Membrane Tumor Necrosis Factor (TNF) Induces p100 Processing via TNF Receptor-2 (TNFR2)

Tumor necrosis factor (TNF) elicits its biological activities by stimulation of two receptors, TNFR1 and TNFR2, both belonging to the TNF receptor superfamily. Whereas TNFR1-mediated signal transduction has been intensively studied and is understood in detail, especially with respect to activation of the classical NFκB pathway, cell death induction, and MAP kinase signaling, TNFR2-associated signal transduction is poorly defined. Here, we demonstrate in various tumor cell lines and primary T-cells that TNFR2, but not TNFR1, induces activation of the alternative NFκB pathway. In accord with earlier findings demonstrating that only membrane TNF, but not soluble TNF, properly activates TNFR2, we further show by use of TNFR1- and TNFR2-specific mutants of soluble TNF and membrane TNF that soluble ligand trimers fail to activate the alternative NFκB pathway. In accord with the known inhibitory role of TRAF2 in the alternative NFκB pathway, TNFR2-, but not TNFR1-specific TNF induced depletion of cytosolic TRAF2. Thus, we identified activation of the alternative NFκB pathway as a TNF signaling effect that can be specifically assigned to TNFR2 and membrane TNF.     

Azadirachtin Interacts with the Tumor Necrosis Factor (TNF) Binding Domain of Its Receptors and Inhibits TNF-induced Biological Responses

The role of azadirachtin, an active component of a medicinal plant Neem (Azadirachta indica), on TNF-induced cell signaling in human cell lines was investigated. Azadirachtin blocks TNF-induced activation of nuclear factor κB (NF-κB) and also expression of NF-κB-dependent genes such as adhesion molecules and cyclooxygenase 2. Azadirachtin inhibits the inhibitory subunit of NF-κB (IκBα) phosphorylation and thereby its degradation and RelA (p65) nuclear translocation. It blocks IκBα kinase (IKK) activity ex vivo, but not in vitro. Surprisingly, azadirachtin blocks NF-κB DNA binding activity in transfected cells with TNF receptor-associated factor (TRAF)2, TNF receptor-associated death domain (TRADD), IKK, or p65, but not with TNFR, suggesting its effect is at the TNFR level. Azadirachtin blocks binding of TNF, but not IL-1, IL-4, IL-8, or TNF-related apoptosis-inducing ligand (TRAIL) with its respective receptors. Anti-TNFR antibody or TNF protects azadirachtin-mediated down-regulation of TNFRs. Further, in silico data suggest that azadirachtin strongly binds in the TNF binding site of TNFR. Overall, our data suggest that azadirachtin modulates cell surface TNFRs thereby decreasing TNF-induced biological responses. Thus, azadirachtin exerts an anti-inflammatory response by a novel pathway, which may be beneficial for anti-inflammatory therapy.     

Role of the TRAF Binding Site and NF-κB Activation in Epstein-Barr Virus Latent Membrane Protein 1-Induced Cell Gene Expression

In this study, we investigated the induction of cellular gene expression by the Epstein-Barr Virus (EBV) latent membrane protein 1 (LMP1). Previously, LMP1 was shown to induce the expression of ICAM-1, LFA-3, CD40, and EBI3 in EBV-negative Burkitt lymphoma (BL) cells and of the epidermal growth factor receptor (EGF-R) in epithelial cells. We now show that LMP1 expression also increased Fas and tumor necrosis factor receptor-associated factor 1 (TRAF1) in BL cells. LMP1 mediates NF-κB activation via two independent domains located in its C-terminal cytoplasmic tail, a TRAF-interacting site that associates with TRAF1, -2, -3, and -5 through a PXQXT/S core motif and a TRADD-interacting site. In EBV-transformed B cells or transiently transfected BL cells, significant amounts of TRAF1, -2, -3, and -5 are associated with LMP1. In epithelial cells, very little TRAF1 is expressed, and only TRAF2, -3, and -5, are significantly complexed with LMP1. The importance of TRAF binding to the PXQXT/S motif in LMP1-mediated gene induction was studied by using an LMP1 mutant that contains alanine point mutations in this motif and fails to associate with TRAFs. This mutant, LMP1(P204A/Q206A), induced 60% of wild-type LMP1 NF-κB activation and had approximately 60% of wild-type LMP1 effect on Fas, ICAM-1, CD40, and LFA-3 induction. In contrast, LMP1(P204A/Q206A) was substantially more impaired in TRAF1, EBI3, and EGF-R induction. Thus, TRAF binding to the PXQXT/S motif has a nonessential role in up-regulating Fas, ICAM-1, CD40, and LFA-3 expression and a critical role in up-regulating TRAF1, EBI3, and EGF-R expression. Further, D1 LMP1, an LMP1 mutant that does not aggregate failed to induce TRAF1, EBI3, Fas, ICAM-1, CD40, and LFA-3 expression confirming the essential role for aggregation in LMP1 signaling. Overexpression of a dominant form of IκBα blocked LMP1-mediated TRAF1, EBI3, Fas, ICAM-1, CD40, and LFA-3 up-regulation, indicating that NF-κB is an important component of LMP1-mediated gene induction from both the TRAF- and TRADD-interacting sites.     

