IκB kinase α phosphorylation of TRAF4 downregulates innate immune signaling

Despite their homology, IκB kinase α (IKKα) and IKKβ have divergent roles in NF-κB signaling. IKKβ strongly activates NF-κB while IKKα can downregulate NF-κB under certain circumstances. Given this, identifying independent substrates for these kinases could help delineate their divergent roles. Peptide substrate array technology followed by bioinformatic screening identified TRAF4 as a substrate for IKKα. Like IKKα, TRAF4 is atypical within its family because it is the only TRAF family member to negatively regulate innate immune signaling. IKKα's phosphorylation of serine-426 on TRAF4 was required for this negative regulation. Binding to the Crohn's disease susceptibility protein, NOD2, is required for TRAF4 phosphorylation and subsequent inhibition of NOD2 signaling. Structurally, serine-426 resides within an exaggerated β-bulge in TRAF4 that is not present in the other TRAF proteins, and phosphorylation of this site provides a structural basis for the atypical function of TRAF4 and its atypical role in NOD2 signaling.     

Selective degradation of the IκB kinase （IKK） by autophagy

Proteasome-mediated degradation and autophagy are the two major pathways mediating the turnover of cellular proteins. The proteasomal pathway is known to be a highly specific and regulated process mediating the degradation of short-lived proteins such as many important factors involved in cellular signaling. In contrast, it is generally thought that autophagy is rather nonselective as it is responsible for the bulk degradation of long-lived proteins and organelles. Challenging this general view, in this issue of Cell Research, Qing et al. report that selective degradation of the IκB kinase （IKK） triggered by the loss of Hsp90 function is mediated by autophagy [1].     

Pharmacophore modeling and hybrid virtual screening for the discovery of novel IκB kinase 2 (IKK2) inhibitors

IKK2 (IκB kinase 2) inhibitors have been identified as potential drug candidates in the treatment of various immune/inflammatory disorders as well as cancer. So far more than one hundred small molecule inhibitors against IKK2 have been reported publicly. In this investigation, pharmacophore modeling was carried out to clarify the essential structure-activity relationship for the known IKK2 inhibitors. One of the established pharmacophore hypotheses, namely Hypo8, which has the best prediction ability to an external test data set, was suggested as a template for virtual screening. Evaluation of the performances of Hypo8 and a hybrid method (Hypo81docking) in virtual screening indicated that the use of the hybrid virtual screening considerably increased the hit rate and enrichment factor. The hybrid method was therefore adopted for screening several commercially available chemical databases, including Specs, NCI, Maybridge and Chinese Nature Product Database (CNPD), for novel potent IKK2 inhibitors. The hit compounds were subsequently subjected to filtering by Lipinski's rule of five. Finally some of the final hit compounds were selected and suggested for further experimental investigations.     

IκB kinase β (IKKβ) does not mediate feedback inhibition of the insulin signalling cascade

In the present study, we have examined whether IKKβ [IκB (inhibitor of nuclear factor κB) kinase β] plays a role in feedback inhibition of the insulin signalling cascade. Insulin induces the phosphorylation of IKKβ, in vitro and in vivo, and this effect is dependent on intact signalling via PI3K (phosphoinositide 3-kinase), but not PKB (protein kinase B). To test the hypothesis that insulin activates IKKβ as a means of negative feedback, we employed a variety of experimental approaches. First, pharmacological inhibition of IKKβ via BMS-345541 did not potentiate insulin-induced IRS1 (insulin receptor substrate 1) tyrosine phosphorylation, PKB phosphorylation or 2-deoxyglucose uptake in differentiated 3T3-L1 adipocytes. BMS-345541 did not prevent insulin-induced IRS1 serine phosphorylation on known IKKβ target sites. Secondly, adenovirus-mediated overexpression of wild-type IKKβ in differentiated 3T3-L1 adipocytes did not suppress insulin-stimulated 2-deoxyglucose uptake, IRS1 tyrosine phosphorylation, IRS1 association with the p85 regulatory subunit of PI3K or PKB phosphorylation. Thirdly, insulin signalling was not potentiated in mouse embryonic fibroblasts lacking IKKβ. Finally, insulin treatment of 3T3-L1 adipocytes did not promote the recruitment of IKKβ to IRS1, supporting our findings that IKKβ, although activated by insulin, does not promote direct serine phosphorylation of IRS1 and does not contribute to the feedback inhibition of the insulin signalling cascade.     

