A novel ubiquitination-dependent protein kinase activity was identified that specifically phosphorylates IκBα at serine residues 32 and 36. This kinase requires the ubiquitin (Ub)-activating enzyme (E1), a specific Ub carrier protein (E2) of the Ubc4/Ubc5 family, and Ub for its activity. The ubiquitination of IκBα is a prerequisite for its phosphorylation, and this process is essential for the degradation of IκBα and the subsequent activation of NF-κB. The kinase activity is activated by a prior ubiquitination event, indicating that ubiquitination serves a novel regulatory function that does not involve proteolysis. The study shows that the ubiquitination of IκBα is regulated by signal-induced phosphorylation at S32 and S36, and that the phosphorylation of IκBα is dependent on the presence of Ubc4/Ubc5, Ub, and E1. The results demonstrate that the ubiquitination of IκBα is a critical step in the activation of NF-κB and that the kinase activity is regulated by a complex involving Ubc4/Ubc5, Ub, and E1. The study also shows that the phosphorylation of IκBα is dependent on the presence of Ubc4/Ubc5, Ub, and E1, and that the ubiquitination of IκBα is required for the activation of the kinase. The findings suggest that the ubiquitination of IκBα is a key regulatory step in the NF-κB signaling pathway.A novel ubiquitination-dependent protein kinase activity was identified that specifically phosphorylates IκBα at serine residues 32 and 36. This kinase requires the ubiquitin (Ub)-activating enzyme (E1), a specific Ub carrier protein (E2) of the Ubc4/Ubc5 family, and Ub for its activity. The ubiquitination of IκBα is a prerequisite for its phosphorylation, and this process is essential for the degradation of IκBα and the subsequent activation of NF-κB. The kinase activity is activated by a prior ubiquitination event, indicating that ubiquitination serves a novel regulatory function that does not involve proteolysis. The study shows that the ubiquitination of IκBα is regulated by signal-induced phosphorylation at S32 and S36, and that the phosphorylation of IκBα is dependent on the presence of Ubc4/Ubc5, Ub, and E1. The results demonstrate that the ubiquitination of IκBα is a critical step in the activation of NF-κB and that the kinase activity is regulated by a complex involving Ubc4/Ubc5, Ub, and E1. The study also shows that the phosphorylation of IκBα is dependent on the presence of Ubc4/Ubc5, Ub, and E1, and that the ubiquitination of IκBα is required for the activation of the kinase. The findings suggest that the ubiquitination of IκBα is a key regulatory step in the NF-κB signaling pathway.