Expression of the EBV-encoded LMP1 induces a plethora of activities in target cells. These include the oncogenic transformation of rodent fibroblast cell lines, up-regulation of various cell surface markers and antiapoptotic proteins, cytokine production, and differentiation blockade in epithelial cells. Furthermore, LMP1 expression is essential for EBV-induced B-cell immortalization in vitro. The signalling pathways which may mediate these phenomena have recently attracted much attention, but the precise organization of LMP1 signal transduction remains unknown. LMP1 expression leads to the rapid activation of the transcription factor NF-κB, an effect mediated independently by two domains in the cytoplasmic C terminus of the protein: CTAR1 (aa 187 to 231) and CTAR2 (aa 351 to 386). More recent studies indicate that LMP1 also mediates activation of a Ras/mitogen-activated protein kinase (MAPK)-dependent pathway (46
) as well as of the JNK cascade. LMP1-induced JNK/AP-1 activation maps entirely to the CTAR2 domain and occurs with kinetics that mirror those of NF-κB activation (10
The effects of CTAR1 on NF-κB could be attributed to its ability to bind molecules of the TRAF family. Indeed, the membrane-proximal LMP1 domain strongly associates with TRAF1 and TRAF3 but is also capable of binding TRAF2 and TRAF5 (2
). The latter proteins are of particular interest, as they mediate NF-κB activation by CD40 and certain other TNFR family members. This could also possibly account for the ability of LMP1 to mimic many of the effects of CD40 ligation on cell growth, cytokine production, and induction of cell surface markers (7
). Unlike CTAR1, CTAR2 does not directly bind TRAFs; however, a dominant-negative TRAF2 mutant has been shown to partially inhibit CTAR2-induced NF-κB (9
). This phenomenon can be explained by the ability of CTAR2 to interact with TRADD (25
). TRADD was first identified by virtue of its association with the intracellular death domain of TNFRI in response to TNF-α cross-linking, where it acts as a platform for the recruitment of other proteins, one of which is TRAF2, and this interaction leads to NF-κB activation (23
The organization and molecular components of LMP1-mediated JNK signalling are, however, unknown. In this study we have dissected the cytoplasmic C tail of LMP1 and found that sequences critical for JNK activation are localized in the extreme C terminus of CTAR2. Thus, deletion of the last 8 aa (aa 378 to 386) abrogates the ability of LMP1 to signal on the JNK axis. Interestingly, the same sequences appear to be important for CTAR2-mediated NF-κB induction. The significance of this extreme C-terminal LMP1 domain for signalling is further evidence by the observation that single point mutations within aa 379 to 385 severely impair CTAR2-mediated NF-κB and JNK activation in HEK 293 cells. Importantly, deletion of the last 8 aa also abrogates the interaction of the LMP1 C terminus with TRADD, suggesting that this adapter protein may be critical for LMP1 signalling. To investigate the contribution of TRADD to LMP1-induced JNK activation, we have used a chimeric molecule consisting of the extracellular and transmembrane domains of CD2 fused to the cytoplasmic C terminus of LMP1 (CD2.192-LMP1). We have found that induction of JNK activity following receptor cross-linking in CD2.192-LMP1-transfected cells is synergistically augmented by low levels of TRADD expression. TRADD can also potentiate CTAR2-mediated NF-κB (reference 25
and our unpublished observations). Taken together, these data provide functional evidence for the contribution of this death domain protein in LMP1 signalling and confirm a role for TRADD in JNK activation.
This observation inevitably raises the question of a possible role for TRAF2 in LMP1-induced JNK activation downstream of TRADD. TRAF2 recruitment to the TNFRI-TRADD complex has been shown to mediate JNK as well as NF-κB activation following TNFRI cross-linking (35
). In addition, transient overexpression of TRAF2 induces JNK activity in the absence of TNFR aggregation (43
). Further evidence to support a role for TRAF2 in JNK signalling emerges from recent findings suggesting that CD40, which directly binds TRAF2, is a potent activator of JNK (51
) and that lymphocytes from TRAF2 dominant-negative transgenic mice are impaired in CD40L-induced JNK activation (34
To determine the contribution of this molecule to LMP1-induced JNK signalling, we have used a N-terminally deleted TRAF2 mutant [TRAF2Δ(6-86)] which exerts a potent, dominant-negative effect on NF-κB and JNK activity mediated by transient CD40 expression or TNF-α treatment (47
) (Fig. B and D). This dominant-negative TRAF2 was transfected in HEK 293 cells in the presence of CD2.192-LMP1; following CD2 receptor cross-linking, only a partial inhibition of JNK activation was observed, and similar results were obtained when TRAF2Δ(6-86) was coexpressed with wild-type LMP1 or LMP1AxAxA
(Fig. A and C and data not shown). Interestingly, expression of TRAF2Δ(6-86) in 293 cells also confers only a partial blockade of CTAR2-mediated NF-κB activation (28
). While these data demonstrate that TRAF2 is a component of CTAR2 signalling, the inability of dominant-negative TRAF2 to completely abolish these signals may indicate an additional contribution(s) from another TRADD-associated protein(s). The preapoptotic protein RIP, for example, interacts with TNFRI-bound TRADD without disrupting the TRADD-TRAF2 complex, and its overexpression induces both JNK and NF-κB activation (21
). The role of RIP in LMP1 CTAR2-mediated signalling is presently unknown. Alternatively, it is possible that TRAF2 is bound in a stable complex with other proteins and that large amounts or prolonged incubations following transfection are required for TRAF2Δ(6-86) to displace endogenous wild-type TRAF2. Indeed, a number of TRAF2-interacting proteins have been identified, such as TRAF1, TANK/I-TRAF, and cellular inhibitors of apoptosis (c-IAPs), among others (4
), which may influence TRAF2 heterocomplex stability and signalling. Consistent with this possibility is the observation that an increase in the amount of TRAF2Δ(6-86) from 2.5 to 5 μg significantly decreased JNK activation induced by expression of 1 μg of LMP1, from 45 to 60% (Fig. C). A similar requirement for large amounts of dominant-negative TRAF2 to elicit a significant inhibitory effect on JNK activation induced by TNF-α has been previously described (22
). Thus, differences in the affinity and/or stoichiometry of TRADD/TRAF2-associated factors may be responsible for the ability of dominant-negative mutated TRAF2 to completely block TNFRI but not LMP1 CTAR2 signals.
