Advertisement

NF-kappa B pp 339-354 | Cite as

Generation of a Proteolytic Signal: E3/E2-Mediated Polyubiquitination of IκBα

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1280)

Abstract

A key regulatory node in NF-κB signaling is the removal of the IκBα inhibitor, whose levels are tightly controlled by the ubiquitin–proteasome system. In response to signal activation and transmission, ubiquitin E1, E2, and E3 enzymes are employed to generate a lysine 48-linked ubiquitin chain that triggers degradation of IκBα by the proteasome. In this chapter we describe an in vitro biochemical approach to reconstitute the ubiquitination system. To do so, we detail methods for the preparation of the relevant enzymes and substrate, as well as for the execution of the reaction with high efficiency. This sensitive and highly reproducible readout can be applied to the study of proteins, small molecules, and other factors that modulate IκBα ubiquitination, thereby producing outcomes that impact NF-κB signaling to advance the course of improving human health.

Key words

NF-κB IκBα ubiquitin E3 ubiquitin ligase SCF Nedd8 Cdc34 UbcH5c Polyubiquitination 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgements

We thank J. Nayak and other members of the Pan lab for their assistance with protocols and careful reading of the method. We are grateful to J. Hurwitz and I. Tappin for assistance with baculovirus preparation. R.A.C. was supported by NIH fellowship 1F30DK095572-01. Z.-Q.P. is the receipt of the 2013 Jiangsu special medical expert award. This work was supported by Public Health Service grants GM61051 and CA095634 to Z.-Q. P.

References

  1. 1.
    Skaug B, Jiang X, Chen ZJ (2009) The role of ubiquitin in NF-kappaB regulatory pathways. Annu Rev Biochem 78:769–796CrossRefPubMedGoogle Scholar
  2. 2.
    Chen ZJ, Parent L, Maniatis T (1996) Site-specific phosphorylation of IkappaBalpha by a novel ubiquitination-dependent protein kinase activity. Cell 84:853–862CrossRefPubMedGoogle Scholar
  3. 3.
    Scherer DC, Brockman JA, Chen Z et al (1995) Signal-induced degradation of I kappa B alpha requires site-specific ubiquitination. Proc Natl Acad Sci U S A 92:11259–11263CrossRefPubMedCentralPubMedGoogle Scholar
  4. 4.
    Chen Z, Hagler J, Palombella VJ et al (1995) Signal-induced site-specific phosphorylation targets I kappa B alpha to the ubiquitin-proteasome pathway. Genes Dev 9:1586–1597CrossRefPubMedGoogle Scholar
  5. 5.
    Tan P, Fuchs SY, Chen A et al (1999) Recruitment of a ROC1-CUL1 ubiquitin ligase by Skp1 and HOS to catalyze the ubiquitination of I kappa B alpha. Mol Cell 3:527–533CrossRefPubMedGoogle Scholar
  6. 6.
    Yamoah K, Oashi T, Sarikas A et al (2008) Autoinhibitory regulation of SCF-mediated ubiquitination by human cullin 1’s C-terminal tail. Proc Natl Acad Sci U S A 105:12230–12235CrossRefPubMedCentralPubMedGoogle Scholar
  7. 7.
    Duda DM, Borg LA, Scott DC et al (2008) Structural insights into NEDD8 activation of cullin-RING ligases: conformational control of conjugation. Cell 134:995–1006CrossRefPubMedCentralPubMedGoogle Scholar
  8. 8.
    Wu K, Kovacev J, Pan Z-Q (2010) Priming and extending: a UbcH5/Cdc34 E2 handoff mechanism for polyubiquitination on a SCF substrate. Mol Cell 37:784–796CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Iwai K (2012) Diverse ubiquitin signaling in NF-κB activation. Trends Cell Biol 22:355–364CrossRefPubMedGoogle Scholar
  10. 10.
    Xu S, Patel P, Abbasian M et al (2005) In vitro SCFβ-Trcp1-mediated IκBα ubiquitination assay for high-throughput screen. Methods Enzymol 399:729–740CrossRefPubMedGoogle Scholar
  11. 11.
    Soucy TA, Smith PG, Milhollen MA et al (2009) An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature 458:732–736CrossRefPubMedGoogle Scholar
  12. 12.
    Ceccarelli DF, Tang X, Pelletier B et al (2011) An allosteric inhibitor of the human Cdc34 ubiquitin-conjugating enzyme. Cell 145:1075–1087CrossRefPubMedGoogle Scholar
  13. 13.
    Shen M, Schmitt S, Buac D et al (2013) Targeting the ubiquitin-proteasome system for cancer therapy. Expert Opin Ther Targets 17(9):1091–1108CrossRefPubMedCentralPubMedGoogle Scholar
  14. 14.
    Gazdoiu S, Yamoah K, Wu K et al (2007) Human Cdc34 employs distinct sites to coordinate attachment of ubiquitin to a substrate and assembly of polyubiquitin chains. Mol Cell Biol 27:7041–7052CrossRefPubMedCentralPubMedGoogle Scholar
  15. 15.
    Ohta T, Michel JJ, Schottelius AJ et al (1999) ROC1, a homolog of APC11, represents a family of cullin partners with an associated ubiquitin ligase activity. Mol Cell 3:535–541CrossRefPubMedGoogle Scholar
  16. 16.
    Zheng N, Schulman B, Song L et al (2002) Structure of the Cul 1–Rbx 1–Skp 1–F boxSkp 2 SCF ubiquitin ligase complex. Nature 416:703–709CrossRefPubMedGoogle Scholar
  17. 17.
    Li T, Pavletich NP, Schulman BA et al (2005) High-level expression and purification of recombinant SCF ubiquitin ligases. Methods Enzymol 398:125–142CrossRefPubMedGoogle Scholar
  18. 18.
    Mercurio F (1997) IKK-1 and IKK-2: cytokine-activated IB kinases essential for NF-B activation. Science (New York, NY) 278:860–866CrossRefGoogle Scholar
  19. 19.
    Delhase M, Hayakawa M, Chen Y et al (1999) Positive and negative regulation of IkappaB kinase activity through IKKbeta subunit phosphorylation. Science (New York, NY) 284:309–313CrossRefGoogle Scholar
  20. 20.
    Kovacev J, Wu K, Spratt DE et al (2014) A snapshot at ubiquitin chain elongation: lysine 48-tetra-ubiquitin slows down ubiquitination. J Biol Chem 289(10):7068–7081CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Oncological SciencesThe Icahn School of Medicine at Mount SinaiNew YorkUSA

Personalised recommendations

Cite protocol

Buy options