Tumor necrosis factor receptor-associated factor (TRAF) 2 and its role in TNF signaling

Tumor necrosis factor (TNF) is the prototypic member of the TNF ligand family and has a key role in the regulation of inflammatory processes. TNF exerts its functions by interaction with the death domain-containing TNF-receptor 1 (TNF-R1) and the non-death domain-containing TNF-receptor 2 (TNF-R2), both members of a receptor family complementary to the TNF ligand family. Due to the prototypic features of the TNF receptors and their importance for the regulation of inflammation, the signal transduction mechanisms utilized by these receptors have been extensively studied. Several proteins that interact directly or indirectly with the cytoplasmic domains of TNF-R1 and TNF-R2 have been identified in the recent years giving ideas how these receptors are connected to the apoptotic pathway and the signaling cascades leading to activation of NF-kappaB and JNK. Of special interest are TNF receptor-associated factor (TRAF) 1 and 2, which defines a novel group of adaptor proteins involved in signal transduction by most members of the TNF receptor family, of IL-1 receptor and IL-17 receptor as well as some members of the TOLL-like receptor family. TRAF 2 is currently the best-characterized TRAF family member, having a key role in mediating TNF-R1-induced activation of NF-kappaB and JNK. Moreover, recent studies suggest that TRAF 2 represents an integration point for pro- and antiapoptotic signals. This review focuses on the molecular mechanisms that underlay signal initiation by TNF-R1 and TNF-R2, with particular consideration of the role of TRAF 2, and highlights the importance of this molecule for the integration of such antagonizing pathways as death induction and NF-kappaB-mediated surviving signals.     

Cutting edge: TNFR-associated factor (TRAF) 6 is essential for MyD88-dependent pathway but not toll/IL-1 receptor domain-containing adaptor-inducing IFN-beta (TRIF)-dependent pathway in TLR signaling

Signaling pathways from TLRs are mediated by the Toll/IL-1R (TIR) domain-containing adaptor molecules. TNF receptor-associated factor (TRAF) 6 is thought to activate NF-kappaB and MAPKs downstream of these TIR domain-containing proteins to induce production of inflammatory cytokines. However, the precise role of TRAF6 in signaling from individual TLRs has not been appropriately addressed. We analyzed macrophages from TRAF6-deficient mice and made the following observations. In the absence of TRAF6, 1) ligands for TLR2, TLR5, TLR7, and TLR9 failed to induce activation of NF-kappaB and MAPKs or production of inflammatory cytokines; 2) TLR4 ligand-induced cytokine production was remarkably reduced and activation of NF-kappaB and MAPKs was observed, albeit with delayed kinetics; and 3) in contrast with previously reported findings, TLR3 signaling was not affected. These results indicate that TRAF6 is essential for MyD88-dependent signaling but is not required for TIR domain-containing adaptor-inducing IFN-beta (TRIF)-dependent signaling.     

Differential requirements for tumor necrosis factor receptor-associated factor family proteins in CD40-mediated induction of NF-kappaB and Jun N-terminal kinase activation.

CD40 is a member of the tumor necrosis factor receptor family that mediates a number of important signaling events in B-lymphocytes and some other types of cells through interaction of its cytoplasmic (ct) domain with tumor necrosis factor receptor-associated factor (TRAF) proteins. Alanine substitution and truncation mutants of the human CD40ct domain were generated, revealing residues critical for binding TRAF2, TRAF3, or both of these proteins. In contrast to TRAF2 and TRAF3, direct binding of TRAF1, TRAF4, TRAF5, or TRAF6 to CD40 was not detected. However, TRAF5 could be recruited to wild-type CD40 in a TRAF3-dependent manner but not to a CD40 mutant (Q263A) that selectively fails to bind TRAF3. CD40 mutants with impaired binding to TRAF2, TRAF3, or both of these proteins completely retained the ability to activate NF-kappaB and Jun N-terminal kinase (JNK), implying that CD40 can stimulate TRAF2- and TRAF3-independent pathways for NF-kappaB and JNK activation. A carboxyl-truncation mutant of CD40 lacking the last 32 amino acids required for TRAF2 and TRAF3 binding, CD40(Delta32), mediated NF-kappaB induction through a mechanism that was suppressible by co-expression of TRAF6(DeltaN), a dominant-negative version of TRAF6, but not by TRAF2(DeltaN), implying that while TRAF6 does not directly bind CD40, it can participate in CD40 signaling. In contrast, TRAF6(DeltaN) did not impair JNK activation by CD40(Delta32). Taken together, these findings reveal redundancy in the involvement of TRAF family proteins in CD40-mediated NF-kappaB induction and suggest that the membrane-proximal region of CD40 may stimulate the JNK pathway through a TRAF-independent mechanism.     

