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.     

Tumor necrosis factor receptor-associated factor (TRAF) 1 regulates CD40-induced TRAF2-mediated NF-kappaB activation

To investigate CD40 signaling complex formation in living cells, we used green fluorescent protein (GFP)-tagged CD40 signaling intermediates and confocal life imaging. The majority of cytoplasmic TRAF2-GFP and, to a lesser extent, TRAF3-GFP, but not TRAF1-GFP or TRAF4-GFP, translocated into CD40 signaling complexes within a few minutes after CD40 triggering with the CD40 ligand. The inhibitor of apoptosis proteins cIAP1 and cIAP2 were also recruited by TRAF2 to sites of CD40 signaling. An excess of TRAF2 allowed recruitment of TRAF1-GFP to sites of CD40 signaling, whereas an excess of TRAF1 abrogated the interaction of TRAF2 and CD40. Overexpression of TRAF1, however, had no effect on the interaction of TRADD and TRAF2, known to be important for tumor necrosis factor receptor 1 (TNF-R1)-mediated NF-kappaB activation. Accordingly, TRAF1 inhibited CD40-dependent but not TNF-R1-dependent NF-kappaB activation. Moreover, down-regulation of TRAF1 with small interfering RNAs enhanced CD40/CD40 ligand-induced NF-kappaB activation but showed no effect on TNF signaling. Because of the trimeric organization of TRAF proteins, we propose that the stoichiometry of TRAF1-TRAF2 heteromeric complexes ((TRAF2)2-TRAF1 versus TRAF2-(TRAF1)2) determines their capability to mediate CD40 signaling but has no major effect on TNF signaling.     

A novel mechanism of TRAF signaling revealed by structural and functional analyses of the TRADD-TRAF2 interaction

TRAF proteins are major mediators for the cell activation, cell survival, and antiapoptotic functions of the TNF receptor superfamily. They can be recruited to activated TNF receptors either by direct interactions with the receptors or indirectly via the adaptor protein TRADD. We now report the structure of the TRADD-TRAF2 complex, which is highly distinct from receptor-TRAF2 interactions. This interaction is significantly stronger and we show by an in vivo signaling assay that TRAF2 signaling is more readily initiated by TRADD than by direct receptor-TRAF2 interactions. TRADD is specific for TRAF1 and TRAF2, which ensures the recruitment of clAPs for the direct inhibition of caspase activation in the signaling complex. The stronger affinity and unique specificity of the TRADD-TRAF2 interaction are crucial for the suppression of apoptosis and provide a mechanistic basis for the perturbation of TRAF recruitment in sensitizing cell death induction.     

TRAF1 is a substrate of caspases activated during tumor necrosis factor receptor-alpha-induced apoptosis

TRAF family proteins are signal-transducing adapter proteins that interact with the cytosolic domains of tumor necrosis factor (TNF) family receptors. Here we show that TRAF1 (but not TRAF2-6) is cleaved by certain caspases in vitro and during TNF-alpha- and Fas-induced apoptosis in vivo. (160)LEVD(163) was identified as the caspase cleavage site within TRAF1, generating two distinct fragments. Significant enhancement of TNF receptor-1 (CD120a)- and, to a lesser extent, Fas (CD95)-mediated apoptosis was observed when overexpressing the C-terminal TRAF1 fragment in HEK293T and HT1080 cells. The same fragment was capable of potently suppressing TNF receptor-1- and TRAF2-mediated nuclear factor-kappaB activation in reporter gene assays, providing a potential mechanism for the enhancement of TNF-mediated apoptosis. Cell death induced by other death receptor-independent stimuli such as cisplatin, staurosporine, and UV irradiation did not result in cleavage of TRAF1, and overexpression of the C-terminal TRAF1 fragment did not enhance cell death in these cases. TRAF1 cleavage was markedly reduced in cells that contain little procaspase-8 protein, suggesting that this apical protease in the TNF/Fas death receptor pathway is largely responsible. These data identify TRAF1 as a specific target of caspases activated during TNF- and Fas-induced apoptosis and illustrate differences in the repertoire of protease substrates cleaved during activation of different apoptotic pathways.     

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.     

Tumor necrosis factor alpha-induced activation of c-jun N-terminal kinase is mediated by TRAF2.

