TANK is a negative regulator of Toll-like receptor signaling and is critical for the prevention of autoimmune nephritis.
ABSTRACT: The intensity and duration of immune responses are controlled by many proteins that modulate Toll-like receptor (TLR) signaling. TANK has been linked to positive regulation of the transcription factors IRF3 and NF-kappaB. Here we demonstrate that TANK is not involved in interferon responses and is a negative regulator of proinflammatory cytokine production induced by TLR signaling. TLR-induced polyubiquitination of the ubiquitin ligase TRAF6 was upregulated in Tank(-/-) macrophages. Notably, Tank(-/-) mice spontaneously developed fatal glomerulonephritis owing to deposition of immune complexes. Autoantibody production in Tank(-/-) mice was abrogated by antibiotic treatment or the absence of interleukin 6 (IL-6) or the adaptor MyD88. Our results demonstrate that constitutive TLR signaling by intestinal commensal microflora is suppressed by TANK.
Project description:The signaling of Toll-like receptors (TLRs) is the host's first line of defense against microbial invasion. The mitochondrion is emerging as a critical platform for antiviral signal transduction. The regulatory role of mitochondria for TLR signaling remains to be explored. Here, we show that the mitochondrial outer-membrane protein MARCH5 positively regulates TLR7 signaling. Ectopic expression or knockdown of MARCH5 enhances or impairs NF-?B-mediated gene expression, respectively. MARCH5 interacts specifically with TANK, and this interaction is enhanced by R837 stimulation. MARCH5 catalyzes the K63-linked poly-ubiquitination of TANK on its Lysines 229, 233, 280, 302 and 306, thus impairing the ability of TANK to inhibit TRAF6. Mislocalization of MARCH5 abolishes its action on TANK, revealing the critical role of mitochondria in modulating innate immunity. Arguably, this represents the first study linking mitochondria to TLR signaling.
Project description:Toll-like receptor (TLR) ligands that signal via TIR-domain-containing adapter-inducing IFN? (TRIF) activate the I?B kinase (IKK)-related kinases, TRAF associated NF?B activator (TANK)-binding kinase-1 (TBK1) and IKK?, which then phosphorylate IRF3 and induce the production of IFN?. Here we show that TBK1 and IKK? are also activated by TLR ligands that signal via MyD88. Notably, the activation of IKK? is rapid, transient, and it precedes a more prolonged activation of TBK1. The MyD88- and TRIF-dependent signaling pathways activate the IKK-related kinases by two signaling pathways. One is mediated by the canonical IKKs, whereas the other culminates in the autoactivation of the IKK-related kinases. Once activated, TBK1/IKK? then phosphorylate and inhibit the canonical IKKs. The negative regulation of the canonical IKKs by the IKK-related kinases occurs in both the TRIF- and MyD88-dependent TLR pathways, whereas IRF3 phosphorylation is restricted to the TRIF-dependent signaling pathway. We have discovered that the activation of IKK? is abolished, the activation of TBK1 is reduced, and the interaction between the IKK-related kinases and the canonical IKKs is suppressed in TANK(-/-) macrophages, preventing the IKK-related kinases from negatively regulating the canonical IKKs. In contrast, IRF3 phosphorylation and IFN? production was normal in TANK(-/-) macrophages. Our results demonstrate a key role for TANK in enabling the canonical IKKs and the IKK-related kinases to regulate each other, which is required to limit the strength of TLR signaling and ultimately, prevent autoimmunity.
Project description:The differentiation of bone-resorbing osteoclasts is induced by RANKL signaling, and leads to the activation of NF-?B via TRAF6 activation. TRAF family member-associated NF-?B activator (TANK) acts as a negative regulator of Toll-like receptors (TLRs) and B-cell receptor (BCR) signaling by inhibiting TRAF6 activation. Tank(-/-) mice spontaneously develop autoimmune glomerular nephritis in an IL-6-dependent manner. Despite its importance in the TCRs and BCR-activated TRAF6 inhibition, the involvement of TANK in RANKL signaling is poorly understood. Here, we report that TANK is a negative regulator of osteoclast differentiation. The expression levels of TANK mRNA and protein were up-regulated during RANKL-induced osteoclastogenesis, and overexpression of TANK in vitro led to a decrease in osteoclast formation. The in vitro osteoclastogenesis of Tank(-/-) cells was significantly increased, accompanied by increased ubiquitination of TRAF6 and enhanced canonical NF-?B activation in response to RANKL stimulation. Tank(-/-) mice showed severe trabecular bone loss, but increased cortical bone mineral density, because of enhanced bone erosion and formation. TANK mRNA expression was induced during osteoblast differentiation and Tank(-/-) osteoblasts exhibited enhaced NF-?B activation, IL-11 expression, and bone nodule formation than wild-type control cells. Finally, wild-type mice transplanted with bone marrow cells from Tank(-/-) mice showed trabecular bone loss analogous to that in Tank(-/-) mice. These findings demonstrate that TANK is critical for osteoclastogenesis by regulating NF-?B, and is also important for proper bone remodeling.
