Skip to main content

Thank you for visiting nature.com. 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.

  • Article
  • Published:

An APC/C inhibitor stabilizes cyclin B1 by prematurely terminating ubiquitination

Nature Chemical Biology volume 8, pages 383–392 (2012)Cite this article

Subjects

Abstract

The anaphase-promoting complex/cyclosome (APC) is a ubiquitin ligase that is required for exit from mitosis. We previously showed that tosyl arginine methyl ester (TAME) inhibits APC-dependent proteolysis by competing with the C-terminal isoleucine-arginine tail of the APC activator cell division cycle 20 (Cdc20) for APC binding. Here we show that in the absence of APC substrates, TAME ejects Cdc20 from the APC by promoting Cdc20 autoubiquitination in its N-terminal region. Cyclin B1 antagonizes TAME's effect by promoting binding of free Cdc20 to the APC and by suppressing Cdc20 autoubiquitination. Nevertheless, TAME stabilizes cyclin B1 in Xenopus extracts by two mechanisms. First, it reduces the kcat of the APC-Cdc20–cyclin B1 complex without affecting the Km, slowing the initial ubiquitination of unmodified cyclin B1. Second, as cyclin B1 becomes ubiquitinated, it loses its ability to promote Cdc20 binding to the APC in the presence of TAME. As a result, cyclin B1 ubiquitination terminates before reaching the threshold necessary for proteolysis.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: TAME-induced Cdc20 dissociation from the APC in mitotic Xenopus extracts requires APC-dependent ubiquitination.
Figure 2: TAME induces Cdc20 dissociation from the APC by promoting Cdc20 ubiquitination.
Figure 3: Ubiquitination of Cdc20 upstream of the CRY box reduces its binding affinity for the APC.
Figure 4: CycB-NT promotes Cdc20 binding to the APC and suppresses Cdc20 ubiquitination.
Figure 5: TAME causes premature termination of cycB-NT ubiquitination.
Figure 6: UBE2S extends ubiquitin chains on cycB-NT in the presence of TAME in a single substrate-binding cycle and promotes substrate degradation in TAME-treated extract.
Figure 7: Schematic illustration of TAME's mechanism.

Similar content being viewed by others

References

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

    Article  CAS  PubMed  Google Scholar 

  2. Verma, R. et al. Ubistatins inhibit proteasome-dependent degradation by binding the ubiquitin chain. Science 306, 117–120 (2004).

    Article  CAS  PubMed  Google Scholar 

  3. 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  PubMed  PubMed Central  Google Scholar 

  4. Pesin, J.A. & Orr-Weaver, T.L. Regulation of APC/C activators in mitosis and meiosis. Annu. Rev. Cell Dev. Biol. 24, 475–499 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Hayes, M.J. et al. Early mitotic degradation of Nek2A depends on Cdc20-independent interaction with the APC/C. Nat. Cell Biol. 8, 607–614 (2006).

    Article  CAS  PubMed  Google Scholar 

  6. Kimata, Y., Baxter, J.E., Fry, A.M. & Yamano, H. A role for the Fizzy/Cdc20 family of proteins in activation of the APC/C distinct from substrate recruitment. Mol. Cell 32, 576–583 (2008).

    Article  CAS  PubMed  Google Scholar 

  7. da Fonseca, P.C. et al. Structures of APC/C(Cdh1) with substrates identify Cdh1 and Apc10 as the D-box co-receptor. Nature 470, 274–278 (2011).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Passmore, L.A. et al. Doc1 mediates the activity of the anaphase-promoting complex by contributing to substrate recognition. EMBO J. 22, 786–796 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Vodermaier, H.C., Gieffers, C., Maurer-Stroh, S., Eisenhaber, F. & Peters, J.M. TPR subunits of the anaphase-promoting complex mediate binding to the activator protein CDH1. Curr. Biol. 13, 1459–1468 (2003).

    Article  CAS  PubMed  Google Scholar 

  11. Burton, J.L. & Solomon, M.J. D box and KEN box motifs in budding yeast Hsl1p are required for APC-mediated degradation and direct binding to Cdc20p and Cdh1p. Genes Dev. 15, 2381–2395 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Hilioti, Z., Chung, Y.S., Mochizuki, Y., Hardy, C.F. & Cohen-Fix, O. The anaphase inhibitor Pds1 binds to the APC/C-associated protein Cdc20 in a destruction box-dependent manner. Curr. Biol. 11, 1347–1352 (2001).

    Article  CAS  PubMed  Google Scholar 

  13. Kraft, C., Vodermaier, H.C., Maurer-Stroh, S., Eisenhaber, F. & Peters, J.M. The WD40 propeller domain of Cdh1 functions as a destruction box receptor for APC/C substrates. Mol. Cell 18, 543–553 (2005).

