I kappa B kinase (IKK)-associated protein 1, a common component of the heterogeneous IKK complex

Activation of the transcription factor NF-kappa B is controlled by the sequ ential phosphorylation, ubiquitination, and degradation of its inhibitory s ubunit, I kappa B. We recently purified a large multiprotein complex, the I kappa B kinase (IKK) signalsome, which contains two regulated I kappa B ki nases, IKK1 and IKK2, that can each phosphorylate I kappa B alpha and I kap pa B beta. The IKK signalsome contains several additional proteins presumab ly required for the regulation of the NT kappa B signal transduction cascad e in vivo. In this report, we demonstrate reconstitution of I kappa B kinas e activity in vitro by using purified recombinant IKK1 and IKK2. Recombinan t IKK1 or IKK2 forms homo- or heterodimers, suggesting the possibility that similar IKK complexes exist in vivo. Indeed, in HeLa cells we identified t wo distinct IKK complexes, one containing IKK1-IKK2 heterodimers and the ot her containing IKK2 homodimers, which display differing levels of activatio n following tumor necrosis factor alpha stimulation. To better elucidate th e nature of the IKK signalsome, we set out to identify IKK-associated prote ins. To this end, we purified and cloned a novel component common to both c omplexes, named IKK-associated protein 1 (IKKAP1). In vitro, IKKAP1 associa ted specifically with IKK2 but not IKK1. Functional analyses revealed that binding to IKK2 requires sequences contained within the N-terminal domain o f IKKAP1. Mutant versions of IKKAP1, which either lack the N-terminal IKK2- binding domain or contain only the IKK2-binding domain, disrupt the NF-kapp a B signal transduction pathway, IKKAP1 therefore appears to mediate an ess ential step of the NF-kappa B signal transduction cascade. Heterogeneity of IKK complexes in vivo may provide a mechanism for differential regulation of NF-kappa B activation.