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.     

IκB激酶(IKK)结构与功能研究进展

真核细胞核转录因子Rel/NF·κB家族广泛存在于从昆虫至人类细胞中,参与细胞的分化、发育、凋亡、黏附及炎症反应。通常NF·κB与其抑制蛋白IκB结合以无活性的方式滞留于胞浆,当多种外部信号作用于细胞时,活化的NF·κB进入细胞核内发挥其功能。目前,对NF·κB激活的信号传导机制已基本明确,IκB激酶(IκB Kinase,IKK)在其中起重要作用,本文综述了近年来对IKK结构、功能及其调控的最新进展。     

Hsp90 inhibition results in autophagy-mediated proteasome-independent degradation of IκB kinase （IKK）

Autophagic and proteasomal proteolysis are two major pathways for degradation of cellular constituents. Current models suggest that autophagy is responsible for the nonselective bulk degradation of long-lived proteins and organelles while the proteasome specifically degrades short-lived proteins including misfolded proteins caused by the absence of Hsp90 function. Here, we show that the IκB kinase （IKK）, an essential activator of NF-κB, is selectively degraded by autophagy when Hsp90 is inhibited by geldanamycin （GA）, a specific Hsp90 inhibitor showing highly effective anti-tumor activity. We find that in this case inactivation of ubiquitination or proteasome fails to block IKK degradation. However, inhibition of autophagy by an autophagy inhibitor or knockout of Atg5, a key component of the autophagy pathway, significantly rescues IKK from GA-induced degradation. These findings provide the first evidence that an Hsp90 client may be degraded by a mechanism different from the proteasome pathway and establish a molecular link among Hsp90, NF-κB and autophagy     

Regulation of IκB kinase by GβL through recruitment of the protein phosphatases

G protein β-like (GβL) is a member of WD repeat-containing family which are involved in various intracellular signaling events. In our previous report, we demonstrated that GβL regulates TNFα-stimulated NF-κB signaling by interacting with and inhibiting phosphorylation of IκB kinase. However, GβL itself does not seem to regulate IKK directly, because it contains no functional domains except WD domains. Here, using immunoprecipitation and proteomic analyses, we identified protein phosphatase 4 as a new binding partner of GβL. We also found that GβL interacts with PP2A and PP6, other members of the same phosphatase family. By interacting with protein phosphatases, which do not directly bind to IKKβ, GβL mediates the association of phosphatases with IKKβ. Overexpression of protein phosphatases inhibited TNFκ-induced activation of NF-κB signaling, which is an effect similar to that of GβL overexpression. Down-regulation of GβL by small interfering RNA diminished the inhibitory effect of phosphatases, resulting in restoration of NF-κB signaling. Thus, we propose that GβL functions as a negative regulator of NF-κB signaling by recruiting protein phosphatases to the IKK complex.     

Diacylglycerol kinase α enhances protein kinase Cζ-dependent phosphorylation at Ser311 of p65/RelA subunit of nuclear factor-κB

We recently reported that diacylglycerol kinase (DGK) α enhanced tumor necrosis factor-α (TNF-α)-induced activation of nuclear factor-κB (NF-κB). However, the signaling pathway between DGKα and NF-κB remains unclear. Here, we found that small interfering RNA-mediated knockdown of DGKα strongly attenuated protein kinase C (PKC) ζ-dependent phosphorylation of a large subunit of NF-κB, p65/RelA, at Ser311 but not PKCζ-independent phosphorylation at Ser468 or Ser536. Moreover, knockdown and overexpression of PKCζ suppressed and synergistically enhanced DGKα-mediated NF-κB activation, respectively. These results strongly suggest that DGKα positively regulates TNF-α-dependent NF-κB activation via the PKCζ-mediated Ser311 phosphorylation of p65/RelA.     