Project description:Upon stimulation by pathogen-associated inflammatory signals, TANK-binding kinase 1 (TBK1) induces type I interferon expression and modulates nuclear factor ?B (NF-?B) signaling. Here, we describe the 2.4 Å-resolution crystal structure of nearly full-length TBK1 in complex with specific inhibitors. The structure reveals a dimeric assembly created by an extensive network of interactions among the kinase, ubiquitin-like, and scaffold/dimerization domains. An intact TBK1 dimer undergoes K63-linked polyubiquitination on lysines 30 and 401, and these modifications are required for TBK1 activity. The ubiquitination sites and dimer contacts are conserved in the close homolog inhibitor of ?B kinase ? (IKK?) but not in IKK?, a canonical IKK that assembles in an unrelated manner. The multidomain architecture of TBK1 provides a structural platform for integrating ubiquitination with kinase activation and IRF3 phosphorylation. The structure of TBK1 will facilitate studies of the atypical IKKs in normal and disease physiology and further the development of more specific inhibitors that may be useful as anticancer or anti-inflammatory agents.
Project description:TRAF family member-associated NF-?B activator (TANK) is a negative regulator of canonical NF-?B signaling in the Toll-like receptor- and B-cell receptor-mediated signaling pathways. However, functions of TANK in viral infection-mediated NF-?B activation remain unclear. Here, we reported that TANK was cleaved by encephalomyocarditis virus 3C at the 197 and 291 glutamine residues, which depends on its cysteine protease activity. In addition, encephalomyocarditis virus 3C impaired the ability of TANK to inhibit TRAF6-mediated NF-?B signaling. Interestingly, we found that several viral proteases encoded by the foot and mouth disease virus, porcine reproductive and respiratory syndrome virus, and equine arteritis virus also cleaved TANK. Our results suggest that TANK is a novel target of some viral proteases, indicating that some positive RNA viruses have evolved to utilize their major proteases to regulate NF-?B activation.
Project description:It is well established that polymorphisms of the caspase activation and recruitment domain 15 (CARD15) gene, a major risk factor in Crohn's disease (CD), lead to loss of nucleotide-binding oligomerization domain 2 (NOD2) function. However, a molecular explanation of how such loss of function leads to increased susceptibility to CD has remained unclear. In a previous study exploring this question, we reported that activation of NOD2 in human dendritic cells by its ligand, muramyl dipeptide (MDP), negatively regulates Toll-like receptor (TLR)-mediated inflammatory responses. Here we show that NOD2 activation results in increased interferon regulatory factor 4 (IRF4) expression and binding to tumor necrosis factor receptor associated factor 6 (TRAF6) and RICK (receptor interacting serine-threonine kinase). We then show that such binding leads to IRF4-mediated inhibition of Lys63-linked polyubiquitination of TRAF6 and RICK and thus to downregulation of nuclear factor (NF)-?B activation. Finally, we demonstrate that protection of mice from the development of experimental colitis by MDP or IRF4 administration is accompanied by similar IRF4-mediated effects on polyubiquitination of TRAF6 and RICK in colonic lamina propria mononuclear cells. These findings thus define a mechanism of NOD2-mediated regulation of innate immune responses to intestinal microflora that could explain the relation of CARD15 polymorphisms and resultant NOD2 dysfunction to CD.
Project description:Signal transmission by the noncanonical IkappaB kinases (IKKs), TANK-binding kinase 1 (TBK1) and IKK?, requires interaction with adapter proteins such as TRAF associated NF-?B activator (TANK). Although increased expression or dysregulation of both kinases has been described for a variety of human cancers, this study shows that deregulated expression of the TANK protein is frequently occurring in glioblastomas (GBMs). The functional relevance of TANK was analyzed in a panel of GBM-derived cell lines and revealed that knockdown of TANK arrests cells in the S-phase and prohibits tumor cell migration. Deregulated TANK expression affects several signaling pathways controlling cell proliferation and the inflammatory response. Interference with stoichiometrically assembled signaling complexes by overexpression or silencing of TANK prevented constitutive interferon-regulatory factor 3 (IRF3) phosphorylation. Knockdown of TANK frequently prevents constitutive activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). TANK-mediated ERK1/2 activation is independent from the canonical MAP kinase or ERK kinase (MEK) 1/2-mediated pathway and utilizes an alternative pathway that uses a TBK1/IKK?/Akt signaling axis, thus identifying a novel pathway suitable to block constitutive ERK1/2 activity.
