IKK Epsilon Research Articles

Microtubule disruption upon CNS damage triggers mitotic entry via TNF signaling activation.

Repair after traumatic injury often starts with mitotic activation around the lesion edges. Early midline cells in the Drosophila embryonic CNS can enter into division following the traumatic disruption of microtubules. We demonstrate that microtubule disruption activates non-canonical TNF signaling by phosphorylation of TGF-β activated kinase 1 (Tak1) and its target IkappaB kinase (Ik2), culminating in Dorsal/NfkappaB nuclear translocation and Jra/Jun expression. Tak1 and Ik2 are necessary for the damaged-induced divisions. Microtubule disruption caused by Tau accumulation is also reported in Alzheimer's disease (AD). Human Tau expression in Drosophila midline cells is sufficient to induce Tak1 phosphorylation, Dorsal and Jra/Jun expression, and entry into mitosis. Interestingly, activation of Tak1 and Tank binding kinase 1 (Tbk1), the human Ik2 ortholog, and NfkappaB upregulation are observed in AD brains.

IKK Epsilon Deficiency Attenuates Angiotensin II-Induced Abdominal Aortic Aneurysm Formation in Mice by Inhibiting Inflammation, Oxidative Stress, and Apoptosis.

Abdominal aortic aneurysm (AAA) is a vascular disorder that is considered a chronic inflammatory disease. However, the precise molecular mechanisms involved in AAA have not been fully elucidated. Recently, significant progress has been made in understanding the function and mechanism of action of inhibitor of kappa B kinase epsilon (IKKε) in inflammatory and metabolic diseases. The angiotensin II- (Ang II-) induced or pharmacological inhibitors were established to test the effects of IKKε on AAA in vivo. After mice were continuously stimulated with Ang II for 28 days, morphologically, we found that knockout of IKKε reduced AAA formation and drastically reduced maximal diameter and severity. We also observed a decrease in elastin degradation and medial destruction, which were independent of systolic blood pressure or plasma cholesterol concentrations. Western blot analyses and immunohistochemical staining were carried out to measure IKKε expression in AAA tissues and cell lines. AAA phenotype of mice was measured by ultrasound and biochemical indexes. In zymography, immunohistology staining, immunofluorescence staining, and reactive oxygen species (ROS) analysis, TUNEL assay was used to examine the effects of IKKε on AAA progression in AAA mice. IKKε deficiency significantly inhibited inflammatory macrophage infiltration, matrix metalloproteinase (MMP) activity, ROS production, and vascular smooth muscle cell (VSMC) apoptosis. We used primary mouse aortic VSMC isolated from apolipoprotein E (Apoe) −/− and Apoe−/−IKKε−/− mice. Mechanistically, IKKε deficiency blunted the activation of the ERK1/2 pathway. The IKKε inhibitor, amlexanox, has the same impact in AAA. Our results demonstrate a critical role of IKKε in AAA formation induced by Ang II in Apoe−/− mice. Targeting IKKε may constitute a novel therapeutic strategy to prevent AAA progression.

Molecular immune mechanisms of HPV-infected HaCaT cells in vitro based on toll-like receptors signaling pathway.

ObjectiveTo explore the molecular immune mechanism of HPV‐infected HaCaT cells in vitro based on TLRs signaling pathway by analyzing the effects of interfering TLRs on inflammatory and immune factors in the signaling pathway.MethodsFCM was used to analyze the proportion of Th1, Th2, Th17, and Treg cells in blood samples. HPV‐infected HaCaT cells were divided into five groups: A, B, C, D, and E. Group A added TLR3 antagonist, group B added TLR9 antagonist, group C added equivalent saline, group D added IRF3 agonist, and group E added IRF3 inhibitor. Immunohistochemistry was used to analyze the expression of TLR3 and TLR9 in HaCaT cell model; ELISA was used to analyze the expression of inflammatory factors IL‐2, TNF‐a, and IFN‐beta; WB was used to analyze the expression of TRAF3, IKK epsilon, and TBK1; RT‐PCR was used to analyze the expression of IRF3 and IRF7 in each cell model.ResultsThe proportion of blood immune cells in patients with HPV infection was Th1, Th17, Th2, and Treg, with statistical significance (P < .05); the expression of TLR3 and TLR9 in HPV‐infected cells was higher than that in negative control group, with statistical significance (P < .05); TLR3 was higher than TLR9, with no significant difference (P > .05); the expression of IL‐2, TNF‐alpha, IFN‐beta in each group, TLR3, and TLR9 was higher than that in negative control group (P < .05). The expression of TRAF3, IKK epsilon, and TBK1 in the control group was higher than that in the TLR3 and TLR9 inhibitor groups, and the expression of IRF3 and IRF7 in the TLR9 inhibitor group was higher than that in the TLR3 inhibitor group (P < .05); the expression of IRF3 and IRF7 in the TLR3i and TLR9i inhibitor groups was lower than that in the TLR3 inhibitor group (P < .05). Compared with the control group, IRF3a group was higher than the control group, IRF3i group was lower than the control group, the difference was statistically significant (P < .05).ConclusionTLR3 and TLR9, the key factors of TLRs, are highly expressed in HaCaT cells infected with HPV. Through TLRs‐IKK‐e‐IRFs‐IFN signaling pathway, they can induce high expression of inflammatory factors, IKK‐e, IRFs, and IFN, and improve immunity.

