Comparision of Drosophila and mammalian TRAF genes

The complete murine TRAF2 gene was obtained using a lambda phage and PCR cloning strategy. The gene was found to consist of ten coding exons and one 5' non-coding exon spread over 28 kbp of DNA. The basic structure of the human TRAF5 and TRAF6 genes obtained by analysis of the genomic DNA database is also reported. Comparison of these three gene structures, along with those previously described for TRAF1, TRAF3 and TRAF4, reveals the evolutionary relationship between the six known mammalian TRAFs. The TRAF1/TRAF2 and TRAF3/TRAF5 gene pairs arise from recent independent gene duplications and appear to share a common ancestral gene. Specific TRAF4 and TRAF6 precursor genes arose earlier during evolution, with the divergence of the TRAF6 precursor occuring earliest of all. The Drosophila genome was found to contain three TRAF family genes: Traf1, Traf2 and a previously undescribed member designated Traf3. TRAF-C domain homology indicates that Drosophila Traf3 is likely to have derived from the common precursor for the TRAF 1, 2, 3 and 5 genes, while Drosophila Traf1 and Traf6 have derived from the TRAF4 and TRAF6 precursor genes, respectively. The implication of these results for the functional evolution of TRAFs is discussed. Analysis is also presented of the conservation of the TRAF2A molecule, a TRAF2 alternate splice isoform with an extended RING finger domain previously described in mice. TRAF2A is not encoded by the human or rat TRAF2 genes and no other murine TRAF gene was found to produce a similar alternate splice product. The sequence of murine C57BL/6 TRAF4 differs significantly from the published murine TRAF4 sequence, but appears to represent the actual TRAF4 sequence expressed in many mouse strains (Grech, 2000).

Physical and functional interactions between Drosophila Traf2 and Pelle kinase contribute to Dorsal activation

Activation of NF-kappaB as a consequence of signaling through the Toll and IL-1 receptors is a major element of innate immune responses. A novel intermediate in these signaling pathways has been identified that bridges TRAF6 to MEKK-1. This adapter protein, which has been named ECSIT (evolutionarily conserved signaling intermediate in Toll pathways), is specific for the Toll/IL-1 pathways and is a regulator of MEKK-1 processing. Expression of wild-type ECSIT accelerates processing of MEKK-1, whereas a dominant-negative fragment of ECSIT blocks MEKK-1 processing and activation of NF-kappaB. These results indicate an important role for ECSIT in signaling to NF-kappaB and suggest that processing of MEKK-1 is required for its function in the Toll/IL-1 pathway (Kopp, 1999).

In an initial database analysis of the sequence of murine ECSIT, Drosophila ESTs homologous to this gene were found. Because the Toll signaling pathway is conserved in Drosophila, whether Drosophila ECSIT is involved in insect host defense responses was investigated. Drosophila ECSIT gene was cloned by reverse transcription-polymerase chain reaction (RT-PCR) using cDNA from Schneider insect cells. Untagged and Flag-tagged ECSIT wild-type DNAs were cloned into a Drosophila expression vector and Schneider cells were transfected for immunoprecipitation, transcription reporter assays, and RT-PCR analysis (Kopp, 1999).

Because ECSIT binds TRAF6 in vertebrate cells, the ability of Drosophila ECSIT to bind Traf2, the Drosophila homolog of Traf6, was assayed by immunoprecipitation. FLAG-tagged Drosophila ECSIT was cotransfected with V5 epitope-tagged Traf2 and subjected to immunoprecipitation with anti-Flag antibody. As expected, Drosophila ECSIT did interact with Traf2 in this assay, establishing dECSIT and Traf2 as conserved binding partners in a Drosophila system (Kopp, 1999).

To assay the role of Drosophila ECSIT in insect immunity, Schneider cells were transfected with ECSIT and the activation of a reporter gene containing the diptericin promoter linked to luciferase was assayed. This promoter responds to bacterial infection of Drosophila and contains Rel binding sites and can be activated by transfection of Drosophila ECSIT. The production of two antibacterial peptides, defensin and attacin, was assayed by RT-PCR. Transfection of Drosophila ECSIT induces the production of these peptides as efficiently as LPS or a dominant-active mutant of the Toll receptor (Kopp, 1999).

Signaling through the Toll receptor is required for dorsal/ventral polarity in Drosophila embryos, and also plays an evolutionarily conserved role in the immune response. Upon ligand binding, Toll appears to multimerize and activate the associated kinase, Pelle. However, the immediate downstream targets of Pelle have not been identified. Drosophila Tumor necrosis factor receptor-associated factor 2 (Traf2), a homolog of human TRAF6, physically and functionally interacts with Pelle, and is phosphorylated by Pelle in vitro. The N-terminal RING/zinc-finger domains of Traf2, but not the TRAF domain, interact strongly with both phosphorylated and unphosphorylated Pelle, and activate Dorsal in SL2 cells. Importantly, Traf2 and Pelle cooperate to activate Dorsal synergistically in cotransfected Schneider cells. Deletion of the C-terminal TRAF domain of Traf2 enhances Dorsal activation, perhaps reflecting the much stronger interaction of the mutant protein with phosphorylated, active Pelle. Taken together, these results indicate that Pelle and Traf2 physically and functionally interact, and that the TRAF domain acts as a regulator of this interaction. Traf2 thus appears to be a downstream target of Pelle (Shen, 2001).

