Suppression of NF-κB signaling by KEAP1 regulation of IKKβ activity through autophagic degradation and inhibition of phosphorylation

IκB kinase β (IKKβ) plays a crucial role in biological processes, including immune response, stress response, and tumor development by mediating the activation of various signaling molecules such as NF-κB. Extensive studies on the mechanisms underlying IKK activation have led to the identification of new activators and have facilitated an understanding of the cellular responses related to NF-κB and other target molecules. However, the molecular processes that modulate IKK activity are still unknown. In this study, we show that KEAP1 is a new IKK binding partner, which is responsible for the down-regulation of TNFα-stimulated NF-κB activation. The E(T/S)GE motif, which is found only in the IKKβ subunit of the IKK complex, is essential for interaction with the C-terminal Kelch domain of KEAP1. Reduction of KEAP1 expression by small interfering RNA enhanced NF-κB activity, and up-regulated the expression of NF-κB target genes. Ectopic expression of KEAP1 decreased the expression of IKKβ, which was restored by an autophagy inhibitor. IKK phosphorylation stimulated by TNFα was blocked by KEAP1. Our data demonstrate that KEAP1 is involved in the negative regulation of NF-κB signaling through the inhibition of IKKβ phosphorylation and the mediation of autophagy-dependent IKKβ degradation.     

The azatryptophan-based fluorescent platform for in vitro rapid screening of inhibitors disrupting IKKβ-NEMO interaction

The nuclear factor-κB (NF-κB) plays an important role in inflammatory and immune responses. Aberrant NF-κB signaling is implicated in multiple disorders, including cancer. Targeting the regulatory scaffold subunit IκB kinase γ (IKKγ/NEMO) as therapeutic interventions could be promising due to its specific involvement in canonical NF-κB activation without interfering with non-canonical signaling. In this study, the use of unnatural amino acid substituted IKKβ with unique photophysical activity to sense water environment changes upon interaction with NEMO provides a powerful in vitro screening platform that would greatly facilitate the identification of compounds having the potential to disrupt IKKβ-NEMO interaction, and thus specifically modulate the canonical NF-κB pathway. We then utilized a competitive binding platform to screen the binding ability of a number of potential molecules being synthesized. Our results suggest that a lead compound (−)-PDC-099 is a potent agent with ascertained potency to disrupt IKKβ-NEMO complex for modulating NF-κB canonical pathway.     

Disruption of IkappaB kinase (IKK)-mediated RelA serine 536 phosphorylation sensitizes human multiple myeloma cells to histone deacetylase (HDAC) inhibitors

Post-translational modifications of RelA play an important role in regulation of NF-κB activation. We previously demonstrated that in malignant hematopoietic cells, histone deacetylase inhibitors (HDACIs) induced RelA hyperacetylation and NF-κB activation, attenuating lethality. We now present evidence that IκB kinase (IKK) β-mediated RelA Ser-536 phosphorylation plays a significant functional role in promoting RelA acetylation, inducing NF-κB activation, and limiting HDACI lethality in human multiple myeloma (MM) cells. Immunoblot profiling revealed that although basal RelA phosphorylation varied in MM cells, Ser-536 phosphorylation correlated with IKK activity. Exposure to the pan-HDACIs vorinostat or LBH-589 induced phosphorylation of IKKα/β (Ser-180/Ser-181) and RelA (Ser-536) in MM cells, including cells expressing an IκBα "super-repressor," accompanied by increased RelA nuclear translocation, acetylation, DNA binding, and transactivation activity. These events were substantially blocked by either pan-IKK or IKKβ-selective inhibitors, resulting in marked apoptosis. Consistent with these events, inhibitory peptides targeting either the NF-κB essential modulator (NEMO) binding domain for IKK complex formation or RelA phosphorylation sites also significantly increased HDACI lethality. Moreover, IKKβ knockdown by shRNA prevented Ser-536 phosphorylation and significantly enhanced HDACI susceptibility. Finally, introduction of a nonphosphorylatable RelA mutant S536A, which failed to undergo acetylation in response to HDACIs, impaired NF-κB activation and increased cell death. These findings indicate that HDACIs induce Ser-536 phosphorylation of the NF-κB subunit RelA through an IKKβ-dependent mechanism, an action that is functionally involved in activation of the cytoprotective NF-κB signaling cascade primarily through facilitation of RelA acetylation rather than nuclear translocation.     

Enterovirus 71 2C protein inhibits TNF-α-mediated activation of NF-κB by suppressing IκB kinase β phosphorylation

Enterovirus 71 (EV71), a single, positive-stranded RNA virus, has been regarded as the most important neurotropic enterovirus after the eradication of the poliovirus. EV71 infection can cause hand, foot, and mouth disease or herpangina. Cytokine storm with elevated levels of proinflammatory and inflammatory cytokines, including TNF-α, has been proposed to explain the pathogenesis of EV71-induced disease. TNF-α-mediated NF-κB signaling pathway plays a key role in inflammatory response. We hypothesized that EV71 might also moderate host inflammation by interfering with this pathway. In this study, we tested this hypothesis and identified EV71 2C protein as an antagonist of TNF-α-mediated activation of NF-κB signaling pathway. Expression of 2C protein significantly reduced TNF-α-mediated NF-κB activation in 293T cells as measured by gene reporter and gel mobility shift assays. Furthermore, overexpression of TNFR-associated factor 2-, MEK kinase 1-, IκB kinase (IKK)α-, or IKKβ-induced NF-κB activation, but not constitutively active mutant of IKKβ (IKKβ SS/EE)-induced NF-κB activation, was inhibited by 2C protein. These data together suggested that the activation of IKKβ is most likely targeted by 2C; this notion was further strengthened by immunoblot detection of IKKβ phosphorylation and IκBα phosphorylation and degradation. Coimmunoprecipitation and colocalization of 2C and IKKβ expressed in mammalian cells provided compelling evidence that 2C interacts with IKKβ. Collectively, our data indicate that EV71 2C protein inhibits IKKβ activation and thus blocks NF-κB activation.     