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.     

Long-term Incubation with Proteasome Inhibitors (PIs) Induces IκBα Degradation via the Lysosomal Pathway in an IκB Kinase (IKK)-dependent and IKK-independent Manner

Proteasome inhibitors (PIs) have been reported to induce apoptosis in many types of tumor. Their apoptotic activities have been suggested to be associated with the up-regulation of molecules implicated in pro-apoptotic cascades such as p53, p21^Waf1, and p27^Kip1. Moreover, the blocking of NF-κB nuclear translocation via the stabilization of IκB is an important mechanism of PI-induced apoptosis. However, we found that long-term incubation with PIs (PS-341 or MG132) increased NF-κB-regulated gene expression such as COX-2, cIAP2, XIAP, and IL-8 in a dose- and time-dependent manner, which was mediated by phosphorylation of IκBα and its subsequent degradation via the alternative route, lysosome. Overexpression of the IκBα superrepressor (IκBα-SR) blocked PI-induced NF-κB activation. Treatment with lysosomal inhibitors (ammonium chloride or chloroquine) or inhibitors of cathepsins (Z-FF-FMK or Z-FA-FMK) or knock-down of LC3B expression by siRNAs suppressed PI-induced IκBα degradation. Furthermore, we found that both IKK-dependent and IKK-independent pathways were required for PI-induced IκBα degradation. Pretreatment with IKKβ specific inhibitor, SC-514, partially suppressed IκBα degradation and IL-8 production by PIs. Blockade of IKK activity using insolubilization by heat shock (HS) and knock-down by siRNAs for IKKβ only delayed IκBα degradation up to 8 h after treatment with PIs. In addition, PIs induced Akt-dependent inactivation of GSK-3β. Inactive GSK-3β accelerated PI-induced IκBα degradation. Overexpression of active GSK-3β (S9A) or knock-down of GSK-3β delayed PI-induced IκBα degradation. Collectively, our data demonstrate that long-term incubation with PIs activates NF-κB, which is mediated by IκBα degradation via the lysosome in an IKK-dependent and IKK-independent manner.     

Phosphoinositide 3-kinase activity leads to silica-induced NF-κB activation through interacting with tyrosine-phosphorylated IκB-α and contributing to tyrosine phosphorylation of p65 NF-κB

The role of the subunits of phosphoinositide (PI) 3-kinase in NF-B activation in silica-stimulated RAW 264.7 cells was investigated. Results indicate that PI3-kinase activity was increased in response to silica. The p85 subunit of PI3-kinase interacted with tyrosine-phosphorylated IB- in silica-stimulated cells. PI3-kinase specific inhibitors, such as wortmannin and LY294003, substantially blocked both silica-induced PI3-kinase and NF-B activation. The inhibition of NF-B activation by PI3-kinase inhibitors was also observed in pervanadate-stimulated but not in LPS-stimulated cells. Furthermore, tyrosine phosphorylation of NF-B p65 was enhanced in cells stimulated with silica, pervanadate or LPS, and wortmannin substantially inhibited the phosphorylation event induced by the first two stimulants but not LPS. Antioxidants, such as superoxide dismutase (SOD), N-acetylcysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), blocked silica-induced PI3-kinase activation, suggesting that reactive oxygen species may be important regulatory molecules in NF-B activation by mediating PI3-kinase activation. Our data suggest that p85 and p110 subunits of PI3-kinase play a role in NF-B activation through interaction with tyrosine-phosphorylated IB- and contributing to tyrosine phosphorylation of p65 NF-B.     