Unlike the situation observed with mammalian TRAF6, the TRAF domain of Traf2 does not show a dominant-negative effect, but instead activates Dorsal to a level comparable to that observed with wild-type Traf2. It is possible that an interaction between the exogenously expressed TRAF domain and endogenous Traf2 may be responsible for this seemingly anomalous activation of signaling by the isolated TRAF domain. Mammalian TRAF domains have been shown to be involved in both homotypic and heterotypic aggregation. In TNF-induced NF-kappaB activation, the interaction between the TRAF6 N-terminal domain and zetaPKC (an atypical member of the protein kinase C family) is important for signaling. zetaPKC is unable to interact with the N-terminal domain of TRAF6 in its monomeric form, but dimerization of this domain dramatically stimulates interaction. Likewise, the interaction between the Traf2 N-terminal region and Pelle could be regulated by TRAF domain dimerization (Shen, 2001).

Traf2 is phosphorylated by activated Pelle in vitro. It is not clear how Traf2 functions in signaling to Dorsal, and whether phosphorylation is critical for its activity. Mammalian TRAF6 has been shown to interact with several downstream factors that appear to be required for NF-kappaB activation. Because it is unknown whether TRAF6 is phosphorylated by IRAK (or indeed phosphorylated at all), it is unclear how phosphorylation might affect these, or other, interactions. Likewise, additional studies are necessary to determine whether phosphorylated IRAK interacts with TRAF6, since Pelle is capable of binding Traf2. If so, TRAF6 could link active IRAK to other downstream targets. In any event, this study has provided evidence that a TRAF can be phosphorylated by an upstream kinase, which extends the possible mechanisms by which these molecules can modulate signaling pathways (Shen, 2001).

Traf2 is covalently modified when overexpressed in SL2 cells, and this modification requires the RING-finger domain. Such domains have been shown to be involved in protein ubiquitination, and indeed it may be that all RING-finger proteins may act as E3 ubiquitin ligases. The ladder-like modification of Traf2 that was detected in transfected cells is similar to the pattern expected for ubiquitination, and may target Traf2 and associated proteins (e.g., Pelle) for degradation. IRAK has been shown to be degraded by the proteasome after phosphorylation. Phosphorylated Pelle rapidly disappears from embryos in the 5th hour after egg laying, after completion of Toll signaling and Dorsal activation. It is conceivable that activated, phosphorylated Pelle may also be degraded by the ubiquitin-proteasome pathway by binding to Traf2, which would thus desensitize Pelle signaling after activation of downstream components. However, it is notable that recent studies have shown that TRAF6 is required for synthesis of an unusual polyubiquitin chain required for IKK activation, by a mechanism not involving degradation. Whether this observation is related to the apparent Traf2 self-ubiquitination detected here is not clear (Shen, 2001).

Drosophila Traf1 has also been shown to interact with Pelle in vitro, and to enhance NF-kappaB-mediated expression in mammalian cells cotransfected with Pelle. Unlike Traf2, which interacts with the Pelle catalytic domain and can synergize strongly with Pelle to activate Dorsal, Traf1 interacts with Pelle in its N-terminal death domain and gives rise only to weak activation of NF-kappaB. These results suggest that the two dTRAFs play distinct roles. Supporting this possibility, Traf1, but not Traf2, activates the JNK pathway by binding to the Ste20 kinase, Msn. In addition, microinjection of mRNA encoding Traf1Delta, which encodes a dominant-negative protein in transfections assays in mammalian cultured cells, into Drosophila embryos, fails to affect normal dorsal-ventral patterning. These results imply that the different downstream signaling events that activate Dorsal and JNK may bifurcate at the level of the Pelle-TRAF interaction (Shen, 2001 and references therein).

Two Drosophila tumor necrosis factor receptor-associated factors (TRAF), DTRAF1 and DTRAF2, are proposed to have functions similar to their mammalian counterparts as signal mediators of cell surface receptors. Traf1 is an in vivo regulator of c-Jun N-terminal kinase (JNK) pathway in Drosophila. Traf2 is an upstream activator of nuclear factor-kappaB (NF-kappaB). Ectopic expression of Traf2 induces nuclear translocation of two Drosophila NF-kappaBs, DIF and Relish, consequently activating the transcription of the antimicrobial peptide genes diptericin, diptericin-like protein, and drosomycin. Consistently, the null mutant of Traf2 shows immune deficiencies in which NF-kappaB nuclear translocation and antimicrobial gene transcription against microbial infection are severely impaired (Cha, 2003).

Microbial infection studies have demonstrated the ability of Drosophila to detect pathogens and activate specific signaling pathways, Toll or Imd pathways, which lead to adapted immune responses. In recent years, several families of antimicrobial peptides and their coding genes have been successfully identified: cecropins, attacins, diptericin, defensin, drosomycin, drosocin, and diptericin-like protein (dptlp). Understanding the molecular mechanisms underlying how microbial infection induces expression of these antimicrobial peptides has been the main question to answer in this field. Meanwhile, Traf2 have been identified as a downstream adaptor for Toll receptor (Shen, 2001) and Toll activation leads to immune responses. Therefore, it was suspected that DTRAFs would be involved in this defense mechanism (Cha, 2003).

Three representative antimicrobial genes, diptericin, dptlp, and drosomycin, were chosen as probes to determine the activity of the antimicrobial defense system. To examine whether Drosophila Traf1 and Traf2 have the ability to induce the transcription of diptericin, dptlp, and drosomycin, Traf1 or Traf2 was ectopically expressed in third-instar larvae by using hs-GAL4 driver, and the expression levels of diptericin, dptlp, and drosomycin were monitored by Northern blot analyses (Cha, 2003).