CYLD and the NEMO Zinc Finger Regulate Tumor Necrosis Factor Signaling and Early Embryogenesis

NF-κB essential modulator (NEMO) and cylindromatosis protein (CYLD) are intracellular proteins that regulate the NF-κB signaling pathway. Although mice with either CYLD deficiency or an alteration in the zinc finger domain of NEMO (K392R) are born healthy, we found that the combination of these two gene defects in double mutant (DM) mice is early embryonic lethal but can be rescued by the absence of TNF receptor 1 (TNFR1). Notably, NEMO was not recruited into the TNFR1 complex of DM cells, and consequently NF-κB induction by TNF was severely impaired and DM cells were sensitized to TNF-induced cell death. Interestingly, the TNF signaling defects can be fully rescued by reconstitution of DM cells with CYLD lacking ubiquitin hydrolase activity but not with CYLD mutated in TNF receptor-associated factor 2 (TRAF2) or NEMO binding sites. Therefore, our data demonstrate an unexpected non-catalytic function for CYLD as an adapter protein between TRAF2 and the NEMO zinc finger that is important for TNF-induced NF-κB signaling during embryogenesis.     

TRAF2 Must Bind to Cellular Inhibitors of Apoptosis for Tumor Necrosis Factor (TNF) to Efficiently Activate NF-��B and to Prevent TNF-induced Apoptosis

Tumor necrosis factor (TNF) receptor-associated factor-2 (TRAF2) binds to cIAP1 and cIAP2 (cIAP1/2) and recruits them to the cytoplasmic domain of several members of the TNF receptor (TNFR) superfamily, including the TNF-TNFR1 ligand-receptor complex. Here, we define a cIAP1/2-interacting motif (CIM) within the TRAF-N domain of TRAF2, and we use TRAF2 CIM mutants to determine the role of TRAF2 and cIAP1/2 individually, and the TRAF2-cIAP1/2 interaction, in TNFR1-dependent signaling. We show that both the TRAF2 RING domain and the TRAF2 CIM are required to regulate NF-κB-inducing kinase stability and suppress constitutive noncanonical NF-κB activation. Conversely, following TNFR1 stimulation, cells bearing a CIM-mutated TRAF2 showed reduced canonical NF-κB activation and TNF-induced RIPK1 ubiquitylation. Remarkably, the RING domain of TRAF2 was dispensable for these functions. However, like the TRAF2 CIM, the RING domain of TRAF2 was required for protection against TNF-induced apoptosis. These results show that TRAF2 has anti-apoptotic signaling roles in addition to promoting NF-κB signaling and that efficient activation of NF-κB by TNFR1 requires the recruitment of cIAP1/2 by TRAF2.     

Tumor Necrosis Factor (TNF) Receptor-associated Factor (TRAF)-interacting Protein (TRIP) Negatively Regulates the TRAF2 Ubiquitin-dependent Pathway by Suppressing the TRAF2-Sphingosine 1-Phosphate (S1P) Interaction

