Roles of TNF Receptor-Associated Factor 3 in Signaling to B Lymphocytes by Carboxyl-Terminal Activating Regions 1 and 2 of the EBV-Encoded Oncoprotein Latent Membrane Protein 1

Abstract

TNFR-associated factor (TRAF)3, an adaptor protein that binds the cytoplasmic domains of both CD40 and the EBV-encoded oncoprotein latent membrane protein (LMP)1, is required for positive signaling by LMP1 but not CD40 in B lymphocytes. The present study further investigated how TRAF3 participates in LMP1 signaling. We found that TRAF3 mediates signaling both through direct interactions with the C-terminal activating region (CTAR)1 of LMP1 and through indirect interactions with the CTAR2 region of LMP1 in mouse B cells. Notably, our results demonstrated that the CTAR2 region appears to inhibit the recruitment of TRAF1 and TRAF2 to membrane rafts by the CTAR1 region. Additionally, the absence of TRAF2 in B cells resulted in only a modest reduction in CTAR1-mediated signals and no detectable effect on CTAR2-mediated signals. CTAR1 and CTAR2 cooperated to achieve the robust signaling activity of LMP1 when recruited to the same membrane microdomains in B cells. Interestingly, TRAF3 deficiency completely abrogated the cooperation between CTAR1 and CTAR2, supporting the hypothesis that TRAF3 participates in the physical interaction between CTAR1 and CTAR2 of LMP1. Together, our findings highlight the central importance of TRAF3 in LMP1-mediated signaling, which is critical for EBV persistent infection and EBV-associated pathogenesis.

Roles of TRAF molecules in B lymphocyte function

“…To date, no specific investigation of the role of TRAF5 in LMP1 signal transduction in B cells has been reported. TRAF6 does not appear to interact directly with LMP1 in B cells [21] or MEFs [27]. However, TRAF6 is required for signals through LMP1 in MEFs and epithelial cells [20,27].…”

“…However, initial work defining LMP1 signal transduction was performed mainly in transformed epithelial cell or mouse embryonic fibroblast (MEF) models. Recently, our laboratory has studied the TRAF requirements for LMP1 signaling in mouse B cell lines transfected with chimeric CD40-LMP1, using cell lines made deficient in TRAFs through gene targeting through somatic recombination [6,11,13,21]. Use of these cell lines has helped to clarify the TRAF requirements for LMP1 signaling in B cells.…”

“…The first, containing amino acids 194-232 (CTAR1), contains a TRAF binding site that has been shown to interact with TRAFs 1,2,3 and 5. The second activating domain, residues 351-386, (CTAR2) does not contain a TRAF binding site, though it does contain a PVQLSYYD site that when mutated may increase binding of TRAFs 1 and 2 [21]. The TRAF binding site in CTAR1 is PQQAT (DD).…”

“…Not all LMP1 responses are dependent on TRAF3, as LMP1 in B cells deficient in TRAF3 can still activate ERK1,2, and Akt, and cytokine production is not affected [6]. TRAF5 has also been shown to associate with the PQAAT site in LMP1 [21]. To date, no specific investigation of the role of TRAF5 in LMP1 signal transduction in B cells has been reported.…”

“…More recently BS69 has been described as a potential bridging molecule between LMP1 CTAR2 and TRAF6 [28]. In mouse B cell lines, TRADD does not detectably interact with CTAR2 [21]. It is not yet known whether BS69 or RIP could serve to assist the binding of TRAF6 to LMP1 in B cells.…”