Coordinated regulation of Toll-like receptor and NOD2 signaling by K63-linked polyubiquitin chains

K63 polyubiquitin chains spatially and temporally link innate immune signaling effectors such that cytokine release can be coordinated. Crohn's disease is a prototypical inflammatory disorder in which this process may be faulty as the major Crohn's disease-associated protein, NOD2 (nucleotide oligomerization domain 2), regulates the formation of K63-linked polyubiquitin chains on the I kappa kinase (IKK) scaffolding protein, NEMO (NF-kappaB essential modifier). In this work, we study these K63-linked ubiquitin networks to begin to understand the biochemical basis for the signaling cross talk between extracellular pathogen Toll-like receptors (TLRs) and intracellular pathogen NOD receptors. This work shows that TLR signaling requires the same ubiquitination event on NEMO to properly signal through NF-kappaB. This ubiquitination is partially accomplished through the E3 ubiquitin ligase TRAF6. TRAF6 is activated by NOD2, and this activation is lost with a major Crohn's disease-associated NOD2 allele, L1007insC. We further show that TRAF6 and NOD2/RIP2 share the same biochemical machinery (transforming growth factor beta-activated kinase 1 [TAK1]/TAB/Ubc13) to activate NF-kappaB, allowing TLR signaling and NOD2 signaling to synergistically augment cytokine release. These findings suggest a biochemical mechanism for the faulty cytokine balance seen in Crohn's disease.     

Novel Lys63-linked ubiquitination of IKKβ induces STAT3 signaling

NFκB signaling plays a significant role in human disease, including breast and ovarian carcinoma, insulin resistance, embryonic lethality and liver degeneration, rheumatoid arthritis, aging and Multiple Myeloma (MM). Inhibitor of κB (IκB) kinase β (IKKβ) regulates canonical Nuclear Factor κB (NFκB) signaling in response to inflammation and cellular stresses. NFκB activation requires Lys63-linked (K63-linked) ubiquitination of upstream proteins such as NEMO or TAK1, forming molecular complexes with membrane-bound receptors. We demonstrate that IKKβ itself undergoes K63-linked ubiquitination. Mutations in IKKβ at Lys171, identified in Multiple Myeloma and other cancers, lead to a dramatic increase in kinase activation and K63-linked ubiquitination. These mutations also result in persistent activation of STAT3 signaling. Liquid chromatography (LC)-high mass accuracy tandem mass spectrometry (MS/MS) analysis identified Lys147, Lys418, Lys555 and Lys703 as predominant ubiquitination sites in IKKβ. Specific inhibition of the UBC13-UEV1A complex responsible for K63-linked ubiquitination establishes Lys147 as the predominant site of K63-ubiquitin conjugation and responsible for STAT3 activation. Thus, IKKβ activation leads to ubiquitination within the kinase domain and assemblage of a K63-ubiquitin conjugated signaling platform. These results are discussed with respect to the importance of upregulated NFκB signaling known to occur frequently in MM and other cancers.     

IRAK-1-mediated negative regulation of Toll-like receptor signaling through proteasome-dependent downregulation of TRAF6

TRAF6 plays a crucial role in signal transduction of the Toll-like receptor (TLR). It has been reported that TRAF6 catalyzes the formation of unique Lys63-linked polyubiquitin chains, which do not lead to proteasome-mediated degradation. Here we found that stimulation of J774.1 cells with various TLR ligands led to decreases in TRAF6 protein levels that occurred at a slower rate than IκBα degradation. The decrease in TRAF6 was inhibited by proteasome inhibitors MG-132, lactacystin and N-acetyl-leucyl-leucyl-norleucinal. Among intracellular TLR signaling molecules MyD88, IRAK-4, IRAK-1, TRAF6, and IKKβ, only IRAK-1 expression downregulated TRAF6 in HEK293 cells. The amount of TRAF6 expressed either transiently or stably was also reduced by co-expression of IRAK-1 and no TRAF6 cleavage products were detected. The levels of either a TRAF6 N-terminal deletion mutant or a ubiquitin ligase-defective mutant were not affected by IRAK-1 expression. Downregulation of TRAF6 required the TRAF6-binding site (Glu544, Glu587, Glu706) of IRAK-1 but not its catalytic site (Asp340). Upon IRAK-1 transfection, no significant TRAF6 ubiquitination was detected. Instead, TRAF6-associated IRAK-1 was ubiquitinated with both Lys48- and Lys63-linked polyubiquitin chains. TRAF6 downregulation was inhibited by co-expression of the E3 ubiquitin ligase Pellino 3, whose Lys63-linked polyubiquitination on IRAK-1 is reported to compete with Lys48-linked IRAK-1 polyubiquitination. Expression of IRAK-1 inhibited IκBα phosphorylation in response to TLR2 stimulation. These results indicate that stimulation of TLRs induces proteasome-dependent downregulation of TRAF6. We conclude that TRAF6 associated with ubiquitinated IRAK-1 is degraded together by the proteasome and that IRAK-1 possesses a negative regulatory role on TLR signaling.     