Physical and functional interaction of filamin (actin-binding protein-280) and tumor necrosis factor receptor-associated factor 2

Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) is an intracellular protein involved in signal transduction from TNF receptor I and II and related receptors. TRAF2 is required for TNF-induced activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and TRAF2 can also mediate activation of NF-kappaB. Here we have identified the actin-binding protein Filamin (actin-binding protein-280) as a TRAF2-interacting protein. Filamin binds to the Ring zinc finger domain of TRAF2. Overexpressed Filamin inhibits TRAF2-induced activation of JNK/SAPK and of NF-kappaB. Furthermore, ectopically expressed Filamin inhibits NF-kappaB activation induced via TNF, interleukin-1, Toll receptors, and TRAF6 but not activation induced via overexpression of NIK, a downstream effector in these pathways. Importantly, TNF fails to activate SAPK or NF-kappaB in a human melanoma cell line deficient in Filamin. Reintroduction of Filamin into these cells restores the TNF response. The data imply a role for Filamin in inflammatory signal transduction pathways.     

TIRP,a novel Toll/interleukin-1 receptor (TIR) domain-containing adapter protein involved in TIR signaling

The Toll/interleukin-1 receptor (TIR) family members play important roles in host defense. These receptors signal through TIR domain-containing adapter proteins. In this report, we identified a novel TIR domain-containing adapter protein designated as TIRP. Co-immunoprecipitation experiments suggest that TIRP is associated with IL-1 receptors. TIRP also interacts with kinase-inactive mutants of IRAK and IRAK-4, IRAK-2, IRAK-M, and TRAF6. Overexpression of TIRP activates NF-kappaB and potentiates IL-1 receptor-mediated NF-kappaB activation. A dominant negative mutant of TIRP inhibits IL-1- but not tumor necrosis factor-triggered NF-kappaB activation. Moreover, TIRP-mediated NF-kappaB activation is inhibited by dominant negative mutants of IRAK, IRAK-2, TRAF6, and IKKbeta. Our findings suggest that TIRP is involved in IL-1-triggered NF-kappaB activation and functions upstream of IRAK, IRAK-2, TRAF6, and IKKbeta     

The Drosophila tumor necrosis factor receptor-associated factor-1 (DTRAF1) interacts with Pelle and regulates NFkappaB activity

A member of the tumor necrosis factor (TNF) receptor-associated factor (TRAF) family was identified in Drosophila. DTRAF1 contains 7 zinc finger domains followed by a TRAF domain, similar to mammalian TRAFs and other members of the family identified in data bases from Caenorhabditis elegans, Arabidopsis, and Dictyostelium. Analysis of DTRAF1 binding to different members of the human TNF receptor family showed that this protein can interact through its TRAF domain with the p75 neurotrophin receptor and weakly with the lymphotoxin-beta receptor. DTRAF1 can also self-associate and binds to human TRAF1, TRAF2, and TRAF4. Interestingly, DTRAF1 interacts with human cIAP-1 and cIAP-2 but not with Drosophila DIAP-1 and -2. By itself, DTRAF1 did not induce significant NFkappaB activation when overexpressed in mammalian cells, although it specifically increased NFkappaB induction by TRAF6. In contrast, TRAF2-mediated NFkappaB induction was partially inhibited by DTRAF1. Mutants of DTRAF1 lacking the N-terminal region inhibited NFkappaB induction by either TRAF2 or TRAF6. DTRAF1 specifically associated with the regulatory N-terminal domain of Pelle, a Drosophila homolog of the human kinase interleukin-1 receptor-associated kinase (IRAK). Interestingly, though Pelle and DTRAF1 individually were unable to induce NFkappaB in a human cell line, co-expression of Pelle and DTRAF1 resulted in significant NFkappaB activity. Interactions of DTRAF1 with human TRAF-, TNF receptor-, and IAP-family proteins imply strong evolutionary conservation of TRAF protein structure and function throughout Metazoan evolution.     