Classical NF-kappaB activation negatively regulates noncanonical NF-kappaB-dependent CXCL12 expression

Ligation of the lymphotoxin-β receptor (LTβR) by LIGHT (lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpes virus entry mediator on T cells (TNFSF14)) activates the noncanonical (NC) NF-κB (nuclear factor-κB) pathway and up-regulates CXCL12 gene expression by human umbilical vein endothelial cells (HUVEC). In contrast, TNF only activates classical NF-κB signaling and does not up-regulate CXCL12. To determine whether cross-talk between the classical and NC pathways affects CXCL12 expression, we investigated the effects of TNF on LIGHT signaling in HUVEC. We show here that TNF inhibits both basal and LIGHT-induced CXCL12 expression. Negative regulation by TNF requires the classical NF-κB pathway as inhibition of basal and induced CXCL12 was reversed in HUVEC-expressing dominant negative IκB (inhibitor of NF-κB) kinase (IKK)β (IKKβ(K44M)). TNF did not inhibit the NC NF-κB pathway activation as LIGHT-induced p100 processing to p52 was intact; however, TNF either alone or together with LIGHT up-regulated p100 and RelB expression and induced the nuclear localization of p100-RelB complexes. Enhanced p100 and RelB expression was inhibited by IKKβ(K44M), which led us to question whether the IκB function of elevated p100 mediates the inhibition of CXCL12 expression by TNF. We retrovirally transduced HUVEC to express p100 at a level similar to that up-regulated by TNF; however, basal and LIGHT-induced CXCL12 expression was normal in the transduced cells. In contrast, ectopic RelB expression recapitulated the effects of TNF on NC signaling and inhibited basal and LIGHT-induced CXCL12 expression by HUVEC. Our findings therefore demonstrate that TNF-induced classical NF-κB signaling up-regulates RelB expression that inhibits both basal and NC NF-κB-dependent CXCL12 expression.     

Vaccinia virus protein C6 is a virulence factor that binds TBK-1 adaptor proteins and inhibits activation of IRF3 and IRF7

Recognition of viruses by pattern recognition receptors (PRRs) causes interferon-β (IFN-β) induction, a key event in the anti-viral innate immune response, and also a target of viral immune evasion. Here the vaccinia virus (VACV) protein C6 is identified as an inhibitor of PRR-induced IFN-β expression by a functional screen of select VACV open reading frames expressed individually in mammalian cells. C6 is a member of a family of Bcl-2-like poxvirus proteins, many of which have been shown to inhibit innate immune signalling pathways. PRRs activate both NF-κB and IFN regulatory factors (IRFs) to activate the IFN-β promoter induction. Data presented here show that C6 inhibits IRF3 activation and translocation into the nucleus, but does not inhibit NF-κB activation. C6 inhibits IRF3 and IRF7 activation downstream of the kinases TANK binding kinase 1 (TBK1) and IκB kinase-ε (IKKε), which phosphorylate and activate these IRFs. However, C6 does not inhibit TBK1- and IKKε-independent IRF7 activation or the induction of promoters by constitutively active forms of IRF3 or IRF7, indicating that C6 acts at the level of the TBK1/IKKε complex. Consistent with this notion, C6 immunoprecipitated with the TBK1 complex scaffold proteins TANK, SINTBAD and NAP1. C6 is expressed early during infection and is present in both nucleus and cytoplasm. Mutant viruses in which the C6L gene is deleted, or mutated so that the C6 protein is not expressed, replicated normally in cell culture but were attenuated in two in vivo models of infection compared to wild type and revertant controls. Thus C6 contributes to VACV virulence and might do so via the inhibition of PRR-induced activation of IRF3 and IRF7.     

Differential usage of NF-κB activating signals by IL-1β and TNF-α in pancreatic beta cells

The cytokines interleukin (IL)-1β and tumor necrosis factor (TNF)-α induce β-cell death in type 1 diabetes via NF-κB activation. IL-1β induces a more marked NF-κB activation than TNF-α, with higher expression of genes involved in β-cell dysfunction and death. We show here a differential usage of the IKK complex by IL-1β and TNF-α in β-cells. While TNF-α uses IKK complexes containing both IKKα and IKKβ, IL-1β induces complexes with IKKα only; this effect is achieved by induction of IKKβ degradation via the proteasome. Both IKKγ and activation of the TRAF6-TAK1-JNK pathway are involved in IL-1β-induced IKKβ degradation.     

Beyond NF-κB activation: nuclear functions of IκB kinase α

IκB kinase (IKK) complex, the master kinase for NF-κB activation, contains two kinase subunits, IKKα and IKKβ. In addition to mediating NF-κB signaling by phosphorylating IκB proteins during inflammatory and immune responses, the activation of the IKK complex also responds to various stimuli to regulate diverse functions independently of NF-κB. Although these two kinases share structural and biochemical similarities, different sub-cellular localization and phosphorylation targets between IKKα and IKKβ account for their distinct physiological and pathological roles. While IKKβ is predominantly cytoplasmic, IKKα has been found to shuttle between the cytoplasm and the nucleus. The nuclear-specific roles of IKKα have brought increasing complexity to its biological function. This review highlights major advances in the studies of the nuclear functions of IKKα and the mechanisms of IKKα nuclear translocation. Understanding the nuclear activity is essential for targeting IKKα for therapeutics.