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