Malt1 ubiquitination triggers NF-kappaB signaling upon T-cell activation

Triggering of antigen receptors on lymphocytes is critical for initiating adaptive immune response against pathogens. T-cell receptor (TCR) engagement induces the formation of the Carma1-Bcl10-Malt1 (CBM) complex that is essential for activation of the IkappaB kinase (IKK)/NF-kappaB pathway. However, the molecular mechanisms that link CBM complex formation to IKK activation remain unclear. Here we report that Malt1 is polyubiquitinated upon T-cell activation. Ubiquitin chains on Malt1 provide a docking surface for the recruitment of the IKK regulatory subunit NEMO/IKKgamma. TRAF6 associates with Malt1 in response to T-cell activation and can function as an E3 ligase for Malt1 in vitro and in vivo, mediating lysine 63-linked ubiquitination of Malt1. Multiple lysine residues in the C-terminus of Malt1 serve as acceptor sites for the assembly of polyubiquitin chains. Malt1 mutants that lack C-terminal ubiquitin acceptor lysines are impaired in rescuing NF-kappaB signaling and IL-2 production in Malt1-/- T cells. Thus, our data demonstrate that induced Malt1 ubiquitination is critical for the engagement of CBM and IKK complexes, thereby directing TCR signals to the canonical NF-kappaB pathway.     

TAK1 lysine 158 is required for TGF-β-induced TRAF6-mediated Smad-independent IKK/NF-κB and JNK/AP-1 activation

Lys63-linked TAK1 polyubiquitination plays an essential role in the regulation of TAK1 activation. TRAF6-mediated Lys63-linked polyubiquitylation of TAK1 has been shown to be required for TGF-β-induced TAK1 activation. However, it remains unclear which lysine residue on TAK1 is TRAF6-mediated TAK1 polyubiquitination acceptor site in TGF-β signaling pathway. Here we report that lysine 158 on TAK1 is required for TGF-β-induced TRAF6-mediated TAK1 polyubiquitination and TAK1-mediated IKK, JNK and p38 activation. Notably, in contrast to TAK1 wild-type and K34R mutant, TAK1 K158R mutant co-overexpression with TAB1 failed to induce Lys63-linked TAK1 polyubiquitination. TRAF6-induced K63-linked TAK1 polyubiquitination was blocked by TAK1 K158R mutation, but not by K34R mutation. Furthermore, TGF-β-induced TAK1 polyubiquitination was inhibited by TAK1 K158R mutation, but not by K34R mutation in HeLa cells. Reconstitution of TAK1-deficient mouse embryo fibroblast cells with TAK1 wild-type, K158R mutant, or K34R mutant reveals that TAK1 lysine 158 residue is required for TGF-β-induced IKK, p38 and JNK activation.     

Optineurin negatively regulates TNFalpha- induced NF-kappaB activation by competing with NEMO for ubiquitinated RIP

