Our results provide definite evidence that the cellular pro-apoptotic TRADD protein is a critical signaling mediator of the EBV oncoprotein LMP1, and they show that DNA tumor viruses have developed means to modulate the molecular and functional properties of cellular signaling molecules. We have demonstrated that EBV masks TRADD's pro-apoptotic activity and that this unique viral function is intrinsically encoded by the TRADD-binding domain of the LMP1 molecule. Moreover, this property of the 16 C-terminal amino acids of LMP1 is transferable to other receptors such as TNFR1. EBV exploits TRADD for NF-κB signaling by LMP1-CTAR2, which contributes important growth factor-like signals for efficient proliferation of EBV-transformed B lymphocytes [35
]. Quantitative analysis has revealed that a recombinant maxi-EBV harboring a LMP1(Y384G) mutant has a ~90% reduced potential of inducing long-term B cell proliferation compared to wild-type virus [37
]. LMP1 also transforms rat fibroblasts in culture [38
]. Whereas CTAR1 but not CTAR2 was the essential domain for in vitro transformation of Rat1 cells in one report [39
], other studies supported a critical role of CTAR2 in oncogenic transformation of rat fibroblasts in vitro as well as in vivo [40
]. Notably, tumor formation by LMP1-transformed Rat1 cells in nude mice essentially requires NF-κB activity and a functional CTAR2 domain [41
]. According to our data, it is therefore highly likely that TRADD is critically involved in mediating these functions of CTAR2, extending the biological spectrum of TRADD activity to cell proliferation and transformation.
So far, studies on the role of TRADD in LMP1 signaling were solely based on the overexpression of TRADD and TRADD mutants, or RNAi-mediated knockdown of TRADD [2
]. Such experiments yielded conflicting results with respect to the potential role of TRADD in LMP1 signal transduction (see Introduction). The knockdown of TRADD in HEK293 epithelial cells even suggested that TRADD is fully dispensable for LMP1 signaling [11
], although residual TRADD protein might have rescued pathway activities in RNAi experiments. Alternatively, this result might reflect different cell type–specific functions of TRADD. To be able to investigate TRADD's molecular role in LMP1 signaling in a clean genetic system, we generated a TRADD
knockout by deleting both TRADD
alleles from human DG75 B lymphocytes. This is a biologically relevant cell system, because human B lymphocytes are the target cells of EBV. DG75 cells also express TNFR1 and readily responded to TNFα stimulation with activation of the NF-κB pathway. The knockout of TRADD
abrogated TNFα-mediated NF-κB activation in DG75 cells, demonstrating that TNFR1 activation of the canonical NF-κB pathway critically depends on TRADD and that TRADD signaling is intact in DG75 cells.
The molecular mechanisms by which CTAR2 of LMP1 activates NF-κB are poorly understood. It has been unclear which direct interaction partners of CTAR2 mediate NF-κB activation. TRADD was believed to be dispensable for LMP1 signaling to NF-κB [11
]. TRAF3 and TRAF6 are required for NF-κB activation by CTAR2, but TRAF molecules do not interact with CTAR2 directly [5
]. In addition, knockdown of BS69, a potential mediator of TRAF6 binding to CTAR2, did not affect LMP1 signaling to NF-κB [42
]. Here we demonstrate that the CTAR2 domain of LMP1 recruits IKKβ for signaling and that IKKβ recruitment and, thus, activation by LMP1 depends on TRADD. In TNFR1 signaling, activation of IKKβ can be functionally separated from its recruitment to the signaling complex. IKKβ directly binds to TRAF2, which gets recruited to TNFR1 via TRADD [25
]. The activation of IKKβ requires the interaction of IKKγ, the regulatory subunit of the IKK complex, with ubiquitinated RIP1 [28
]. In contrast, TRAF2, RIP1, and IKKγ are dispensable for LMP1 signaling [10
How does NF-κB signaling at CTAR2 work? We propose a mechanism of IKKβ activation at CTAR2 that involves two pathways: a TRADD-dependent IKKβ recruitment pathway and a parallel activation pathway that is critically mediated by TRAF6 (). Such a mechanism resolves the paradox that TRADD is solely required for CTAR2-triggered NF-κB signaling, whereas TRAF6 is essential for both NF-κB and JNK activation. Because TRAF2 is dispensable for NF-κB induction by LMP1 [10
], a yet-undefined factor must be postulated that bridges IKKβ binding to TRADD in LMP1 signaling. We exclude TRAF3 or TRAF6 as candidates, because TRADD has affinity for only TRAF1 and TRAF2 [43
], and we did not detect TRAF3 or TRAF6 in immunoprecipitations together with TRADD and IKKβ (unpublished data). Potential candidates might include p62/PKCζ [44
]. Despite their role in canonical NF-κB signaling, a physical and/or functional interaction of TRAF3 and TRAF6 with CTAR2 must be independent of TRADD, because both TRAFs are critical for JNK activation by CTAR2, which is still intact in TRADD–/– cells. The transcriptional corepressor BS69 was suggested as the potential mediator of TRAF6 interaction with CTAR2 [42
], although a recent report showed that endogenous BS69 is exclusively located within the nucleus interacting with chromatin [45
]. However, cytoplasmic BS69 protein levels beyond the detection limit of the applied BS69 antibody possibly suffice to mediate LMP1 signaling. It is also conceivable that additional and yet-unknown factors might be involved in mediating the interaction of TRAFs with CTAR2.
