Skip to main content
SpringerLink
Log in

Detection and Analysis of Cell Cycle-Associated APC/C-Mediated Cellular Ubiquitylation In Vitro and In Vivo

  • Protocol
  • First Online:
Book cover Proteostasis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1449))

Abstract

The anaphase-promoting complex or cyclosome (APC/C) is one of the major orchestrators of the cell division cycle in mammalian cells. The APC/C acts as a ubiquitin ligase that triggers sequential ubiquitylation of a significant number of substrates which will be eventually degraded by proteasomes during major transitions of the cell cycle. In this chapter, we present accessible methodologies to assess both in in vitro conditions and in cellular systems ubiquitylation reactions mediated by the APC/C. In addition, we also describe techniques to evidence the changes in protein stability provoked by modulation of the activity of the APC/C. Finally, specific methods to analyze interactors or posttranslational modifications of particular APC/C subunits are also discussed. Given the crucial role played by the APC/C in the regulation of the cell cycle, this review only focuses on its action and effects in actively proliferating cells.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Teixeira LK, Reed SI (2013) Ubiquitin ligases and cell cycle control. Annu Rev Biochem 82:387–414. doi:10.1146/annurev-biochem-060410-105307

    Article  CAS  PubMed  Google Scholar 

  2. Gutierrez GJ, Ronai Z (2006) Ubiquitin and SUMO systems in the regulation of mitotic checkpoints. Trends Biochem Sci 31(6):324–332. doi:10.1016/j.tibs.2006.04.001, S0968-0004(06)00095-8 [pii]

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Mocciaro A, Rape M (2012) Emerging regulatory mechanisms in ubiquitin-dependent cell cycle control. J Cell Sci 125(Pt 2):255–263. doi:10.1242/jcs.091199

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Peters JM (2006) The anaphase promoting complex/cyclosome: a machine designed to destroy. Nat Rev Mol Cell Biol 7(9):644–656. doi:10.1038/nrm1988

    Article  CAS  PubMed  Google Scholar 

  5. Chang L, Barford D (2014) Insights into the anaphase-promoting complex: a molecular machine that regulates mitosis. Curr Opin Struct Biol 29:1–9. doi:10.1016/j.sbi.2014.08.003

    Article  CAS  PubMed  Google Scholar 

  6. Barford D (2015) Understanding the structural basis for controlling chromosome division. Philos Trans A Math Phys Eng Sci 373(2036). doi: 10.1098/rsta.2013.0392

    Google Scholar 

  7. Pines J (2011) Cubism and the cell cycle: the many faces of the APC/C. Nat Rev Mol Cell Biol 12(7):427–438. doi:10.1038/nrm3132

    Article  CAS  PubMed  Google Scholar 

  8. Lu D, Hsiao JY, Davey NE, Van Voorhis VA, Foster SA, Tang C, Morgan DO (2014) Multiple mechanisms determine the order of APC/C substrate degradation in mitosis. J Cell Biol 207(1):23–39. doi:10.1083/jcb.201402041

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Matsumoto ML, Wickliffe KE, Dong KC, Yu C, Bosanac I, Bustos D, Phu L, Kirkpatrick DS, Hymowitz SG, Rape M, Kelley RF, Dixit VM (2010) K11-linked polyubiquitination in cell cycle control revealed by a K11 linkage-specific antibody. Mol Cell 39(3):477–484. doi:10.1016/j.molcel.2010.07.001

    Article  CAS  PubMed  Google Scholar 

  10. Meyer HJ, Rape M (2011) Processive ubiquitin chain formation by the anaphase-promoting complex. Semin Cell Dev Biol 22(6):544–550. doi:10.1016/j.semcdb.2011.03.009

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Song L, Rape M (2011) Substrate-specific regulation of ubiquitination by the anaphase-promoting complex. Cell Cycle 10(1):52–56

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Williamson A, Banerjee S, Zhu X, Philipp I, Iavarone AT, Rape M (2011) Regulation of ubiquitin chain initiation to control the timing of substrate degradation. Mol Cell 42(6):744–757. doi:10.1016/j.molcel.2011.04.022

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Kelly A, Wickliffe KE, Song L, Fedrigo I, Rape M (2014) Ubiquitin chain elongation requires E3-dependent tracking of the emerging conjugate. Mol Cell 56(2):232–245. doi:10.1016/j.molcel.2014.09.010