NF-kappaB essential modulator (NEMO), the regulatory subunit of the IkappaB kinase (IKK) that activates NF-kappaB, is essential for NF-kappaB activation. NEMO was recently found to contain a region that preferentially binds Lys (K)63-linked but not K48-linked polyubiquitin (polyUb) chains, and the ability of NEMO to bind to K63-linked polyUb RIP (receptor-interacting protein) is necessary for efficient tumor necrosis factor alpha (TNFalpha)-induced NF-kappaB activation. Optineurin is a homolog of NEMO, and mutations in the optineurin gene are found in a subset of patients with glaucoma, a neurodegenerative disease involving the loss of retinal ganglion cells. Although optineurin shares considerable homology with NEMO, in resting cells, it is not present in the high-molecular-weight complex containing IKKalpha and IKKbeta, and optineurin cannot substitute for NEMO in lipopolysaccharide (LPS)-induced NF-kappaB activation. On the other hand, the overexpression of optineurin blocks the protective effect of E3-14.7K on cell death caused by the overexpression of TNFalpha receptor 1 (TNFR1). Here we show that optineurin has a K63-linked polyUb-binding region similar to that of NEMO, and like NEMO, it bound K63- but not K48-linked polyUb. Optineurin competitively antagonized NEMO's binding to polyUb RIP, and its overexpression inhibited TNFalpha-induced NF-kappaB activation. This competition occurs at physiologic protein levels because microRNA silencing of optineurin resulted in markedly enhanced TNFalpha-induced NF-kappaB activity. These results reveal a physiologic role for optineurin in dampening TNFalpha signaling, and this role might provide an explanation for its association with glaucoma.     

The TRAF6 ubiquitin ligase and TAK1 kinase mediate IKK activation by BCL10 and MALT1 in T lymphocytes

The CARD domain protein BCL10 and paracaspase MALT1 are essential for the activation of IkappaB kinase (IKK) and NF-kappaB in response to T cell receptor (TCR) stimulation. Here we present evidence that TRAF6 ubiquitin ligase and TAK1 protein kinase mediate IKK activation by BCL10 and MALT1. RNAi-mediated silencing of MALT1, TAK1, TRAF6, and TRAF2 suppressed TCR-dependent IKK activation and interleukin-2 production in T cells. Furthermore, we have reconstituted the pathway from BCL10 to IKK activation in vitro with purified proteins of MALT1, TRAF6, TAK1, and ubiquitination enzymes including Ubc13/Uev1A. We find that a small fraction of BCL10 and MALT1 proteins form high molecular weight oligomers. Strikingly, only these oligomeric forms of BCL10 and MALT1 can activate IKK in vitro. The MALT1 oligomers bind to TRAF6, induce TRAF6 oligomerization, and activate the ligase activity of TRAF6 to polyubiquitinate NEMO. These results reveal an oligomerization --> ubiquitination --> phosphorylation cascade that culminates in NF-kappaB activation in T lymphocytes.     

TRAF6 Autoubiquitination-Independent Activation of the NFκB and MAPK Pathways in Response to IL-1 and RANKL

The adapter protein TRAF6 is critical for mediating signal transduction from members of the IL-1R/TLR and TNFR superfamilies. The TRAF6 RING finger domain functions as an ubiquitin E3 ligase capable of generating non-degradative K63-linked ubiquitin chains. It is believed that these chains serve as docking sites for formation of signaling complexes, and that K63-linked autoubiquitination of TRAF6 is essential for formation and activation of a complex involving the kinase TAK1 and its adapters, TAB1 and TAB2. In order to assess independently the E3 ligase and ubiquitin substrate functions of TRAF6, we generated, respectively, RING domain and complete lysine-deficient TRAF6 mutants. We found that while the TRAF6 RING domain is required for activation of TAK1, it is dispensable for interaction between TRAF6 and the TAK1-TAB1-TAB2 complex. Likewise, lysine-deficient TRAF6 was found to interact with the TAK1-TAB1-TAB2 complex, but surprisingly was also found to be fully competent to activate TAK1, as well as NFκB and AP-1 reporters. Furthermore, lysine-deficient TRAF6 rescued IL-1-mediated NFκB and MAPK activation, as well as IL-6 elaboration in retrovirally-rescued TRAF6-deficient fibroblasts. Lysine-deficient TRAF6 also rescued RANKL-mediated NFκB and MAPK activation, and osteoclastogenesis in retrovirally-rescued TRAF6-deficient bone marrow macrophages. While incapable of being ubiquitinated itself, we demonstrate that lysine-deficient TRAF6 remains competent to induce ubiquitination of IKKγ/NEMO. Further, this NEMO modification contributes to TRAF6-mediated activation of NFκB. Collectively, our results suggest that while TRAF6 autoubiquitination may serve as a marker of activation, it is unlikely to underpin RING finger-dependent TRAF6 function.     