The signaling pathway downstream of TNF receptor (TNFR) is involved in the induction of a wide range of cellular processes, including cell proliferation, activation, differentiation, and apoptosis. TNFR-associated factor 2 (TRAF2) is a key adaptor molecule in TNFR signaling complexes that promotes downstream signaling cascades, such as nuclear factor-κB (NF-κB) and mitogen-activated protein kinase activation. TRAF-interacting protein (TRIP) is a known cellular binding partner of TRAF2 and inhibits TNF-induced NF-κB activation. Recent findings that TRIP plays a multifunctional role in antiviral response, cell proliferation, apoptosis, and embryonic development have increased our interest in exploring how TRIP can affect the TNFR-signaling pathway on a molecular level. In our current study, we demonstrated that TRIP is negatively involved in the TNF-induced inflammatory response through the down-regulation of proinflammatory cytokine production. Here, we demonstrated that the TRAF2-TRIP interaction inhibits Lys⁶³-linked TRAF2 ubiquitination by inhibiting TRAF2 E3 ubiquitin (Ub) ligase activity. The TRAF2-TRIP interaction inhibited the binding of sphingosine 1-phosphate, which is a cofactor of TRAF2 E3 Ub ligase, to the TRAF2 RING domain. Finally, we demonstrated that TRIP functions as a negative regulator of proinflammatory cytokine production by inhibiting TNF-induced NF-κB activation. These results indicate that TRIP is an important cellular regulator of the TNF-induced inflammatory response.     

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.     

Targeting of Tumor Necrosis Factor Receptor 1 to Low Density Plasma Membrane Domains in Human Endothelial Cells

TNFR1 (tumor necrosis factor receptor 1) localizes to caveolae of human endothelial-derived EA.hy926 cells. Transduced TNFR1 molecules lacking amino acid residues 229–244 (spanning the transmembrane/intercellular boundary) are expressed on the cell surface equivalently to full-length TNFR1 molecules but incompletely localize to caveolae. A peptide containing this sequence pulls down CAV-1 (caveolin-1) and TNFR1 from cell lysates but fails to do so following disruption of caveolae with methyl-β-cyclodextrin. We previously reported that methyl-β-cyclodextrin eliminates caveolae and blocks tumor necrosis factor (TNF)-induced internalization of TNFR1 but not TNF-induced activation of NF-κB in EA.hy926 cells. Both CAV-1 and FLOT-2 (flotillin-2), organizing proteins of caveolae and lipid rafts, respectively, associate with caveolae in EA.hy926 cells. Small interfering RNA-mediated knockdown of CAV-1 but not FLOT-2 strikingly reduces caveolae number. Both knockdowns reduce total TNFR1 protein expression, but neither prevents TNFR1 localization to low density membrane domains, TNF-induced internalization of TNFR1, or NF-κB activation by TNF. Both CAV-1 and FLOT-2 knockdowns reduce TNF-mediated activation of stress-activated protein kinase (SAPK). However, both knockdowns reduce expression of TRAF2 (TNF receptor-associated factor-2) protein, and small interfering RNA targeting of TRAF2 also selectively inhibits SAPK activation. We conclude that TNFR1 contains a membrane-proximal sequence that targets the receptor to caveolae/lipid rafts. Neither TNFR1 targeting to nor internalization from these low density membrane domains depends upon CAV-1 or FLOT-2. Furthermore, both NF-κB and SAPK activation appear independent of both TNFR1 localization to low density membrane domains and to TNF-induced receptor internalization.     

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.     

Dynamic Arginine Methylation of Tumor Necrosis Factor (TNF) Receptor-associated Factor 6 Regulates Toll-like Receptor Signaling

Arginine methylation is a common post-translational modification, but its role in regulating protein function is poorly understood. This study demonstrates that, TNF receptor-associated factor 6 (TRAF6), an E3 ubiquitin ligase involved in innate immune signaling, is regulated by reversible arginine methylation in a range of primary and cultured cells. Under basal conditions, TRAF6 is methylated by the methyltransferase PRMT1, and this inhibits its ubiquitin ligase activity, reducing activation of toll-like receptor signaling. In response to toll-like receptor ligands, TRAF6 is demethylated by the Jumonji domain protein JMJD6. Demethylation is required for maximal activation of NF-κB. Loss of JMJD6 leads to reduced response, and loss of PRMT1 leads to basal pathway activation with subsequent desensitization to ligands. In human primary cells, variations in the PRMT1/JMJD6 ratio significantly correlate with TRAF6 methylation, basal activation of NF-κB, and magnitude of response to LPS. Reversible arginine methylation of TRAF6 by the opposing effects of PRMT1 and JMJD6 is, therefore, a novel mechanism for regulation of innate immune pathways.     

