Roles of TNF receptor-associated factor 3 in signaling to B lymphocytes by carboxyl-terminal activating regions 1 and 2 of the EBV-encoded oncoprotein latent membrane protein 1

TNFR-associated factor (TRAF)3, an adaptor protein that binds the cytoplasmic domains of both CD40 and the EBV-encoded oncoprotein latent membrane protein (LMP)1, is required for positive signaling by LMP1 but not CD40 in B lymphocytes. The present study further investigated how TRAF3 participates in LMP1 signaling. We found that TRAF3 mediates signaling both through direct interactions with the C-terminal activating region (CTAR)1 of LMP1 and through indirect interactions with the CTAR2 region of LMP1 in mouse B cells. Notably, our results demonstrated that the CTAR2 region appears to inhibit the recruitment of TRAF1 and TRAF2 to membrane rafts by the CTAR1 region. Additionally, the absence of TRAF2 in B cells resulted in only a modest reduction in CTAR1-mediated signals and no detectable effect on CTAR2-mediated signals. CTAR1 and CTAR2 cooperated to achieve the robust signaling activity of LMP1 when recruited to the same membrane microdomains in B cells. Interestingly, TRAF3 deficiency completely abrogated the cooperation between CTAR1 and CTAR2, supporting the hypothesis that TRAF3 participates in the physical interaction between CTAR1 and CTAR2 of LMP1. Together, our findings highlight the central importance of TRAF3 in LMP1-mediated signaling, which is critical for EBV persistent infection and EBV-associated pathogenesis.     

The EBV transforming protein, latent membrane protein 1, mimics and cooperates with CD40 signaling in B lymphocytes

Latent membrane protein 1 (LMP1) is required for EBV-induced immortalization of human B cells, and expression of the protein in the absence of other viral proteins leads to an activated phenotype in B cells. It has been well documented that LMP1 causes B cells to up-regulate adhesion molecules, such as LFA-1 and ICAM-1, and coactivation molecules, such as B7-1 and CD23, as well as to activate NF-kappaB. Ligation of the endogenous B cell CD40 molecule also induces these and other activated phenotypic changes. Here, we report that expression of LMP1 also activates B cells to secrete Ig and IL-6 and rescues them from B cell receptor-mediated growth arrest analogous to CD40 signaling. Furthermore, an HLA-A2LMP1 chimeric construct demonstrates that the oligomerization of the carboxyl-terminal 200 amino acids of LMP1 is sufficient for B cell signaling. Finally, we demonstrate that LMP1 and CD40 signaling pathways interact cooperatively in inducing B cell effector functions.     

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.     

TNF receptor family member BCMA (B cell maturation) associates with TNF receptor-associated factor (TRAF) 1, TRAF2, and TRAF3 and activates NF-kappa B, elk-1, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase

BCMA (B cell maturation) is a nonglycosylated integral membrane type I protein that is preferentially expressed in mature B lymphocytes. Previously, we reported in a human malignant myeloma cell line that BCMA is not primarily present on the cell surface but lies in a perinuclear structure that partially overlaps the Golgi apparatus. We now show that in transiently or stably transfected cells, BCMA is located on the cell surface, as well as in a perinulear Golgi-like structure. We also show that 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 showed 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, as BCMA is lacking a "death domain" and its overexpression activates NF-kappa B and c-Jun N-terminal kinase, we can reasonably hypothesize that upon binding of its corresponding ligand BCMA transduces signals for cell survival and proliferation.     

