The study identifies a large, multisubunit kinase (molecular mass ~700 kDa) that phosphorylates IκBα at serine residues 32 and 36, which are required for its subsequent ubiquitination and degradation. This kinase activity is dependent on the ubiquitin-activating enzyme (E1), a specific ubiquitin carrier protein (E2) of the Ubc4/Ubc5 family, and ubiquitin (Ub). The kinase is activated by a prior ubiquitination event, suggesting that ubiquitination serves a novel regulatory function. The identification of this kinase and its requirement for ubiquitination provide new targets for inhibiting aberrant NF-κB functions, which are implicated in various pathological conditions.The study identifies a large, multisubunit kinase (molecular mass ~700 kDa) that phosphorylates IκBα at serine residues 32 and 36, which are required for its subsequent ubiquitination and degradation. This kinase activity is dependent on the ubiquitin-activating enzyme (E1), a specific ubiquitin carrier protein (E2) of the Ubc4/Ubc5 family, and ubiquitin (Ub). The kinase is activated by a prior ubiquitination event, suggesting that ubiquitination serves a novel regulatory function. The identification of this kinase and its requirement for ubiquitination provide new targets for inhibiting aberrant NF-κB functions, which are implicated in various pathological conditions.