Site-specific phosphorylation of I kappa B alpha by a novel ubiquitination-dependent protein kinase activity

Signal-induced activation of the transcription factor NF-kappa B requires specific phosphorylation of the inhibitor I kappa B alpha and its subsequent proteolytic degradation. Phosphorylation of serine residues 32 and 36 targets I kappa B alpha to the ubiquitin (Ub)-proteasome pathway. Here we report the identification of a large, multisubunit kinase (molecular mass similar to 700 kDa) that phosphorylates I kappa B alpha at S32 and S36. Remarkably, the activity of this kinase requires the Uh-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 I kappa B alpha. Thus, ubiquitination serves a novel regulatory function that does not involve proteolysis.