HIV-1 Nef induces proinflammatory state in macrophages through its acidic cluster domain: involvement of TNF alpha receptor associated factor 2

Background

HIV-1 Nef is a virulence factor that plays multiple roles during HIV replication. Recently, it has been described that Nef intersects the CD40 signalling in macrophages, leading to modification in the pattern of secreted factors that appear able to recruit, activate and render T lymphocytes susceptible to HIV infection. The engagement of CD40 by CD40L induces the activation of different signalling cascades that require the recruitment of specific tumor necrosis factor receptor-associated factors (i.e. TRAFs). We hypothesized that TRAFs might be involved in the rapid activation of NF-κB, MAPKs and IRF-3 that were previously described in Nef-treated macrophages to induce the synthesis and secretion of proinflammatory cytokines, chemokines and IFNβ to activate STAT1, -2 and -3.

Methodology/Principal Findings

Searching for possible TRAF binding sites on Nef, we found a TRAF2 consensus binding site in the AQEEEE sequence encompassing the conserved four-glutamate acidic cluster. Here we show that all the signalling effects we observed in Nef treated macrophages depend on the integrity of the acidic cluster. In addition, Nef was able to interact in vitro with TRAF2, but not TRAF6, and this interaction involved the acidic cluster. Finally silencing experiments in THP-1 monocytic cells indicate that both TRAF2 and, surprisingly, TRAF6 are required for the Nef-induced tyrosine phosphorylation of STAT1 and STAT2.

Conclusions

Results reported here revealed TRAF2 as a new possible cellular interactor of Nef and highlighted that in monocytes/macrophages this viral protein is able to manipulate both the TRAF/NF-κB and TRAF/IRF-3 signalling axes, thereby inducing the synthesis of proinflammatory cytokines and chemokines as well as IFNβ.     

HOIL-1L Interacting Protein (HOIP) as an NF-κB Regulating Component of the CD40 Signaling Complex

The tumor necrosis factor receptor (TNFR) superfamily mediates signals critical for regulation of the immune system. One family member, CD40, is important for the efficient activation of antibody-producing B cells and other antigen-presenting cells. The molecules and mechanisms that mediate CD40 signaling are only partially characterized. Proteins known to interact with the cytoplasmic domain of CD40 include members of the TNF receptor-associated factor (TRAF) family, which regulate signaling and serve as links to other signaling molecules. To identify additional proteins important for CD40 signaling, we used a combined stimulation/immunoprecipitation procedure to isolate CD40 signaling complexes from B cells and characterized the associated proteins by mass spectrometry. In addition to known CD40-interacting proteins, we detected SMAC/DIABLO, HTRA2/Omi, and HOIP/RNF31/PAUL/ZIBRA. We found that these previously unknown CD40-interacting partners were recruited in a TRAF2-dependent manner. HOIP is a ubiquitin ligase capable of mediating NF-κB activation through the ubiquitin-dependent activation of IKKγ. We found that a mutant HOIP molecule engineered to lack ubiquitin ligase activity inhibited the CD40-mediated activation of NF-κB. Together, our results demonstrate a powerful approach for the identification of signaling molecules associated with cell surface receptors and indicate an important role for the ubiquitin ligase activity of HOIP in proximal CD40 signaling.     

High-affinity interactions of tumor necrosis factor receptor-associated factors (TRAFs) and CD40 require TRAF trimerization and CD40 multimerization.

Signaling by some TNF receptor family members, including CD40, is mediated by TNF receptor-associated factors (TRAFs) that interact with receptor cytoplasmic domains following ligand-induced receptor oligomerization. Here we have defined the oligomeric structure of recombinant TRAF domains that directly interact with CD40 and quantitated the affinities of TRAF2 and TRAF3 for CD40. Biochemical and biophysical analyses demonstrated that TRAF domains of TRAF1, TRAF2, TRAF3, and TRAF6 formed homo-trimers in solution. N-terminal deletions of TRAF2 and TRAF3 defined minimal amino acid sequences necessary for trimer formation and indicated that the coiled coil TRAF-N region is required for trimerization. Consistent with the idea that TRAF trimerization is required for high-affinity interactions with CD40, monomeric TRAF-C domains bound to CD40 significantly weaker than trimeric TRAFs. In surface plasmon resonance studies, a hierarchy of affinity of trimeric TRAFs for trimeric CD40 was found to be TRAF2 > TRAF3 > TRAF1 and TRAF6. CD40 trimerization was demonstrated to be sufficient for optimal NF-kappaB and p38 mitogen activated protein kinase activation through wild-type CD40. In contrast, a higher degree of CD40 multimerization was necessary for maximal signaling in a cell line expressing a mutated CD40 (T254A) that signaled only through TRAF6. The affinities of TRAF proteins for oligomerized receptors as well as different requirements for degree of receptor multimerization appear to contribute to the selectivity of TRAF recruitment to receptor cytoplasmic domains.     

