Generation of a proteolytic signal: E3/E2-mediated polyubiquitination of IκBα.
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.
Department of Oncological Sciences, The Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY, 10029-6574, USA.
Robert A
RA
Chong
Kenneth
K
Wu
Jordan
J
Kovacev
Zhen-Qiang
ZQ
Pan
D002460:Cell Line; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D000072000:NF-KappaB Inhibitor alpha; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D044843:SKP Cullin F-Box Protein Ligases / Q000378:metabolism; D015398:Signal Transduction; D025801:Ubiquitin / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D054875:Ubiquitination
D020672:I-kappa B Proteins; C000606204:NFKBIA protein, human; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D000072000:NF-KappaB Inhibitor alpha; D044763:Ubiquitin-Conjugating Enzymes; D044843:SKP Cullin F-Box Protein Ligases