Molecular mechanisms of TNFR-associated factor 6 (TRAF6) utilization by the oncogenic viral mimic of CD40, latent membrane protein 1 (LMP1)

Abstract

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.

FIGURE 1.

Role of TRAF6 in early LMP1 signaling events. A, diagram of hCD40, LMP1, and hCD40LMP1 receptors. The hCD40LMP1 chimera (right) consists of the extracellular and TM domains of hCD40 (left) and the CY domain of LMP1 (center). hCD40LMP1 was stably transfected into TRAF6^+/+ or TRAF6^−/− A20.2J cells, and clones were expression-matched for hCD40LMP1 using flow cytometry (data not shown). B, TRAF6^+/+ (T6^+/+) or TRAF6^−/− (T6^−/−) cells expressing hCD40LMP1 were stimulated with sorbitol (S) for 15 min or with anti-hCD40 Ab for 0, 10, 30, or 60 min. Western blots show protein levels for phospho-JNK, phospho-p38, and phospho-TAK1, with total JNK and actin as loading controls. C, similar to B, except cells were stimulated with anti-mCD40 Ab (C, positive control) for 10 min or with anti-hCD40 Ab for 0, 10, 30, or 60 min. Western blots show protein levels for pIκBα and total IκBα, with actin as a loading control. Data shown are representative of three independent experiments.

FIGURE 2.

Requirement of the TRAF6 TRAF-C domain in LMP1 signaling. A, structure of TRAF6 and TRAF6ΔTRAF. TRAF6 (T6) consists of several domains: the zinc-binding domains, the coiled-coil domain, and the TRAF-C domain, which is required for CD40 binding. TRAF6ΔTRAF (T6ΔTRAF) lacks the TRAF-C receptor-binding domain. B, unstimulated hCD40LMP1⁺ TRAF6^+/+, TRAF6^−/−, and TRAF6^−/− cells expressing TRAF6 or TRAF6ΔTRAF were subjected to SDS-PAGE. Western blots show the expression levels of TRAF6 and actin as a loading control. C, hCD40LMP1⁺, TRAF6^−/− cells expressing transfected TRAF6, or TRAF6ΔTRAF were stimulated with sorbitol (S) for 15 min or anti-hCD40 Ab for 0, 30, or 60 min. Western blots show levels of phospho-JNK and phospho-p38, with total JNK and actin as loading controls. D, as in C, except immunoblots show levels of pIκBα and total IκBα, with actin as a loading control. Data shown are representative of three independent experiments.

FIGURE 3.

Requirement of TRAF6 in hCD40LMP1-mediated CD80 up-regulation. A, hCD40LMP1⁺ TRAF6^−/− cells or subclones expressing transfected TRAF6 or TRAF6ΔTRAF were stimulated with anti-hCD40 Ab (hCD40LMP1), MOPC-21 (isotype control Ab), or LPS for 72 h prior to staining with an isotype control Ab (black histograms) or anti-CD80 Ab (gray histograms). Unstimulated cells (no stim.) were included as controls. Each condition, including no stimulation, was performed in triplicate, and data shown are representative histograms (y axis = arbitrary cell number and x axis = log scale, where 1 is 10¹, etc.). The numbers inside the individual plots represent the percentage of cells that were positive for CD80 expression. B, graphical representation of A, depicting the triplicate stimulations and error bars. Data shown are representative of four independent experiments.

FIGURE 4.

TRAF6 association with the CTAR subdomains of LMP1. A, schematic representation of hCD40LMP1, hCD40CTAR1, and hCD40CTAR2 receptors. The CY domain of hCD40LMP1 (left) contains two subdomains that allow the binding of signaling complexes: CTAR1 and CTAR2. The hCD40CTAR1 molecule (center) lacks the CTAR2 subdomain, whereas the hCD40CTAR2 molecule (right) lacks the CTAR1 subdomain. B, TRAF6^+/+ CH12.LX cells stably expressing hCD40Δ55 (hCD40 lacking most of the CY domain), hCD40LMP1, hCD40CTAR1, or hCD40CTAR2 were stimulated with anti-hCD40 Ab-coated magnetic beads for 0 min (-) or 45 min (+) or with MOPC-21 Ab-coated beads (isotype control, C) for 30 min. hCD40 was immunoprecipitated using the same beads. Western blots show levels of TRAF6 and hCD40 (hCD40Δ55, ∼35 kDa; hCD40LMP1, ∼72 kDa; hCD40CTAR1, ∼48 kDa; hCD40CTAR2, ∼65 kDa). Data shown are representative of three independent experiments.

FIGURE 5.

Role of the TRAF binding site in TRAF6 recruitment to hCD40LMP1. A, TRAF6^+/+ CH12.LX cells stably expressing hCD40LMP1 or hCD40LMP1 with a mutated TRAF binding site (hCD40LMP1PQAA1) were stimulated with anti-hCD40 Ab-coated beads for 0 min (-) or 30 min (+). hCD40 was immunoprecipitated using the same beads. Western blots show levels of TRAF6 and LMP1. These images were part of the same membrane, but the middle lanes were omitted here for clarity. B, splenic B cells were purified from mCD40^−/−, mCD40LMP1⁺, or mCD40LMP1PQAA1⁺ mice (n = 2 per group). Cells were stimulated for 30 min with anti-mCD40 Ab-coated beads. mCD40 was immunoprecipitated using the same beads. Western blots show levels of TRAF2 (control), TRAF6, and LMP1. Data shown are representative of four independent experiments.