    Article  CAS  PubMed  Google Scholar 

  14. Schwab, M., Neutzner, M., Mocker, D. & Seufert, W. Yeast Hct1 recognizes the mitotic cyclin Clb2 and other substrates of the ubiquitin ligase APC. EMBO J. 20, 5165–5175 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Buschhorn, B.A. et al. Substrate binding on the APC/C occurs between the coactivator Cdh1 and the processivity factor Doc1. Nat. Struct. Mol. Biol. 18, 6–13 (2011).

    Article  CAS  PubMed  Google Scholar 

  16. Burton, J.L., Tsakraklides, V. & Solomon, M.J. Assembly of an APC-Cdh1–substrate complex is stimulated by engagement of a destruction box. Mol. Cell 18, 533–542 (2005).

    Article  CAS  PubMed  Google Scholar 

  17. Eytan, E., Moshe, Y., Braunstein, I. & Hershko, A. Roles of the anaphase-promoting complex/cyclosome and of its activator Cdc20 in functional substrate binding. Proc. Natl. Acad. Sci. USA 103, 2081–2086 (2006).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Dimova, N. et al. APC/C-mediated multiple monoubiquitination provides an alternative degradation signal for cyclin B1. Nat. Cell Biol 14, 168–176 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Townsley, F.M., Aristarkhov, A., Beck, S., Hershko, A. & Ruderman, J.V. Dominant-negative cyclin-selective ubiquitin carrier protein E2-C/UbcH10 blocks cells in metaphase. Proc. Natl. Acad. Sci. USA 94, 2362–2367 (1997).

    Article  CAS  PubMed  Google Scholar 

  21. 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  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  23. 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  PubMed  PubMed Central  Google Scholar 

  24. Kirkpatrick, D.S. et al. Quantitative analysis of in vitro ubiquitinated cyclin B1 reveals complex chain topology. Nat. Cell Biol. 8, 700–710 (2006).

    Article  CAS  PubMed  Google Scholar 

  25. 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  PubMed  PubMed Central  Google Scholar 

  26. 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  CAS  PubMed  Google Scholar 

  27. Wu, T. et al. UBE2S drives elongation of K11-linked ubiquitin chains by the anaphase-promoting complex. Proc. Natl. Acad. Sci. USA 107, 1355–1360 (2010).

    Article  CAS  PubMed  Google Scholar 

  28. Pierce, N.W., Kleiger, G., Shan, S.O. & Deshaies, R.J. Detection of sequential polyubiquitylation on a millisecond timescale. Nature 462, 615–619 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. 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 

  30. Kim, H.S. et al. SIRT2 maintains genome integrity and suppresses tumorigenesis through regulating APC/C activity. Cancer Cell 20, 487–499 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Varetti, G., Guida, C., Santaguida, S., Chiroli, E. & Musacchio, A. Homeostatic control of mitotic arrest. Mol. Cell 44, 710–720 (2011).

    Article  CAS  PubMed  Google Scholar 

  32. 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  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Mansfeld, J., Collin, P., Collins, M.O., Choudhary, J.S. & Pines, J. APC15 drives the turnover of MCC-CDC20 to make the spindle assembly checkpoint responsive to kinetochore attachment. Nat. Cell Biol. 13, 1234–1243 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Burton, J.L. & Solomon, M.J. Mad3p, a pseudosubstrate inhibitor of APCCdc20 in the spindle assembly checkpoint. Genes Dev. 21, 655–667 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Foe, I.T. et al. Ubiquitination of Cdc20 by the APC occurs through an intramolecular mechanism. Curr. Biol. 21, 1870–1877 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Rodrigo-Brenni, M.C. & Morgan, D.O. Sequential E2s drive polyubiquitin chain assembly on APC targets. Cell 130, 127–139 (2007).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank J. Pines (University of Cambridge, UK) for providing the Cdc20K-less construct and J.W. Harper for critically reading the manuscript. This work was supported by US National Institutes of Health grant GM66492 to R.W.K.

Author information

Authors and Affiliations

  1. Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA

    Xing Zeng & Randall W King

Authors
  1. Xing Zeng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Randall W King
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

X.Z. and R.W.K. designed the experiments, X.Z. performed the experiments, and X.Z. and R.W.K. wrote the manuscript.

Corresponding author

Correspondence to Randall W King.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Methods and Supplementary Results (PDF 4060 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zeng, X., King, R. An APC/C inhibitor stabilizes cyclin B1 by prematurely terminating ubiquitination. Nat Chem Biol 8, 383–392 (2012). https://doi.org/10.1038/nchembio.801

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.801

This article is cited by

Search

Advanced search

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