Transmembrane domains 1 and 2 of the latent membrane protein 1 of Epstein-Barr virus contain a lipid raft targeting signal and play a critical role in cytostasis

The latent membrane protein 1 (LMP-1) oncoprotein of Epstein-Barr virus (EBV) is a constitutively active, CD40-like cell surface signaling protein essential for EBV-mediated human B-cell immortalization. Like ligand-activated CD40, LMP-1 activates NF-kappaB and Jun kinase signaling pathways via binding, as a constitutive oligomer, to tumor necrosis factor receptor-associated factors (TRAFs). LMP-1's lipid raft association and oligomerization have been linked to its activation of cell signaling pathways. Both oligomerization and lipid raft association require the function of LMP-1's polytopic multispanning transmembrane domain, a domain that is indispensable for LMP-1's growth-regulatory signaling activities. We have begun to address the sequence requirements of the polytopic hydrophobic transmembrane domain for LMP-1's signaling and biochemical activities. Here we report that transmembrane domains 1 and 2 are sufficient for LMP-1's lipid raft association and cytostatic activity. Transmembrane domains 1 and 2 support NF-kappaB activation, albeit less potently than does the entire polytopic transmembrane domain. Interestingly, LMP-1's first two transmembrane domains are not sufficient for oligomerization or TRAF binding. These results suggest that lipid raft association and oligomerization are mediated by distinct and separable activities of LMP-1's polytopic transmembrane domain. Additionally, lipid raft association, mediated by transmembrane domains 1 and 2, plays a significant role in LMP-1 activation, and LMP-1 can activate NF-kappaB via an oligomerization/TRAF binding-independent mechanism. To our knowledge, this is the first demonstration of an activity's being linked to individual membrane-spanning domains within LMP-1's polytopic transmembrane domain.     

TRAF6 is a critical mediator of signal transduction by the viral oncogene latent membrane protein 1

The oncogenic latent membrane protein 1 (LMP1) of the Epstein-Barr virus recruits tumor necrosis factor-receptor (TNFR)-associated factors (TRAFs), the TNFR-associated death domain protein (TRADD) and JAK3 to induce intracellular signaling pathways. LMP1 serves as the prototype of a TRADD-binding receptor that transforms cells but does not induce apoptosis. Here we show that TRAF6 critically mediates LMP1 signaling to p38 mitogen-activated protein kinase (MAPK) via a MAPK kinase 6-dependent pathway. In addition, NF-kappaB but not c-Jun N-terminal kinase 1 (JNK1) induction by LMP1 involves TRAF6. The PxQxT motif of the LMP1 C-terminal activator region 1 (CTAR1) and tyrosine 384 of CTAR2 together are essential for full p38 MAPK activation and for TRAF6 recruitment to the LMP1 signaling complex. Dominant-negative TRADD blocks p38 MAPK activation by LMP1. The data suggest that entry of TRAF6 into the LMP1 complex is mediated by TRADD and TRAF2. In TRAF6-knockout fibroblasts, significant induction of p38 MAPK by LMP1 is dependent on the ectopic expression of TRAF6. We describe a novel role of TRAF6 as an essential signaling mediator of a transforming oncogene, downstream of TRADD and TRAF2.     

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

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

EB病毒潜伏膜蛋白-1介导的信号传导

EB病毒编码LMP-1介导的信号传导途径已引起人们广泛的注意.它涉及TRAF/TRADD途径,AP-1途径,JAK/STAT及其他途径.就此作一综述,有助于人们认识LMP-1的致瘤效应.     

LMP-1's transmembrane domains encode multiple functions required for LMP-1's efficient signaling

The latent membrane protein-1 (LMP-1) of Epstein-Barr virus (EBV) contributes to the proliferation of infected B lymphocytes by signaling through its binding to cellular signaling molecules. It apparently mimics members of the tumor necrosis factor receptor family, in particular, CD40, by binding a similar set of cellular molecules as does CD40. LMP-1 differs dramatically in its structure from CD40. LMP-1 has six membrane-spanning domains as opposed to CD40's one. LMP-1 also differs from CD40 in its apparent independence of a ligand for its signaling. We have examined the role of LMP-1's membrane-spanning domains in its signaling. Their substitution with six membrane-spanning domains from the LMP-2A protein of EBV yields a derivative which neither coimmunoprecipitates with LMP-1 nor signals to increase the activity of NF-kappaB as does wild-type LMP-1. These observations indicate that LMP-1 has specific sequences in its membrane-spanning domains required for these activities. LMP-1's first and sixth membrane-spanning domains have multiple leucine residues potentially similar to leucine-heptad motifs that can mediate protein-protein interactions in membranes (Gurezka et al., J. Biol. Chem. 274:9265-9270, 1999). Substitution of seven leucines in LMP-1's sixth membrane-spanning domain has no effect on its function, whereas similar substitutions in its first membrane-spanning domain yielded a derivative which aggregates as does wild-type LMP-1 but has only 3% of wild-type's ability to signal through NF-kappaB. Importantly, this derivative complements a mutant of LMP-1 with wild-type membrane-spanning domains but no carboxy-terminal signaling domain. These findings together indicate that the membrane-spanning domains of LMP-1 contribute multiple functions to its signaling.     

