Pursuing different 'TRADDes': TRADD signaling induced by TNF-receptor 1 and the Epstein-Barr virus oncoprotein LMP1

The pro-apoptotic tumor necrosis factor (TNF)-receptor 1-associated death domain protein (TRADD) was initially identified as the central signaling adapter molecule of TNF-receptor 1 (TNFR1). Upon stimulation with the pro-inflammatory cytokine TNFalpha, TRADD is recruited to the activated TNFR1 by direct interaction between the death domains of both molecules. TRADD mediates TNFR1 activation of NF-kappaB and c-Jun N-terminal kinase (JNK), as well as caspase-dependent apoptosis. Surprisingly, TRADD is also recruited by latent membrane protein 1 (LMP1), the major oncoprotein of the human Epstein-Barr tumor virus. By mimicking a constitutively active receptor, LMP1 is essential for B-cell transformation by the virus, activating NF-kappaB, phosphatidylinositol 3-kinase, JAK/STAT and mitogen-activated protein kinase signaling. In contrast to TNFR1, LMP1's interaction with TRADD is independent of a functional death domain. The unique structure of the LMP1-TRADD complex dictates an unusual type of TRADD-dependent NF-kappaB signaling and subverts TRADD's potential to induce apoptosis. This article provides an overview of TNFR1 and LMP1 signal transduction with a focus on TRADD's functions in apoptotic and transforming signaling, incorporating recent results from TRADD RNAi and knockout studies.     

LMP1 signal transduction differs substantially from TNF receptor 1 signaling in the molecular functions of TRADD and TRAF2

The Epstein-Barr virus latent membrane protein 1 (LMP1) binds tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) and the TNFR-associated death domain protein (TRADD). Moreover, it induces NF-kappaB and the c-Jun N-terminal kinase 1 (JNK1) pathway. Thus, LMP1 appears to mimick the molecular functions of TNFR1. However, TNFR1 elicits a wide range of cellular responses including apoptosis, whereas LMP1 constitutes a transforming protein. Here we mapped the JNK1 activator region (JAR) of the LMP1 molecule. JAR overlaps with the TRADD-binding domain of LMP1. In contrast to TNFR1, LMP1 recruits TRADD via the TRADD N-terminus but not the TRADD death domain. Consequently, the molecular function of TRADD in LMP1 signaling differs from its role in TNFR1 signal transduction. Whereas NF-kappaB activation by LMP1 was blocked by a dominant-negative TRADD mutant, LMP1 induces JNK1 independently of the TRADD death domain and TRAF2, which binds to TRADD. Further downstream, JNK1 activation by TNFR1 involves Cdc42, whereas LMP1 signaling to JNK1 is independent of p21 Rho-like GTPases. Although both LMP1 and TNFR1 interact with TRADD and TRAF2, the different topologies of the signaling complexes correlate with substantial differences between LMP1 and TNFR1 signal transduction to JNK1.     

TRADD contributes to tumour suppression by regulating ULF-dependent p19Arf ubiquitylation

Tumour necrosis factor receptor (TNFR)-associated death domain (TRADD) protein is a central adaptor in the TNFR1 signalling complex that mediates both cell death and inflammatory signals. Here, we report that Tradd deficiency in mice accelerated tumour formation in a chemical-induced carcinogenesis model independently of TNFR1 signalling. In vitro, primary cells lacking TRADD were less susceptible to HRas-induced senescence and showed a reduced level of accumulation of the p19(Arf) tumour suppressor protein. Our data indicate that TRADD shuttles dynamically from the cytoplasm into the nucleus to modulate the interaction between p19(Arf) and its E3 ubiquitin ligase ULF, thereby promoting p19(Arf) protein stability and tumour suppression. These results reveal a previously unknown tumour-suppressive role for nuclear TRADD, augmenting its long-established cytoplasmic functions in inflammatory and immune signalling cascades. Our findings also make an important contribution to the rapidly expanding field of p19(Arf) post-translational regulation.     