Caspase-mediated cleavage converts the tumor necrosis factor (TNF) receptor-associated factor (TRAF)-1 from a selective modulator of TNF receptor signaling to a general inhibitor of NF-kappaB activation

The role of tumor necrosis factor (TNF) receptor-associated factor (TRAF)-1 in NF-kappaB activation by various members of the TNF receptor family is not well understood, and conflicting data have been published. Here, we show that TRAF1 differentially affects TRAF2 recruitment and activation of NF-kappaB by members of the TNF receptor family. Interestingly, a naturally occurring caspase-derived cleavage product of TRAF1 solely comprising its TRAF domain (TRAF1-(164-416)) acted as a general inhibitor of NF-kappaB activation. In contrast, a corresponding fragment generated by cleavage of TRAF3 showed no effect in this regard. In accordance with these functional data, TRAF1, but not TRAF3, interacted with the IKK complex via its N-TRAF domain. Endogenous TRAF1 and the overexpressed TRAF domain of TRAF1 were found to be constitutively associated with the IKK complex, whereas endogenous receptor interacting protein was only transiently associated with the IKK complex upon TNF stimulation. Importantly, the caspase-generated TRAF1-fragment, but not TRAF1 itself inhibited IKK activation. Our results suggest that TRAF1 and TRAF1-(164-416) exert their regulatory effects on receptor-induced NF-kappaB activation not only by modulation of TRAF2 receptor interaction but especially TRAF1-(164-416) also by directly targeting the IKK complex.     

TANK Potentiates Tumor Necrosis Factor Receptor-Associated Factor-Mediated c-Jun N-Terminal Kinase/Stress-Activated Protein Kinase Activation through the Germinal Center Kinase Pathway

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are mediators of many members of the TNF receptor superfamily and can activate both the nuclear factor kappaB (NF-kappaB) and stress-activated protein kinase (SAPK; also known as c-Jun N-terminal kinase) signal transduction pathways. We previously described the involvement of a TRAF-interacting molecule, TRAF-associated NF-kappaB activator (TANK), in TRAF2-mediated NF-kappaB activation. Here we show that TANK synergized with TRAF2, TRAF5, and TRAF6 but not with TRAF3 in SAPK activation. TRAF2 and TANK individually formed weak interactions with germinal center kinase (GCK)-related kinase (GCKR). However, when coexpressed, they formed a strong complex with GCKR, thereby providing a potential mechanism for TRAF and TANK synergy in GCKR-mediated SAPK activation, which is important in TNF family receptor signaling. Our results also suggest that TANK can form potential intermolecular as well as intramolecular interactions between its amino terminus and carboxyl terminus. This study suggests that TANK is a regulatory molecule controlling the threshold of NF-kappaB and SAPK activities in response to activation of TNF receptors. In addition, CD40 activated endogenous GCKR in primary B cells, implicating GCK family proteins in CD40-mediated B-cell functions.     

TRAF family proteins interact with the common neurotrophin receptor and modulate apoptosis induction.

The common neurotrophin receptor, p75(NTR), has been shown to signal in the absence of Trk tyrosine kinase receptors, including induction of neural apoptosis and activation of NF-kappaB. However, the mechanisms by which p75(NTR) initiates these intracellular signal transduction pathways are unknown. Here we report interactions between p75(NTR) and the six members of TRAF (tumor necrosis factor receptor-associated factors) family proteins. The binding of different TRAF proteins to p75(NTR) was mapped to distinct regions in p75(NTR). Furthermore, TRAF4 interacted with dimeric p75(NTR), whereas TRAF2 interacted preferentially with monomeric p75(NTR). TRAF2-p75(NTR), TRAF4-p75(NTR), and TRAF6-p75(NTR) interactions modulated p75(NTR)-induced cell death and NF-kappaB activation with contrasting effects. Coexpression of TRAF2 with p75(NTR) enhanced cell death, whereas coexpression of TRAF6 was cytoprotective. Furthermore, overexpression of TRAF4 abrogated the ability of dimerization to prevent the induction of apoptosis normally mediated by monomeric p75(NTR). TRAF4 also inhibited the NF-kappaB response, whereas TRAF2 and TRAF6 enhanced p75(NTR)-induced NF-kappaB activation. These results demonstrate that TRAF family proteins interact with p75(NTR) and differentially modulate its NF-kappaB activation and cell death induction.     

