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NF-kappa B pp 283–295Cite as

Elucidating Dynamic Protein–Protein Interactions and Ubiquitination in NF-κB Signaling Pathways

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

Abstract

The Nuclear factor-kappaB (NF-κB) family of transcription factors plays critical roles in inflammatory responses and host defense; however, uncontrolled NF-κB activation can be deleterious by promoting autoimmune diseases and cancers. Lysine K63 (K63)-linked polyubiquitination has emerged as an important regulatory mechanism in NF-κB signaling by regulating dynamic protein–protein interactions that trigger NF-κB signaling. RIP1 and TRAF6 serve as key substrates of K63-linked polyubiquitin chains in tumor necrosis factor receptor (TNFR) and interleukin-1 receptor (IL-1R) pathways respectively as a mechanism to recruit TAK1 and IKK kinases by associated ubiquitin-binding adaptor molecules. Activation of IKKβ by TAK1 induces IκBα phosphorylation, degradation, and downstream NF-κB activation. The ubiquitin-editing enzyme A20 maintains transient NF-κB activation by opposing the K63-linked polyubiquitination of RIP1 and TRAF6. A20 inducibly interacts with the adaptor molecule TAX1BP1 and the E3 ligases Itch and RNF11 to form an A20 ubiquitin-editing enzyme complex. Notably, loss-of-function somatic mutations or polymorphisms in human A20 are associated with B-cell lymphomas or a variety of autoimmune diseases as a result of dysregulated NF-κB activation. In this chapter, we summarize the protocols routinely used in our laboratories to examine ubiquitination and NF-κB signaling.

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Acknowledgements

This work was supported by National Institutes of Health grants (RO1GM083143 and RO1CA135362 to E.W.H.) and a Stanley J. Glaser Research Award and American Cancer Society Institutional Grant award to N.S. We thank Drs. Alfonso Lavorgna and Soratree Charoenthongtrakul for critical reading of the manuscript.

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Authors and Affiliations

  1. Department of Microbiology and Immunology, Sylvester Comprehensive Cancer Center, Miller School of Medicine, The University of Miami, Miami, FL, 33136, USA

    Noula Shembade

  2. Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, 1650 Orleans Street, Baltimore, MD, 21287, USA

    Edward W. Harhaj

Authors
  1. Noula Shembade
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  2. Edward W. Harhaj
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Correspondence to Noula Shembade or Edward W. Harhaj .

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Editors and Affiliations

  1. Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania, USA

    Michael J. May

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Shembade, N., Harhaj, E.W. (2015). Elucidating Dynamic Protein–Protein Interactions and Ubiquitination in NF-κB Signaling Pathways. In: May, M. (eds) NF-kappa B. Methods in Molecular Biology, vol 1280. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2422-6_16

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  • DOI: https://doi.org/10.1007/978-1-4939-2422-6_16

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2421-9

  • Online ISBN: 978-1-4939-2422-6

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