The Epstein-Barr Virus Oncoprotein Latent Membrane Protein 1 Engages the Tumor Necrosis Factor Receptor-Associated Proteins TRADD and Receptor-Interacting Protein (RIP) but Does Not Induce Apoptosis or Require RIP for NF-κB Activation

A site in the Epstein-Barr virus (EBV) transforming protein LMP1 that constitutively associates with the tumor necrosis factor receptor 1 (TNFR1)-associated death domain protein TRADD to mediate NF-kappaB and c-Jun N-terminal kinase activation is critical for long-term lymphoblastoid cell proliferation. We now find that LMP1 signaling through TRADD differs from TNFR1 signaling through TRADD. LMP1 needs only 11 amino acids to activate NF-kappaB or synergize with TRADD in NF-kappaB activation, while TNFR1 requires approximately 70 residues. Further, LMP1 does not require TRADD residues 294 to 312 for NF-kappaB activation, while TNFR1 requires TRADD residues 296 to 302. LMP1 is partially blocked for NF-kappaB activation by a TRADD mutant consisting of residues 122 to 293. Unlike TNFR1, LMP1 can interact directly with receptor-interacting protein (RIP) and stably associates with RIP in EBV-transformed lymphoblastoid cell lines. Surprisingly, LMP1 does not require RIP for NF-kappaB activation. Despite constitutive association with TRADD or RIP, LMP1 does not induce apoptosis in EBV-negative Burkitt lymphoma or human embryonic kidney 293 cells. These results add a different perspective to the molecular interactions through which LMP1, TRADD, and RIP participate in B-lymphocyte activation and growth.     

Stat1 as a component of tumor necrosis factor alpha receptor 1-TRADD signaling complex to inhibit NF-kappaB activation

Activated tumor necrosis factor alpha (TNF-alpha) receptor 1 (TNFR1) recruits TNFR1-associated death domain protein (TRADD), which in turn triggers two opposite signaling pathways leading to caspase activation for apoptosis induction and NF-kappaB activation for antiapoptosis gene upregulation. Here we show that Stat1 is involved in the TNFR1-TRADD signaling complex, as determined by employing a novel antibody array screening method. In HeLa cells, Stat1 was associated with TNFR1 and this association was increased with TNF-alpha treatment. TNFR1 signaling factors TRADD and Fas-associated death domain protein (FADD) were also found to interact with Stat1 in a TNF-alpha-dependent process. Our in vitro recombinant protein-protein interaction studies demonstrated that Stat1 could directly interact with TNFR1 and TRADD but not with FADD. Interaction between Stat1 and receptor-interacting protein (RIP) or TNFR-associated factor 2 (TRAF2) was not detected. Examination of Stat1-deficient cells showed an apparent increase in TNF-alpha-induced TRADD-RIP and TRADD-TRAF2 complex formation, while interaction between TRADD and FADD was unaffected. As a consequence, TNF-alpha-mediated I-kappaB degradation and NF-kappaB activation were markedly enhanced in Stat1-deficient cells, whereas overexpression of Stat1 in 293T cells blocked NF-kappaB activation by TNF-alpha. Thus, Stat1 acts as a TNFR1-signaling molecule to suppress NF-kappaB activation.     

The viral oncoprotein LMP1 exploits TRADD for signaling by masking its apoptotic activity

The tumor necrosis factor (TNF)-receptor 1–associated death domain protein (TRADD) mediates induction of apoptosis as well as activation of NF-κB by cellular TNF-receptor 1 (TNFR1). TRADD is also recruited by the latent membrane protein 1 (LMP1) oncoprotein of Epstein-Barr virus, but its role in LMP1 signaling has remained enigmatic. In human B lymphocytes, we have generated, to our knowledge, the first genetic knockout of TRADD to investigate TRADD's role in LMP1 signal transduction. Our data from TRADD-deficient cells demonstrate that TRADD is a critical signaling mediator of LMP1 that is required for LMP1 to recruit and activate I-κB kinase β (IKKβ). However, in contrast to TNFR1, LMP1-induced TRADD signaling does not induce apoptosis. Searching for the molecular basis for this observation, we characterized the 16 C-terminal amino acids of LMP1 as an autonomous and unique virus-derived TRADD-binding domain. Replacing the death domain of TNFR1 by LMP1′s TRADD-binding domain converts TNFR1 into a nonapoptotic receptor that activates NF-κB through a TRAF6-dependent pathway, like LMP1 but unlike wild-type TNFR1. Thus, the unique interaction of LMP1 with TRADD encodes the transforming phenotype of viral TRADD signaling and masks TRADD's pro-apoptotic function.     