Ubiquitin-specific protease 4 (USP4) targets TRAF2 and TRAF6 for deubiquitination and inhibits TNFα-induced cancer cell migration

TRAF [TNF (tumour necrosis factor)-receptor-associated factor] 2 and 6 are essential adaptor proteins for the NF-κB (nuclear factor κB) signalling pathway, which play important roles in inflammation and immune response. Polyubiquitination of TRAF2 and TRAF6 is critical to their activities and functions in TNFα- and IL (interleukin)-1β-induced NF-κB activation. However, the regulation of TRAF2 and TRAF6 by deubiquitination remains incompletely understood. In the present study, we identified USP (ubiquitin-specific protease) 4 as a novel deubiquitinase targeting TRAF2 and TRAF6 for deubiquitination. We found that USP4 specifically interacts with TRAF2 and TRAF6, but not TRAF3. Moreover, USP4 associates with TRAF6 both in vitro and in vivo, independent of its deubiquitinase activity. The USP domain is responsible for USP4 to interact with TRAF6. Ectopic expression of USP4 inhibits the TRAF2- and TRAF6-stimulated NF-κB reporter gene and negatively regulates the TNFα-induced IκBα (inhibitor of NF-κBα) degradation and NF-κB activation. Knockdown of USP4 significantly increased TNFα-induced cytokine expression. Furthermore, we found that USP4 deubiquitinates both TRAF2 and TRAF6 in vivo and in vitro in a deubiquitinase activity-dependent manner. Importantly, the results of the present study showed that USP4 is a negative regulator of TNFα- and IL-1β-induced cancer cell migration. Taken together, the present study provides a novel insight into the regulation of the NF-κB signalling pathway and uncovers a previously unknown function of USP4 in cancer.     

Modulation of life and death by the tumor necrosis factor receptor-associated factors (TRAFs)

The TNF receptor-associated factor (TRAF) family is a group of adapter proteins that link a wide variety of cell surface receptors. Including the TNF and IL-1 receptor superfamily to diverse signaling cascades, which lead to the activation of NF-kappaB and mitogen-activated protein kinases. In addition, TRAFs interact with a variety of proteins that regulate receptor-induced cell death or survival. Thus, TRAF-mediated signals may directly induce cell survival or interfere with the death receptor-induced apoptosis.     

Tumor necrosis factor receptor-associated factor 2 (TRAF2)-deficient B lymphocytes reveal novel roles for TRAF2 in CD40 signaling

CD40 function is initiated by tumor necrosis factor (TNF) receptor-associated factor (TRAF) adapter proteins, which play important roles in signaling by numerous receptors. Characterizing roles of individual TRAFs has been hampered by limitations of available experimental models and the poor viability of most TRAF-deficient mice. Here, B cell lines made deficient in TRAF2 using a novel homologous recombination system reveal new roles for TRAF2. We demonstrate that TRAF2 participates in synergy between CD40 and B cell antigen receptor signals, and in CD40-mediated, TNF-dependent IgM production. We also find that TRAF2 participates in the degradation of TRAF3 associated with CD40 signaling, a role that may limit inhibitory actions of TRAF3. Finally, we show that TRAF2 and TRAF6 have overlapping functions in CD40-mediated NF-kappaB activation and CD80 up-regulation. These findings demonstrate previously unappreciated roles for TRAF2 in signaling by TNF receptor family members, using an approach that facilitates the analysis of genes critical to the viability of whole organisms.     

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.