Project description:Tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) is a key regulator of the activation of transcription factor NF-?B by the interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) superfamily. Recruitment of TRAF6 to the receptor-associated IRAK1-IRAK4-MyD88 adaptor protein complex induces lysine 63 (K63) autopolyubiquitination of TRAF6, which leads to further recruitment of downstream regulators, such as TAB2/3 and TAK1, and subsequently triggers NF-?B activation. Here, we identified the putative E2 ubiquitin-conjugating (UBC) enzyme UBE2O as a novel negative regulator of TRAF6-dependent NF-?B signaling. We found that UBE2O binds to TRAF6 to inhibit its K63-polyubiquitination, and to prevent the activation of NF-?B by IL-1? and lipopolysaccharides (LPS). We further show that the inhibitory effect of UBE2O is independent of its carboxy-terminal UBC domain. In contrast, we found that UBE2O acts to disrupt the IL-1?-induced association of TRAF6 with MyD88. These results provide novel insight into the regulation of signaling by IL-1R/TLR and TRAF6.
Project description:Beta interferon (IFN-?) is involved in a wide range of cellular functions, and its secretion must be tightly controlled to inhibit viral spreading while minimizing cellular damage. Intracellular viral replication triggers cellular signaling cascades leading to the activation of the transcription factors NF-?B and interferon regulatory factor 3 (IRF3) and IRF7 (IRF3/7), which synergistically bind to the IFN-? gene promoter to induce its expression. The mitochondrial antiviral signaling protein (MAVS) is a governing adaptor protein that mediates signaling communications between virus-sensing proteins and transcription factors. The activity of MAVS in the regulation of IFN-? secretion is affected by many cellular factors. However, the mechanism of MAVS-mediated IRF3/7 activation is not completely understood. Here, we identified a highly conserved DLAIS motif at amino acid positions 438 to 442 of MAVS that is indispensable for IRF3/7 activation. Specifically, the L439S and A440R mutations suppress IRF3/7 activation. Pulldown experiments using wild-type and mutant MAVS showed that mindbomb E3 ubiquitin protein ligase 2 (MIB2) binds to the DLAIS motif. Furthermore, the DLAIS motif was found to be critical for MIB2 binding, the ligation of K63-linked ubiquitin to TANK-binding kinase 1, and phosphorylation-mediated IRF3/7 activation. Our results suggest that MIB2 plays a putative role in MAVS-mediated interferon signaling.Mitochondrial antiviral signaling protein (MAVS) mediates signaling from virus-sensing proteins to transcription factors for the induction of beta interferon. However, the mechanism underlying activation of MAVS-mediated interferon regulatory factors 3 and 7 (IRF3/7) is not completely understood. We found a highly conserved DLAIS motif in MAVS that is indispensable for IRF3/7 activation through TANK-binding kinase 1 (TBK1) and identified it as the binding site for mindbomb E3 ubiquitin protein ligase 2 (MIB2). The mutations that targeted the DLAIS motif abolished MIB2 binding, attenuated the K63-linked ubiquitination of TBK1, and decreased the phosphorylation-mediated activation of IRF3/7.
Project description:BACKGROUND:In response to viral infection, the innate immune system recognizes viral nucleic acids and then induces production of proinflammatory cytokines and type I interferons (IFNs). Toll-like receptor 7 (TLR7) and TLR9 detect viral RNA and DNA, respectively, in endosomal compartments, leading to the activation of nuclear factor kappaB (NF-kappaB) and IFN regulatory factors (IRFs) in plasmacytoid dendritic cells. During such TLR signaling, TNF receptor-associated factor 6 (TRAF6) is essential for the activation of NF-kappaB and the production of type I IFN. In contrast, RIG-like helicases (RLHs), cytosolic RNA sensors, are indispensable for antiviral responses in conventional dendritic cells, macrophages, and fibroblasts. However, the contribution of TRAF6 to the detection of cytosolic viral nucleic acids has been controversial, and the involvement of TRAF6 in IRF activation has not been adequately addressed. PRINCIPAL FINDINGS:Here we first show that TRAF6 plays a critical role in RLH signaling. The absence of TRAF6 resulted in enhanced viral replication and a significant reduction in the production of IL-6 and type I IFNs after infection with RNA virus. Activation of NF-kappaB and IRF7, but not that of IRF3, was significantly impaired during RLH signaling in the absence of TRAF6. TGFbeta-activated kinase 1 (TAK1) and MEKK3, whose activation by TRAF6 during TLR signaling is involved in NF-kappaB activation, were not essential for RLH-mediated NF-kappaB activation. We also demonstrate that TRAF6-deficiency impaired cytosolic DNA-induced antiviral responses, and this impairment was due to defective activation of NF-kappaB and IRF7. CONCLUSIONS/SIGNIFICANCE:Thus, TRAF6 mediates antiviral responses triggered by cytosolic viral DNA and RNA in a way that differs from that associated with TLR signaling. Given its essential role in signaling by various receptors involved in the acquired immune system, TRAF6 represents a key molecule in innate and antigen-specific immune responses against viral infection.