Schematic Model of TRADD's Role in Nonapoptotic Signaling Induced by the TRADD-Binding Domain of LMP1
Upon overexpression, TRADD is a highly apoptotic protein [22
]. In TNFR1 signaling, TRADD mediates the activation of caspase-dependent apoptosis through FADD [22
]. In contrast, LMP1 is a transforming protein with anti-apoptotic properties that does not induce programmed cell death, even if NF-κB signaling is blocked () [5
]. We showed here that TRADD is essential for NF-κB induction by CTAR2, a domain that delivers signals critical for long-term proliferation of EBV-transformed B lymphocytes [35
]. Thus, TRADD has a unique biological role in LMP1-induced cell proliferation and, therefore, contributes to B cell transformation by EBV. How does LMP1 achieve to exploit TRADD without inducing apoptosis? Our results demonstrated that aa 371–386 of LMP1 encompass an autonomous and unique viral TRADD-binding domain that encodes the nonapoptotic properties of TRADD signaling. This domain has no sequence homology to any known TRADD-binding site of cellular receptors or signaling molecules. Accordingly, the interaction between LMP1 and TRADD does not require a functional death domain in either of the two molecules. Our data demonstrated that this unique molecular structure of the LMP1–TRADD complex is intrinsically determined by the TRADD-binding site of LMP1 and can be transferred together with the nonapoptotic phenotype of TRADD signaling to TNFR1. Therefore, the unique interaction of TRADD with the TRADD-binding site of LMP1 must prevent apoptosis induction by TRADD. Accordingly, we could not detect an interaction of TRADD with the apoptosis mediator FADD in the presence of LMP1 (unpublished data). Sequences within the TRADD death domain, which are required for apoptosis induction, might be masked by components of the LMP1–TRADD complex. Based on our domain-swapping experiments, we can further exclude the possibility that signals originating elsewhere at the LMP1 signaling domain, for instance at CTAR1, are required to suppress apoptosis induction by TRADD, because the 16 C-terminal amino acids of LMP1 did not induce apoptosis in the context of wild-type TNFR1.
We also showed that LMP1 and its anticipated functional analogue, CD40, substantially differ in their receptor-proximal mechanisms activating signal transduction. Very much in contrast to LMP1, CD40 does not require TRADD for signaling. Thus, although both molecules share similarities regarding their role in supporting B-cell proliferation [46
], they work through different receptor-proximal signaling molecules. This is in line with previous observations that LMP1 assembles a more efficient signaling complex than CD40 and that LMP1, but not CD40, signaling is dependent on TRAF3 [20
In summary, our experiments defined an essential and unique role for TRADD in signaling of the viral oncoprotein LMP1. The viral TRADD-binding site of LMP1 determines the nonapoptotic and TRAF6-dependent type of TRADD signaling which leads to the activation of NF-κB. Thus, the human DNA tumor virus EBV has developed strategies to alter the functional and molecular properties of cellular signaling molecules to exploit them for its own purpose of cell transformation.