Activation of the heterodimeric IκB kinase α (IKKα)-IKKβ complex is directional: IKKα regulates IKKβ under both basal and stimulated conditions

Signal-induced nuclear expression of the eukaryotic NF-κB transcription factor involves the stimulatory action of select mitogen-activated protein kinase kinase kinases on the IκB kinases (IKKα and IKKβ) which reside in a macromolecular signaling complex termed the signalsome. While genetic studies indicate that IKKβ is the principal kinase involved in proinflammatory cytokine-induced IκB phosphorylation, the function of the equivalently expressed IKKα is less clear. Here we demonstrate that assembly of IKKα with IKKβ in the heterodimeric signalsome serves two important functions: (i) in unstimulated cells, IKKα inhibits the constitutive IκB kinase activity of IKKβ; (ii) in activated cells, IKKα kinase activity is required for the induction of IKKβ. The introduction of kinase-inactive IKKξ, activation loop mutants of IKKα, or IKKα antisense RNA into 293 or HeLa cells blocks NIK (NF-κB-inducing kinase)-induced phosphorylation of the IKKβ activation loop occurring in functional signalsomes. In contrast, catalytically inactive mutants of IKKβ do not block NIK-mediated phosphorylation of IKKα in these macromolecular signaling complexes. This requirement for kinase-proficient IKKξ to activate IKKβ in heterodimeric IKK signalsomes is also observed with other NF-κB inducers, including tumor necrosis factor alpha, human T-cell leukemia virus type 1 Tax, Cot, and MEKK1. Conversely, the θ isoform of protein kinase C, which also induces NF- κB/Rel, directly targets IKKβ for phosphorylation and activation, possibly acting through homodimeric IKKβ complexes. Together, our findings indicate that activation of the heterodimeric IKK complex by a variety of different inducers proceeds in a directional manner and is dependent on the kinase activity of IKKα to activate IKKβ.