MEKK1 activates both IκB kinase α and IκB kinase β

A critical step in the signal-induced activation of the transcription factor NF-KB is the site-specific phosphorylation of its inhibitor, I kappa B, that targets the latter for degradation by the ubiquitin-proteasome pathway. We have previously shown that mitogen-activated protein kinase/ERK kinase kinase 1 (MEKK1) can induce both this site specific phosphorylation of I kappa B alpha at Ser-32 and Ser-36 in vivo and the activity of a high molecular weight I kappa B kinase complex in vitro. Subsequently, others have identified two proteins, I kappa B kinase alpha (IKK-alpha) and I kappa B kinase beta (IKK-beta), that are present in a tumor necrosis factor alpha-inducible, high molecular weight I kappa B kinase complex. These kinases are believed to directly phosphorylate I kappa B based on the examination of the kinase activities of IKK immunoprecipitates, but more rigorous proof of this has yet to be demonstrated. We show herein that recombinant IKK-alpha and IKK-beta can, in fact, directly phosphorylate I kappa B alpha at Ser-32 and Ser-36, as well as homologous residues in I kappa B beta in vitro, and thus are bona fide I kappa B kinases. We also show that MEKK1 can induce the activation of both IKK-alpha and IKK-beta in vivo. Finally, we show that IKK-alpha is present in the MEKK1-inducible, high molecular weight I kappa B kinase complex and treatment of this complex with MEKK1 induces phosphorylation of IKK-alpha in vitro. We conclude that IKK-alpha and IKK-beta can mediate the NF-KB-inducing activity of MEKK1.