The transcription of diptericin, dptlp, and drosomycin was increased by ectopic expression of Traf2 in the absence of microbial infection. However, the expression levels of diptericin, dptlp, and drosomycin were not altered by Traf1 overexpression. In addition, Traf2-induced expression of diptericin and dptlp is completely inhibited in a relish (rel, Drosophila NF-kappaB)-null mutant background, whereas drosomycin expression is partially inhibited by the same mutation. The partial inhibition of the drosomycin expression by rel mutation suggests that the involvement of another Drosophila NF-kappaB, such as Dif, in antimicrobial response gene transcription. These results strongly suggest that Traf2, but not Traf1, functions downstream of microbial sensory receptors, Toll or Imd, and upstream of the NF-kappaBs to regulate Drosophila immune responses (Cha, 2003).

To further confirm the results, transgenic fly lines that have a GFP or a LacZ reporter gene fused to the drosomycin or the diptericin promoter, respectively, were used, allowing observation of the reporter gene activity, which reflects the drosomycin or diptericin gene expression level. The drosomycin-GFP reporter activity is dramatically increased in the microbe-infected larva compared to the uninfected control. As expected, Traf2 overexpression alone in the absence of microbial infection strongly induces drosomycin-GFP reporter gene activity. Further dissection analyses show that drosomycin-GFP and diptericin-LacZ reporter activities are highly induced in the fat body, which is a representative target tissue for immune responses in Drosophila. However, Traf1 overexpression fails to induce the reporter activities in both whole larvae and their fat bodies, further confirming the noninvolvement of Traf1 in the immune responses of Drosophila (Cha, 2003).

In order to confirm that the Traf2-induced immune responses are mediated by Dif and Relish, which are Drosophila NF-kappaBs specifically activated by Toll and Imd pathways, respectively, the subcellular localization of Dif and Relish was determened by using their specific antibodies (Cha, 2003).

Dif and Relish are dispersed in the cytoplasm of fat body cells in the absence of microbial infection. In contrast to this, either the microbial infection or overexpression of Traf2 fully induces the nuclear translocation of both Dif and Relish, demonstrating that both Dif and Relish participate in the Traf2-mediated immune responses. However, the subcellular localization of Dif and Relish is not altered by Traf1 induction, further confirming that Traf1 is not involved in the NF-kappaB signaling pathway. These data clearly demonstrated that Traf2, but not Traf1, has the capability to induce transcriptional activation of immune response genes by specifically activating NF-kappaBs (Cha, 2003).

The Traf2-null mutant, Traf2ex1, was generated by P-element excision method. RT-PCR analysis shows that the homozygous Traf2ex1 mutant fails to produce Traf2 mRNA. Intriguingly, the mutant flies manage to develop into adults and show no morphological defects. To determine whether the Traf2ex1 mutant shows a deficiency in immune responses, the transcriptional induction level of diptericin and drosomycin was examined after microbial infection. The null mutation of Traf2 drastically disrupts the transcriptional induction of diptericin and drosomycin when compared to the wild-type control. However, Traf1-null mutation (Traf1ex1) has no effect on the induction of diptericin and drosomycin gene expression after microbial infection. The nuclear translocation of Dif and Relish was examined in the Traf2-null mutant. Consistent with Northern blot analysis, the nuclear translocation of Dif and Relish induced by microbial infections is impaired in the Traf2ex1 mutant. These results support the position that Traf2, but not Traf1, is critical for the NF-kappaB-mediated Drosophila innate immune responses (Cha, 2003).

In the mammalian system, when interleukin-1 (IL-1) receptor, a Toll-like receptor, is stimulated by binding of its ligand, IL-1 receptor associated kinase (IRAK) is recruited to the IL-1 receptor complex and phosphorylated. Consequently, the receptor associated IRAK (Drosophila homolog: Pelle) binds to TRAF6, which evokes a strong activation of the NF-kappaB signaling pathway. The importance of TRAF6 in the activation of this pathway has been confirmed by various experiments. For example, overexpression of TRAF6 can lead to NF-kappaB activation, and a dominant-negative mutant of TRAF6 inhibits IL-1-induced NF-kappaB activation (Cha, 2003).

Between the two Drosophila homologs of mammalian TRAFs, the TRAF domain of Traf2 is most closely related to that of mammalian TRAF6. Based on this structural similarity, there have been reports that Traf2 contributes to dorsal activation and immune responses by activating NF-kappaB in a cell culture system (Kopp, 1999; Shen, 2001). Thus, in agreement with these results, it has been demonstrated that Traf2 can activate Drosophila NF-kappaBs and their downstream target genes diptericin, dptlp and drosomycin. Also, it has been suggested that Traf1 is involved in the NF-kappaB-mediated immune response (Zapata, 2000). However, in the present study, Traf1 does not induce NF-kappaB activation and the consequent NF-kappaB-dependent immune responses in vivo. These data suggest that Traf2 is a highly specific signal mediator activating the NF-kappaB signaling pathway (Cha, 2003).

Although overexpression of Traf2 is sufficient to activate the NF-kappaB signaling pathway and induce innate immune responses, the Traf2-null mutation could not completely block the processes. This suggests the presence of other signaling pathway(s) that bypass Traf2 to transmit the exogenous microbial signals to NF-kappaBs. Further studies with the Traf2-null mutant are required to elucidate the unknown signaling mechanism (Cha, 2003).

