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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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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