Tumor necrosis factor alpha (TNF alpha) a pro-inflammatory cytokine is an endogenous mediator of septic shock, inflammation, anti-viral responses and apoptotic cell death. TNF alpha elicits its complex biological responses through the individual or cooperative action of two TNF receptors of mol. wt 55 kDa (TNF-RI) and mol. wt 75 kDa (TNF-RII). To determine signaling events specific for TNF-RII we fused the extracellular domain of the mouse CD4 antigen to the intracellular domain of TNF-RII. Crosslinking of the chimeric receptor using anti-CD4 antibodies initiates exclusively TNF-RII-mediated signals. Our findings show that: (i) TNF-RII is able to activate two members of the MAP kinase family: extracellular regulated kinase (ERK) and c-jun N-terminal kinase (JNK); (ii) TRAF2, a molecule that binds TNF-RII and associates indirectly with TNF-RI, is sufficient to activate JNK upon overexpression; (iii) dominant-negative TRAF2 blocks TNF alpha-mediated JNK activation and (iv) TRAF2 signals the activation of JNK and NF-kappaB through different pathways. Our findings suggest that TNF alpha-mediated JNK activation in fibroblasts is independent of the cell death pathway and that TRAF2 occupies a key role in TNF receptor signaling to JNK.     

Fas-associated death domain protein and caspase-8 are not recruited to the tumor necrosis factor receptor 1 signaling complex during tumor necrosis factor-induced apoptosis

Death receptors are a subfamily of the tumor necrosis factor (TNF) receptor subfamily. They are characterized by a death domain (DD) motif within their intracellular domain, which is required for the induction of apoptosis. Fas-associated death domain protein (FADD) is reported to be the universal adaptor used by death receptors to recruit and activate the initiator caspase-8. CD95, TNF-related apoptosis-inducing ligand (TRAIL-R1), and TRAIL-R2 bind FADD directly, whereas recruitment to TNF-R1 is indirect through another adaptor TNF receptor-associated death domain protein (TRADD). TRADD also binds two other adaptors receptor-interacting protein (RIP) and TNF-receptor-associated factor 2 (TRAF2), which are required for TNF-induced NF-kappaB and c-Jun N-terminal kinase activation, respectively. Analysis of the native TNF signaling complex revealed the recruitment of RIP, TRADD, and TRAF2 but not FADD or caspase-8. TNF failed to induce apoptosis in FADD- and caspase-8-deficient Jurkat cells, indicating that these apoptotic mediators were required for TNF-induced apoptosis. In an in vitro binding assay, the intracellular domain of TNF-R1 bound TRADD, RIP, and TRAF2 but did not bind FADD or caspase-8. Under the same conditions, the intracellular domain of both CD95 and TRAIL-R2 bound both FADD and caspase-8. Taken together these results suggest that apoptosis signaling by TNF is distinct from that induced by CD95 and TRAIL. Although caspase-8 and FADD are obligatory for TNF-mediated apoptosis, they are not recruited to a TNF-induced membrane-bound receptor signaling complex as occurs during CD95 or TRAIL signaling, but instead must be activated elsewhere within the cell.     

TNF signaling: early events and phosphorylation

Tumor necrosis factor-alpha (TNF) is a major mediator of apoptosis as well as immunity and inflammation. Inappropriate production of TNF or sustained activation of TNF signaling has been implicated in the pathogenesis of a wide spectrum of human diseases, including cancer, osteoporosis, sepsis, diabetes, and autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease. TNF binds to two specific receptors, TNF-receptor type I (TNF-R1, CD120a, p55/60) and TNF-receptor type II (TNF-R2, CD120b, p75/80). Signaling through TNF-R1 is extremely complex, leading to both cell death and survival signals. Many findings suggest an important role of phosphorylation of the TNF-R1 by number of protein kinases. Role of TNF-R2 phosphorylation on its signaling properties is understood less than TNF-R1. Other cellular substrates as TRADD adaptor protein, TRAF protein family and RIP kinases are reviewed in relation to TNF receptor-mediated apoptosis or survival pathways and regulation of their actions by phosphorylation.     