Bendless modulates JNK-mediated cell death and migration in Drosophila

The TNF-JNK pathway is a highly conserved signaling pathway that regulates a wide spectrum of biological processes including cell death and migration. To further delineate this pathway, a genetic screen was carried out for dominant modifiers of the cell death phenotype triggered by ectopic expression of Eiger (Egr), the Drosophila TNF ortholog. This study shows that Bendless (Ben), an E2 ubiquitin-conjugating enzyme, modulates Egr-induced JNK activation and cell death through dTRAF2. Furthermore, Ben physically interacts with dTRAF2 and regulates Egr-induced dTRAF2 polyubiquitination. Finally, Ben is required for JNK-dependent tumor progression, cell migration, oxidative stress resistance and longevity. These results indicate that Ben constitutes an essential component of the evolutionarily conserved TNF-JNK pathway that modulates cell death and invasion, tumor progression, stress response and lifespan in metazoans (Ma, 2013)

Interactions of TRAFs with receptors

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. Interactions between p75(NTR) and the six members of TRAF (tumor necrosis factor receptor-associated factors) family proteins are reported in this study. The binding of different TRAF proteins to p75(NTR) was mapped to distinct regions in p75(NTR). Furthermore, TRAF4 interacts with dimeric p75(NTR), whereas TRAF2 interacts preferentially with monomeric p75(NTR). TRAF2-p75(NTR), TRAF4-p75(NTR), and TRAF6-p75(NTR) interactions modulate p75(NTR)-induced cell death and NF-kappaB activation with contrasting effects. Coexpression of TRAF2 with p75(NTR) enhances cell death, whereas coexpression of TRAF6 is cytoprotective. Furthermore, overexpression of TRAF4 abrogates the ability of dimerization to prevent the induction of apoptosis normally mediated by monomeric p75(NTR). TRAF4 also inhibits the NF-kappaB response, whereas TRAF2 and TRAF6 enhance 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 (Yeh, 1999).

BCMA (B cell maturation, also known as tumor necrosis factor receptor superfamily, member 17) is a nonglycosylated integral membrane type I protein that is preferentially expressed in mature B lymphocytes. In a human malignant myeloma cell line, BCMA is not primarily present on the cell surface but lies in a perinuclear structure that partially overlaps the Golgi apparatus. In transiently or stably transfected cells, BCMA is located on the cell surface, as well as in a perinulear Golgi-like structure. Overexpression of BCMA in 293 cells activates NF-kappa B, Elk-1, the c-Jun N-terminal kinase, and the p38 mitogen-activated protein kinase. Coimmunoprecipitation experiments performed in transfected cells show that BCMA associates with TNFR-associated factor (TRAF) 1, TRAF2, and TRAF3 adaptor proteins. Analysis of deletion mutants of the intracytoplasmic tail of BCMA showed that the 25-aa protein segment, from position 119 to 143, conserved between mouse and human BCMA, is essential for its association with the TRAFs and the activation of NF-kappa B, Elk-1, and c-Jun N-terminal kinase. BCMA belongs structurally to the TNFR family. Its unique TNFR motif corresponds to a variant motif present in the fourth repeat of the TNFRI molecule. This study confirms that BCMA is a functional member of the TNFR superfamily. Furthermore, since BCMA is lacking a 'death domain' and its overexpression activates NF-kappa B and c-Jun N-terminal kinase, it can reasonably be hypothesized that upon binding of its corresponding ligand BCMA transduces signals for cell survival and proliferation (Hatzoglou, 2000).

TRAF structure and the specificity of TRAF signaling

TNF-induced activation of the transcription factor NF-kappaB and the c-jun N-terminal kinase (JNK/SAPK) requires TNF receptor-associated factor 2 (TRAF2). The NF-kappaB-inducing kinase (NIK) associates with TRAF2 and mediates TNF activation of NF-kappaB. NIK interacts with additional members of the TRAF family and this interaction requires the conserved 'WKI' motif within the TRAF domain. The role of NIK in JNK activation by TNF was examined. Whereas overexpression of NIK potently induces NF-kappaB activation, it fails to stimulate JNK activation. A kinase-inactive mutant of NIK is a dominant negative inhibitor of NF-kappaB activation but does not suppress TNF- or TRAF2-induced JNK activation. Thus, TRAF2 is the bifurcation point of two kinase cascades leading to activation of NF-kappaB and JNK, respectively (Song, 1997).

The diverse biological effects of the tumor necrosis factor (TNF) receptor superfamily are believed to be mediated in part through TNF receptor-associated factors (TRAFs), a family of cytoplasmic adaptor proteins that can activate intracellular signaling pathways, including the nuclear factor-kappaB (NF-kappaB) and c-Jun N-terminal kinase (JNK) pathways. TRAFs 2, 5, and 6 strongly activate both pathways when overexpressed; however, TRAF 3 (a close homolog of TRAF 5) does not significantly activate either pathway. The current study addresses the structural basis for this difference by substituting corresponding domains of TRAF 5 into TRAF 3 and testing activation of both pathways. A small region of TRAF 5 (the first zinc finger and 10 residues of the second zinc finger) is sufficient to convert TRAF 3 into an activator of both pathways. Also, an intact zinc ring finger is required for NF-kappaB activation but not JNK activation. In agreement with this finding, TRAF 2A, a TRAF 2 splice variant with an altered ring finger, is a specific activator of JNK. These findings suggest that different domains of TRAFs may be involved in NF-kappaB and JNK signaling. Also, alternative splicing of TRAFs may represent a novel mechanism whereby TNF family receptors can mediate distinct downstream effects in different tissues (Dadgostar, 1998).