    Article  CAS  PubMed  Google Scholar 

  14. Meyer HJ, Rape M (2014) Enhanced protein degradation by branched ubiquitin chains. Cell 157(4):910–921. doi:10.1016/j.cell.2014.03.037

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Lu Y, Wang W, Kirschner MW (2015) Specificity of the anaphase-promoting complex: a single-molecule study. Science 348(6231):1248737. doi:10.1126/science.1248737

    Article  PubMed  PubMed Central  Google Scholar 

  16. Puram SV, Bonni A (2011) Novel functions for the anaphase-promoting complex in neurobiology. Semin Cell Dev Biol 22(6):586–594. doi:10.1016/j.semcdb.2011.03.006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Zhang Z, Yang J, Kong EH, Chao WC, Morris EP, da Fonseca PC, Barford D (2013) Recombinant expression, reconstitution and structure of human anaphase-promoting complex (APC/C). Biochem J 449(2):365–371. doi:10.1042/BJ20121374

    Article  CAS  PubMed  Google Scholar 

  18. Chang L, Zhang Z, Yang J, McLaughlin SH, Barford D (2014) Molecular architecture and mechanism of the anaphase-promoting complex. Nature 513(7518):388–393. doi:10.1038/nature13543

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Frye JJ, Brown NG, Petzold G, Watson ER, Grace CR, Nourse A, Jarvis MA, Kriwacki RW, Peters JM, Stark H, Schulman BA (2013) Electron microscopy structure of human APC/C(CDH1)-EMI1 reveals multimodal mechanism of E3 ligase shutdown. Nat Struct Mol Biol 20(7):827–835. doi:10.1038/nsmb.2593

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Brown NG, Watson ER, Weissmann F, Jarvis MA, VanderLinden R, Grace CR, Frye JJ, Qiao R, Dube P, Petzold G, Cho SE, Alsharif O, Bao J, Davidson IF, Zheng JJ, Nourse A, Kurinov I, Peters JM, Stark H, Schulman BA (2014) Mechanism of polyubiquitination by human anaphase-promoting complex: RING repurposing for ubiquitin chain assembly. Mol Cell 56(2):246–260. doi:10.1016/j.molcel.2014.09.009

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Brown NG, VanderLinden R, Watson ER, Qiao R, Grace CR, Yamaguchi M, Weissmann F, Frye JJ, Dube P, Ei Cho S, Actis ML, Rodrigues P, Fujii N, Peters JM, Stark H, Schulman BA (2015) RING E3 mechanism for ubiquitin ligation to a disordered substrate visualized for human anaphase-promoting complex. Proc Natl Acad Sci U S A 112(17):5272–5279. doi:10.1073/pnas.1504161112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Gutierrez GJ, Tsuji T, Chen M, Jiang W, Ronai ZA (2010) Interplay between Cdh1 and JNK activity during the cell cycle. Nat Cell Biol 12(7):686–695, doi:ncb2071 [pii] 10.1038/ncb2071

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Rosner M, Schipany K, Hengstschläger M (2013) Merging high-quality biochemical fractionation with a refined flow cytometry approach to monitor nucleocytoplasmic protein expression throughout the unperturbed mammalian cell cycle. Nat Protoc 8(3):602–626. doi:10.1038/nprot.2013.011

    Article  PubMed  Google Scholar 

  24. Gutierrez GJ, Tsuji T, Cross JV, Davis RJ, Templeton DJ, Jiang W, Ronai ZA (2010) JNK-mediated phosphorylation of Cdc25C regulates cell cycle entry and G(2)/M DNA damage checkpoint. J Biol Chem 285(19):14217–14228, doi:M110.121848 [pii] 10.1074/jbc.M110.121848

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Chen M, Gutierrez GJ, Ronai ZA (2011) Ubiquitin-recognition protein Ufd1 couples the endoplasmic reticulum (ER) stress response to cell cycle control. Proc Natl Acad Sci U S A 108(22):9119–9124, doi:1100028108 [pii] 10.1073/pnas.1100028108

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Chen M, Gutierrez GJ, Ronai ZA (2012) The anaphase-promoting complex or cyclosome supports cell survival in response to endoplasmic reticulum stress. PLoS One 7(4):e35520, doi:PONE-D-11-23151 [pii] 10.1371/journal.pone.0035520

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Reimann JD, Freed E, Hsu JY, Kramer ER, Peters JM, Jackson PK (2001) Emi1 is a mitotic regulator that interacts with Cdc20 and inhibits the anaphase promoting complex. Cell 105(5):645–655

