Functional isoforms of I kappa B kinase alpha (IKK alpha) lacking leucine zipper and helix-loop-helix domains reveal that IKK alpha and IKK beta havedifferent activation requirements

The activity of the NF-kappa B family of transcription factors is regulated principally by phosphorylation and subsequent degradation of their inhibit ory I kappa B subunits. Site-specific serine phosphorylation of I kappa Bs by two I kappa B kinases (IKK alpha [also known as CHUK] and IKK beta) targ ets them for proteolysis. IKK alpha and -beta have a unique structure, with an amino-terminal serine-threonine kinase catalytic domain and carboxy-pro ximal helix-loop-helix (HLH) and leucine zipper-like (LZip) amphipathic alp ha-helical domains. Here, we describe the properties of two novel cellular isoforms of IKK alpha: IKK alpha-Delta H and IKK alpha-Delta LH. IKK alpha- Delta H and IKK alpha-Delta LH are differentially spliced isoforms of the I KK alpha mRNA lacking its HLH domain and both its LZip and HLH domains, res pectively. IKK alpha is the major RNA species in most murine cells and tiss ues, except for activated T lymphocytes and the brain, where the alternativ ely spliced isoforms predominate. Remarkably, IKK alpha-Delta H and IKK alp ha-Delta LH, like IKK alpha, respond to tumor necrosis factor alpha stimula tion to potentiate NF-kappa B activation in HEK293 cells. A mutant, catalyt ically inactive form of IKK alpha blocked IKK alpha-, IKK alpha-Delta H-, a nd IKK alpha-Delta LH-mediated NF-kappa B activation. Akin to IKK alpha, it s carboxy-terminally truncated isoforms associated with the upstream activa tor NIK (NF-kappa B-inducing kinase). In contrast to IKK alpha, IKK alpha-D elta LH failed to associate with either itself, IKK alpha, IKK beta, or NEM O-IKK gamma-IKKAP1, while IKK alpha-Delta H complexed with IKK beta and IKK alpha but not with NEMO. Interestingly, each IKK alpha isoform rescued HEK 293 cells from the inhibitory effects of a dominant-negative NEMO mutant, w hile IKK alpha could not. IKK alpha-Delta Cm, a recombinant mutant of IKK a lpha structurally akin to IKK alpha-Delta LH, was equally functional in the se assays, but in sharp contrast, IKK beta-Delta Cm, a structurally analogo us mutant of IKK beta, was inactive. Our results demonstrate that the funct ional roles of seemingly analogous domains in IKK alpha and IKK beta need n ot be equivalent and can also exhibit different contextual dependencies. Th e existence of cytokine-inducible IKK alpha-Delta H and IKK alpha-Delta LH isoforms illustrates potential modes of NF-kappa B activation, which are no t subject to the same in vivo regulatory constraints as either TKK alpha or IKK beta.