Abstract
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.
Highlights
NF-B2 describes a family of transcription factors that regulate the inducible expression of many genes essential for innate and adaptive immunity, inflammation, and cell survival
NF-B essential modulator (NEMO) and IKK are essential for tumor necrosis factor (TNF)-induced NF-B activation, and we recently demonstrated that NEMO and IKK␣ are sufficient for interleukin (IL)-1-induced signaling
The IKK NEMO-binding domain (NBD) Is Required for TNF- but Not IL-1-induced NF-B Activation—To determine the effects of selectively disrupting the IKK-NEMO interaction on classical NF-B signaling and IKK complex formation, we generated retroviral constructs encoding both WT IKK and a truncation mutant lacking the NBD (IKK⌬NBD) (Fig. 1A)
Summary
Reagents and Cell Culture—Recombinant human IL-1␣ was obtained from Peprotech (Rocky Hill, NJ). Pellets were resuspended and swollen for 30 min on ice in 100 l of Buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 2 mM NaF, 2 mM -glycerol phosphate, and complete mini protease inhibitors), incubated a further 5 min on ice in 0.1% Nonidet P-40, and vortexed and centrifuged (3800 ϫ g) for 1 min. After 30 min, a final volume 0.1% Nonidet P-40 was added to the swollen pellet, and cells were lysed with 15 strikes of a 2-ml Dounce homogenizer on ice. one-fourth volume of 5ϫ gel filtration buffer (100 mM Tris, pH 7.5, 50% glycerol, 2.5 mM EDTA, 750 mM NaCl) was added to the lysate before being centrifuged for 10 min at 425 ϫ g. Fractions containing the IKK complex were immunoprecipitated using either anti-NEMO or anti-IKK␣, and the resulting precipitates were immunoblotted as described above
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