TNF) Receptor-associated Factor 6 Signaling Research Articles

TRAF6 signaling in dendritic cells plays protective role against infectious colitis by limiting C. rodentium infection through the induction of Th1 and Th17 responses

Tumor necrosis factor receptor-associated factor 6 (TRAF6) plays a pivotal role in the induction of inflammatory responses not only in innate immune cells but also in non-immune cells, leading to the activation of adaptive immunity. Signal transduction mediated by TRAF6, along with its upstream molecule MyD88 in intestinal epithelial cells (IECs) is crucial for the maintenance of mucosal homeostasis following inflammatory insult. The IEC-specific TRAF6-deficient (TRAF6ΔIEC) and MyD88-deficient (MyD88ΔIEC) mice exhibit increased susceptibility to DSS-induced colitis, emphasizing the critical role of this pathway. Moreover, MyD88 also plays a protective role in Citrobacter rodentium (C. rodentium) infection-induced colitis. However, its pathological role of TRAF6 in infectious colitis remains unclear. To investigate the site-specific roles of TRAF6 in response to enteric bacterial pathogens, we infected TRAF6ΔIEC and dendritic cell (DC)-specific TRAF6-deficient (TRAF6ΔDC) mice with C. rodentium and found that the pathology of infectious colitis was exacerbated with significantly decreased survival rates in TRAF6ΔDC mice, but not in TRAF6ΔIEC mice, compared to those in control mice. TRAF6ΔDC mice showed increased bacterial burdens, marked disruption of epithelial and mucosal structures with increased infiltration of neutrophils and macrophages, and elevated cytokine levels in the colon at the late stages of infection. The frequencies of IFN-γ producing Th1 cells and IL-17A producing Th17 cells in the colonic lamina propria were significantly reduced in TRAF6ΔDC mice. Finally, we demonstrated that TRAF6-deficient DCs failed to produce IL-12 and IL-23 in response to C. rodentium stimulation, and to induce both Th1 and Th17 cells in vitro. Thus, TRAF6 signaling in DCs, but not in IECs, protects against colitis induced by C. rodentium infection by producing IL-12 and IL-23 that induce Th1 and Th17 responses in the gut.

Home Dust Mites Promote MUC5AC Hyper-Expression by Modulating the sNASP/TRAF6 Axis in the Airway Epithelium.

House dust mites (HDMs) are a common source of respiratory allergens responsible for allergic asthma and innate immune responses in human diseases. Since HDMs are critical factors in the triggering of allergen-induced airway mucosa from allergic asthma, we aimed to investigate the mechanisms of Toll-like receptors (TLR) in the signaling of the HDM extract that is involved in mucus hypersecretion and airway inflammation through the engagement of innate immunity. Previously, we reported that the somatic nuclear autoantigenic sperm protein (sNASP)/tumor necrosis factor receptor-associated factor 6 (TRAF6) axis controls the initiation of TLRs to maintain the homeostasis of the innate immune response. The present study showed that the HDM extract stimulated the biogenesis of Mucin 5AC (MUC5AC) in bronchial epithelial cells via the TLR2/4 signaling pathway involving MyD88 and TRAF6. Specifically, sNASP binds to TRAF6 in unstimulated bronchial epithelial cells to prevent the activation of TRAF6-depenedent kinases. Upon on HDMs’ stimulation, sNASP is phosphorylated, leading to the activation of TRAF6 downstream of the p38 MAPK and NF-κB signaling pathways. Further, NASP-knockdown enhanced TRAF6 signaling and MUC5AC biogenesis. In the HDM-induced mouse asthma model, we found that the HDM extract promoted airway hyperresponsiveness (AHR), MUC5AC, and allergen-specific IgE production as well as IL-5 and IL-13 for recruiting inflammatory cells. Treatment with the PEP-NASP peptide, a selective TRAF6-blocking peptide, ameliorated HDM-induced asthma in mice. In conclusion, this study indicated that the sNASP/TRAF6 axis plays a regulatory role in asthma by modulating mucus overproduction, and the PEP-NASP peptide might be a potential target for asthma treatment.

TRAF6-overexpressing dendritic cells loaded with MUC1 peptide enhance anti-tumor activity in B16-MUC1 melanoma-bearing mice

Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) signaling is a critical positive mechanism for the development, homeostasis and activation of immune cells. We investigated the effect of TRAF6 overexpression on dendritic cells (DCs) maturation. TRAF6-overexpressing DCs had increased expression of costimulatory molecules, major histocompatibility complex (MHC) molecules and IL-12 expression. This indicated that TRAF6 promoted the maturation of DCs and indirectly promoted Th1 activation. The antitumor activities between TRAF6-overexpressing DCs and control DCs were compared by administering DCs pulsed with mucin 1 (MUC1) Ag peptide in a therapeutic human MUC1-overexpressing mouse B16 melanoma cells (B16-MUC1) model. Administration of TRAF6-overexpressing DCs significantly inhibited the growth of B16-MUC1 tumors, accompanied by an increase in MUC1-specific Th1 responses and Tc1 responses, as well as a decrease in Tregs levels. TRAF6 signaling has been found to be involved in DCs maturation and Th1 activation in vitro, as well as therapeutic tumor models in vivo, indicating that TRAF6-overexpressing DCs may be a promising approach for cancer immunotherapy.

Pathogenic Functions of Tumor Necrosis Factor Receptor-Associated Factor 6 Signaling Following Traumatic Brain Injury.

Neuroinflammation contributes to delayed (secondary) neurodegeneration following traumatic brain injury (TBI). Tumor necrosis factor receptor-associated factor 6 (TRAF6) signaling may promote post-TBI neuroinflammation, thereby exacerbating secondary injury. This study investigated the pathogenic functions of TRAF6 signaling following TBI in vivo and in vitro. A rat TBI model was established by air pressure contusion while lipopolysaccharide (LPS) exposure was used to induce inflammatory-like responses in cultured astrocytes. Model rats were examined for cell-specific expression of TRAF6, NF-κB, phosphorylated (p)-NF-κB, MAPKs (ERK, JNK, and p38), p-MAPKs, chemokines (CCL2 and CXCL1), and chemokine receptors (CCR2 and CXCR2) by immunofluorescence, RT-qPCR, western blotting, and ELISA, for apoptosis by TUNEL staining, and spatial cognition by Morris water maze testing. These measurements were compared between TBI model rats receiving intracerebral injections of TRAF6-targeted RNAi vector (AAV9-TRAF6-RNAi), empty vector, MAPK/NF-κB inhibitors, or vehicle. Primary astrocytes were stimulated with LPS following TRAF6 siRNA or control transfection, and NF-κB, MAPKs, chemokine, and chemokine receptor expression levels evaluated by western blotting and ELISA. TRAF6 was expressed mainly in astrocytes and neurons of injured cortex, peaking 3 days post-TBI. Knockdown by AAV9-TRAF6-RNAi improved spatial learning and memory, decreased TUNEL-positive cell number in injured cortex, and downregulated expression levels of p-NF-κB, p-ERK, p-JNK, p-p38, CCL2, CCR2, CXCL1, and CXCR2 post-TBI. Inhibitors of NF-κB, ERK, JNK, and p38 significantly suppressed CCL2, CCR2, CXCL1, and CXCR2 expression following TBI. Furthermore, TRAF6-siRNA inhibited LPS-induced NF-κB, ERK, JNK, p38, CCL2, and CXCL1 upregulation in cultured astrocytes. Targeting TRAF6-MAPKs/NF-κB-chemokine signaling pathways may provide a novel therapeutic approach for reducing post-TBI neuroinflammation and concomitant secondary injury.

Melatonin Inhibits Osteoclastogenesis and Bone Loss in Ovariectomized Mice by Regulating PRMT1-Mediated Signaling.

Melatonin, a pineal gland hormone, has been suggested to treat postmenopausal osteoporosis due to its inhibitory effect on osteoclast differentiation. We previously reported that protein arginine methyltransferase 1 (PRMT1) was an important mediator of receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. However, the relationship between melatonin and PRMT1 in osteoclast differentiation and estrogen deficiency-induced osteoporosis is unclear. In this study, we investigated the inhibitory mechanisms of melatonin in vitro and in vivo by focusing on PRMT1. Melatonin treatment effectively blocked RANKL-induced osteoclastogenesis by inhibiting PRMT1 and asymmetric dimethylarginine (ADMA) expression. RANKL-induced tumor necrosis factor receptor-associated factor 6 (TRAF6) and the phosphorylation of JNK were also suppressed by melatonin, and TRAF6 siRNA attenuated RANKL-induced p-JNK and PRMT1 production. Melatonin inhibited the transcriptional activity of NF-κB by interfering with the binding of PRMT1 and NF-κB subunit p65 in RANKL-treated bone marrow-derived macrophages. Our results also revealed that melatonin inhibits RANKL-induced PRMT1 expression through receptors-independent pathway. Thus, the anti-osteoclastogenic effect of melatonin was mediated by a cascade of inhibition of RANKL-induced TRAF6, JNK, PRMT1, and NF-κB signaling in melatonin receptors-independent pathway. In vivo, ovariectomy caused significant decreases in bone mineral density, but melatonin treatment alleviated the ovariectomized (OVX)-induced bone loss by inhibiting bone resorption. Furthermore, the expression PRMT1 and TRAP mRNA was upregulated in OVX-femurs, but effectively suppressed by melatonin injection. These findings suggest that melatonin inhibited osteoclast differentiation and estrogen deficiency-induced osteoporosis by suppressing RANKL-induced TRAF6, JNK, PRMT1, and NF-κB signaling cascades in melatonin receptors-independent pathway.

BCL3 regulates RANKL-induced osteoclastogenesis by interacting with TRAF6 in bone marrow-derived macrophages.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an essential component of the signaling complex that mediates osteoclastogenesis. As an adaptor protein of E3 ligase function, TRAF6 regulates NF-κB signaling via TAK1 and I-κB kinase (IKK) activation. Here, we investigated novel mechanisms by which TRAF6 signaling is regulated under receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. A yeast two-hybrid screen system identified cellular factors that interact with TRAF6. The interactions were confirmed by glutathione S-transferase pull-down and co-immunoprecipitation assays, followed by immuno-blotting. The role of TRAF6 in bone growth and remodeling was determined by osteoclast differentiation and bone-resorption pit assays. Regulatory mechanisms were examined by co-immunoprecipitation, immuno-blotting, real-time polymerase chain reaction, and luciferase reporter assays. We show that B-cell chronic lymphatic leukemia protein 3 (BCL3) interacts with TRAF6 through its ankyrin-repeat domain and inhibits osteoclastogenesis in bone marrow derived macrophages (BMDMs). Further, TRAF6 interacts with CYLD to mediate BCL3 deubiquitination, which facilitates the cytoplasmic accumulation of BCL3 and represses BCL3 and p50 complex-mediated cyclin D1 transcription. TRAF6 promotes RANKL-induced osteoclastogenesis by regulating novel non-canonical NF-κB signaling via BCL3 deubiquitination, indicating that BCL3 provides valuable insights into bone loss-associated diseases.

Characterization of the black carp TRAF6 signaling molecule in innate immune defense

Tumor necrosis factor receptor-associated factor 6 (TRAF6) plays a vital role in the innate immune response of higher vertebrates. To elucidate its function in teleost fish, TRAF6 homologue of black carp (Mylopharyngodon piceus) has been cloned and characterized in this study. Black carp TRAF6 (bcTRAF6) transcription in Mylopharyngodon piceus fin (MPF) cells was up-regulated in response to both poly (I:C) treatment and viral infection, but was suppressed by LPS stimulation. bcTRAF6 migrated around 72 KDa in immunoblot analysis and was identified as a cytosolic protein suggested to be associated with vesicles scattering in the cytoplasm. Reporter assay demonstrated that NF-κB instead of IFN was activated by bcTRAF6; and EPC cells expressing bcTRAF6 presented the same cytopathic effect (CPE) ratio to that of control cells. When co-expressed with bcMAVS, bcTRAF6 was redistributed and overlapped with the subcellular location of bcMAVS. It was interesting that bcMAVS mediated the IFN induction was up-regulated by low input of bcTRAF6 but down-regulated by high input of bcTRAF6. Taken together, the data generated in this paper supported the conclusion that bcTRAF6 associated with bcMAVS and was recruited into bcMAVS mediated signaling during host innate immune response.

Interaction of Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6) and Vav3 in the Receptor Activator of Nuclear Factor κB (RANK) Signaling Complex Enhances Osteoclastogenesis

The signaling pathway downstream of stimulation of receptor activator of nuclear factor κB (RANK) by RANK ligand is crucial for osteoclastogenesis. RANK recruits TNF receptor-associated factor 6 (TRAF6) to TRAF6-binding sites (T6BSs) in the RANK cytoplasmic tail (RANKcyto) to trigger downstream osteoclastogenic signaling cascades. RANKcyto harbors an additional highly conserved domain (HCR) that also activates crucial signaling during RANK-mediated osteoclastogenesis. However, the functional cross-talk between T6BSs and the HCR in the RANK signaling complex remains unclear. To characterize the cross-talk between T6BSs and the HCR, we screened TRAF6-interacting proteins using a proteomics approach. We identified Vav3 as a novel TRAF6 binding partner and evaluated the functional importance of the TRAF6-Vav3 interaction in the RANK signaling complex. We demonstrated that the coiled-coil domain of TRAF6 interacts directly with the Dbl homology domain of Vav3 to form the RANK signaling complex independent of the TRAF6 ubiquitination pathway. TRAF6 is recruited to the RANKcyto mutant, which lacks T6BSs, via the Vav3 interaction; conversely, Vav3 is recruited to the RANKcyto mutant, which lacks the IVVY motif, via the TRAF6 interaction. Finally, we determined that the TRAF6-Vav3 interaction resulting from cross-talk between T6BSs and the IVVY motif in RANKcyto enhances downstream NF-κB, MAPK, and NFATc1 activation by further strengthening TRAF6 signaling, thereby inducing RANK-mediated osteoclastogenesis. Thus, Vav3 is a novel TRAF6 interaction partner that functions in the activation of cooperative signaling between T6BSs and the IVVY motif in the RANK signaling complex.

The tyrosine kinase Syk differentially regulates Toll-like receptor signaling downstream of the adaptor molecules TRAF6 and TRAF3.

Toll-like receptors (TLRs) are a major family of pattern recognition receptors, and they play a crucial role in innate immune responses. Activation of TLR4 signaling at the plasma membrane by its ligand lipopolysaccharide (LPS) stimulates a proinflammatory pathway dependent on the E3 ubiquitin ligase TRAF6 (tumor necrosis factor receptor-associated factor 6) and the kinase TAK1 (transforming growth factor β-activated kinase 1), whereas TLR4 signaling at endosomes stimulates the production of type I interferons (IFNs) through a pathway that depends on TRAF3 and the kinase TBK1 (TANK-binding kinase-1). We found that the nonreceptor tyrosine kinase Syk partially mediated the endocytosis of TLR4, but it also played a dual role in TLR4-mediated signaling. LPS-dependent stimulation of TLR4 in Syk-deficient macrophages led to enhanced activation of TAK1 and increased production of proinflammatory cytokines compared to that in wild-type macrophages. In contrast, Syk-deficient macrophages exhibited decreased TLR4-dependent activation of TBK1 signaling and production of type I IFNs. We found that Syk was present in both TRAF6- and TRAF3-containing signaling complexes; however, the LPS-dependent, lysine 63-linked ubiquitination of TRAF6 and TRAF3 was oppositely regulated by Syk. We identified the domains of Syk that interacted with TRAF3, TRAF6, TAK1, and TBK1, factors activated by multiple TLRs, which suggests that Syk may act as a common regulator of various TLR responses. Together, our results demonstrate the opposing regulatory roles of Syk in TLR-mediated TRAF6 and TRAF3 signaling pathways, which suggests that Syk may fine-tune the innate immune response to lessen inflammation.