Cytokine-induced activation of mixed lineage kinase 3 requires TRAF2 and TRAF6

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates multiple mitogen-activated protein kinase (MAPK) pathways in response to growth factors, stresses and the pro-inflammatory cytokine, tumor necrosis factor (TNF). MLK3 is required for optimal activation of stress activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) signaling by TNF, however, the mechanism by which MLK3 is recruited and activated by the TNF receptor remains poorly understood. Here we report that both TNF and interleukin-1β (IL-1β) stimulation rapidly activate MLK3 kinase activity. We observed that TNF stimulates an interaction between MLK3 and TNF receptor associated factor (TRAF) 2 and IL-1β stimulates an interaction between MLK3 and TRAF6. RNA interference (RNAi) of traf2 or traf6 dramatically impairs MLK3 activation by TNF indicating that TRAF2 and TRAF6 are critically required for MLK3 activation. We show that TNF also stimulates ubiquitination of MLK3 and MLK3 can be conjugated with lysine 48 (K48)- and lysine 63 (K63)-linked polyubiquitin chains. Our results suggest that K48-linked ubiquitination directs MLK3 for proteosomal degradation while K63-linked ubiquitination is important for MLK3 kinase activity. These results reveal a novel mechanism for MLK3 activation by the pro-inflammatory cytokines TNF and IL-1β.     

TNF and IL-1 exhibit distinct ubiquitin requirements for inducing NEMO–IKK supramolecular structures

Nuclear factor κB (NF-κB) essential modulator (NEMO), a regulatory component of the IκB kinase (IKK) complex, controls NF-κB activation through its interaction with ubiquitin chains. We show here that stimulation with interleukin-1 (IL-1) and TNF induces a rapid and transient recruitment of NEMO into punctate structures that are anchored at the cell periphery. These structures are enriched in activated IKK kinases and ubiquitinated NEMO molecules, which suggests that they serve as organizing centers for the activation of NF-κB. These NEMO-containing structures colocalize with activated TNF receptors but not with activated IL-1 receptors. We investigated the involvement of nondegradative ubiquitination in the formation of these structures, using cells deficient in K63 ubiquitin chains or linear ubiquitin chain assembly complex (LUBAC)-mediated linear ubiquitination. Our results indicate that, unlike TNF, IL-1 requires K63-linked and linear ubiquitin chains to recruit NEMO into higher-order complexes. Thus, different mechanisms are involved in the recruitment of NEMO into supramolecular complexes, which appear to be essential for NF-κB activation.     

Activation of IKK by TNFalpha requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO

The receptor interacting protein kinase 1 (RIP1) is essential for the activation of nuclear factor kappaB (NF-kappaB) by tumor necrosis factor alpha (TNFalpha). Here, we present evidence that TNFalpha induces the polyubiquitination of RIP1 at Lys-377 and that this polyubiquitination is required for the activation of IkappaB kinase (IKK) and NF-kappaB. A point mutation of RIP1 at Lys-377 (K377R) abolishes its polyubiquitination as well as its ability to restore IKK activation in a RIP1-deficient cell line. The K377R mutation of RIP1 also prevents the recruitment of TAK1 and IKK complexes to TNF receptor. Interestingly, polyubiquitinated RIP1 recruits IKK through the binding between the polyubiquitin chains and NEMO, a regulatory subunit of the IKK complex. Mutations of NEMO that disrupt its polyubiquitin binding also abolish IKK activation. These results reveal the biochemical mechanism underlying the essential signaling function of NEMO and provide direct evidence that signal-induced site-specific ubiquitination of RIP1 is required for IKK activation.     

Phosphorylation and ubiquitination of the IkappaB kinase complex by two distinct signaling pathways

The IkappaB kinase (IKK) complex serves as the master regulator for the activation of NF-kappaB by various stimuli. It contains two catalytic subunits, IKKalpha and IKKbeta, and a regulatory subunit, IKKgamma/NEMO. The activation of IKK complex is dependent on the phosphorylation of IKKalpha/beta at its activation loop and the K63-linked ubiquitination of NEMO. However, the molecular mechanism by which these inducible modifications occur remains undefined. Here, we demonstrate that CARMA1, a key scaffold molecule, is essential to regulate NEMO ubiquitination upon T-cell receptor (TCR) stimulation. However, the phosphorylation of IKKalpha/beta activation loop is independent of CARMA1 or NEMO ubiquitination. Further, we provide evidence that TAK1 is activated and recruited to the synapses in a CARMA1-independent manner and mediate IKKalpha/beta phosphorylation. Thus, our study provides the biochemical and genetic evidence that phosphorylation of IKKalpha/beta and ubiquitination of NEMO are regulated by two distinct pathways upon TCR stimulation.     