Zika Virus Non-Structural Protein NS5 Inhibits the RIG-I Pathway and Interferon Lambda 1 Promoter Activation by Targeting IKK Epsilon.

The Zika virus (ZIKV) is a member of the Flaviviridae family and an important human pathogen. Most pathogenic viruses encode proteins that interfere with the activation of host innate immune responses. Like other flaviviruses, ZIKV interferes with the expression of interferon (IFN) genes and inhibits IFN-induced antiviral responses. ZIKV infects through epithelial barriers where IFN-λ1 is an important antiviral molecule. In this study, we analyzed the effects of ZIKV proteins on the activation of IFN-λ1 promoter. All ZIKV proteins were cloned and transiently expressed. ZIKV NS5, but no other ZIKV protein, was able to interfere with the RIG-I signaling pathway. This inhibition took place upstream of interferon regulatory factor 3 (IRF3) resulting in reduced phosphorylation of IRF3 and reduced activation of IFN-λ1 promoter. Furthermore, we showed that ZIKV NS5 interacts with the protein kinase IKKε, which is likely critical to the observed inhibition of phosphorylation of IRF3.

Air Pollution Exposure during Pregnancy and Lactation Induces Obesity and Glucose Intolerance in the Offspring

Air pollution has been a very significant environmental cause of diseases globally. Fine particulate matter (PM2.5) is one of the most toxic substances in air pollution and is associated with type 2 diabetes mellitus risk. However, there is a gap in knowledge regarding the effects of air pollution exposure on insulin resistance and obesity development. We aimed to investigate whether 3 months of PM2.5 exposure induces obesity, hypothalamic leptin resistance and inflammation in adult mice. Also, we aimed to determine whether PM2.5 exposure during pregnancy and lactation changes food intake (FI), energy expenditure, body composition and weight, glucose, and insulin metabolism in the offspring. Three months of PM2.5 exposure decreased O2 consumption measured by CLAMS, and increased body weight and fat mass in adult mice without FI changes. These mice presented hypothalamic leptin resistance with decreased STAT3 phosphorylation and enhanced IKK epsilon (IKKe) expression measured by immunofluorescence and Real-Time PCR. Knocking down IKKe, partially reversed this phenotype. PM2.5 exposure during pregnancy and lactation increased body weight and fat mass of male offspring, observed by a MicroPet CT analysis, accompanied by decreased FI and increased respiratory exchange ratio. Male offspring presented higher serum insulin levels and glucose intolerance measured by GTT despite normal insulin tolerance measured by ITT. Female offspring had no phenotype alterations, except by the light reduced food intake. In conclusion, PM2.5 exposure during adulthood induced obesity, partially due to a marked reduction in energy expenditure caused by hypothalamic leptin resistance. Elevated inflammation may explain that with a significant role of IKKe in the hypothalamus. Air pollution exposure during pregnancy and lactation had a remarkable effect inducing obesity in the male offspring due to an energy unbalance, which may lead to increased insulin levels and glucose intolerance. Disclosure O.P. Zord o: None. L.W. Claro: None. P. Saldiva: None. J. Donato: None. M. Veras: None. P.O. Prada: None.

