The ubiquitous transcription factor NF-kappaB is tightly regulated by its inhibitor IkappaB, which sequesters NF-kappaB in the cytoplasm of quiescent cells. Upon stimulation of cells with NF-kappaB agonists, IkappaB is rapidly phosphorylated by a large protein kinase complex, and subsequently degraded by the ubiquitin-proteasome pathway. The degradation of IkappaB liberates NF-kappaB which translocates into the nucleus to turn on numerous target genes, many of which are involved in immune and inflammatory responses, viral pathogenesis, and apoptosis. Despite the critical importance of the ubiquitin-mediated degradation of IkappaB in the NF-kappaB signaling pathway, little is known about the mechanisms and the ubiquitination enzymes involved. We have established an in vitro system that faithfully recapitulates the specificity and inducibility of IkappaB ubiquitination in vivo. In this application, we propose to utilize the in vitro system to identify the enzymes that catalyze the signal-induced ubiquitination of IkappaB, and to study the mechanisms involved. Specifically, we plan to 1) identify and characterize the ubiquitin-IkappaB ligase (E3-IkappaB); 2) identify and characterize the IkappaB ubiquitin-conjugating enzyme (E2-IkappaB); 3) study the mechanisms and specificity of IkappaB ubiquitination using a reconstituted system. The proposed study should lead to the discovery of an ubiquitination machinery that governs NF-kappaB signaling, and provide important insights into the fundamental question of how specificity of selective protein ubiquitination is achieved.
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