Members of the tumor necrosis factor receptor family as well as other receptors achieve their diverse biological effects through the activation of intracellular signals including the c-Jun N-terminal kinase (JNK) pathway. Such signals are believed to be delivered through mediators known as TNF receptor-associated factors (TRAFs). Although the N-terminal zinc finger region of TRAFs has been shown to be essential for downstream signaling, there is no indication yet as to the nature of its role or of the factors that distinguish the N terminus of TRAF 3, which does not activate JNK in the systems examined thus far, from those of other TRAFs, which do activate this pathway. Among the known TRAFs, localization to the insoluble cell pellet fraction consistently correlates with JNK activation and both characteristics are shown to map to the TRAF N terminus. Furthermore, it is demonstrated that forced localization of TRAF 3 to the cell membrane is sufficient to convert this molecule into an activator of JNK. This suggests that one of the roles of the TRAF N terminus may be to participate in interactions that promote the recruitment of TRAFs to the membrane and that this localization effect plays an important role in TRAF-mediated JNK activation (Dadgostar, 2000).

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 can 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 retain 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), mediates NF-kappaB induction through a mechanism that is 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) does 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 (Leo, 1999).

Signalling downstream of TRAFs in the activation of NF-kappaB

TNF receptor-associated factor (TRAF) proteins are candidate signal transducers that associate with the cytoplasmic domains of members of the tumor necrosis factor (TNF) receptor superfamily. The role of TRAFs in the TNF-R2 and CD40 signal transduction pathways, resulting in the activation of transcription factor NF-kappaB, was investigated. Overexpression of TRAF2, but not TRAF1 or TRAF3, is sufficient to induce NF-kappaB activation. A truncated derivative of TRAF2 lacking an amino-terminal RING finger domain is a dominant-negative inhibitor of NF-kappa B activation mediated by TNF-R2 and CD40. Thus, TRAF2 is a common mediator of TNF-R2 and CD40 signaling (Rothe, 1995).

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are signal transducers for several members of the TNF receptor superfamily. A novel member of the TRAF family has been identified by degenerate oligonucleotide PCR amplification; the protein contains a zinc RING finger motif and zinc finger motif, a coiled-coil region, and a C-terminal 'TRAF' homology domain. In vitro translated TRAF5 binds to the cytoplasmic region of the lymphotoxin-beta receptor (LT-betaR) but not to several other related receptors, including CD40, both TNF receptors, Fas, and nerve growth factor receptor. TRAF5 and LT-betaR coimmunoprecipitate when overexpressed in COS7 cells. TRAF5 mRNA expression is found in all visceral organs and overlaps with LT-betaR. These features distinguish TRAF5 from the other members of the TRAF family. The transcription factor NF-kappaB is activated in HEK293 cells by overexpression of full-length TRAF5 but not a truncated form lacking the zinc binding region. Furthermore, overexpression of LT-betaR in HEK293 cells also results in activation of NF-kappaB, which is partially inhibited by the truncated TRAF5 mutant. These results show TRAF5 is functionally similar to TRAF2 in that both mediate activation NF-kappaB and implicate TRAF5 as a signal transducer for LT-betaR (Nakano, 1996).

Activation of NF-kappaB as a consequence of signaling through the Toll and IL-1 receptors is a major element of innate immune responses. A novel intermediate in these signaling pathways bridges TRAF6 to MEKK-1. This adapter protein, which has been named ECSIT (evolutionarily conserved signaling intermediate in Toll pathways: (see Drosophila ECSIT), is specific for the Toll/IL-1 pathways and is a regulator of MEKK-1 processing. Expression of wild-type ECSIT accelerates processing of MEKK-1, whereas a dominant-negative fragment of ECSIT blocks MEKK-1 processing and activation of NF-kappaB. These results indicate an important role for ECSIT in signaling to NF-kappaB and suggest that processing of MEKK-1 is required for its function in the Toll/IL-1 pathway (Carothers, 1999).

Signalling downstream of TRAFs in JNK pathway

CD40 activates nuclear factor kappa B (NF kappa B) and the mitogen-activated protein kinase (MAPK) subfamily, including extracellular signal-regulated kinase (ERK). The CD40 cytoplasmic tail interacts with tumor necrosis factor receptor-associated factor (TRAF)2, TRAF3, TRAF5, and TRAF6. These TRAF proteins, with the exception of TRAF3, are required for NF kappa B activation. Transient expression of TRAF6 stimulates both ERK and NF kappa B activity in the 293 cell line. Coexpression of the dominant-negative H-Ras does not affect TRAF6-mediated ERK activity, suggesting that TRAF6 may activate ERK along a Ras-independent pathway. The deletion mutant of TRAF6 lacking the NH2-terminal domain acts as a dominant-negative mutant to suppress ERK activation by full-length CD40. The deletion mutation also acts to suppress prominently ERK activation by a deletion mutant of CD40 containing only the binding site for TRAF6 in the cytoplasmic tail (CD40 delta 246). Transient expression of the dominant-negative H-Ras significantly suppresses ERK activation by full-length CD40, but marginally suppresses ERK activation by CD40 delta 246, compatible with the possibility that TRAF6 is a major transducer of ERK activation by CD40 delta 246, whose activity is mediated by a Ras-independent pathway. These results suggest that CD40 activates ERK by both a Ras-dependent pathway and a Ras-independent pathway in which TRAF6 could be involved (Kashiwada, 1998).

