., Xie, S. & Sorger, P. K. Chromosome segregation and genomic stability. Curr. Opin. Genet. Dev. 14, 120–125 (2004)

    Article  CAS  Google Scholar 

  3. Kops, G. J., Weaver, B. A. & Cleveland, D. W. On the road to cancer: aneuploidy and the mitotic checkpoint. Nature Rev. Cancer 5, 773–785 (2005)

    Article  CAS  Google Scholar 

  4. Peters, J. M. The anaphase promoting complex/cyclosome: a machine designed to destroy. Nature Rev. Mol. Cell Biol. 7, 644–656 (2006)

    Article  CAS  Google Scholar 

  5. Musacchio, A. & Hardwick, K. G. The spindle checkpoint: structural insights into dynamic signalling. Nature Rev. Mol. Cell Biol. 3, 731–741 (2002)

    Article  CAS  Google Scholar 

  6. Bharadwaj, R. & Yu, H. The spindle checkpoint, aneuploidy, and cancer. Oncogene 23, 2016–2027 (2004)

    Article  CAS  Google Scholar 

  7. Nasmyth, K. How do so few control so many? Cell 120, 739–746 (2005)

    Article  CAS  Google Scholar 

  8. Hoyt, M. A., Totis, L. & Roberts, B. T. S. cerevisiae genes required for cell cycle arrest in response to loss of microtubule function. Cell 66, 507–517 (1991)

    Article  CAS  Google Scholar 

  9. Li, R. & Murray, A. W. Feedback control of mitosis in budding yeast. Cell 66, 519–531 (1991)

    Article  CAS  Google Scholar 

  10. Weiss, E. & Winey, M. The Saccharomyces cerevisiae spindle pole body duplication gene MPS1 is part of a mitotic checkpoint. J. Cell Biol. 132, 111–123 (1996)

    Article  CAS  Google Scholar 

  11. Karess, R. Rod-Zw10-Zwilch: a key player in the spindle checkpoint. Trends Cell Biol. 15, 386–392 (2005)

    Article  CAS  Google Scholar 

  12. 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 

  13. 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 

  14. Mapelli, M. et al. Determinants of conformational dimerization of Mad2 and its inhibition by p31comet. EMBO J. 25, 1273–1284 (2006)

    Article  CAS  Google Scholar 

  15. Kops, G. J. et al. ZW10 links mitotic checkpoint signaling to the structural kinetochore. J. Cell Biol. 169, 49–60 (2005)

    Article  CAS  Google Scholar 

  16. Reddy, S. K., Rape, M., Marganski, W. A. & Kirschner, M. W. Ubiquitination by the anaphase-promoting complex drives spindle checkpoint inactivation. Nature advance online publication, doi:10.1038/nature05734 (this issue).

  17. Silva, J. M. et al. Second-generation shRNA libraries covering the mouse and human genomes. Nature Genet. 37, 1281–1288 (2005)

    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. Geley, S. et al. Anaphase-promoting complex/cyclosome-dependent proteolysis of human cyclin A starts at the beginning of mitosis and is not subject to the spindle assembly checkpoint. J. Cell Biol. 153, 137–148 (2001)

    Article  CAS  Google Scholar 

  20. den Elzen, N. & Pines, J. Cyclin A is destroyed in prometaphase and can delay chromosome alignment and anaphase. J. Cell Biol. 153, 121–136 (2001)

    Article  CAS  Google Scholar 

  21. Rape, M. & Kirschner, M. W. Autonomous regulation of the anaphase-promoting complex couples mitosis to S-phase entry. Nature 432, 588–595 (2004)

    Article  ADS  CAS  Google Scholar 

  22. Rape, M., Reddy, S. K. & Kirschner, M. W. The processivity of multiubiquitination by the APC determines the order of substrate degradation. Cell 124, 89–103 (2006)

    Article  CAS  Google Scholar 

  23. Ferrell, J. E. & Xiong, W. Bistability in cell signaling: How to make continuous processes discontinuous, and reversible processes irreversible. Chaos 11, 227–236 (2001)

    Article  ADS  CAS  Google Scholar 

  24. Markevich, N. I., Hoek, J. B. & Kholodenko, B. N. Signaling switches and bistability arising from multisite phosphorylation in protein kinase cascades. J. Cell Biol. 164, 353–359 (2004)

    Article  CAS  Google Scholar 

  25. Takizawa, C. G. & Morgan, D. O. Control of mitosis by changes in the subcellular location of cyclin-B1-Cdk1 and Cdc25C. Curr. Opin. Cell Biol. 12, 658–665 (2000)

    Article  CAS  Google Scholar 

  26. Stegmeier, F. & Amon, A. Closing mitosis: the functions of the Cdc14 phosphatase and its regulation. Annu. Rev. Genet. 38, 203–232 (2004)

    Article  CAS  Google Scholar 

Download references


We thank S. Taylor, H. Yu, W. Earnshaw and J. Jin for gifts of reagents; M. Vidal for providing access to their BioRobot platform; S. Lyman and R. King for communicating unpublished results and assistance with the development of the Taxol screening assay; C. Shamu for access to the ICCB-Longwood screening facilities; S. Reddy for helpful comments throughout the course of the work; and T. Westbrook and A. Smogorzewska for their critical reading of the manuscript. F.S. is a fellow of the Helen Hay Whitney Foundation. M.R. is a Human Frontiers Science Program Long-Term Fellow. The siRNA and ICCB-Longwood resources used were funded in part by a NCI grant (T. Mitchison). M.E.S. is an American Cancer Society Postdoctoral Fellow. X.L.A. is an NIH pre-doctoral fellow. M.W.K. thanks the National Institute of General Medical Sciences for its support for the grant Cell Cycle Regulation. This work was supported by grants from NIH and DOD to S.J.E. and by grants from the NIH to J.W.H. S.J.E. is an investigator of the Howard Hughes Medical Institute.

Author information

Authors and Affiliations


Corresponding authors

Correspondence to Marc W. Kirschner, J. Wade Harper or Stephen J. Elledge.

Ethics declarations

Competing interests

Reprints and permissions information is available at The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Methods, Supplementary Figures 1-11 with Legends, and Supplementary Tables 2 and 3. (PDF 4063 kb)

Supplementary Table 1

This file contains Supplementary Table 1. This file contains detailed information on the Ubiquitin-Proteasome Pathway (UPP) shRNA library. (XLS 526 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing