Expanding TRAF function: TRAF3 as a tri-faced immune regulator

Tumour necrosis factor receptor (TNFR)-associated factor (TRAF) proteins are essential components of signalling pathways activated by TNFR or Toll-like receptor (TLR) family members. Acting alone or in combination, the seven known TRAFs control many biological processes, including cytokine production and cell survival. The function of one TRAF in particular, TRAF3, remained elusive for many years. Recent work has revealed that TRAF3 is a highly versatile regulator that positively controls type I interferon production, but negatively regulates mitogen-activated protein kinase activation and alternative nuclear factor-κB signalling. In this Review, we discuss our current understanding of the role of TRAF3 in TNFR and TLR signalling pathways, and its role in disease.     

The Structure and Ubiquitin Binding Properties of TRAF RING Heterodimers

Tumour necrosis factor (TNF) receptor associated factor (TRAF) family members share a common domain architecture, but play non-redundant physiological roles in cell signalling. At the N terminus, most TRAFs have a RING domain, followed by a series of Zinc finger (ZF) domains. The RING domain of TRAF6 dimerizes, and the RING homodimer together with the first ZF assembles ubiquitin chains that form a platform which facilitates activation of downstream kinases. The RING dimer interface is conserved amongst TRAF proteins, suggesting that functional heterodimers could be possible. Here we report the structure of the TRAF5-TRAF6 RING heterodimer, which accounts for the stability of the heterodimer as well as its ability to assemble ubiquitin chains. We also show that the RING domain of TRAF6 heterodimerizes with TRAF3 and TRAF2, and demonstrate that the linker helix and first ZF of TRAF2 can cooperate with TRAF6 to promote chain assembly. Collectively our results suggest that TRAF RING homo- and hetero-dimers have the potential to bridge interaction of nearby TRAF trimers and modulate TRAF-mediated signalling.     

Smurf2 is a TRAF2 binding protein that triggers TNF-R2 ubiquitination and TNF-R2-induced JNK activation

TRAF2 plays a central role in TNF-induced signalling to NF-κB and JNK/p38 MAPK. To better understand the molecular mechanisms that mediate this dual function of TRAF2, we performed a yeast two-hybrid screening for TRAF2 interacting proteins using the Sos recruitment system. This resulted in the identification of the E3 ubiquitin ligase Smurf2 as a TRAF2 binding protein. TRAF2 overexpression was shown to trigger Smurf2 ubiquitination and the formation of a TNF-R2/Smurf2 complex. Smurf2 on its turn promoted TNF-R2 ubiquitination and the relocalization of TNF-R2 as well as TRAF2 to a detergent-insoluble cell fraction. This was associated with enhanced TNF-R2-induced JNK activation, whereas TNF-R2-induced NF-κB activation remained unaffected. These results suggest an important role for Smurf2 binding to TRAF2 in determining specific signalling outputs of TNF-R2.     

A diverse family of proteins containing tumor necrosis factor receptor-associated factor domains

We have identified three new tumor necrosis factor-receptor associated factor (TRAF) domain-containing proteins in humans using bioinformatics approaches, including: MUL, the product of the causative gene in Mulibrey Nanism syndrome; USP7 (HAUSP), an ubiquitin protease; and SPOP, a POZ domain-containing protein. Unlike classical TRAF family proteins involved in TNF family receptor (TNFR) signaling, the TRAF domains (TDs) of MUL, USP7, and SPOP are located near the NH(2) termini or central region of these proteins, rather than carboxyl end. MUL and USP7 are capable of binding in vitro via their TDs to all of the previously identified TRAF family proteins (TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, and TRAF6), whereas the TD of SPOP interacts weakly with TRAF1 and TRAF6 only. The TD of MUL also interacted with itself, whereas the TDs of USP7 and SPOP did not self-associate. Analysis of various MUL and USP7 mutants by transient transfection assays indicated that the TDs of these proteins are necessary and sufficient for suppressing NF-kappaB induction by TRAF2 and TRAF6 as well as certain TRAF-binding TNF family receptors. In contrast, the TD of SPOP did not inhibit NF-kappaB induction. Immunofluorescence confocal microscopy indicated that MUL localizes to cytosolic bodies, with targeting to these structures mediated by a RBCC tripartite domain within the MUL protein. USP7 localized predominantly to the nucleus, in a TD-dependent manner. Data base searches revealed multiple proteins containing TDs homologous to those found in MUL, USP7, and SPOP throughout eukaryotes, including yeast, protists, plants, invertebrates, and mammals, suggesting that this branch of the TD family arose from an ancient gene. We propose the moniker TEFs (TD-encompassing factors) for this large family of proteins.     

YopJ targets TRAF proteins to inhibit TLR-mediated NF-kappaB, MAPK and IRF3 signal transduction

The Yersinia pestis virulence factor YopJ is a potent inhibitor of the NF-kappaB and MAPK signalling pathways, however, its molecular mechanism and relevance to pathogenesis are the subject of much debate. In this report, we characterize the effects of this type III effector protein on bone fide signalling events downstream of Toll-like receptors (TLRs), critical sensors in innate immunity. YopJ inhibited TLR-mediated NF-kappaB and MAP kinase activation, as suggested by previous studies. In addition, induction of the TLR-mediated interferon response was blocked by YopJ, indicating that YopJ also inhibits IRF3 signalling. Examination of the NF-kappaB signalling pathway in detail suggested that YopJ acts at the level of TAK1 (MAP3K7) activation. Further studies revealed a YopJ-dependent decrease in the ubiquitination of TRAF3 and TRAF6. These data support the hypothesis that YopJ is a deubiquitinating protease that acts on TRAF proteins to prevent or remove the K63-polymerized ubiquitin conjugates required for signal transduction. Our data do not directly address the alternative hypothesis that YopJ is an acetyltransferase that acts on the activation loop of IKK and MKK proteins, but support the conclusion that the critical function of YopJ is to deubiquinate TRAF proteins.     

T3JAM, a novel protein that specifically interacts with TRAF3 and promotes the activation of JNK(1)

Previous studies suggest that localization of tumor necrosis factor receptor (TNFR)-associated factor (TRAF) family members is important for regulating their signal transduction. During a screen for TRAF3-associated proteins that potentially alter TRAF3 subcellular localization and enable signal transduction, we identified a novel protein, T3JAM (TRAF3-interacting Jun N-terminal kinase (JNK)-activating modulator). This protein associates specifically with TRAF3 but not other TRAF family members. Coexpression of T3JAM with TRAF3 recruits TRAF3 to the detergent-insoluble fraction. More importantly, T3JAM and TRAF3 synergistically activate JNK but not nuclear factor (NF)-kappaB. Our studies indicate that T3JAM may function as an adapter molecule that specifically regulates TRAF3-mediated JNK activation.     

MIP-T3, a novel protein linking tumor necrosis factor receptor-associated factor 3 to the microtubule network

In this study, we report the identification of a novel tumor necrosis factor receptor-associated factor 3 (TRAF3)-interacting protein designated MIP-T3. MIP-T3 is a 83-kDa protein with no significant homology to known mammalian proteins. MIP-T3 mRNA and TRAF3 mRNA are ubiquitously expressed, and TRAF3 is the only TRAF protein to interact with MIP-T3. The MIP-T3-TRAF3 interaction requires the coiled-coil TRAF-N domain of TRAF3. To our knowledge, this is the first case of a TRAF-binding protein that interacts with a single member of the TRAF family specifically through a TRAF-N coiled-coil domain. MIP-T3 binds to Taxol-stabilized microtubules and to tubulin in vitro, and MIP-T3 recruits TRAF3 to microtubules when both proteins are overexpressed in HeLa cells. In a 293 cell line stably expressing CD40, TRAF3 is released from the TRAF3.MIP-T3 complex and recruited to the CD40 receptor upon CD40 ligand stimulation. MIP-T3 may provide a novel mechanism in sequestering TRAF3 to the cytoskeletal network.     

HSP70 interacts with TRAF2 and differentially regulates TNFα signalling in human colon cancer cells

Members of tumour necrosis factor (TNF) family usually trigger both survival and apoptotic signals in various cell types. Heat shock proteins (HSPs) are conserved proteins implicated in protection of cells from stress stimuli. However, the mechanisms of HSPs in TNFα‐induced signalling pathway have not been fully elucidated. We report here that HSP70 over‐expression in human colon cancer cells can inhibit TNFα‐induced NFκB activation but promote TNFα‐induced activation of c‐Jun N‐terminal kinase (JNK) through interaction with TNF receptor (TNFR)‐associated factor 2 (TRAF2). We provide evidence that HSP70 over‐expression can sequester TRAF2 in detergent‐soluble fractions possibly through interacting with TRAF2, leading to reduced recruitment of receptor‐interacting protein (RIP1) and IκBα kinase (IKK) signalosome to the TNFR1–TRADD complex and inhibited NFκB activation after TNFα stimuli. In addition, we found that HSP70–TRAF2 interaction can promote TNFα‐induced JNK activation. Therefore, our study suggests that HSP70 may differentially regulate TNFα‐induced activation of NFκB and JNK through interaction with TRAF2, contributing to the pro‐apoptotic roles of HSP70 in TNFα‐induced apoptosis of human colon cancer cells.     

Epstein-Barr Virus-Encoded Latent Membrane Protein 1 Activates the JNK Pathway through Its Extreme C Terminus via a Mechanism Involving TRADD and TRAF2

The transforming Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) activates signalling on the NF-κB axis through two distinct domains in its cytoplasmic C terminus, namely, CTAR1 (amino acids [aa] 187 to 231) and CTAR2 (aa 351 to 386). The ability of CTAR1 to activate NF-κB appears to be attributable to the direct interaction of tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2), while recent work indicates that CTAR2-induced NF-κB is mediated through its association with TNF receptor-associated death domain (TRADD). LMP1 expression also results in activation of the c-Jun N-terminal kinase (JNK) (also known as stress-activated protein kinase) cascade, an effect which is mediated exclusively through CTAR2 and can be dissociated from NF-κB induction. The organization and signalling components involved in LMP1-induced JNK activation are not known. In this study we have dissected the extreme C terminus of LMP1 and have identified the last 8 aa of the protein (aa 378 to 386) as being important for JNK signalling. Using a series of fine mutants in which single amino acids between codons 379 and 386 were changed to glycine, we have found that mutations of Pro³⁷⁹, Glu³⁸¹, Ser³⁸³, or Tyr³⁸⁴ diminish the ability of LMP1 CTAR2 to engage JNK signalling. Interestingly, this region was also found to be essential for CTAR2-mediated NF-κB induction and coincides with the LMP1 amino acid sequences shown to bind TRADD. Furthermore, we have found that LMP1-mediated JNK activation is synergistically augmented by low levels of TRADD expression, suggesting that this adapter protein is critical for LMP1 signalling. TRAF2 is known to associate with TRADD, and expression of a dominant-negative N-terminal deletion TRAF2 mutant was found to partially inhibit LMP1-induced JNK activation in 293 cells. In addition, the TRAF2-interacting protein A20 blocked both LMP1-induced JNK and NF-κB activation, further implicating TRAF2 in these phenomena. While expression of a kinase-inactive mutated NF-κB-inducing kinase (NIK), a mitogen-activated protein kinase kinase kinase which also associates with TRAF2, impaired LMP1 signalling on the NF-κB axis, it did not inhibit LMP1-induced JNK activation, suggesting that these two pathways may bifurcate at the level of TRAF2. These data further define a role for TRADD and TRAF2 in JNK activation and confirm that LMP1 utilizes signalling mechanisms used by the TNF receptor/CD40 family to elicit its pleiotropic activities.     

TNFR-associated factor family protein expression in normal tissues and lymphoid malignancies

TNFR-associated factors (TRAFs) constitute a family of adapter proteins that associate with particular TNF family receptors. Humans and mice contain six TRAF genes, but little is known about their in vivo expression at the single cell level. The in vivo locations of TRAF1, TRAF2, TRAF5, and TRAF6 were determined in human and mouse tissues by immunohistochemistry. Striking diversity was observed in the patterns of immunostaining obtained for each TRAF family protein, suggesting their expression is independently regulated in a cell type-specific manner. Dynamic regulation of TRAFs was observed in cultured PBLs, where anti-CD3 Abs, mitogenic lectins, and ILs induced marked increases in the steady-state levels of TRAF1, TRAF2, TRAF5, and TRAF6. TRAF1 was also highly inducible by CD40 ligand in cultured germinal center B cells, whereas TRAF2, TRAF3, TRAF5, and TRAF6 were relatively unchanged. Analysis of 83 established human tumor cell lines by semiquantitative immunoblotting methods revealed tendencies of certain cancer types to express particular TRAFs. For example, expression of TRAF1 was highly restricted, with B cell lymphomas consistently expressing this TRAF family member. Consistent with results from tumor cell lines, immunohistochemical analysis of 232 non-Hodgkin lymphomas revealed TRAF1 overexpression in 112 (48%) cases. TRAF1 protein levels were also elevated in circulating B cell chronic lymphocytic leukemia specimens (n = 49) compared with normal peripheral blood B cells (p = 0.01), as determined by immunoblotting. These findings contribute to an improved understanding of the cell-specific roles of TRAFs in normal tissues and provide evidence of altered TRAF1 expression in lymphoid malignancies.     

TRAF7 potentiates MEKK3-induced AP1 and CHOP activation and induces apoptosis

The tumor necrosis factor receptor-associated factor (TRAF) protein family members are critically involved in activation of NF-kappaB, JNK, and p38 activation triggered by tumor necrosis factor (TNF) receptor family members and toll/interleukin-1 receptor (TIR)-containing receptors. TRAF proteins (except for TRAF1) contain an N-terminal RING finger domain that is essential for their functions. In this report, we identified a protein designated as TRAF7, which contains a RING finger domain and a zinc finger domain that are mostly conserved with those of TRAFs. TRAF7 also contains seven WD40 repeats at its C terminus. TRAF7 specifically interacted with MEKK3 and potentiated MEKK3-mediated AP1 and CHOP activation. Depletion of TRAF7 by antisense RNA inhibited MEKK3-mediated AP1 and CHOP activation. Moreover, overexpression of TRAF7 induced caspase-dependent apoptosis. Domain mapping experiments indicated that TRAF7 potentiated MEKK3-mediated AP1 and CHOP activation and induced apoptosis through distinct domains. Our studies identified a novel TRAF family member that is involved in MEKK3 signaling and apoptosis.     

TRAF family proteins link PKR with NF-kappa B activation

The double-stranded RNA (dsRNA)-dependent protein kinase PKR activates NF-kappa B via the I kappa B kinase (IKK) complex, but little is known about additional molecules that may be involved in this pathway. Analysis of the PKR sequence enabled us to identify two putative TRAF-interacting motifs. The viability of such an interaction was further suggested by computer modeling. Here, we present evidence of the colocalization and physical interaction between PKR and TRAF family proteins in vivo, as shown by immunoprecipitation and confocal microscopy experiments. This interaction is induced upon PKR dimerization. Most importantly, we show that the binding between PKR and TRAFs is functionally relevant, as observed by the absence of NF-kappa B activity upon PKR expression in cells genetically deficient in TRAF2 and TRAF5 or after expression of TRAF dominant negative molecules. On the basis of sequence information and mutational and computer docking analyses, we favored a TRAF-PKR interaction model in which the C-terminal domain of TRAF binds to a predicted TRAF interaction motif present in the PKR kinase domain. Altogether, our data suggest that TRAF family proteins are key components located downstream of PKR that have an important role in mediating activation of NF-kappa B by the dsRNA-dependent protein kinase.     

Siah ubiquitin ligase is structurally related to TRAF and modulates TNF-alpha signaling.

Members of the Siah (seven in absentia homolog) family of RING domain proteins are components of E3 ubiquitin ligase complexes that catalyze ubiquitination of proteins. We have determined the crystal structure of the substrate-binding domain (SBD) of murine Siah1a to 2.6 A resolution. The structure reveals that Siah is a dimeric protein and that the SBD adopts an eight-stranded beta-sandwich fold that is highly similar to the TRAF-C region of TRAF (TNF-receptor associated factor) proteins. The TRAF-C region interacts with TNF-alpha receptors and TNF-receptor associated death-domain (TRADD) proteins; however, our findings indicate that these interactions are unlikely to be mimicked by Siah. The Siah structure also reveals two novel zinc fingers in a region with sequence similarity to TRAF. We find that the Siah1a SBD potentiates TNF-alpha-mediated NF-kappa B activation. Therefore, Siah proteins share important similarities with the TRAF family of proteins, including their overall domain architecture, three-dimensional structure and functional activity.     

4-1BB and Ox40 Are Members of a Tumor Necrosis Factor (TNF)-Nerve Growth Factor Receptor Subfamily That Bind TNF Receptor-Associated Factors and Activate Nuclear Factor κB

Members of the tumor necrosis factor (TNF)-nerve growth factor (NGF) receptor family have been shown to be important costimulatory molecules for cellular activation. 4-1BB and Ox40 are two recently described members of this protein family which are expressed primarily on activated T cells. To gain insight into the signaling pathways employed by these factors, yeast two-hybrid library screens were performed with the cytoplasmic domains of 4-1BB and Ox40 as baits. TNF receptor-associated factor 2 (TRAF2) was identified as an interacting protein in both screens. The ability of both 4-1BB and Ox40 to interact with TRAF2 was confirmed in mammalian cells by coimmunoprecipitation studies. When the binding of the receptors to other TRAF proteins was investigated, 4-1BB and Ox40 displayed distinct binding patterns. While 4-1BB bound TRAF2 and TRAF1, Ox40 interacted with TRAF3 and TRAF2. Using deletion and alanine scanning analysis, we defined the elements in the cytoplasmic domains of both receptors that mediate these interactions. The 4-1BB receptor was found to have two independent stretches of acidic residues that can mediate association of the TRAF molecules. In contrast, a single TRAF binding domain was identified in the cytoplasmic tail of Ox40. The cytoplasmic domains of both receptors were shown to activate nuclear factor κB in a TRAF-dependent manner. Taken together, our results indicate that 4-1BB and Ox40 bind TRAF proteins to initiate a signaling cascade leading to activation of nuclear factor κB.     

SUMOylation Is Required for Optimal TRAF3 Signaling Capacity

TNF receptor–associated factors (TRAFs) are multifunctional adaptor proteins involved in temporal and spatial coordination of signals necessary for normal immune function. Here, we report that TRAF3, a TRAF family member with a key role in Toll-like and TNF family receptor signaling and suppressor of lymphomagenesis, is post-translationally modified by the small ubiquitin-related modifier (SUMO). Through yeast two-hybrid and co-immunoprecipitation assays we have identified Ubc9, the SUMO conjugating enzyme, as a novel TRAF3-interacting protein. We show that Ubc9-dependent SUMOylation of TRAF3 modulates optimal association with the CD40 receptor, thereby influencing TRAF3 degradation and non-canonical NF-κB activation upon CD40 triggering. Collectively, our findings describe a novel post-translational modification of a TRAF family member and reveal a link between SUMOylation and TRAF-mediated signal transduction.