NEMO-binding domains of both IKKalpha and IKKbeta regulate IkappaB kinase complex assembly and classical NF-kappaB activation.
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.
- SourceAvailable from: Benjamin R Tenoever[Show abstract] [Hide abstract]
ABSTRACT: The ability of a cell to combat an intracellular pathogen requires a mechanism to recognize the threat and elicit a transcriptional response against it. In the context of virus infection, the cell must take measures to inhibit viral replication, meanwhile, convey warning signals to neighboring cells of the imminent threat. This immune response is predominantly mediated by the production of cytokines, notably, interferon beta (IFNβ). IFNβ signaling results in the transcriptional induction of over one hundred antiviral gene products whose timely expression renders infected cells more capable of inhibiting virus replication, while providing the uninfected cells with the reinforcements to generate a less permissive cellular environment. Induction of IFNβ and many aspects of the antiviral response pivot on the function of the IKK and IKK-related kinases. Despite sharing high levels of homology and some degree of functional redundancy, the classic IKK kinases: IKKα and IKKβ, and the IKK-related kinases: TBK1 and IKKɛ, perform distinct roles in regulating the host antiviral defense. These kinases serve as molecular operators in their cooperative ability to integrate incoming cellular cues and act on a range of essential antiviral transcription factors to reshape the cellular transcriptome during infection.Viruses 01/2010; 2(1):55-72. DOI:10.3390/v2010055 · 3.28 Impact Factor
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ABSTRACT: The classical nuclear factor kappaB (NF-kappaB) signaling pathway is under the control of the IkappaB kinase (IKK) complex, which consists of IKK-1, IKK-2, and NF-kappaB essential modulator (NEMO). This complex is responsible for the regulation of cell proliferation, survival, and differentiation. Dysregulation of this pathway is associated with several human diseases, and as such, its inhibition offers an exciting opportunity for therapeutic intervention. NEMO binding domain (NBD) peptides inhibit the binding of recombinant NEMO to IKK-2 in vitro. However, direct evidence of disruption of this binding by NBD peptides in biological systems has not been provided. Using a cell system, we expanded on previous observations to show that NBD peptides inhibit inflammation-induced but not basal cytokine production. We report that these peptides cause the release of IKK-2 from an IKK complex and disrupt NEMO-IKK-2 interactions in cells. We demonstrate that by interfering with NEMO-IKK-2 interactions, NBD peptides inhibit IKK-2 phosphorylation, without affecting signaling intermediates upstream of the IKK complex of the NF-kappaB pathway. Furthermore, in a cell-free system of IKK complex activation by TRAF6 (TNF receptor-associated factor 6), we show that these peptides inhibit the ability of this complex to phosphorylate downstream substrates, such as p65 and inhibitor of kappaB alpha (IkappaB alpha). Thus, consistent with the notion that NEMO regulates IKK-2 catalytic activity by serving as a scaffold, appropriately positioning IKK-2 for activation by upstream kinase(s), our findings provide novel insights into the molecular mechanisms by which NBD peptides exert their anti-inflammatory effects in cells.Journal of Biological Chemistry 02/2010; 285(18):13498-506. DOI:10.1074/jbc.M109.099895 · 4.60 Impact Factor
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ABSTRACT: The signaling and adaptor protein Homer3 plays a role in controlling immune homeostasis and self-reactivity. Homer3 is recruited to the immune synapse (IS) following TCR ligation, although the mechanisms regulating this subcellular localization are unknown. We show that Homer3 specifically associates with a novel ubiquitin-like domain in the IkappaB kinase (IKK) beta subunit of the IKK complex. Homer3 associates with IKKbeta in T cells and colocalizes with the IKK complex at the IS. However, Homer3 is not required for IKK activation, as NF-kappaB signaling is intact in Homer3-deficient T cells. Instead, the IKK complex recruits Homer3 to the IS following TCR engagement, and we present evidence that this association regulates actin dynamics in T cells. These findings identify a novel interaction between two major signaling proteins and reveal an unexpected NF-kappaB-independent function for the IKK complex in regulating the subcellular localization of Homer3.The Journal of Immunology 09/2010; 185(5):2665-9. DOI:10.4049/jimmunol.0903488 · 5.36 Impact Factor