Role of tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) in distinct and overlapping CD40 and TNF receptor 2/CD120b-mediated B lymphocyte activation

Members of the tumor necrosis factor receptor (TNFR) family play a variety of roles in the regulation of lymphocyte activation. An important TNFR family member for B cell activation is CD40. CD40 signals stimulate B cell TNF-alpha secretion, which subsequently signals via TNFR2 (CD120b) to enhance B cell activation. Although the function of the pro-apoptotic and pro-inflammatory receptor TNFR1 (CD120a) has been the subject of much research, less is understood about the distinct contributions of CD120b to cell activation and how it stimulates downstream events. Members of the tumor necrosis factor receptor family bind various members of the cytoplasmic adapter protein family, the tumor necrosis factor receptor-associated factors (TRAFs), during signaling. Both CD40 and CD120b bind TNF receptor-associated factor 2 (TRAF2) upon ligand stimulation. Wild type and TRAF2-deficient B cells expressing CD40 or the hybrid molecule (human) CD40 (mouse)-CD120b were examined. CD40- and CD120b-mediated IgM secretion were partly TRAF2-dependent, but only CD40 required TRAF2 for c-Jun N-terminal kinase activation. CD40 and CD120b used primarily divergent mechanisms to activate NF-kappaB, exemplifying how TNFR family members can use diverse mechanisms to mediate similar downstream events.     

The IVVY Motif and Tumor Necrosis Factor Receptor-associated Factor (TRAF) Sites in the Cytoplasmic Domain of the Receptor Activator of Nuclear Factor κB (RANK) Cooperate to Induce Osteoclastogenesis

Receptor activator of NF-κB (RANK) activation by RANK ligand (RANKL) mediates osteoclastogenesis by recruiting TNF receptor-associated factors (TRAFs) via three cytoplasmic motifs (motif 1, PFQEP^369–373; motif 2, PVQEET^559–564; and motif 3, PVQEQG^604–609) to activate the NF-κB and MAPK signaling pathways. RANK also has a TRAF-independent motif (IVVY^535–538), which is dispensable for the activation of TRAF-induced signaling pathways but essential for osteoclast lineage commitment by inducing the expression of nuclear factor of activated T-cells c1 (NFATc1) to regulate osteoclast gene expression. Notably, TNF/IL-1-mediated osteoclastogenesis requires RANK ligand assistance, and the IVVY motif is also critical for TNF/IL-1-mediated osteoclastogenesis by rendering osteoclast genes responsive to these two cytokines. Here we show that the two types of RANK cytoplasmic motifs have to be on the same RANK molecule to mediate osteoclastogenesis, suggesting a functional cooperation between them. Subsequent osteoclastogenesis assays with TNF or IL-1 revealed that, although all three TRAF motifs play roles in TNF/IL-1-mediated osteoclastogenesis, motifs 2 and 3 are more potent than motif 1. Accordingly, inactivation of motifs 2 and 3 blocksTNF/IL-1-mediated osteoclastogenesis. Mechanistically, double mutation of motifs 2 and 3, similar to inactivation of the IVVY motif, abrogates the expression of nuclear factor of activated T-cells c1 and osteoclast genes in assays reflecting RANK-initiated and TNF/IL-1-mediated osteoclastogenesis. In contrast, double inactivation of motifs 2 and 3 did not affect the ability of RANK to activate the NF-κB and MAPK signaling pathways. Collectively, these results indicate that the RANK IVVY motif cooperates with the TRAF-binding motifs to promote osteoclastogenesis, which provides novel insights into the molecular mechanism of RANK signaling in osteoclastogenesis.     