Regulation of TRAF2 signaling by self-induced degradation

Receptors belonging to the tumor necrosis factor receptor (TNF-R) family utilize cytoplasmic adapter proteins called TNF-R-associated factors (TRAFs) as key elements in their signaling pathways. However, it is not yet clear how individual TRAFs regulate signaling by this large and growing receptor family. Signaling via the TNF-R family member CD40 has recently been shown to result in recruitment of TRAF2 to plasma membrane detergent-resistant microdomains (lipid rafts) as well as to subsequently initiate TRAF2 degradation. As TRAF2 associates with most members of the TNF-R family, we wished to determine how this degradation occurs. We show here that CD40-mediated TRAF2 degradation requires the zinc-binding RING domain of TRAF2 and is preceded by TRAF2 ubiquitination, suggesting that the TRAF2 RING may promote ubiquitination although the RING itself is not a target of ubiquitination. Several approaches show that ubiquitination and proteasomal activity are integral to TRAF2 degradation, and inhibition of this process potentiates CD40 signaling.     

TNF-alpha induced c-IAP1/TRAF2 complex translocation to a Ubc6-containing compartment and TRAF2 ubiquitination

Signaling through tumor necrosis factor receptor 2 (TNF-R2) results in ubiquitination of TRAF2 by the E3 c-IAP1. In this report, we confirm that TRAF2 translocates to a Triton X-100 (TX)-insoluble compartment upon TNF-R2 engagement. Moreover, TRAF2 ubiquitination occurs in this compartment, from which TRAF2 is degraded in a proteasome-dependent manner. Confocal microscopy demonstrated that the TX-insoluble compartment is perinuclear and co-localizes with endoplasmic reticulum (ER) markers. The ER transmembrane Ubc6 bound to c-IAP1 and served as a cognate E2 for c-IAP1's E3 activity in vitro. Furthermore, Ubc6 co-localized with translocated TRAF2/c-IAP1 in the ER-associated compartment in vivo, and a catalytically inactive Ubc6 mutant inhibited TNF-alpha-induced, TNF-R2-dependent TRAF2 degradation. These results indicate that upon TNF-R2 signaling, translocation of TRAF2 and c-IAP1 to an ER-associated, Ubc6-containing perinuclear compartment is required for the ubiquitination of TRAF2 by c-IAP1. Therefore, the ER plays a key role in the TNF-R-mediated signal transduction cascade by acting as a site of assembly for E2/E3/substrate complexes.     

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.     

Protein kinase PKN1 associates with TRAF2 and is involved in TRAF2-NF-kappaB signaling pathway

PKN1 is a fatty acid and Rho-activated serine/threonine protein kinase whose catalytic domain is highly homologous to protein kinase C (PKC) family. In yeast two-hybrid screening for PKN1 binding proteins, we identified tumor necrosis factor alpha (TNFalpha) receptor-associated factor 2 (TRAF2). TRAF2 is one of the major mediators of TNF receptor superfamily transducing TNF signal to various functional targets, including activation of NF-kappaB, JNK, and apoptosis. FLAG-tagged PKN1 was co-immunoprecipitated with endogenous TRAF2 from HEK293 cell lysate, and in vitro binding assay using the deletion mutants of TRAF2 showed that PKN1 directly binds to the TRAF domain of TRAF2. PKN1 has the TRAF2-binding consensus sequences PXQX (S/T) at amino acid residues 580-584 (PIQES), and P580AQ582A mutant was not co-immunoprecipitated with TRAF2. Furthermore, the reduced expression of PKN1 by RNA interference (RNAi) down-regulated TRAF2-induced NF-kappaB activation in HEK293T cells. These results suggest that PKN1 is involved in TRAF2-NF-kappaB signaling pathway.     

TRAF7的研究进展

TRAFs家族是一类多功能蛋白,最初是作为TNFR介导的信号通路中的转导分子而被发现的。TRAFs作为信号接头蛋白和调节分子,参与了TNFR、TLRs、NLRs和RLRs等多种受体介导的信号通路。TRAF7是最新发现的TRAF家族成员,因其保守的RING结构域,而具有E3泛素连接酶活性。此外,TRAF7还以其独特机制参与了MAP激酶、TNFR及TLR2介导的信号通路的转导,以及细胞应激、分化和凋亡等重要生理过程的调控,与乳腺癌、脑膜瘤等多种疾病的发生密切相关。结合最新研究进展对TRAF7的结构、功能及其参与的生物学过程进行综述。