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