Reciprocal Interaction between TRAF6 and Notch Signaling Regulates Adult Myofiber Regeneration upon Injury

Skeletal muscle is a postmitotic tissue that repairs and regenerates through activation of a population of stem-cell-like satellite cells. However, signaling mechanisms governing adult skeletal muscle regeneration remain less understood. In the present study, we have investigated the role of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), an adaptor protein involved in receptor-mediated activation of multiple signaling pathways in regeneration of adult myofibers. Skeletal muscle-specific depletion of TRAF6 in mice (TRAF6(mko)) improved regeneration of myofibers upon injury with a concomitant increase in the number of satellite cells and activation of the Notch signaling pathway. Ex vivo cultures of TRAF6(mko) myofiber explants demonstrated an increase in the proliferative capacity of myofiber-associated satellite cells accompanied by an upregulation of Notch ligands. Deletion of TRAF6 also inhibited the activity of transcription factor NF-κB and the expression of inflammatory cytokines and augmented the M2c macrophage phenotype in injured muscle tissues. Collectively, our study demonstrates that specific inhibition of TRAF6 improves satellite cell activation and skeletal muscle regeneration through upregulation of Notch signaling and reducing the inflammatory repertoire.

Mechanism of TRAF6 Intranuclear Translocation

Proper cellular response to external signals requires exact spatiotemporal positioning of intracellular components. Adaptor molecules are used to assemble protein complexes to facilitate signaling pathways. We studied the interplay between two adaptor molecules, tumor necrosis factor receptor associated factor‐6 (TRAF6) and Syntenin (Syn‐1). TRAF6 transduces signaling for the TNF and the Toll‐like/Interleukin receptor families. Many studies focused on cytoplasmic TRAF6, but few report on the inhibition of TRAF6 and the mechanisms by which it enters the nucleus. We find that overexpression of Syn‐1 not only attenuates TRAF6 signaling, but causes nuclear localization in both HEK293 and RAW246.7 cells. TRAF6 is an E3‐ligase whose activity we previously reported is maintained by auto‐ubiquitination and an open conformation. Knockdown of Syn‐1 potentiates TRAF6 activation and ubiquitination. Syn‐1 exhibits reduced effect on TRAF6 mutants that cannot be ubiquitinated. This implies that Syn‐1 binds to TRAF6 in a closed state. Protein domain deletions in both TRAF6 and Syn‐1 show that the full‐length molecules are not necessary for interaction. Mutagenesis or deletion of a newly identified TRAF Interaction motif (TIM) in Syn‐1 reveals differences in how Syn‐1 affects TRAF6 signaling and localization. Syn‐1 also localizes to the nucleus when overexpressed with either wild type or a constitutively active TRAF6. Lastly, Syn‐1 binds to the transcription factor Sox4 resulting in Sox4‐Syn‐1 relocalization to the nucleus. We have observed that overexpression of Sox4 also induces TRAF6 nuclear translocation, providing a mechanism for TRAF6 entry into the nucleus.

TRAF6-mediated regulation of the PI3 kinase (PI3K)–Akt–GSK3β cascade is required for TNF-induced cell survival

We recently demonstrated that the tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) helps maintenance of cell survival by regulating glycogen synthase kinase 3β (GSK3β) activity during TNF signaling. However, the molecular linkage between TRAF6 and GSK3β signaling is unknown. Herein, we showed that TRAF6 positively regulated cell survival by modulating PI3K–Akt–GSK3β cascades. In 3T3 cells lacking TRAF6, but not those lacking TRAF2, TNF stimulation led to prolonged hyperphosphorylation of Akt, which coincided with the activation of upstream PI3K. Pharmacologically blocking PI3K significantly inhibited Akt and GSK3β phosphorylation. Importantly, PI3K inhibition rescued cell death in TRAF6-null 3T3 cells. These data suggested TRAF6 regulates TNF-mediated cell survival through PI3K–Akt–GSK3β cascades.

Blockage of TRAF6 Signaling Inhibits Osteoclast Formation and Tumor Cell through Blocking the NF-kB and JNK Pathways in Multiple Myeloma.

