NEMO-binding Domains of Both IKKα and IKKβ Regulate IκB Kinase Complex Assembly and Classical NF-κB Activation

Proinflammatory NF-kappa B activation requires the I kappa B (inhibitor of NF-kappa B) kinase (IKK) complex that contains two catalytic subunits named IKK alpha and IKK beta and a regulatory subunit named NF-kappa B essential modulator (NEMO). NEMO and IKK beta are essential for tumor necrosis factor (TNF)-induced NF-kappa B activation, and we recently demonstrated that NEMO and IKK alpha are sufficient for interleukin (IL)-1-induced signaling. IKK alpha and IKK beta both contain a functional NEMO-binding domain (NBD); however, the role of NEMO association with each kinase in NF-kappa B signaling and IKK complex formation remains unclear. To address this question, we stably reconstituted IKK alpha(-/-) and IKK beta(-/-) murine embryonic fibroblasts (MEFs) with wild-type (WT) or NBD-deficient (Delta NBD) versions of IKK alpha and IKK beta, respectively. TNF-induced classical NF-kappa B activation in IKK beta(-/-) MEFs was rescued by IKK beta(WT) but not IKK beta(Delta NBD), whereas neither IKK beta(WT) nor IKK beta(Delta NBD) affected IL-1-induced NF-kappa B signaling. As previously described, classical NF-kappa B transcriptional activity was absent in IKK alpha(-/-) cells. Reconstitution with either IKK alpha(WT) or IKK alpha(Delta NBD) rescued both IL-1 and TNF-induced transcription, demonstrating that NEMO association is not required for IKK alpha-dependent regulation of NF-kappa B-dependent transcription. Stably expressed IKK alpha(WT) or IKK beta(WT) associated with endogenous IKKs and NEMO in IKK alpha(-/-) or IKK beta(-/-) MEFs, respectively, resulting in formation of the heterotrimeric IKK alpha-IKK beta-NEMO complex. In contrast, although the IKK alpha(Delta NBD) and IKK beta(Delta NBD) mutants associated with endogenous IKKs containing an NBD, these dimeric endogenous IKK-IKK Delta NBD complexes did not associate with NEMO. These findings therefore demonstrate that formation of the heterotrimeric IKK alpha-IKK beta-NEMO holocomplex absolutely requires two intact NEMO-binding domains.