Structurally distinct recognition motifs in lymphotoxin-beta receptor and CD40 for tumor necrosis factor receptor-associated factor (TRAF)-mediated signaling

Lymphotoxin-beta receptor (LTbetaR) and CD40 are members of the tumor necrosis factor family of signaling receptors that regulate cell survival or death through activation of NF-kappaB. These receptors transmit signals through downstream adaptor proteins called tumor necrosis factor receptor-associated factors (TRAFs). In this study, the crystal structure of a region of the cytoplasmic domain of LTbetaR bound to TRAF3 has revealed an unexpected new recognition motif, 388IPEEGD393, for TRAF3 binding. Although this motif is distinct in sequence and structure from the PVQET motif in CD40 and PIQCT in the regulator TRAF-associated NF-kappaB activator (TANK), recognition is mediated in the same binding crevice on the surface of TRAF3. The results reveal structurally adaptive "hot spots" in the TRAF3-binding crevice that promote molecular interactions driving specific signaling after contact with LTbetaR, CD40, or the downstream regulator TANK.     

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.     

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.     

Role of TNF receptor-associated factor 2 in the activation of IgM secretion by CD40 and CD120b

TNFR-associated factors (TRAFs) participate in the signaling of many TNFR family members, including CD40, CD120a (TNFR1), and CD120b (TNFR2). Previously, we found that a dominant-negative TRAF2 molecule inhibits CD40-mediated Ab secretion by the mouse B cell line CH12.LX. However, disruption of the TRAF2 binding site in the cytoplasmic domain of CD40 does not diminish the ability of CD40 to stimulate Ab secretion, nor is this mutation able to circumvent the inhibition of Ab secretion by dominant-negative TRAF2. Here we demonstrate that CD40-induced TNF stimulates IgM production through CD120b and that CD120b signaling is required for optimal CD40-induced IgM secretion. Furthermore, although both CD40 and CD120b can bind TRAF2, TRAF2-dependent CD40 signals cannot substitute for TRAF2-dependent CD120b signals in the activation of IgM secretion. Our results indicate a potentially important role for CD120b in the activation of IgM secretion and that TRAF2 is used by CD40 and CD120b in distinct ways.     

CD40 signaling through a newly identified tumor necrosis factor receptor-associated factor 2 (TRAF2) binding site

Tumor necrosis factor receptor-associated factors (TRAFs) belong to a family of adapter proteins that are involved in tumor necrosis factor receptor superfamily signaling. It has been shown that the recruitment of TRAFs to the CD40 cytoplasmic tail is essential for CD40-mediated B cell responses. However, it has also been shown that some early B cell responses, such as up-regulation of cell surface molecules and B cell proliferation are only marginally impaired by the disruption of previously defined TRAF binding sites (Ahonen, C., Manning, E., Erickson, L. D., O'Connor, B. P., Lind, E. F., Pullen, S. S., Kehry, M. R., and Noelle, R. J. (2002) Nat. Immunol. 3, 451-456; and Manning, E., Pullen, S. S., Souza, D. J., Kehry, M., and Noelle, R. J. (2002) Eur. J. Immunol. 32, 39-49). In this report, we identify a second TRAF2 binding site in the CD40 C terminus. The binding motif "SVQE" fits into the major TRAF2 binding consensus sequence, and its disruption resulted in the loss of remaining CD40 functions. Hence, like CD30, the CD40 cytoplasmic tail contains two distinct and functionally important TRAF2 binding sites.     

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 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.     

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.     

Synergistic B cell activation by CD40 and the B cell antigen receptor: role of B lymphocyte antigen receptor-mediated kinase activation and tumor necrosis factor receptor-associated factor regulation

Optimal activation of B-lymphocytes depends both upon expression of various cell surface receptors and adequate integration of signaling pathways. This requires signals generated upon recognition of antigen by the B lymphocyte antigen receptor (BCR) as well as additional signals provided by cognate interaction with T helper cells, including the CD40-CD154 interaction. Engagement of both the BCR and CD40 results in synergistic activation of B cells. Previous studies identified tumor necrosis factor receptor-associated factor (TRAF)-2 and TRAF3 in the CD40-signaling pathway together with BCR-activated protein kinase D (PKD) as important cooperative factors in this synergy. To better understand the role of these factors in bridging the BCR and CD40 signaling pathways, BCR signal regulation of TRAF function was examined. Results show that phosphorylation of TRAF2 is increased upon BCR but not CD40 engagement and that of the potentially phosphorylated residues of TRAF2, tyrosine 484 is crucial for BCR-CD40 synergy. Additionally, wild type or constitutively active Bruton's tyrosine kinase (Btk) enhanced, whereas the xid mutant form of Btk prevented, BCR-CD40 synergy. These effects were dependent upon TRAF2 and PKD activity. These findings suggest a model in which Btk contributes to the enhancement of the CD40 response by TRAF2 in a PKD-dependent manner.     

Intracellular localization and transcriptional regulation of tumor necrosis factor (TNF) receptor-associated factor 4 (TRAF4).

To gain insight in the subcellular localization of tumor necrosis factor receptor-associated factor (TRAF4) we analyzed GFP chimeras of full-length TRAF4 and various deletion mutants derived thereof. While TRAF4-GFP (T4-GFP) was 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 were both localized predominantly to the nucleus. Passive nuclear localization of T4(259-470) can be ruled out as the TRAF domain of TRAF4 was sufficient to form high molecular weight complexes. T4(259-470) recruited full-length TRAF4 into the nucleus whereas TRAF4 was 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 was 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, we investigated whether this pathway is also involved in the regulation of the TRAF4 gene. Indeed, in primary T-cells and Jurkat cells stimulated with the NF-kappaB inducers TNF or phorbol 12-myristate 13-acetate (PMA), TRAF4-mRNA was 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 was completely inhibited. In cells deficient for RIP (receptor interactive protein), an essential signaling intermediate of TNF-dependent NF-kappaB activation, TNF-, but not PMA-induced up-regulation of TRAF4 was blocked. These data suggest that activation of the NF-kappaB pathway is involved in up-regulation of TRAF4 in T-cells.     