Tumor necrosis factor (TNF)-induced activation of the c-jun N-terminal kinase (JNK, also known as SAPK; stress-activated protein kinase) requires TNF receptor-associated factor 2 (TRAF2). The apoptosis signal-regulating kinase 1 (ASK1) is activated by TNF and stimulates JNK activation. ASK1 interacts with members of the TRAF family and is activated by TRAF2, TRAF5, and TRAF6 overexpression. A truncated derivative of TRAF2, which inhibits JNK activation by TNF, blocks TNF-induced ASK1 activation. A catalytically inactive mutant of ASK1 is a dominant-negative inhibitor of TNF- and TRAF2-induced JNK activation. In untransfected mammalian cells, ASK1 rapidly associates with TRAF2 in a TNF-dependent manner. Thus, ASK1 is a mediator of TRAF2-induced JNK activation (Nishitoh, 1998).

Tumor necrosis factor-alpha (TNF), a major inflammatory cytokine, generates a wide variety of cellular responses via key cytoplasmic adaptor molecules named TNF receptor-associated factors (TRAFs). TRAF2, TRAF5 and TRAF6 associate with apoptosis signal-regulating kinase 1 (ASK1), and a catalytically-inactive ASK1 mutant blocks stress-activated protein kinase (SAPK)/Jun NH2-terminal kinase (JNK) activation by these TRAFs. A truncated derivative of TRAF2, which inhibits SAPK activation by TNF, blocks TNF-induced ASK1 activation. Furthermore, protection from TNF-induced cell death conferred by an ASK1 mutant is dependent upon TRAF2. Hence, ASK1 is a common mediator of TRAF-regulated SAPK and apoptosis signaling, and the TRAF2 - ASK1 connection completes the signaling cascade from TNF to SAPK/JNK activation (Hoeflich, 1999).

Interleukin-1 (IL-1) and tumor necrosis factor (TNF-alpha) stimulate transcription factors AP-1 and NF-kappaB through activation of the MAP kinases JNK and p38 and the IkappaB kinase (IKK), respectively. The TNF-alpha and IL-1 signals are transduced through TRAF2 and TRAF6, respectively. Overexpressed TRAF2 or TRAF6 activate JNK, p38, or IKK in the absence of extracellular stimulation. By replacing the carboxy-terminal TRAF domain of TRAF2 and TRAF6 with repeats of the immunophilin FKBP12, it has been demonstrated that their effector domains are composed of their amino-terminal Zn and RING fingers. Oligomerization of the TRAF2 effector domain results in specific binding to MEKK1, a protein kinase capable of JNK, p38, and IKK activation, and induction of TNF-alpha and IL-1 responsive genes. TNF-alpha also enhances the binding of native TRAF2 to MEKK1 and stimulates the kinase activity of the latter. Thus, TNF-alpha and IL-1 signaling is based on oligomerization of TRAF2 and TRAF6 leading to activation of effector kinases (Baud, 1999).

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. A TRAF-interacting molecule, TRAF-associated NF-kappaB activator (TANK), is involved in TRAF2-mediated NF-kappaB activation. TANK synergizes with TRAF2, TRAF5, and TRAF6 but not with TRAF3 in SAPK activation. TRAF2 and TANK individually form 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. These 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 activates endogenous GCKR in primary B cells, implicating GCK family proteins in CD40-mediated B-cell functions (Chin, 1999).

Transcriptional regulation of TRAFs

To understand how the TNF receptor-associated factor 1 (TRAF1) is transcriptionally regulated, in vitro DNA binding assays, promoter-reporter gene assays, and RNase protection assays were performed with the human TRAF1 gene. Binding of NF-kappaB to three of five putative binding sites within the human TRAF1 promoter was found in electrophoretic mobility shift assay studies, and analysis of TRAF1 gene promoter luciferase constructs confirmed the functional importance of these elements. Moreover, triggering of TNF-R1, CD40, and the interleukin-1 receptor results in transcription of the TRAF1 gene, whereas receptors that are not activators or only poor activators of NF-kappaB in HeLa cells failed to show a significant TRAF1 induction. Because it has been shown that members of the TRAF family are involved in activation of NF-kappaB and the c-Jun N-terminal kinase (JNK) by the interleukin-1 receptor and members of the TNF receptor superfamily, a role of TRAF1 in receptor cross-talk and/or feedback regulation of activated receptor signaling complexes can be suggested. In fact, TNF-induced activation of JNK is prolonged in transfectants overexpressing TRAF1, whereas overexpression of a deletion mutant of TRAF1 in which the N-terminal part had been replaced by the green fluorescent protein interfers with TNF-induced activation of NF-kappaB and JNK (Schwenzer, 1999).

Expression, regulation and signaling downstream of TRAF4

TRAF4 is observed throughout mouse embryogenesis, most notably during ontogenesis of the central (CNS) and peripheral (PNS) nervous system, and of nervous tissues of sensory organs. TRAF4 is preferentially expressed by post-mitotic undifferentiated neurons. Interestingly, TRAF4 remains expressed in the adult hippocampus and olfactory bulb, known to contain multipotential cells responsible for neoneurogenesis (Masson, 1998).