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.     

Characterization of LMP-1's association with TRAF1, TRAF2, and TRAF3.

The latent membrane protein 1 (LMP-1) of Epstein-Barr virus (EBV) contributes to the immortalizing activity of EBV in primary, human B lymphocytes. LMP-1 is targeted to the plasma membrane, where it influences signaling pathways of infected cells. LMP-1 has been found to associate with members of the tumor necrosis factor receptor-associated factor (TRAF) family of proteins. As with LMP-1, the TRAF molecules have been shown to participate in cell signaling pathways. We have characterized and mapped in detail a region of LMP-1 that associates with TRAF1, TRAF2, and TRAF3. TRAF3 alone associates with LMP-1 in a yeast two-hybrid assay, whereas all three TRAF molecules associate with LMP-1 under various conditions when they are assayed in extracts of human cells. TRAF1, TRAF2, and TRAF3 appear to associate independently with LMP-1 but bind an overlapping target site. TRAF3 associates with LMP-1 most avidly and can compete with TRAF1 and TRAF2 for binding to LMP-1. TRAF2 associates with truncated derivatives of the carboxy terminus of LMP-1 more efficiently than with the intact terminus, indicating that LMP-1's conformation may regulate its association with TRAF2. Finally, point mutations that decrease LMP-1's association with the three TRAF molecules to 3 to 20% of wild-type levels do not detectably affect otherwise intact LMP-1's induction of NF-kappaB activity. Therefore, these associations are not necessary for the majority of intact LMP-1's induction of this signaling pathway.     

AIP1/DAB2IP, a novel member of the Ras-GAP family, transduces TRAF2-induced ASK1-JNK activation

Previously we have shown that ASK-interacting protein 1 (AIP1, also known as DAB2IP), a novel member of the Ras-GAP protein family, mediates TNF-induced activation of ASK1-JNK signaling pathway. However, the mechanism by which TNF signaling is coupled to AIP1 is not known. Here we show that AIP1 is localized on the plasma membrane in resting endothelial cells (EC) in a complex with TNFR1. TNF binding induces release of AIP1 from TNFR1, resulting in cytoplasmic translocation and concomitant formation of an intracellular signaling complex comprised of TRADD, RIP1, TRAF2, and AIPl. A proline-rich region (amino acids 796-807) is critical for maintaining AIP1 in a closed form, which associates with a region of TNFR1 distinct from the death domain, the site of TNFR1 association with TRADD. An AIP1 mutant with deletion of this proline-rich region constitutively binds to TRAF2 and ASK1. A PERIOD-like domain (amino acids 591-719) of AIP1 binds to the intact RING finger of TRAF2, and specifically enhances TRAF2-induced ASK1 activation. At the same time, the binding of AIP1 to TRAF2 inhibits TNF-induced IKK-NF-kappaB signaling. Taken together, our data suggest that AIP1 is a novel transducer in TNF-induced TRAF2-dependent activation of ASK1 that mediates a balance between JNK versus NF-kappaB signaling.     

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.     

STAP-2 negatively regulates both canonical and noncanonical NF-kappaB activation induced by Epstein-Barr virus-derived latent membrane protein 1

The signal-transducing adaptor protein 2 (STAP-2) is a recently identified adaptor protein that contains a pleckstrin homology (PH) and Src homology 2 (SH2)-like domains, as well as a proline-rich domain in its C-terminal region. In previous studies, we demonstrated that STAP-2 binds to MyD88 and IKK-alpha or IKK-beta and modulates NF-kappaB signaling in macrophages. In the present study, we found that ectopic expression of STAP-2 inhibited Epstein-Barr virus (EBV) LMP1-mediated NF-kappaB signaling and interleukin-6 expression. Indeed, STAP-2 associated with LMP1 through its PH and SH2-like domains, and these proteins interacted with each other in EBV-positive human B cells. We found, furthermore, that STAP-2 regulated LMP1-mediated NF-kappaB signaling through direct or indirect interactions with the tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3) and TNFR-associated death domain (TRADD) proteins. STAP-2 mRNA was induced by the expression of LMP1 in human B cells. Furthermore, transient expression of STAP-2 in EBV-positive human B cells decreased cell growth. Finally, STAP-2 knockout mouse embryonic fibroblasts showed enhanced LMP1-induced cell growth. These results suggest that STAP-2 acts as an endogenous negative regulator of EBV LMP1-mediated signaling through TRAF3 and TRADD.     