Molecular mechanisms of TNFR-associated factor 6 (TRAF6) utilization by the oncogenic viral mimic of CD40, latent membrane protein 1 (LMP1)

Latent membrane protein 1 (LMP1), encoded by Epstein-Barr virus, is required for EBV-mediated B cell transformation and plays a significant role in the development of posttransplant B cell lymphomas. LMP1 has also been implicated in exacerbation of autoimmune diseases such as systemic lupus erythematosus. LMP1 is a constitutively active functional mimic of the tumor necrosis factor receptor superfamily member CD40, utilizing tumor necrosis factor receptor-associated factor (TRAF) adaptor proteins to induce signaling. However, LMP1-mediated B cell activation is amplified and sustained compared with CD40. We have previously shown that LMP1 and CD40 use TRAFs 1, 2, 3, and 5 differently. TRAF6 is important for CD40 signaling, but the role of TRAF6 in LMP1 signaling in B cells is not clear. Although TRAF6 binds directly to CD40, TRAF6 interaction with LMP1 in B cells has not been characterized. Here we tested the hypothesis that TRAF6 is a critical regulator of LMP1 signaling in B cells, either as part of a receptor-associated complex and/or as a cytoplasmic adaptor protein. Using TRAF6-deficient B cells, we determined that TRAF6 was critical for LMP1-mediated B cell activation. Although CD40-mediated TRAF6-dependent signaling does not require the TRAF6 receptor-binding domain, we found that LMP1 signaling required the presence of this domain. Furthermore, TRAF6 was recruited to the LMP1 signaling complex via the TRAF1/2/3/5 binding site within the cytoplasmic domain of LMP1.     

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.     

Identification of TIFA as an adapter protein that links tumor necrosis factor receptor-associated factor 6 (TRAF6) to interleukin-1 (IL-1) receptor-associated kinase-1 (IRAK-1) in IL-1 receptor signaling

Tumor necrosis factor receptor-associated factor 6 (TRAF6) transduces signals from members of the Toll/interleukin-1 (IL-1) receptor family by interacting with IL-1 receptor-associated kinase-1 (IRAK-1) after IRAK-1 is released from the receptor-MyD88 complex upon IL-1 stimulation. However, the molecular mechanisms underlying regulation of the IRAK-1/TRAF6 interaction are largely unknown. We have identified TIFA, a TRAF-interacting protein with a forkhead-associated (FHA) domain. The FHA domain is a motif known to bind directly to phosphothreonine and phosphoserine. In transient transfection assays, TIFA activates NFkappaBeta and c-Jun amino-terminal kinase. However, TIFA carrying a mutation that abolishes TRAF6 binding or mutations in the FHA domain that are known to abolish FHA domain binding to phosphopeptide fails to activate NFkappaBeta and c-Jun amino-terminal kinase. TIFA, when overexpressed, binds both TRAF6 and IRAK-1 and significantly enhances the IRAK-1/TRAF6 interaction. Furthermore, analysis of endogenous proteins indicates that TIFA associates with TRAF6 constitutively, whereas it associates with IRAK-1 in an IL-1 stimulation-dependent manner in vivo. Thus, TIFA is likely to mediate IRAK-1/TRAF6 interaction upon IL-1 stimulation.     

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.     

Two carboxyl-terminal activation regions of Epstein-Barr virus latent membrane protein 1 activate NF-kappaB through distinct signaling pathways in fibroblast cell lines

Latent membrane protein 1 (LMP1), an Epstein-Barr virus transforming protein, is able to activate NF-kappaB through its carboxyl-terminal activation region 1 (CTAR1) and 2 (CTAR2), but the exact role of each domain is not fully understood. Here we show that LMP1 activates NF-kappaB in different NF-kappaB essential modulator (NEMO)-defective cell lines, but not in cells lacking both IkappaB kinase 1 (IKK1) and 2 (IKK2). Mutational studies reveal that CTAR1, but not CTAR2, mediates NEMO-independent NF-kappaB activation and that this process largely depends on IKK1. Retroviral expression of LMP1 mutants in cells lacking either functional NF-kappaB inducing kinase (NIK), NEMO, IKK1, or IKK2 further illustrates distinct signals from the two activation regions of LMP1 for persistent NF-kappaB activation. One originates in CTAR2, operates through the canonical NEMO-dependent pathway, and induces NFKB2 p100 production; the second signal originates in CTAR1, utilizes NIK and IKK1, and induces the processing of p100. Our results thus help clarify how two functional domains of LMP1 persistently activate NF-kappaB through distinct signaling pathways.     