Nuclear and cytoplasmic shuttling of TRADD induces apoptosis via different mechanisms

The adapter protein tumor necrosis factor receptor (TNFR)1-associated death domain (TRADD) plays an essential role in recruiting signaling molecules to the TNFRI receptor complex at the cell membrane. Here we show that TRADD contains a nuclear export and import sequence that allow shuttling between the nucleus and the cytoplasm. In the absence of export, TRADD is found within nuclear structures that are associated with promyelocytic leukemia protein (PML) nuclear bodies. In these structures, the TRADD death domain (TRADD-DD) can activate an apoptosis pathway that is mechanistically distinct from its action at the membrane-bound TNFR1 complex. Apoptosis by nuclear TRADD-DD is promyelocytic leukemia protein dependent, involves p53, and is inhibited by Bcl-xL but not by caspase inhibitors or dominant negative FADD (FADD-DN). Conversely, apoptosis induced by TRADD in the cytoplasm is resistant to Bcl-xL, but sensitive to caspase inhibitors and FADD-DN. These data indicate that nucleocytoplasmic shuttling of TRADD leads to the activation of distinct apoptosis mechanisms that connect the death receptor apparatus to nuclear events.     

The role of TRADD in death receptor signaling

TRADD (TNFR1-associated death domain protein) was initially identified as an adaptor molecule that transduces the signal downstream of the TNFR1 (tumor necrosis factor receptor 1). TNFR1 belongs to the so-called death receptor (DR) family of receptors that depending on the context can induce either apoptosis or proliferation, as well as NF-κB and MAP kinase activation. The receptors of this group contain death domain (DD) that is necessary for the induction of apoptosis. This review summarizes the recent advances in the field of DR signaling and in particular the role of TRADD.     

The role of TRADD in death receptor signaling

TRADD (TNFR1-associated death domain protein) was initially identified as an adaptor molecule that transduces the signal downstream of the TNFR1 (tumor necrosis factor receptor 1). TNFR1 belongs to the so-called death receptor (DR) family of receptors that depending on the context can induce either apoptosis or proliferation, as well as NF-κB and MAP kinase activation. The receptors of this group contain death domain (DD) that is necessary for the induction of apoptosis. This review summarizes the recent advances in the field of DR signaling and in particular the role of TRADD.     

Solution structure of the tumor necrosis factor receptor-1 death domain.

Tumor necrosis factor receptor-1 death domain (TNFR-1 DD) is the intracellular functional domain responsible for the receptor signaling activities. The solution structure of the R347K mutant of TNFR-1 DD was solved by NMR spectroscopy. A total of 20 structures were calculated by means of hybrid distance geometry-simulated annealing using a total of 1167 distance constraints and 117 torsion angle constraints. The atomic rms distribution about the mean coordinate positions for the 20 structures for residues composing the secondary structure region is 0.40 A for the backbone atoms and 1.09 A for all atoms. The structure consists of six antiparallel alpha-helices arranged in a similar fashion to the other members of the death domain superfamily. The secondary structure and three-dimensional structure of R347K TNFR1-DD are very similar to the secondary structure and deduced topology of the R347A TNFR1-DD mutant. Mutagenesis studies identified critical residues located in alpha2 and part of alpha3 and alpha4 that are crucial for self-interaction and interaction with TRADD. Structural superposition with previously solved proteins in the death domain superfamily reveals that the major differences between the structures reside in alpha2, alpha3, and alpha4. Interestingly, these regions correspond to the binding sites of TNFR1-DD, providing a structural basis for the specificity of death domain interactions and its subsequent signaling event.     

