These experiments indicate that TES2/CTAR2 signaling is similar but not identical to TNFR1 death domain signaling. TES2/CTAR2 and TNFR1 directly interact with the TRADD death domain and stably associate with TRADD when their cytoplasmic tails are aggregated. The six hydrophobic transmembrane domains of each LMP1 molecule mediate constitutive oligomeric aggregation in the plasma membrane (Fig. ), whereas trimeric TNF-α ligand mediates trimerization of TNFR1 in the plasma membrane (20
). Both LMP1 and trimerized TNFR1 associate with TRADD, recruit TRAF2, activate NIK and IκB kinases, phosphorylate IκB, and activate NF-κB (1
). LMP1 TES2/CTAR2 and TNFR1 also share the ability to activate c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) (14
The studies reported here also delineate differences between LMP1 TES2/CTAR2 and TNFR1 that advance our understanding of how LMP1 and TNFR1 alter cell growth and survival. Eleven amino acids of LMP1 are sufficient for engaging the TRADD death domain to activate NF-κB, whereas the TNFR1 death domain that engages the TRADD death domain is about 70 residues (53
). Further, while TRADD residues 296 to 299 or 300 to 302 are critical for TRADD interaction with itself or with TNFR1 as well as for NF-κB activation or for apoptosis (22
), these TRADD residues are not required for synergy with TES2/CTAR2 in NF-κB activation. A mutant TRADD that is deleted for the C-terminal residues 294 to 312 and for most of the N-terminal TRAF interaction domain even had a partial dominant negative effect on TES2/CTAR2 mediated NF-κB activation. Since the C-terminal residues of the TRADD are not required for LMP1 to synergize with TRADD in NF-κB activation, this TRADD mutant likely binds to TES2/CTAR2 and fails to signal because of the absence of both a wild-type death domain and a wild-type TRAF recruitment domain. The importance of TRADD residues 296 to 299 or 300 to 302 for TNFR1 interaction and for downstream effects and their lack of importance for TES2/CTAR2 signaling are consistent with a model in which the smaller TES2/CTAR2 domain interacts with part of the TRADD death domain and thereby propagates only a subset of the TNFR1 inducible TRADD effects.
While TNFR1 signaling through TRADD results in death domain-mediated recruitment of FADD and FADD-initiated apoptosis, LMP1 TES2/CTAR2 signaling through TRADD is deficient in induction of apoptosis. Preliminary results from yeast two-hybrid analyses reveal a low-level LMP1 TES2/CTAR2 association with FADD. Further, F-LMP1 immunoprecipitation from LCLs results in little or no specific coprecipitation of FADD (27
). TNFR1-induced apoptosis is accentuated by blocking de novo protein synthesis with cycloheximide, by expression of a nondegradable IκBα, or by other interventions that inhibit NF-κB or JNK/SAPK pathways (3
). Neither cycloheximide treatment nor inhibition of NF-κB by expression of a nondegradable IκBα enabled LMP1 TES2/CTAR2 to cause apoptosis. Thus, either LMP1 TES2/CTAR2 is intrinsically unable to transmit a proapoptotic signal through TRADD or TES2/CTAR2 activates an antiapoptotic pathway that is independent of NF-κB or new protein synthesis. LMP1 thus appears able to specifically signal through TRADD and separate NF-κB and JNK/SAPK activation from the induction of apoptosis. Separation of TRADD-mediated effects has been previously described for a TRADD mutant that induces apoptosis but not NF-κB activation (45
Another difference between LMP1 TES2/CTAR2 and TNFR1 is the ability of TES2/CTAR2 to directly interact with another death domain containing protein the Fas receptor-interacting protein RIP (17
). RIP interaction was diminished by the Y384
-to-ID TES2/CTAR2 mutation that substantially diminishes TRADD interaction, NF-κB activation, and LCL outgrowth (28
). Although TES2/CTAR2 associates with RIP at a lower level than TRADD and does not synergize with RIP in NF-κB activation, the RIP interaction is substantial and formally opens the possibility that RIP or another death domain protein is, in addition to TRADD, important in signaling from TES2/CTAR2. RIP is critical for TNFR1-mediated NF-κB activation in Jurkat cells and mouse fibroblasts (33
), and other roles are likely. The RIP kinase domain has no known function, and RIP-deficient mice exhibit runting, neonatal lethality, and lymphoid defects that are not fully explained by defects in TNF-α signaling (33
). In sum, the interaction of RIP with TES2/CTAR2, the association of RIP with LMP1 in LCLs, the additive effect of RIP with LMP1 TES2/CTAR2 in NF-κB activation, and the strong negative effect of the YYD-to-ID mutation on these activities as well as on growth transformation are consistent with the possibility that RIP has a role in LMP1 signaling that mediates growth transformation.
A quite surprising aspect of the experiments reported here was the finding that LMP1 differs strikingly from TNFR1 in not requiring RIP for NF-κB activation in Jurkat cells (33
). LMP1 TES2/CTAR2 activates NF-κB as well in a RIP-deficient Jurkat cell line as in a wild-type Jurkat cell line. The simplest model to explain this discrepancy is that RIP is a critical part of the TNFR1-TRADD signaling complex that activates NF-κB but is not an essential mediator of NF-κB activation downstream of TRADD. In RIP-deficient cells, the TES2/CTAR2-TRADD complex activates NF-κB by recruitment of TRAFs to the TRADD N terminus, as evidenced by the dominant negative effect of a N-terminal TRAF2 deletion mutant on TES2/CTAR2-mediated NF-κB activation (28
These and previous experiments indicate that LMP1 uses TNFR signaling molecules to accomplish an EBV-specific task (9
). The LMP1 amino terminus and six transmembrane domains constitutively aggregate the C-terminal cytoplasmic domains independently of TNF-α ligand (Fig. ). TES1/CTAR1 associates with TRAF1, -2, -3, and -5, activates NF-κB, and contributes to initial resting B-lymphocyte growth transformation, while TES2/CTAR2 associates with TRADD and RIP, activates NF-κB and JNK/SAPK, and enables long-term lymphoblastoid cell outgrowth.