EBV latent membrane protein 1 activates Akt, NFkappaB, and Stat3 in B cell lymphomas

Latent membrane protein 1 (LMP1) is the major oncoprotein of Epstein-Barr virus (EBV). In transgenic mice, LMP1 promotes increased lymphoma development by 12 mo of age. This study reveals that lymphoma develops in B-1a lymphocytes, a population that is associated with transformation in older mice. The lymphoma cells have deregulated cell cycle markers, and inhibitors of Akt, NFkappaB, and Stat3 block the enhanced viability of LMP1 transgenic lymphocytes and lymphoma cells in vitro. Lymphoma cells are independent of IL4/Stat6 signaling for survival and proliferation, but have constitutively activated Stat3 signaling. These same targets are also deregulated in wild-type B-1a lymphomas that arise spontaneously through age predisposition. These results suggest that Akt, NFkappaB, and Stat3 pathways may serve as effective targets in the treatment of EBV-associated B cell lymphomas.     

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 first homolog of a TRAF7 (TNF receptor-associated factor 7) gene in a mollusk, Crassostrea hongkongensis

Tumor necrosis factor (TNF) receptor-associated factor 7 (TRAF7) is one of several adaptor proteins that are critically involved in the activation of TLR-dependent NF-κB signaling. In this report, the first mollusk TRAF7 (designated ChTRAF7) homolog was isolated from Crassostrea hongkongensis by screening a suppression subtractive library. The full-length cDNA, 2290 bp in length, encodes a putative protein of 686 amino acids that contains a RING finger domain, an adjacent zinc finger domain, and seven WD40 repeats. ChTRAF7 is ubiquitously expressed in various tissues including digestive gland, mantle, gill, heart, hemocytes, muscle, and gonads, with the highest expression observed in gonads. Temporal expression of ChTRAF7 following bacterial infection shows that expression of ChTRAF7 in hemocytes decreases from 2 to 12 h post-challenge, and then recovered to the original level after 24 h. These results indicate that ChTRAF7 may play an important role in signal transduction in the immune response of oysters.     

The latent membrane protein 1 of Epstein-Barr virus (EBV) primes EBV latency cells for type I interferon production

Epstein-Barr virus (EBV) latency has been associated with a variety of human cancers. Latent membrane protein 1 (LMP-1) is one of the key viral proteins required for transformation of primary B cells in vitro and establishment of EBV latency. We have previously shown that LMP-1 induces the expression of several interferon (IFN)-stimulated genes and has antiviral effect (Zhang, J., Das, S. C., Kotalik, C., Pattnaik, A. K., and Zhang, L. (2004) J. Biol. Chem. 279, 46335-46342). In this report, a novel mechanism related to the antiviral effect of LMP-1 is identified. We show that EBV type III latency cells, in which LMP-1 is expressed, are primed to produce robust levels of endogenous IFNs upon infection of Sendai virus. The priming action is due to the expression of LMP-1 but not EBV nuclear antigen 2 (EBNA-2). The signaling events from the C-terminal activator regions of LMP-1 are essential to prime cells for high IFN production. LMP-1-mediated activation of NF-kappaB is apparently necessary and sufficient for LMP-1-mediated priming effect in DG75 cells, a human B cell line. IFN regulatory factor 7 (IRF-7) that can be activated by LMP-1 is also implicated in the priming action. Taken together, these data strongly suggest that LMP-1 may prime EBV latency cells for IFN production and that the antiviral property of LMP-1 may be an intrinsic part of EBV latency program, which may assist the establishment and/or maintenance of viral latency.     

