Functional Isoforms of IκB Kinase α (IKKα) Lacking Leucine Zipper and Helix-Loop-Helix Domains Reveal that IKKα and IKKβ Have Different Activation Requirements

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Abstract
The activity of the NF-κB family of transcription factors is regulated principally by phosphorylation and subsequent degradation of their inhibitory IκB subunits. Site-specific serine phosphorylation of IκBs by two IκB kinases (IKKα [also known as CHUK] and IKKβ) targets them for proteolysis. IKKα and -β have a unique structure, with an amino-terminal serine-threonine kinase catalytic domain and carboxy-proximal helix-loop-helix (HLH) and leucine zipper-like (LZip) amphipathic α-helical domains. Here, we describe the properties of two novel cellular isoforms of IKKα: IKKα-ΔH and IKKα-ΔLH. IKKα-ΔH and IKKα-ΔLH are differentially spliced isoforms of the IKKα mRNA lacking its HLH domain and both its LZip and HLH domains, respectively. IKKα is the major RNA species in most murine cells and tissues, except for activated T lymphocytes and the brain, where the alternatively spliced isoforms predominate. Remarkably, IKKα-ΔH and IKKα-ΔLH, like IKKα, respond to tumor necrosis factor alpha stimulation to potentiate NF-κB activation in HEK293 cells. A mutant, catalytically inactive form of IKKα blocked IKKα-, IKKα-ΔH-, and IKKα-ΔLH-mediated NF-κB activation. Akin to IKKα, its carboxy-terminally truncated isoforms associated with the upstream activator NIK (NF-κB-inducing kinase). In contrast to IKKα, IKKα-ΔLH failed to associate with either itself, IKKα, IKKβ, or NEMO-IKKγ-IKKAP1, while IKKα-ΔH complexed with IKKβ and IKKα but not with NEMO. Interestingly, each IKKα isoform rescued HEK293 cells from the inhibitory effects of a dominant-negative NEMO mutant, while IKKα could not. IKKα-ΔCm, a recombinant mutant of IKKα structurally akin to IKKα-ΔLH, was equally functional in these assays, but in sharp contrast, IKKβ-ΔCm, a structurally analogous mutant of IKKβ, was inactive. Our results demonstrate that the functional roles of seemingly analogous domains in IKKα and IKKβ need not be equivalent and can also exhibit different contextual dependencies. The existence of cytokine-inducible IKKα-ΔH and IKKα-ΔLH isoforms illustrates potential modes of NF-κB activation, which are not subject to the same in vivo regulatory constraints as either IKKα or IKKβ.