TRAF-4 was discovered because of its expression in breast cancers and is a member of the tumor necrosis factor (TNF) receptor-associated factor (TRAF) family of putative signal-transducing proteins. In vitro binding assays have demonstrated that TRAF-4 interacts with the cytosolic domain of the lymphotoxin-beta receptor (LT beta R) and weakly with the p75 nerve growth factor receptor (NGFR) but not with TNFR1, TNFR2, Fas, or CD40. Immunofluorescence analysis of TRAF-4 in transfected cells demonstrates localization to cytosol but not nucleus. Immunohistochemical assays of normal human adult tissues reveals prominent cytosolic immunostaining in thymic epithelial cells and lymph node dendritic cells but not in lymphocytes or thymocytes, paralleling the reported patterns of LT beta R expression. The basal cell layer of most epithelia in the body was very strongly TRAF-4 immunopositive, including epidermis, nasopharynx, respiratory tract, salivary gland, and esophagus. Similar findings were obtained in 12- to 18-week human fetal tissue, indicating a highly restricted pattern of expression: even during development in the mammary gland, epithelial cells of the terminal ducts were strongly TRAF-4 immunopositive whereas myoepithelial cells and most of the mammary epithelial cells lining the extralobular ducts were TRAF-4 immunonegative. Of 84 primary breast cancers evaluated, only 7 expressed TRAF-4. Ductal carcinoma in situ (DCIS) lesions were uniformly TRAF-4 immunonegative. In the prostate, the basal cells were strongly immunostained for TRAF-4, whereas the secretory epithelial cells were TRAF-4 negative. Basal cells in prostate hypertrophy and prostatic intraepithelial neoplasia were strongly TRAF-4 positive, but none of the 32 primary and 16 metastatic prostate cancer specimens examined contained TRAF-4-positive malignant cells. Although TRAF-4 is also expressed in some types of mesenchymal cells, these findings suggest that TRAF-4 is a marker of normal epithelial stem cells, the expression of which often ceases on differentiation and malignant transformation (Krajewska, 1998).

TRAF4 is one of six identified members of the family of TNFR-associated factors. While the other family members have been found to play important roles in the development and maintenance of a normal immune system, the importance of TRAF4 has remained unclear. To address this issue, TRAF4-deficient mice were generated. Despite widespread expression of TRAF4 in the developing embryo, as well as in the adult, lack of TRAF4 expression results in a localized, developmental defect of the upper respiratory tract. TRAF4-deficient mice are born with a constricted upper trachea at the site of the tracheal junction with the larynx. This narrowing of the proximal end of the trachea results in respiratory air flow abnormalities and increases rates of pulmonary inflammation. These data demonstrate that TRAF4 is required to regulate the anastomosis of the upper and lower respiratory systems during development (Shiels, 2000).

To gain insight in the subcellular localization of tumor necrosis factor receptor-associated factor (TRAF4), GFP chimeras were examined of full-length TRAF4 and various deletion mutants derived thereof. While TRAF4-GFP (T4-GFP) is clearly localized in the cytoplasm, the N-terminal deletion mutant, T4(259-470), comprising the TRAF domain of the molecule, and a C-terminal deletion mutant consisting mainly of the RING and zinc finger domains of TRAF4 are both localized predominantly to the nucleus. Passive nuclear localization of T4(259-470) can be ruled out since the TRAF domain of TRAF4 is sufficient to form high molecular weight complexes. T4(259-470) recruits full-length TRAF4 into the nucleus whereas TRAF4 is unable to change the nuclear localization of T4(259-470). Thus, it seems that individual T4(259-470) mutant molecules are sufficient to direct the respective TRAF4-T4(259-470) heteromeric complexes into the nucleus. In cells forming cell-cell contacts, TRAF4 is recruited to the sites of contact via its C-TRAF domain. The expression of some TRAF proteins is regulated by the NF-kappaB pathway. Thus, whether this pathway is also involved in the regulation of the TRAF4 gene was investigated. Indeed, in primary T-cells and Jurkat cells stimulated with the NF-kappaB inducers TNF or phorbol 12-myristate 13-acetate (PMA), TRAF4-mRNA is rapidly up-regulated. In Jurkat T-cells deficient for I-kappaB kinase gamma (IKKgamma, also known as NEMO), an essential component of the NF-kappaB-inducing-IKK complex, induction of TRAF4, is completely inhibited. In cells deficient for RIP (receptor interactive protein), an essential signaling intermediate of TNF-dependent NF-kappaB activation, TNF-induced (but not PMA-induced) up-regulation of TRAF4 is blocked. These data suggest that activation of the NF-kappaB pathway is involved in up-regulation of TRAF4 in T-cells (Glauner, 2002).