Parameters that define Suppressor of IKK epsilon (SIKE): cytoskeletal protein interactions revealed through determination of binding affinities and SIKE's dimer interface

Upon pathogen challenge multiple receptors both inside and on the surface of the cell, recognize pathogen associated molecular patterns (PAMPs) and initiate the production of proinflammatory, antiviral, and apoptotic responses. Pathways converge at key hubs that serve to amplify and regulate the signals, and are often responsible for determining the downstream response. TANK Binding Kinase 1 (TBK1) serves as a catalytic hub in the antiviral TLR3 mediated innate immune pathway. Suppressor of IKK epsilon (SIKE) is a recently identified high affinity alternative substrate of TBK1. It was initially found to inhibit TBK1 activation of type 1 interferon production. Upon subsequent study, it was found that SIKE was phosphorylated at six serine residues by TBK1. This phosphorylation of SIKE corresponds to the activation of the antiviral response, and releases SIKE from the SIKE:TBK1 interaction. The primary function of SIKE remains unknown. Examination of SIKE's interaction network has established direct interactions with cytoskeletal proteins including tubulin and α‐actinin. Migration assays have shown that chronic myelogenous leukemia (CML) cells in which SIKE has been knocked out migrate at a slower rate. Together, these studies suggest that SIKE plays a role in cytoskeletal rearrangements associated with innate immune responses such as migration and phagocytosis. The goal of this study is to define the interaction surface of SIKE as well as the binding affinities associated with these interactions. A quartz crystal microbalance with dissipation (QCM‐D) assay was developed to obtain binding affinities for the SIKE:cytoskeletal protein complexes. A gold sensor was utilized and functionalized with protein G and α‐His antibody to which 6xHis‐SIKE was immobilized. Increasing concentrations of binding partner were flowed over the chip to develop a binding curve. Prior to examining SIKE:cytoskeletal protein interactions, SIKE's oligomeric state was defined by crosslinking studies. Crosslinking with bis(sulfosuccinimidyl)suberate (BS3) shows that SIKE is a dimer. Chemical crosslinking followed by tandem mass spectrometry was employed to determine the dimeric interface of the SIKE dimer. BS3 was used in an excess of 20 and 100X. Data were analyzed with Mascot using xComb strategy. Residues 13, 66, 68, 110, and 169 were found to be accessible to BS3 modification, while in the dimer only residues 13, 110, 119, and 120 were found to be accessible. Residues 110 and 119 are found in multiple crosslinked peptides in both dimer and monomer samples. Residues 178 and 195 were found in crosslinked peptides unique to the dimer. These data have been applied to SIKE dimer models, generated by computational docking experiments to select for models consistent with the crosslinking restraints. These data define the dimer interface of SIKE as well as the surfaces available for SIKE cytoskeletal protein interactions. Together, these studies begin to reveal parameters that determine SIKE's interactions and the protein surfaces available to mediate these interactions.Support or Funding InformationStudies were funded by USD SURE, Beckman Foundation, NIH‐NIAID R21.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Suppressor of IKK epsilon (SIKE) acts as a bridge between innate immune signalling pathway and cytoskeletal rearrangements

Innate immunity coordinates the first line of defense against invading pathogens. This involves the recruitment and subsequent migration of nearby immune cells to the site of infection, allowing the pathogen to be consumed via phagocytosis to sequester the infection. These processes require the rapid disassembly, rearrangement, and repolymerization of the network of structural proteins known as the cytoskeleton. Migration and phagocytosis are one of the functional consequences of innate immune receptor triggered signaling pathways. Signals coalesce at catalytic hubs, such as kinases, which serve to amplify and regulate the signal. In the anti‐viral response, TANK binding kinase 1 (TBK1) functions as a catalytic hub. Suppressor of IKK epsilon (SIKE) acts as a high affinity, alternate TBK1 substrate, although SIKE's downstream function is not yet defined. The primary goal of this work was to assess the effect of SIKE on cytoskeletal rearrangements in migration and phagocytosis. For these studies, a CRISPR knockout cell line was derived, SIKE‐CR, from chronic myelogenous leukemia (CML) cell line KBM‐7 named HAP1. Wound healing assays of HAP1 parental cells and SIKE‐CR illustrated that the absence of SIKE significantly decreased cell migration. In the mouse macrophage RAW 264.7 cell line, SIKE expression was knocked down via lentiviral delivery of shRNA targeting SIKE. Phagocytosis was assessed in WT and SIKE knockdown cells via uptake of latex beads labeled with IgG‐FITC. Reduced SIKE expression increased phagocytic uptake. To determine SIKE interactions with cytoskeletal proteins, co‐immunoprecipitations (cIP), in vitro precipitation (IVP) reactions, and enzyme‐linked immunosorbent assays (ELISA) were completed and demonstrated SIKE:tubulin and SIKE:α‐actinin complexes. Together, these studies establish an interaction between SIKE and cytoskeletal proteins and suggest that SIKE functions in cytoskeletal rearrangement associated with migration and phagocytosis.Support or Funding InformationWork supported by NIH grant R21AI107447 and USD SURE.