    Article  CAS  PubMed  Google Scholar 

  28. Wang W, Kirschner MW (2013) Emi1 preferentially inhibits ubiquitin chain elongation by the anaphase-promoting complex. Nat Cell Biol 15(7):797–806. doi:10.1038/ncb2755

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Pfleger CM, Kirschner MW (2000) The KEN box: an APC recognition signal distinct from the D box targeted by Cdh1. Genes Dev 14(6):655–665

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Pfleger CM, Lee E, Kirschner MW (2001) Substrate recognition by the Cdc20 and Cdh1 components of the anaphase-promoting complex. Genes Dev 15(18):2396–2407. doi:10.1101/gad.918201

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Zeng X, Sigoillot F, Gaur S, Choi S, Pfaff KL, Oh DC, Hathaway N, Dimova N, Cuny GD, King RW (2010) Pharmacologic inhibition of the anaphase-promoting complex induces a spindle checkpoint-dependent mitotic arrest in the absence of spindle damage. Cancer Cell 18(4):382–395. doi:10.1016/j.ccr.2010.08.010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Clark E, Spector DH (2015) Studies on the contribution of human cytomegalovirus UL21a and UL97 to viral growth and inactivation of the anaphase promoting complex/cyclosome (APC/C) E3 ubiquitin ligase reveal a unique cellular mechanism for down-modulation of the APC/C subunits APC1, APC4, and APC5. J Virol. doi:10.1128/JVI.00403-15

    Google Scholar 

  33. Zeng X, King RW (2012) An APC/C inhibitor stabilizes cyclin B1 by prematurely terminating ubiquitination. Nat Chem Biol 8(4):383–392. doi:10.1038/nchembio.801

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Sackton KL, Dimova N, Zeng X, Tian W, Zhang M, Sackton TB, Meaders J, Pfaff KL, Sigoillot F, Yu H, Luo X, King RW (2014) Synergistic blockade of mitotic exit by two chemical inhibitors of the APC/C. Nature 514(7524):646–649. doi:10.1038/nature13660

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

Research performed at the laboratory of Pathophysiological Cell Signaling is funded by the following bodies: FWO (G0C7514N grant), BELSPO (IAP-VII/07 program), VUB Research Council (new PI grant), and Innoviris (Brains Back to Brussels program to GJG). CC thanks financial support from the European Union’s Seventh Framework Program (FP7) 2007–2013 under grant agreement no. 264257. The authors would also like to acknowledge networking support by the Proteostasis COST Action (BM1307).

Author information

Author notes

    Authors and Affiliations

    1. Department of Structural Biology, Vlaams Instituut voor Biotechnologie (VIB), Vrije Universiteit Brussel (VUB), Pleinlaan 2, Brussels, 1050, Belgium

      Cesyen Cedeño

    2. Laboratory of Pathophysiological Cell Signaling (PACS), Department of Biology, Faculty of Sciences and Bioengineering Sciences, Vrije Universiteit Brussel (VUB), Pleinlaan 2, Brussels, 1050, Belgium

      Esther La Monaca, Mara Esposito & Gustavo J. Gutierrez

    Authors
    1. Cesyen Cedeño
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Esther La Monaca
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Mara Esposito
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. Gustavo J. Gutierrez
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding author

    Correspondence to Gustavo J. Gutierrez .

    Editor information

    Editors and Affiliations

    1. Computational and Experimental Biology Group Department of Health Promotion and Chronic Diseases, National Health Institute Dr. Ricardo Jorge, INSA, I.P., Lisboa, Portugal

      Rune Matthiesen

    Rights and permissions

    Reprints and permissions

    Copyright information

    © 2016 Springer Science+Business Media New York

    About this protocol

    Cite this protocol

    Cedeño, C., La Monaca, E., Esposito, M., Gutierrez, G.J. (2016). Detection and Analysis of Cell Cycle-Associated APC/C-Mediated Cellular Ubiquitylation In Vitro and In Vivo. In: Matthiesen, R. (eds) Proteostasis. Methods in Molecular Biology, vol 1449. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3756-1_15

    Download citation

    • DOI: https://doi.org/10.1007/978-1-4939-3756-1_15

    • Published:

    • Publisher Name: Humana Press, New York, NY

    • Print ISBN: 978-1-4939-3754-7

    • Online ISBN: 978-1-4939-3756-1

    • eBook Packages: Springer Protocols

    Publish with us

    Policies and ethics

    Search

    Navigation