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.     

LMP1, a viral relative of the TNF receptor family,signals principally from intracellular compartments

Latent membrane protein 1 (LMP1) is an Epstein-Barr virus (EBV)-encoded, ligand-independent receptor that mimics CD40. We report here that LMP1 signals principally from intracellular compartments. LMP1 associates simultaneously with lipid rafts and with its signaling molecules, tumor necrosis factor-receptor (TNF-R)-associated factors (TRAFs) and TNF-R1-associated death domain protein (TRADD) intracellularly, although it can be detected at low levels at the plasma membrane, indicating that most of LMP1's signaling complex resides in intracellular compartments. LMP1's signaling is independent of its accumulation at the plasma membrane in different cells, and as demonstrated by a mutant of LMP1 which has significantly reduced localization at the plasma membrane yet signals as efficiently as does wild-type LMP1. The fusion of the transmembrane domain of LMP1 to signaling domains of CD40, TNF-R1 and Fas activates their signaling; we demonstrate that a fusion of LMP1 with CD40 recruits TRAF2 intracellularly. Our results imply that members of the TNF-R family can signal from intracellular compartments containing lipid rafts and may do so when they act in autocrine loops.     

Apoptosis-inducing agents cause rapid shedding of tumor necrosis factor receptor 1 (TNFR1). A nonpharmacological explanation for inhibition of TNF-mediated activation

Several chemical compounds not known to interact with tumor necrosis factor (TNF) signal transducing proteins inhibit TNF-mediated activation of vascular endothelial cells (EC). Four structurally diverse agents, arachidonyl trifluoromethylketone, staurosporine, sodium salicylate, and C6-ceramide, were studied. All four agents caused EC apoptosis at concentrations that inhibited TNF-induced IkappaBalpha degradation. However, evidence of apoptosis was not evident until after several (e.g. 3-12) hours of treatment, whereas 2 h of treatment was sufficient to inhibit TNF responses. IL-1-induced IkappaBalpha degradation was unaffected by these treatments. Inhibition of TNF signaling could not be prevented with either of the broad spectrum caspase inhibitors zVADfmk or yVADcmk. The inhibition of p38 kinase with SB203580 prevented the inhibition of TNF signaling by all agents except arachidonyl trifluoromethylketone. No changes in the levels or molecular weights of the adaptor proteins TRADD (TNF receptor-associated death domain), RIP (receptor-interacting protein), or TRAF2 (TNF receptor-associated factor-2) were caused by apoptogenic drugs. However, TNF receptor 1 (TNFR1) surface expression was significantly reduced by all four agents. Furthermore, TNF-dependent recruitment of TRADD to surface TNFR1 was also inhibited. These data suggest that several putative inhibitors of TNF signaling work by triggering apoptosis and that an early event coincident with the initiation of apoptosis, preceding evidence of injury, is loss of TNFR1. Consistent with this hypothesis, cotreatment of EC with the metalloproteinase inhibitor Tapi (TNF-alpha proteinase inhibitor) blocked the reduction in surface TNFR1 by apoptogenic drugs and prevented inhibition of TNF-induced IkappaBalpha degradation without blocking apoptosis. TNFR1 loss could be a mechanism to limit inflammation in response to apoptotic cell death.     

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.     

Human activation-induced cytidine deaminase is induced by IL-4 and negatively regulated by CD45: implication of CD45 as a Janus kinase phosphatase in antibody diversification

Activation-induced cytidine deaminase (AID) plays critical roles in Ig class switch recombination and V(H) gene somatic hypermutation. We investigated the role of IL-4 in AID mRNA induction, the signaling transduction involved in IL-4-mediated AID induction, and the effect of CD45 on IL-4-dependent AID expression in human B cells. IL-4 was able to induce AID expression in human primary B cells and B cell lines, and IL-4-induced AID expression was further enhanced by CD40 signaling. IL-4-dependent AID induction was inhibited by a dominant-negative STAT6, indicating that IL-4 induced AID expression via the Janus kinase (JAK)/STAT6 signaling pathway. Moreover, triggering of CD45 with anti-CD45 Abs can inhibit IL-4-induced AID expression, and this CD45-mediated AID inhibition correlated with the ability of anti-CD45 to suppress IL-4-activated JAK1, JAK3, and STAT6 phosphorylations. Thus, in humans, IL-4 alone is sufficient to drive AID expression, and CD40 signaling is required for optimal AID production; IL-4-induced AID expression is mediated via the JAK/STAT signaling pathway, and can be negatively regulated by the JAK phosphatase activity of CD45. This study indicates that the JAK phosphatase activity of CD45 can be induced by anti-CD45 Ab treatment, and this principle may find clinical application in modulation of JAK activation in immune-mediated diseases.     

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.     

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.