Juncture of the cytoskeleton and innate immune signaling pathways: the role of Suppressor of IKK epsilon (SIKE)

The innate immune system rapidly responds to challenges by pathogens via activation of an inflammatory response. As a convergence point for multiple inflammatory and anti‐viral signaling pathways, TANK binding kinase 1 (TBK1) serves as a catalytic hub to initiate host defenses. Suppressor of IKK epsilon (SIKE) is a newly identified TBK1 substrate. The goal of this project was to identify the function of SIKE within the host's anti‐viral response. Co‐immunoprecipitation – tandem MS/MS analyses of the SIKE interaction network identified several interactions with cytoskeletal components. Immunofluorescence assays of endogenous SIKE with cytoskeletal markers and colocalization analyses indicated that SIKE colocalized with tubulin, actin, and a‐actinin. Reciprocal immunoprecipitation (RcIPs) studies confirmed the SIKE:tubulin and SIKE:a‐actinin interaction that appeared enhanced following dsRNA stimulation, a mimic of viral infection. ELISA assays were used to characterize the binding affinity between SIKE and the cytoskeletal proteins under unstimulated and simulated viral‐stimulated conditions. The SIKE:actin interaction was not detected by RcIP. In vitro immunoprecipitation assays mapped direct interactions between SIKE:tubulin and SIKE:a‐actinin and were used to further probe for a weak SIKE:actin interaction. Assays employed purified cytoskeletal proteins and full‐length (residues 1–207), N‐terminal (residues 1–112), C – terminal (residues 113–207) or a phosphomimetic (S6E) of a 6x‐His‐tagged SIKE construct. Together these studies establish an interaction between SIKE and cytoskeletal proteins that may provide a direct link between innate immune signaling and cytoskeletal components.Support or Funding InformationThis work was supported by NIH grant R21AI107447 and the University of San Diego SURE program.

ID: 219: DAPK1: A novel regulator of RIG-I identified by Kinome-Wide siRNA screening

The pattern recognition receptor RIG-I is a pivotal sensor of viral infections. Its activation by 5′-triphosphorylated- or double-stranded-RNA leads to subsequent signaling via MAVS, TBK1 and IKK epsilon resulting in IRF3 nuclear translocation. Activated IRF3 induces transcription of type I and type III interferons and several interferon stimulated genes. Despite intensive investigations on the RIG-I signaling pathway, its regulatory network still remains largely elusive. To gain more insight into the complex regulation of this pathway a kinome-wide siRNA screen was performed. The primary screen revealed over 100 siRNAs that significantly altered the translocation of IRF3 to the nucleus upon RIG-I stimulation. The top 50 candidates were further analyzed in three independent validation screens based on IRF3-sensitive promoter reporter assays or Rift-valley-fever virus replication. Taking all three validation screens into account, 21 novel regulators of the RIG-I signaling pathway could be identified. Relevance of the identified hits in regulating the host-cell antiviral defense was demonstrated by analyzing cytokine profiles and the impact on Influenza A virus replication. In the course of this screen, DAPK1 was identified as an inhibitor of RIG-I mediated IRF3 activation. Extensive mapping experiments revealed a minimal construct, including the kinase domain, to be sufficient for inhibiting IRF3 reporter activation in over-expression experiments. Furthermore, interaction studies revealed binding of DAPK1 to ligand-activated RIG-I, suggesting that a DAPK1 mediated phosphorylation of RIG-I inhibits its activity. In fact, in an in vitro kinase assays we could demonstrate that RIG-I is a substrate of DAPK1.