Suppression of NF-κB and NF-κB-Regulated Gene Expression by Apigenin through IκBα and IKK Pathway in TRAMP Mice

Aberrant Nuclear Factor-κappaB (NF-κB) activation due to rapid IκBα turnover and high basal IκBα kinase (IKK) activity has been frequently observed in prostate cancer. Apigenin, a naturally occurring plant flavone, exhibits anti-proliferative, anti-inflammatory and anti-carcinogenic activities by inhibiting NF-κB pathway, through a mechanism not fully understood. We found that apigenin feeding in microgram doses (bioavailable in humans) inhibited prostate tumorigenesis in TRAMP mice by interfering with NF-κB signaling. Apigenin feeding to TRAMP mice (20 and 50 μg/mouse/day, 6 days/week for 20 weeks) exhibited significant decrease in tumor volumes of the prostate and completely abolished metastasis, which correlated with inhibition of NF-κB activation and binding to the DNA. Apigenin intake blocked phosphorylation and degradation of IκBα by inhibiting IKK activation, which in turn led to suppression of NF-κB activation. The expression of NF-κB-regulated gene products involved in proliferation (cyclin D1, and COX-2), anti-apoptosis (Bcl-2 and Bcl-xL), and angiogenesis (vascular endothelial growth factor) were also downregulated after apigenin feeding. These events correlated with the induction of apoptosis in tumor cells, as evident by increased cleaved caspase-3 labeling index in the dorsolateral prostate. Our results provide convincing evidence that apigenin inhibits IKK activation and restores the expression of IκBα, preventing it’s phosphorylation in a fashion similar to that elicited by IKK and proteasomal inhibitors through suppression of NF-κB signaling pathway.     

Interaction of β2-glycoprotein I with lipopolysaccharide leads to Toll-like receptor 4 (TLR4)-dependent activation of macrophages

β(2)-Glycoprotein I (β(2)GPI) is an abundant plasma protein that binds to the surface of cells and particles expressing negatively charged lipids, but its physiological role remains unknown. Antibodies to β(2)GPI are found in patients with anti-phospholipid syndrome, a systemic autoimmune disease associated with vascular thrombosis and pregnancy morbidity. Although it has been suggested that anti-β(2)GPI antibodies activate endothelial cells and monocytes by signaling through TLR4, it is unclear how anti-β(2)GPI antibodies and/or β(2)GPI interact with TLR4. A number of mammalian proteins (termed "endogenous Toll-like receptor (TLR) ligands") have been reported to bind to TLR4, but, in most cases, subsequent studies have shown that LPS interaction with these proteins is responsible for TLR activation. We hypothesized that, like other endogenous TLR ligands, β(2)GPI interacts specifically with LPS and that this interaction is responsible for apparent TLR4 activation by β(2)GPI. Here, we show that both LPS and TLR4 are required for β(2)GPI to bind to and activate macrophages. Untreated β(2)GPI stimulated TNF-α production in TLR4-sufficient (but not TLR4-deficient) macrophages. In contrast, neither polymyxin B-treated nor delipidated β(2)GPI stimulated TNF-α production. Furthermore, β(2)GPI bound to LPS in a specific and dose-dependent manner. Finally, untreated β(2)GPI bound to the surface of TLR4-sufficient (but not TLR4-deficient) macrophages. Polymyxin B treatment of β(2)GPI abolished macrophage binding. Our findings suggest a potential new biological activity for β(2)GPI as a protein that interacts specifically with LPS and point to the need to evaluate newly discovered endogenous TLR ligands for potential interactions with LPS.     

Irradiation leads to sensitization of hepatocytes to TNF-α-mediated apoptosis by upregulation of IκB expression

This study aimed to reveal the pathophysiological signalling responsible for radiation-induced sensitization of hepatocytes to TNF-α-mediated apoptosis. IκB was upregulated in irradiated hepatocytes. Administration of IκB antisense oligonucleotides prior to irradiation inhibited occurrence of apoptosis after TNF-α administration. Caspases-8, -9 and -3 activities were increased in irradiated hepatocytes and downregulation of apoptosis by IκB antisense oligonucleotides was mediated by suppression of caspases-9 and -3 activation but not of caspase-8 activation, suggesting that radiation-induced sensitization of hepatocytes to TNF-α-mediated apoptosis additionally requires changes upstream of caspase-8 activation. Herein, upregulation of FLIP may play a crucial role. Cleavage of bid, upregulation of bax, downregulation of bcl-2 and release of cytochrome c after TNF-α-administration depend on radiation-induced upregulation of IκB, thus demonstrating an apoptosis permitting effect of IκB. H. Gürleyen and H. Christiansen contributed equally to this work.