A role for NF-kappaB essential modifier/IkappaB kinase-gamma (NEMO/IKKgamma) ubiquitination in the activation of the IkappaB kinase complex by tumor necrosis factor-alpha

NEMO (NF-kappaB essential modifier)/IKKgamma (IkappaB kinase-gamma) is required for the activation of the IkappaB kinase complex (IKK) by inflammatory stimuli such as tumor necrosis factor (TNF-alpha). Here we show that TNF-alpha stimulates the ubiquitination of NEMO in a manner that does not appear to target it for degradation and that is impaired by mutations in the NEMO zinc finger. Mutations of the zinc finger are found in patients with hypohidrotic ectodermal dysplasia with immunodeficiency (HED-ID) and lead to the impairment of TNF-alpha-stimulated IKK phosphorylation and activation. In addition, the ubiquitination of NEMO is mediated by c-IAP1, an inhibitor of apoptosis protein that is a component of the TNF receptor signaling complex. Thus, the ubiquitination of NEMO mediated by c-IAP1 likely plays an important role in the activation of IKK by TNF-alpha. Also, defective NEMO ubiquitination may be responsible for the impaired cellular NF-kappaB signaling found in patients with HED-ID.     

Activation of the IkappaB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain

TRAF6 is a signal transducer in the NF-kappaB pathway that activates IkappaB kinase (IKK) in response to proinflammatory cytokines. We have purified a heterodimeric protein complex that links TRAF6 to IKK activation. Peptide mass fingerprinting analysis reveals that this complex is composed of the ubiquitin conjugating enzyme Ubc13 and the Ubc-like protein Uev1A. We find that TRAF6, a RING domain protein, functions together with Ubc13/Uev1A to catalyze the synthesis of unique polyubiquitin chains linked through lysine-63 (K63) of ubiquitin. Blockade of this polyubiquitin chain synthesis, but not inhibition of the proteasome, prevents the activation of IKK by TRAF6. These results unveil a new regulatory function for ubiquitin, in which IKK is activated through the assembly of K63-linked polyubiquitin chains.     

Interleukin-1-induced NF-kappaB activation is NEMO-dependent but does not require IKKbeta

Activation of NF-kappaB by the pro-inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) requires the IkappaB kinase (IKK) complex, which contains two kinases named IKKalpha and IKKbeta and a critical regulatory subunit named NEMO. Although we have previously demonstrated that NEMO associates with both IKKs, genetic studies reveal that only its interaction with IKKbeta is required for TNF-induced NF-kappaB activation. To determine whether NEMO and IKKalpha can form a functional IKK complex capable of activating the classical NF-kappaB pathway in the absence of IKKbeta, we utilized a panel of mouse embryonic fibroblasts (MEFs) lacking each of the IKK complex subunits. This confirmed that TNF-induced IkappaBalpha degradation absolutely requires NEMO and IKKbeta. In contrast, we consistently observed intact IkappaBalpha degradation and NF-kappaB activation in response to IL-1 in two separate cell lines lacking IKKbeta. Furthermore, exogenously expressed, catalytically inactive IKKbeta blocked TNF- but not IL-1-induced IkappaBalpha degradation in wild-type MEFs, and reconstitution of IKKalpha/beta double knockout cells with IKKalpha rescued IL-1- but not TNF-induced NF-kappaB activation. Finally, we have shown that incubation of IKKbeta-deficient MEFs with a cell-permeable peptide that blocks the interaction of NEMO with the IKKs inhibits IL-1-induced NF-kappaB activation. Our results therefore demonstrate that NEMO and IKKalpha can form a functional IKK complex that activates the classical NF-kappaB pathway in response to IL-1 but not TNF. These findings further suggest NEMO differentially regulates the fidelity of the IKK subunits activated by distinct upstream signaling pathways.