We have recently shown that silencing of tumor necrosis factor receptor-associated factor 6 (TRAF6) with a C-terminal siRNA inhibits proliferation and increases apoptosis of multiple myeloma (MM) tumor cells. In addition, TRAF6 ubiquitin ligase is also essential for receptor activator of nuclear factor kappa B ligand (RANKL) signaling and osteoclast differentiation. Based on TRAF6, CD40, and RANKL sequences and the TRAF6 interaction domain with CD40 or RANKL resides between residues 333 to 508, we cloned a sequence representing a 167 amino acid sequence from this domain in order to produce a TRAF6 dominant negative fragment (TRAF6dn) from the NIH gene bank (U78798) into the PCRII-TOPO vector. Subsequently, we re-cloned this fragment into an expression vector (pLenti6.2-hTRAF6dn). Expression of the TRAF6 dominant negative peptide was confirmed by Western blot analysis. We used human MM monocytes isolated by anti-CD14 micro-bead affinity column from MM patients' peripheral blood (PB) or bone marrow (BM). In order to quantify osteoclast formation, the cells were fixed and stained for tartrate resistant acid phosphatase following seven days of culture. The BM and PB CD14+ cells were cultured on slide-culture dishes at a density of 2 × 105 cells per well. The cells infected with the pLenti6.2-hTRAF6dn or with the control vector, pLenti6.2/GW/EmGFP, were treated with 50ng/ml RANKL and 10ng/ml mCSF at the beginning of the culture period, and these factors were added again during a medium change after three days of incubation. We found that the TRAF6dn vector significantly inhibited osteoclast cell formation of CD14+ cells induced by RANKL and mCSF in a concentration dependent fashion compared with the control group. Furthermore, we examined c-Jun, a component of the transcription factor complex AP-1, which binds and activates transcription at TRE/AP-1 elements. The results showed that total endogenous c-Jun is reduced after TRAF6dn blocks TRAF6 signaling whereas cells infected with the control vector showed no changes in c-Jun. We further examined the effects of TRAF6dn on MM cell growth and apoptosis. Both tumor cells from fresh MM BM and the U266 and MM1s cell lines showed decreased cell proliferation and increased apoptosis in the presence of the TRAF6dn vector at 72 hours whereas the control vector had no effect on MM tumor cell growth or apoptosis. Furthermore, the TRAF6dn vector led to marked decreases in phospho-NF-kB protein levels compared to the control vector. Thus, we have demonstrated that inhibition of TRAF6 with a dominant negative construct both inhibits MM cell growth as well as osteoclast formation, and also reduces NF-kB activation and c-Jun levels which likely results in decreased activation of TRE/AP-1 elements. These studies suggest that the inhibition of TRAF6 may be an excellent therapeutic target for multiple myeloma since its inhibition results in suppression of tumor growth as well as osteoclast formation.

Site-specific Lys-63-linked Tumor Necrosis Factor Receptor-associated Factor 6 Auto-ubiquitination Is a Critical Determinant of IκB Kinase Activation

Tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) is a key mediator in proximal signaling of the interleukin-1/Toll-like receptor and the TNF receptor superfamily. Analysis of TRAF6-deficient mice revealed a fundamental role of TRAF6 in osteoclastogenesis; however, the molecular mechanism underlying TRAF6 signaling in this biological process is not understood. Recent biochemical evidence has indicated that TRAF6 possesses ubiquitin ligase activity that controls the activation of IKK and NF-kappaB. Because these studies are primarily based on cell-free systems, the role of the ubiquitin ligase activity of TRAF6 and its auto-ubiquitination to initiate the NF-kappaB pathway in vivo remain elusive. Here we show that an intact RING domain of TRAF6 in conjunction with the E2 enzyme Ubc13/Uev1A is necessary for Lys-63-linked auto-ubiquitination of TRAF6 and for its ability to activate IKK and NF-kappaB. Furthermore, a RING mutant of TRAF6 abolishes its ability to induce receptor activator of NF-kappaB-independent osteoclast differentiation and nuclear accumulation of the transcription factor NFATc1. Notably, we map the auto-ubiquitination site of TRAF6 to a single Lys residue, which if mutated renders TRAF6 unable to activate transforming growth factor-beta-activated kinase 1 and IKK and to cause spontaneous osteoclast differentiation. Additionally, we provide biochemical and in vivo evidence that TRAF6 serves as an E3 to directly ubiquitinate NEMO. Reconstituting TRAF6-deficent cells with various TRAF6 mutants, we clearly demonstrate the requirement for the TRAF6 RING domain and site-specific auto-ubiquitination of TRAF6 to activate IKK in response to interleukin-1. These data establish a signaling cascade in which regulated site-specific Lys-63-linked TRAF6 auto-ubiquitination is the critical upstream mediator of IKK.