p70S6K is an intracellular serine/threonine kinase that mediates cell cycle progression and gene transcription. Immunofluorescent staining shows in factor-dependent hematopoietic M-07e cells that p70S6K is localized both in the cytosol and, after cytokine stimulation, also in the nucleus. It is hypothesized that the p70S6K might interact with a transcription factor in the nucleus or with other proteins in the cytosol, in addition to the S6 protein. By screening a yeast two-hybrid HeLa cDNA library with full-length p70S6K cDNA as bait, tumor necrosis factor receptor-associated factor (TRAF) 4 was identified as a new binding partner for this kinase. TRAF4 is a member of the TRAF family of putative signal-transducing proteins. Members of this family are capable of negatively regulating apoptotic pathways by inducing the expression of genes that promote cell survival. Immunoprecipitation experiments showed that stimulation of receptors of the tumor necrosis factor (TNF) family induces the formation of TRAF4/p70S6K complexes. Transfection studies showed that TRAF4 functions in p70S6K activation: TNF induces phosphorylation of S6, the main intracellular substrate of the kinase, in cells stably expressing TRAF4, but not in TRAF4-negative cells. In addition to its role in p70S6K activation, an anti-apoptotic role for TRAF4 is postulated, because the agonistic anti-Fas antibody CH-11 induces apoptosis in untransfected HEK-293 cells, but not in TRAF4-expressing HEK-293 cells. In conclusion: (1) TNF-receptor activation leads to activation of the p70S6K; (2) TRAF4 is a mediator in this TNF-induced signaling pathway, and (3) TRAF4 inhibits Fas-induced apoptosis (Fleckenstein, 2003).

TRAF4 belongs to the tumor necrosis factor receptor-associated factor (TRAF) family of proteins but, unlike other family members, has not yet been clearly associated to any specific receptor or signaling pathway. To investigate the biological function of TRAF4, traf4-deficient mice were generated by gene disruption. The traf4 gene mutation is embryonic lethal but with great individual variation, since approximately one third of the homozygous mutant embryos die in utero around embryonic day 14, whereas the others reach adulthood. Surviving mutant mice manifest numerous developmental abnormalities, notably, 100% of homozygous mutant mice suffer respiratory disorder and wheezing caused by tracheal ring disruption. Additional malformations concern mainly the axial skeleton: the ribs, sternum, tail, and vertebral arches are affected, with various degrees of penetrance. Traf4-deficient mice also exhibit a high incidence of spina bifida, a defect likened to neural tube defects (NTD) that are common congenital malformations in humans. Altogether, these results demonstrate that TRAF4 is required during embryogenesis in key biological processes including the formation of the trachea, the development of the axial skeleton, and the closure of the neural tube. Considering the normal expression pattern of TRAF4 in neural tissues, it is concluded that TRAF4 participates in neurulation in vivo (Regnier, 2002).

The role of p53 in tumor suppression partly relies on its ability to transcriptionally regulate target genes involved in the initiation of cell cycle arrest or the activation of programmed cell death. In recent years many genes have been identified as p53-regulated genes; however, no single target gene has been shown to be required for the full apoptotic effect. TRAF4 has been identified as a p53-regulated gene in a microarray screen using a Murine 11K Affymetrix GeneChip hybridized with cRNA from the p53 temperature-sensitive cell line, Vm10. TRAF4 is a member the TRAF family of adaptor proteins that mediate cellular signaling by binding to various members of the tumor necrosis family receptor superfamily and interleukin-1/Toll-like receptor super-family. In contrast to its other family members, TRAF4 has not been shown to bind to a member of the tumor necrosis factor receptor superfamily in vivo, nor has it been shown to regulate signaling pathways common to its other family members. Therefore the role of TRAF4 in a signaling pathway has not yet been established and requires further study. TRAF4 is specifically regulated by p53 in response to temperature sensitive p53, overexpression of p53 by use of an adenovirus, and stabilization of p53 in response to DNA damage. The murine TRAF4 promoter contains a functional p53 DNA-binding site approximately 1 kilobase upstream of the initiating methionine. TRAF4 localizes to the cytoplasm and appears to remain in the cytoplasm following DNA damage. Interestingly, the overexpression of TRAF4 induces apoptosis and suppresses colony formation. These data suggest a correlation that the orphan adaptor protein TRAF4 may play a role in p53-mediated proapoptotic signaling in the response to cellular stress (Sax, 2003).

The angiogenic factors TNFalpha and HIV-1 Tat activate an NAD(P)H oxidase in endothelial cells, which operates upstream of c-Jun N-terminal kinase (JNK), a MAPK involved in the determination of cell fate. To further understand oxidant-related signaling pathways, lung and endothelial cell libraries were screened for interaction partners of p47(phox), and the orphan adapter TNF receptor-associated factor 4 (TRAF4) was recovered. Domain analysis suggests a tail-to-tail interaction between the C terminus of p47(phox) and the conserved TRAF domain of TRAF4. In addition, TRAF4, like p47(phox), is recovered largely in the cytoskeleton/membrane fraction. Coexpression of p47(phox) and TRAF4 increases oxidant production and JNK activation, whereas each alone had minimal effect. In addition, a fusion between p47(phox) and the TRAF4 C terminus constitutively activates JNK, and this activation is decreased by the antioxidant N-acetyl cysteine. In contrast, overexpression of the p47(phox) binding domain of TRAF4 blocks endothelial cell JNK activation by TNFalpha and HIV-1 Tat, suggesting an uncoupling of p47(phox) from upstream signaling events. A secondary screen of endothelial cell proteins for TRAF4-interacting partners yields a number of proteins known to control cell fate. It is concluded that endothelial cell agonists such as TNFalpha and HIV-1 Tat initiate signals that enter basic signaling cassettes at the level of TRAF4 and an NAD(P)H oxidase. It is speculated that endothelial cells may target endogenous oxidant production to specific sites critical to cytokine signaling as a mechanism for increasing signal specificity and decreasing toxicity of these reactive species (Xu, 2002).

TNF-receptor-associated factor 1: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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