Exploring Suppressor of IKK Epsilon's Interaction with Cytoskeletal Proteins

The innate immune system rapidly responds to challenges by pathogens via activation of an inflammatory response. As a convergence point for multiple inflammatory and anti-viral signaling pathways, TANK binding kinase 1 (TBK1) serves as a catalytic hub to initiate host defenses. Suppressor of IKK epsilon (SIKE) is a newly identified TBK1 substrate that, due to its high affinity for the kinase, shifts TBK1's catalytic function towards SIKE phosphorylation and reduces type I interferon production via interferon regulatory factor 3/7 phosphorylation. The goal of this project was to identify the function of SIKE within the host's anti-viral response. Co-immunoprecipitation – tandem MS/MS analyses of the SIKE interaction network identified several interactions with cytoskeletal components. Immunofluorescence assays of endogenous SIKE with cytoskeletal markers and colocalization analyses indicated that SIKE colocalized with actin and a-actinin, but not focal adhesion kinase. Reciprocal immunoprecipitation (RcIPs) studies were used to confirm and further delineate the SIKE:actin interaction. RcIPs employed epitope tagged actin and full-length (residues 1-207), N-terminal (residues 1-112) or C –terminal (residues 113-207) FLAG-tagged SIKE. To determine the basal versus viral induced SIKE:actin interaction, studies were completed +/- double stranded RNA stimulation to mimic a viral infection. These studies support a role for SIKE in cytoskeletal rearrangements, such as cell migration and formation/resolution of RNA granules, that contribute to the antiviral response. This work was supported by NIH grant R21AI107447.

IKKε is key to induction of insulin resistance in the hypothalamus, and its inhibition reverses obesity.

IKK epsilon (IKKε) is induced by the activation of nuclear factor-κB (NF-κB). Whole-body IKKε knockout mice on a high-fat diet (HFD) were protected from insulin resistance and showed altered energy balance. We demonstrate that IKKε is expressed in neurons and is upregulated in the hypothalamus of obese mice, contributing to insulin and leptin resistance. Blocking IKKε in the hypothalamus of obese mice with CAYMAN10576 or small interfering RNA decreased NF-κB activation in this tissue, relieving the inflammatory environment. Inhibition of IKKε activity, but not TBK1, reduced IRS-1(Ser307) phosphorylation and insulin and leptin resistance by an improvement of the IR/IRS-1/Akt and JAK2/STAT3 pathways in the hypothalamus. These improvements were independent of body weight and food intake. Increased insulin and leptin action/signaling in the hypothalamus may contribute to a decrease in adiposity and hypophagia and an enhancement of energy expenditure accompanied by lower NPY and increased POMC mRNA levels. Improvement of hypothalamic insulin action decreases fasting glycemia, glycemia after pyruvate injection, and PEPCK protein expression in the liver of HFD-fed and db/db mice, suggesting a reduction in hepatic glucose production. We suggest that IKKε may be a key inflammatory mediator in the hypothalamus of obese mice, and its hypothalamic inhibition improves energy and glucose metabolism.

57: Pivotal role of the IKK-related kinase IKK epsilon to modulate IL29 or type I IFN induction during hepatitis C virus infection

Acute HCV infection can induce IL-29, a type III IFN, in hepatocytes but the mechanism of this induction is not known. Here, kinetics of IL-29 induction was analysed in the Huh7.25/CD81 cells infected with cell-culture HCV JFH1. IL-29 was produced and secreted early in infection (day 4) and able to activate the JAK/STAT-mediated induction of ISGs while IFN β was either absent or produced at low levels and at later times (6–7 days). Involvement of the RIG-I/MAVS or TLR3 pathways in IL-29 induction was ruled out since the HCV NS3/4A sensitive-MAVS was fully cleaved at day 2 post infection and since IFN was not induced in polyIC-treated cells. However, IRF3 and IRF7 were activated at day 4 of infection, as shown by IRF3-Gal4 and IRF7-Gal4 assays. We also found that JFH1 infection triggered induction of IRF7, the IKK-related kinase IKK e and IL8 between day 2 and day 4. Importantly, infection of human primary hepatocytes with JFH1 or with HCV of genotype 1b or 3 confirmed the data observed in the Huh7.25/CD81. We then infected the cells in presence of MRT67307, a specific inhibitor of IKK e /TBK1, previously used to demonstrate the ability of these non-canonical IKKs to impair NF-KB activation by the canonical IKKs. MRT67307 triggered increase in the induction of IL8, an NF-KB-dependent cytokine, as expected, but strinkingly it inhibits strongly induction of IL29 while that of IFN β was increased. IFN β , however, represented only a minor fraction of the IFN produced by these cells, suggesting the presence of other type I IFN isotypes. Taken together, these results suggest that the balance between type I and type III IFN induction during HCV infection can be modulated as a function of inflammation related to NF-KB activation and pinpoint a role for the IKK-related kinases, such as IKK e , in this process.

