NEMO-binding domains of both IKKα and IKKβ regulate IκB kinase complex assembly and classical NF-κB activation

Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania 19104, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 09/2009; 284(40):27596-608. DOI: 10.1074/jbc.M109.047563
Source: PubMed


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

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    • "NEMO is approximately 50 kDa in size, containing two coiled-coil domains along with LZ and zinc finger motifs. Whereas IKKα and IKKβ are responsible for the catalytic activity of the complex, NEMO does not perform any enzymatic functions, but serves as a regulatory hub [29]. While the three individual subunits together form a complex of 210 kDa, the purified IKK complex is estimated to be 700–900 kDa in size. "
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