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.     

Characterization of the roles of TNF receptor-associated factor 6 in CD40-mediated B lymphocyte effector functions

Signaling through CD40 in B cells leads to B cell proliferation, Ig and IL-6 secretion, isotype switching, and up-regulation of surface molecules. TNF receptor-associated factor (TRAF) proteins associate with the cytoplasmic tail of CD40 and act as adapter molecules. Of the six TRAFs identified to date, TRAFs 2, 3, 5, and 6 are reported to associate directly with the cytoplasmic tail of CD40, but previous studies have principally examined transient overexpression of TRAF6 in cells that do not normally express CD40. Thus, we examined the role of TRAF6 in CD40-mediated B lymphocyte effector functions using two approaches. We produced and stably expressed in mouse B cell lines a human CD40 molecule with two cytoplasmic domain point mutations (hCD40EEAA); this mutant fails to bind TRAF6, while showing normal association with TRAFs 2 and 3. We also inducibly expressed in B cells a transfected "dominant-negative" TRAF6 molecule which contains only the C-terminal TRAF-binding domain of TRAF6. Using both molecules, we found that TRAF6 association with CD40 is important for CD40-induced IL-6 and Ig secretion, and that TRAF6 mediates its effects on CD40-stimulated Ig secretion principally through its effects on IL-6 production by the B cell. TRAF6 association with CD40 was also found to be important for B7-1 up-regulation, but not for up-regulation of other surface molecules. Interestingly, however, although we could show TRAF6-dependent CD40-mediated activation of NF-kappaB in 293 kidney epithelial cells, no such effect was seen in B cells, suggesting that TRAF6 has cell-type-specific functions.     

Differential regulation of CD40-mediated TNF receptor-associated factor degradation in B lymphocytes

Engagement of CD40 on murine B cells by its ligand CD154 induces the binding of TNFR-associated factors (TRAFs) 1, 2, 3, and 6, followed by the rapid degradation of TRAFs 2 and 3. TRAF degradation occurs in response to signaling by other TNFR superfamily members, and is likely to be a normal regulatory component of signaling by this receptor family. In this study, we found that receptor-induced TRAF degradation limits TRAF2-dependent CD40 signals to murine B cells. However, TRAFs 1 and 6 are not degraded in response to CD40 engagement, despite their association with CD40. To better understand the mechanisms underlying differential TRAF degradation, mixed protein domain TRAF chimeras were analyzed in murine B cells. Chimeras containing the TRAF2 zinc (Zn) domains induced effective degradation, if attached to a TRAF domain that binds to the PXQXT motif of CD40. However, the Zn domains of TRAF3 and TRAF6 could not induce degradation in response to CD40, regardless of the TRAF domains to which they were attached. Our data indicate that TRAF2 serves as the master regulator of TRAF degradation in response to CD40 signaling, and this function is dependent upon both the TRAF Zn domains and receptor binding position.     

CD40 signaling through tumor necrosis factor receptor-associated factors (TRAFs). Binding site specificity and activation of downstream pathways by distinct TRAFs.

Tumor necrosis factor receptor-associated factors (TRAFs) associate with the CD40 cytoplasmic domain and initiate signaling after CD40 receptor multimerization by its ligand. We used saturating peptide-based mutational analyses of the TRAF1/TRAF2/TRAF3 and TRAF6 binding sequences in CD40 to finely map residues involved in CD40-TRAF interactions. The core binding site for TRAF1, TRAF2, and TRAF3 in CD40 could be minimally substituted. The TRAF6 binding site demonstrated more amino acid sequence flexibility and could be optimized. Point mutations that eliminated or enhanced binding of TRAFs to one or both sites were made in CD40 and tested in quantitative CD40-TRAF binding assays. Sequences flanking the core TRAF binding sites were found to modulate TRAF binding, and the two TRAF binding sites were not independent. Cloned stable transfectants of human embryonic kidney 293 cells that expressed wild type CD40 or individual CD40 mutations were used to demonstrate that both TRAF binding sites were required for optimal NF-kappaB and c-Jun N-terminal kinase activation. In contrast, p38 mitogen-activated protein kinase activation was primarily dependent upon TRAF6 binding. These studies suggest a role in CD40 signaling for competitive TRAF binding and imply that CD40 responses reflect an integration of signals from individual TRAFs.     

Recruitment of CD40 and tumor necrosis factor receptor-associated factors 2 and 3 to membrane microdomains during CD40 signaling

Signals delivered to antigen-presenting cells through CD40 are critical for the activation of immune responses. Intracellular tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are key elements of the signal transduction pathways of many TNF receptor family members, including CD40. We show for the first time that engagement of CD40 in intact B cells induces the rapid translocation of TRAF2 from the cytoplasm to the plasma membrane. We found that CD40 engagement also results in its recruitment, together with TRAF2 and TRAF3, to membrane microdomains, regions of the plasma membrane enriched in signaling molecules such as the Src family kinases. Using a membrane-permeable chelator of zinc or a mutant TRAF2 molecule, we show that the putative zinc-binding domains of TRAFs contribute to their recruitment to microdomains and to the downstream activation of c-Jun N-terminal kinase. We suggest that the zinc RING and zinc finger domains of TRAFs are required for communication between CD40 and microdomain-associated signaling molecules and may serve a similar role in the signal transduction pathways of other TNF receptor family members.