Pellino 1 is required for interleukin-1 (IL-1)-mediated signaling through its interaction with the IL-1 receptor-associated kinase 4 (IRAK4)-IRAK-tumor necrosis factor receptor-associated factor 6 (TRAF6) complex

The signaling pathway downstream of the mammalian interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) is evolutionally conserved with that mediated by the Drosophila Toll protein. Toll initiates its signal through the adapter molecule Tube and the serine-threonine kinase Pelle. Pelle is highly homologous to members of the IL-1R-associated kinase (IRAK) family in mammals. Recently, a novel Pelle-interacting protein called Pellino was identified in Drosophila. We now report a mammalian counterpart of Pellino, termed Pellino 1, which is required for NF kappa B activation and IL-8 gene expression in response to IL-1, probably through its signal-dependent interaction with IRAK4, IRAK, and the tumor necrosis factor receptor-associated factor 6 (TRAF6). The Pellino 1-IRAK-IRAK4-TRAF6 signaling complex is likely to be intermediate, located between the IL-1 receptor complex and the TAK1 complex in the IL-1 pathway.     

An atypical tumor necrosis factor (TNF) receptor-associated factor-binding motif of B cell-activating factor belonging to the TNF family (BAFF) receptor mediates induction of the noncanonical NF-kappaB signaling pathway

BAFF receptor (BAFFR) is a member of the TNF receptor (TNFR) superfamily that regulates the survival and maturation of B cells. BAFFR exerts its signaling function by inducing activation of NF-kappaB, although the underlying mechanism has not been well defined. By using a chimeric BAFFR, we show that BAFFR preferentially induces the noncanonical NF-kappaB signaling pathway. This specific function of BAFFR is mediated by a sequence motif, PVPAT, which is homologous to the TRAF-binding site (PVQET) present in CD40, a TNFR known to induce both the canonical and noncanonical NF-kappaB pathways. Mutation of this putative TRAF-binding motif within BAFFR abolishes its interaction with TRAF3 as well as its ability to induce noncanonical NF-kappaB. Interestingly, modification of the PVPAT sequence to the typical TRAF-binding sequence, PVQET, is sufficient to render the BAFFR capable of inducing strong canonical NF-kappaB signaling. Further, this functional acquisition of the modified BAFFR is associated with its stronger and more rapid association with TRAF3. These findings suggest that the PVPAT sequence of BAFFR not only functions as a key signaling motif of BAFFR but also determines its signaling specificity in the induction of the noncanonical NF-kappaB pathway.     

Tumor necrosis factor (TNF)-induced germinal center kinase-related (GCKR) and stress-activated protein kinase (SAPK) activation depends upon the E2/E3 complex Ubc13-Uev1A/TNF receptor-associated factor 2 (TRAF2)

Tumor necrosis factor (TNF)-induced activation of apoptosis signal-regulating kinase 1 (ASK1) and germinal center kinases (GCKs) and the subsequent activation of stress-activated protein kinases (SAPKs and c-Jun NH(2)-terminal kinases) requires TNF receptor-associated factor 2 (TRAF2). Although the TRAF2 TRAF domain binds ASK1, GCK, and the highly related kinase GCKR, the RING finger domain is needed for their activation. Here, we report that TNF activates GCKR and the SAPK pathway in a manner that depends upon TRAF2 and Ubc13, a member along with Uev1A of a dimeric ubiquitin-conjugating enzyme complex. Interference with Ubc13 function or expression inhibits both TNF- and TRAF2-mediated GCKR and SAPK activation, but has a minimal effect on ASK1 activation. TNF signaling leads to TRAF2 polyubiquitination and oligomerization and to the oligomerization, ubiquitination, and activation of GCKR, all of which are sensitive to the disruption of Ubc13 function. These results indicate that the assembly of a TRAF2 lysine 63-linked polyubiquitin chain by Ubc13/Uev1A is required for TNF-mediated GCKR and SAPK activation, but may not be required for ASK1 activation.