Molecular characterization of CD40 signaling intermediates

Signal transduction through the CD40 receptor is initiated by binding of its trimeric ligand and propagated by interactions of tumor necrosis factor receptor-associated factor (TRAF) proteins with the multimerized CD40 cytoplasmic domain. Using defined multimeric constructs of the CD40 cytoplasmic domain expressed as either soluble or myristoylated proteins, we have addressed the extent of receptor multimerization needed to initiate signal transduction and identified components of CD40 signaling complexes. Signal transduction in human embryonic kidney 293 cells, measured by nuclear factor kappaB activation, was observed in cells expressing soluble trimeric CD40 cytoplasmic domain and to a lesser extent in cells expressing dimeric CD40 cytoplasmic domain. Nuclear factor kappaB activation was strongest in cells expressing myristoylated trimeric CD40 cytoplasmic domain. Signal transduction through trimeric CD40 cytoplasmic domains with various point mutations in the TRAF binding sites was similar to signal transduction through analogous full-length receptors. Transiently expressed soluble trimeric CD40 cytoplasmic domain was isolated as complexes that contained TRAF2, TRAF3, TRAF5, TRAF6, and the inhibitor of apoptosis protein (c-IAP1). Association of c-IAP1 with the CD40 cytoplasmic domain complex was indirect and dependent on the presence of an intact TRAF1/2/3 binding site. These results suggest that extracellular ligation of CD40 can be bypassed and that soluble trimerized CD40 complexes can be isolated and used to identify components that link CD40 with signaling pathways.     

Cutting edge: contrasting roles of TNF receptor-associated factor 2 (TRAF2) and TRAF3 in CD40-activated B lymphocyte differentiation.

In B lymphocytes, CD40 signals contribute to the activation of Ab secretion, isotype switching, T cell costimulation, and immunological memory. TRAF proteins appear to be important components of the CD40 signal transduction complex, but their roles in the activation of B cell effector functions are poorly understood. We examined the contributions of TNF receptor-associated factor 2 (TRAF2) and TRAF3 to CD40-activated differentiation in mouse B cells transfected with inducible TRAF and dominant-negative TRAF cDNAs. We find that binding of TRAF2 and TRAF3 to CD40 is not required for the induction of Ab secretion, but that both TRAF molecules can regulate the activation process. We demonstrate a negative regulatory role for TRAF3 and that this activity is dependent on the availability of an intact TRAF3-binding site in the cytoplasmic domain of CD40. In contrast, TRAF2 appears to play a positive role in B cell differentiation, and this activity is apparent even when its binding site on CD40 is disrupted.     

Hepatitis C Virus Core Protein Binds to the Cytoplasmic Domain of Tumor Necrosis Factor (TNF) Receptor 1 and Enhances TNF-Induced Apoptosis

The hepatitis C virus (HCV) core protein is known to be a multifunctional protein, besides being a component of viral nucleocapsids. Previously, we have shown that the core protein binds to the cytoplasmic domain of lymphotoxin β receptor, which is a member of tumor necrosis factor receptor (TNFR) family. In this study, we demonstrated that the core protein also binds to the cytoplasmic domain of TNFR 1. The interaction was demonstrated both by glutathione S-transferase fusion protein pull-down assay in vitro and membrane flotation method in vivo. Both the in vivo and in vitro binding required amino acid residues 345 to 407 of TNFR 1, which corresponds to the “death domain” of this receptor. We have further shown that stable expression of the core protein in a mouse cell line (BC10ME) or human cell lines (HepG2 and HeLa cells) sensitized them to TNF-induced apoptosis, as determined by the TNF cytotoxicity or annexin V apoptosis assay. The presence of the core protein did not alter the level of TNFR 1 mRNA in the cells or expression of TNFR 1 on the cell surface, suggesting that the sensitization of cells to TNF by the viral core protein was not due to up-regulation of TNFR 1. Furthermore, we observed that the core protein blocked the TNF-induced activation of RelA/NF-κB in murine BC10ME cells, thus at least partially accounting for the increased sensitivity of BC10ME cells to TNF. However, NF-κB activation was not blocked in core protein-expressing HeLa or HepG2 cells, implying another mechanism of TNF sensitization by core protein. These results together suggest that the core protein can promote cell death during HCV infection via TNF signaling pathways possibly as a result of its interaction with the cytoplasmic tail of TNFR 1. Therefore, TNF may play a role in HCV pathogenesis.