Phospho-SXXE/D motif mediated TNF receptor 1-TRADD death domain complex formation for T cell activation and migration

In TNF-treated cells, TNFR1, TNFR-associated death domain protein (TRADD), Fas-associated death domain protein, and receptor-interacting protein kinase proteins form the signaling complex via modular interaction within their C-terminal death domains. In this paper, we report that the death domain SXXE/D motifs (i.e., S381DHE motif of TNFR1-death domain as well as S215LKD and S296LAE motifs of TRADD-death domain) are phosphorylated, and this is required for stable TNFR1-TRADD complex formation and subsequent activation of NF-κB. Phospho-S215LKD and phospho-S296LAE motifs are also critical to TRADD for recruiting Fas-associated death domain protein and receptor-interacting protein kinase. IκB kinase β plays a critical role in TNFR1 phosphorylation of S381, which leads to subsequent T cell migration and accumulation. Consistently, we observed in inflammatory bowel disease specimens that TNFR1 was constitutively phosphorylated on S381 in those inflammatory T cells, which had accumulated in high numbers in the inflamed mucosa. Therefore, SXXE/D motifs found in the cytoplasmic domains of many TNFR family members and their adaptor proteins may serve to function as a specific interaction module for the α-helical death domain signal transduction.     

A mechanism for death receptor discrimination by death adaptors

The death domain and death effector domain are two common motifs that mediate protein-protein interactions between components of cell death signaling complexes. The mechanism by which these domains engage their binding partners has been explored by extensive mutagenesis of two death adaptors, FADD and TRADD, suggesting that a death adaptor can discriminate its intended binding partners from other proteins harboring similar motifs. Death adaptors are found to utilize one of two topologically conserved surfaces for protein-protein interaction, whether that partner is another adaptor or its cognate receptor. These surfaces are topologically related to the interaction between death domains observed in the x-ray crystal structure of the Drosophila adaptor Tube bound to Pelle kinase. Comparing the topology of protein-protein interactions for FADD complexes to TRADD complexes reveals that FADD uses a Tube-like surface in each of its death motifs to engage either CD95 or TRADD. TRADD reverses these roles, employing a Pelle-like surface to interact with either receptor TNFR1 or adaptor FADD. Since death adaptors display a Tube-like or Pelle-like preference for engaging their binding partners, Tube/Pelle-like pairing provides a mechanism for death adaptor discrimination of death receptors.     

The adaptor protein TRADD is essential for TNF-like ligand 1A/death receptor 3 signaling

TNFR-associated death domain protein (TRADD) is a key effector protein of TNFR1 signaling. However, the role of TRADD in other death receptor (DR) signaling pathways, including DR3, has not been completely characterized. Previous studies using overexpression systems suggested that TRADD is recruited to the DR3 complex in response to the DR3 ligand, TNF-like ligand 1A (TL1A), indicating a possible role in DR3 signaling. Using T cells from TRADD knockout mice, we demonstrate in this study that the response of both CD4(+) and CD8(+) T cells to TL1A is dependent upon the presence of TRADD. TRADD knockout T cells therefore lack the appropriate proliferative response to TL1A. Moreover, in the absence of TRADD, both the stimulation of MAPK signaling and activation of NF-κB in response to TL1A are dramatically reduced. Unsurprisingly, TRADD is required for recruitment of receptor interacting protein 1 and TNFR-associated factor 2 to the DR3 signaling complex and for the ubiquitination of receptor interacting protein 1. Thus, our findings definitively establish an essential role of TRADD in DR3 signaling.     