Correction for Wang et al., IKK epsilon kinase is crucial for viral G protein-coupled receptor tumorigenesis

Correction for Wang et al., IKK epsilon kinase is crucial for viral G protein-coupled receptor tumorigenesis

The NLRP4-DTX4 axis: a key suppressor of TBK1 and innate antiviral signaling

The NLRP4-DTX4 axis: a key suppressor of TBK1 and innate antiviral signaling

Interferon Regulatory Factor 3 Is Regulated by a Dual Phosphorylation-dependent Switch

The transcription factor interferon regulatory factor 3 (IRF-3) regulates genes in the innate immune response. IRF-3 is activated through phosphorylation by the kinases IKK epsilon and/or TBK1. Phosphorylation results in IRF-3 dimerization and removal of an autoinhibitory structure to allow interaction with the coactivators CBP/p300. The precise role of the different phosphorylation sites has remained controversial. Using purified proteins we show that TBK1 can directly phosphorylate full-length IRF-3 in vitro. Phosphorylation at residues in site 2 (Ser(396)-Ser(405)) alleviates autoinhibition to allow interaction with CBP (CREB-binding protein) and facilitates phosphorylation at site 1 (Ser(385) or Ser(386)). Phosphorylation at site 1 is, in turn, required for IRF-3 dimerization. The data support a two-step phosphorylation model for IRF-3 activation mediated by TBK1.

IKKɛ Regulates F Actin Assembly and Interacts with Drosophila IAP1 in Cellular Morphogenesis

Differentiated cells assume complex shapes through polarized cell migration and growth. These processes require the restricted organization of the actin cytoskeleton at limited subcellular regions. IKK epsilon is a member of the IkappaB kinase family, and its developmental role has not been clear. Drosophila IKK epsilon was localized to the ruffling membrane of cultured cells and was required for F actin turnover at the cell margin. In IKK epsilon mutants, tracheal terminal cells, bristles, and arista laterals, which require accurate F actin assembly for their polarized elongation, all exhibited aberrantly branched morphology. These phenotypes were sensitive to a change in the dosage of Drosophila inhibitor of apoptosis protein 1 (DIAP1) and the caspase DRONC without apparent change in cell viability. In contrast to this, hyperactivation of IKK epsilon destabilized F actin-based structures. Expression of a dominant-negative form of IKK epsilon increased the amount of DIAP1. The results suggest that at the physiological level, IKK epsilon acts as a negative regulator of F actin assembly and maintains the fidelity of polarized elongation during cell morphogenesis. This IKK epsilon function involves the negative regulation of the nonapoptotic activity of DIAP1.

Association of the Adaptor TANK with the IκB Kinase (IKK) Regulator NEMO Connects IKK Complexes with IKKε and TBK1 Kinases

Canonical activation of NF-kappa B is mediated via phosphorylation of the inhibitory I kappa B proteins by the I kappa B kinase complex (IKK). IKK is composed of a heterodimer of the catalytic IKK alpha and IKK beta subunits and a presumed regulatory protein termed NEMO (NF-kappa B essential modulator) or IKK gamma. NEMO/IKK gamma is indispensable for activation of the IKKs in response to many signals, but its mechanism of action remains unclear. Here we identify TANK (TRAF family member-associated NF-kappa B activator) as a NEMO/IKK gamma-interacting protein via yeast two-hybrid analyses. This interaction is confirmed in mammalian cells, and the domains required are mapped. TANK was previously shown to assist NF-kappa B activation in a complex with TANK-binding kinase 1 (TBK1) or IKK epsilon, two kinases distantly related to IKK alpha/beta, but the underlying mechanisms remained unknown. Here we show that TBK1 and IKK epsilon synergize with TANK to promote interaction with the IKKs. The TANK binding domain within NEMO/IKK gamma is required for proper functioning of this IKK subunit. These results indicate that TANK can synergize with IKK epsilon or TBK1 to link them to IKK complexes, where the two kinases may modulate aspects of NF-kappa B activation.