In this context it is also of interest that CD40 but not LMP1 CTAR1 can activate the JNK/AP-1 pathway in 293 cells (10
), a phenomenon which may reflect differences in the interaction of TRAFs with the cytoplasmic tails of CD40 and LMP1. Thus, despite a common PxQxT TRAF-binding motif, TRAF1 interacts with LMP1 CTAR1 directly but with the CD40 cytoplasmic tail only indirectly (6
). In addition, while TRAF2 strongly binds CD40, it interacts only weakly with CTAR1 (6
), and there is some evidence that CTAR1 and CD40 signalling may be quantitatively and qualitatively different (11a
). Alternatively, these data may indicate a disruption in the wiring of signals leading to JNK activation downstream of CTAR1/TRAF2.
The contribution of TRAF2 in CTAR2-mediated signalling is further emphasized by the ability of the TRAF2-interacting proteins A20 and NIK to influence NF-κB and/or JNK activation from this LMP1 C-terminal domain. The NF-κB-inducible zinc finger A20 protein inhibits TNF-α-mediated NF-κB and AP-1 transactivation, presumably by interfering with TRAF2 signal transduction. Indeed, A20 has been shown to block TRAF2-induced NF-κB activation (55
). The ability of A20 to also inhibit interleukin-1-induced NF-κB activation (26
), which is mediated by TRAF6 (3
), suggests that A20 may function as a promiscuous inhibitor of TRAF activities. In this context, our data demonstrating that A20 expression suppresses both LMP1-induced NF-κB and JNK activation while dominant-negative TRAF2 has only a partial effect may indicate an additional role for other TRAF family members in CTAR2-mediated signalling. Interestingly, overexpression of TANK has also been shown to confer a more potent inhibitory effect on CTAR2-mediated NF-κB than the dominant-negative TRAF2 mutant (28
). Unlike A20, the TRAF2-interacting protein kinase NIK appears to regulate LMP1-induced NF-κB but not JNK activation. Thus, expression of the kinase-inactive NIK mutant [NIK(KK429-430AA)] significantly impaired wild-type LMP1-, CTAR1-, and CTAR2-mediated NF-κB but had no effect on JNK signalling. This observation coupled with the reported ability of a dominant-negative SEK to block LMP1-induced JNK but not NF-κB (10
) suggests that these two signalling pathways bifurcate at the level of TRAF2.
Thus, the organization of LMP1 signalling so far appears to be similar but not identical to that of CD40 or TNFRI (reviewed in reference 8
). The CTAR1 domain, which binds TRAF1, TRAF2, and TRAF3, mediates low NF-κB activity via a CTAR1-TRAF2-NIK connection but fails to induce JNK in 293 cells. NIK may in turn activate the recently identified IκB kinase (IKK), which induces phosphorylation and degradation of IκBα and release of functional NF-κB (38
). Indeed, LMP1 appears to activate NF-κB through phosphorylation of IκBα (10
). CTAR2 mimics TNFRI by exploiting TRADD as its signalling adapter. Recruitment of TRAF2 to the LMP1-TRADD complex may modulate JNK/AP-1 and NF-κB signalling but not to the same extent as in TNFRI. Induction of NF-κB may occur via a NIK-dependent cascade similar to that of CTAR1, while the signalling component leading to SEK–JNK–AP-1 activation downstream of TRAF2 is presently unknown. Expression of A20 disrupts both JNK and NF-κB signals. Additional TRADD-interacting molecules which regulate JNK activation from CTAR2 may exist.
The identification of the signalling mechanisms used by the EBV-encoded LMP1 so far reveals important similarities with the pathways activated by TNFR or CD40 cross-linking and may explain its ability to recapitulate many of the functions of this receptor superfamily. However, the present study also highlights interesting differences in the nature of TRADD-dependent effects mediated via CTAR2, which may have important implications for the transforming ability of LMP1.