Competitive control of independent programs of tumor necrosis factor receptor-induced cell death by TRADD and RIP1

Stimulation of tumor necrosis factor receptor 1 (TNFR1) can initiate several cellular responses, including apoptosis, which relies on caspases, necrotic cell death, which depends on receptor-interacting protein kinase 1 (RIP1), and NF-kappaB activation, which induces survival and inflammatory responses. The TNFR-associated death domain (TRADD) protein has been suggested to be a crucial signal adaptor that mediates all intracellular responses from TNFR1. However, cells with a genetic deficiency of TRADD are unavailable, precluding analysis with mature immune cell types. We circumvented this problem by silencing TRADD expression with small interfering RNA. We found that TRADD is required for TNFR1 to induce NF-kappaB activation and caspase-8-dependent apoptosis but is dispensable for TNFR1-initiated, RIP1-dependent necrosis. Our data also show that TRADD and RIP1 compete for recruitment to the TNFR1 signaling complex and the distinct programs of cell death. Thus, TNFR1-initiated intracellular signals diverge at a very proximal level by the independent association of two death domain-containing proteins, RIP1 and TRADD. These single transducers determine cell fate by triggering NF-kappaB activation, apoptosis, and nonapoptotic death signals through separate and competing signaling pathways.     

Inhibition of receptor internalization by monodansylcadaverine selectively blocks p55 tumor necrosis factor receptor death domain signaling

The 55-kDa receptor for tumor necrosis factor (TR55) triggers multiple signaling cascades initiated by adapter proteins like TRADD and FAN. By use of the primary amine monodansylcadaverine (MDC), we addressed the functional role of tumor necrosis factor (TNF) receptor internalization for intracellular signal distribution. We show that MDC does not prevent the interaction of the p55 TNF receptor (TR55) with FAN and TRADD. Furthermore, the activation of plasmamembrane-associated neutral sphingomyelinase activation as well as the stimulation of proline-directed protein kinases were not affected in MDC-treated cells. In contrast, activation of signaling enzymes that are linked to the "death domain" of TR55, like acid sphingomyelinase and c-Jun-N-terminal protein kinase as well as TNF signaling of apoptosis in U937 and L929 cells, are blocked in the presence of MDC. The results of our study suggest a role of TR55 internalization for the activation of select TR55 death domain signaling pathways including those leading to apoptosis.     

Hepatitis C virus core protein inhibits tumor necrosis factor alpha-mediated apoptosis by a protective effect involving cellular FLICE inhibitory protein

We have previously shown that hepatitis C virus (HCV) core protein modulates multiple cellular processes, including those that inhibit tumor necrosis factor alpha (TNF-alpha)-mediated apoptosis. In this study, we have investigated the signaling mechanism for inhibition of TNF-alpha-mediated apoptosis in human hepatoma (HepG2) cells expressing core protein alone or in context with other HCV proteins. Activation of caspase-3 and the cleavage of DNA repair enzyme poly(ADP-ribose) polymerase were inhibited upon TNF-alpha exposure in HCV core protein-expressing HepG2 cells. In vivo protein-protein interaction studies displayed an association between TNF receptor 1 (TNFR1) and TNFR1-associated death domain protein (TRADD), suggesting that the core protein does not perturb this interaction. A coimmunoprecipitation assay also suggested that HCV core protein does not interfere with the TRADD-Fas-associated death domain protein (FADD)-procaspase-8 interaction. Further studies indicated that HCV core protein expression inhibits caspase-8 activation by sustaining the expression of cellular FLICE (FADD-like interleukin-1beta-converting enzyme)-like inhibitory protein (c-FLIP). Similar observations were also noted upon expression of core protein in context to other HCV proteins expressed from HCV full-length plasmid DNA or a replicon. A decrease in endogenous c-FLIP by specific small interfering RNA induced TNF-alpha-mediated apoptotic cell death and caspase-8 activation. Taken together, our results suggested that the TNF-alpha-induced apoptotic pathway is inhibited by a sustained c-FLIP expression associated with the expression of HCV core protein, which may play a role in HCV-mediated pathogenesis.     

Solution structure of N-TRADD and characterization of the interaction of N-TRADD and C-TRAF2, a key step in the TNFR1 signaling pathway

TRADD is a multifunctional signaling adaptor protein that is recruited to TNFR1 upon ligand binding. The C-terminal of TRADD comprises the "death domain" that is responsible for association of TNFR1 and other death domain-containing proteins such as FADD and RIP. The N-terminal domain (N-TRADD) promotes the recruitment of TRAF2 to TNFR1 by binding to the C-terminal of TRAF2, leading to the activation of JNK/AP1 and NF-kappa B. The solution structure of N-TRADD was determined, revealing a novel protein fold. A combination of NMR, BIAcore, and mutagenesis experiments was used to help identify the site of interaction of N-TRADD with C-TRAF2, providing a framework for future attempts to selectively inhibit the TNF signaling pathways.     

Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD.

Keratin 8 and 18 (K8/18) are the major components of intermediate filament (IF) proteins of simple or single-layered epithelia. Recent data show that normal and malignant epithelial cells deficient in K8/18 are nearly 100 times more sensitive to tumor necrosis factor (TNF)-induced cell death. We have now identified human TNF receptor type 1 (TNFR1)-associated death domain protein (TRADD) to be the K18-interacting protein. Among IF proteins tested in two-hybrid systems, TRADD specifically bound K18 and K14, type I (acidic) keratins. The COOH-terminal region of TRADD interacted with the coil Ia of the rod domain of K18. Endogenous TRADD coimmunoprecipitated with K18, and colocalized with K8/18 filaments in human mammary epithelial cells. Overexpression of the NH2 terminus (amino acids 1-270) of K18 containing the TRADD-binding domain as well as overexpression of K8/18 in SW13 cells, which are devoid of keratins, rendered the cells more resistant to killing by TNF. We also showed that overexpressed NH2 termini of K18 and K8/18 were associated with endogenous TRADD in SW13 cells, resulting in the inhibition of caspase-8 activation. These results indicate that K18 may sequester TRADD to attenuate interactions between TRADD and activated TNFR1 and moderate TNF-induced apoptosis in simple epithelial cells.     

The adaptor protein TRADD activates distinct mechanisms of apoptosis from the nucleus and the cytoplasm

TNFR1 associated death domain protein (TRADD) contains an N-terminal TRAF binding domain and a C-terminal death domain along with nuclear import and export sequences that cause shuttling between the cytoplasm and nucleus. The death domain of TRADD contains the nuclear import sequence and expression of the core death domain (nuclear TRADD) results in exclusive nuclear localization and activation of a distinct apoptotic pathway. Cytoplasmic TRADD activates apoptosis through Fas-associated death domain protein (FADD) and caspase-8 activation that was blocked by caspase inhibitors or dominant-negative FADD. These inhibitors did not inhibit death induced by nuclear TRADD, which could only be inhibited by combining caspase inhibitors and a serine protease inhibitor. The pathway activated by nuclear TRADD requires caspase-9 catalytic activity. However, apoptosis activating factor deficiency confers only partial protection from death. This pathway represents an alternate means by which TRADD can regulate cell death independently of FADD and caspase-8 that occurs from the nucleus rather than the cytoplasm.     

Tumor necrosis factor receptor-1-induced neuronal death by TRADD contributes to the pathogenesis of Japanese encephalitis

While a number of studies have documented the neurotropism of Japanese encephalitis virus (JEV), little is known regarding the molecular mechanism of neuronal death following viral infection. The tumor necrosis factor receptor (TNFR)-associated death domain (TRADD) has been suggested to be the crucial signal adaptor that mediates all intracellular responses from TNFR-1. Using mouse (Neuro2a) and human (SK-N-SH) neuroblastoma cell lines, we have shown that the altered expression of TNFR-1 and TRADD following JEV infection regulates the downstream apoptotic cascades. Activation of TRADD led to mitochondria-mediated neuronal apoptosis. As TRADD-knockout animals or deficient cell lines are unavailable, it has been difficult to definitively address the physiological role of TRADD in diseases pathology following JEV infection. We circumvented this problem by silencing TRADD expression with small-interfering RNA (siRNA) and have found that TRADD is required for TNFR-1-initiated neuronal apoptosis following in vitro infection with JEV. Interestingly, siRNA against TRADD also decreased the viral load in Neuro2a cells. Furthermore, siRNA against TRADD increased the survival of JEV-infected mice by altering the expression of pro apoptotic versus antiapoptotic molecules. These studies show that the engagement of TNFR-1 and TRADD following JEV infection plays a crucial role in neuronal apoptosis.