Site-specific phosphorylation of IκBα by a novel ubiquitination- dependent protein kinase activity

Signal-induced activation of the transcription factor NF-κB requires specific phosphorylation of the inhibitor IκBα and its subsequent proteolytic degradation. Phosphorylation of serine residues 32 and 36 targets IκBα to the ubiquitin (Ub)-proteasome pathway. Here we report the identification of a large, multisubunit kinase (molecular mass ~700 kDa) that phosphorylates IκBα at S32 and S36. Remarkably, the activity of this kinase requires the Ub-activating enzyme (E1), a specific Ub carrier protein (E2) of the Ubc4/Ubc5 family, and Ub. We also show that a ubiquitination event in the kinase complex is a prerequisite for specific phosphorylation of IκBα. Thus, ubiquitination serves a novel regulatory function that does not involve proteolysis.