Elucidating dynamic protein-protein interactions and ubiquitination in NF-κB signaling pathways.
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.
Department of Microbiology and Immunology, Sylvester Comprehensive Cancer Center, Miller School of Medicine, The University of Miami, Miami, FL, 33136, USA, nshembade@med.miami.edu.
Noula
N
Shembade
Edward W
EW
Harhaj
D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D004268:DNA-Binding Proteins; D004789:Enzyme Activation; D005347:Fibroblasts / Q000378:metabolism; D051550:I-kappa B Kinase / Q000378:metabolism; D047468:Immunoprecipitation / Q000379:methods; D016393:Lymphoma, B-Cell / Q000378:metabolism; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D015398:Signal Transduction; D048029:TNF Receptor-Associated Factor 6 / Q000378:metabolism; D054875:Ubiquitination
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D016328:NF-kappa B; C404508:RNF11 protein, human; D048029:TNF Receptor-Associated Factor 6; D051550:I-kappa B Kinase