Cooperation between TNF Receptor-Associated Factors 1 and 2 in CD40 Signaling

Abstract

TNFR-associated factor 1 (TRAF1) is unique among the TRAF family, lacking most zinc-binding features, and showing marked up-regulation following activation signals. However, the biological roles that TRAF1 plays in immune cell signaling have been elusive, with many reports assigning contradictory roles to TRAF1. The overlapping binding site for TRAFs 1, 2, and 3 on many TNFR superfamily molecules, together with the early lethality of mice deficient in TRAFs 2 and 3, has complicated the quest for a clear understanding of the functions of TRAF1. Using a new method for gene targeting by homologous recombination in somatic cells, we produced and studied signaling by CD40 and its viral oncogenic mimic, latent membrane protein 1 (LMP1) in mouse B cell lines lacking TRAF1, TRAF2, or both TRAFs. Results indicate that TRAFs 1 and 2 cooperate in CD40-mediated activation of the B cell lines, with a dual deficiency leading to a markedly greater loss of function than that of either TRAF alone. In the absence of TRAF1, an increased amount of TRAF2 was recruited to lipid rafts, and subsequently, more robust degradation of TRAF2 and TRAF3 was induced in response to CD40 signaling. In contrast, LMP1 did not require either TRAFs 1 or 2 to induce activation. Taken together, our findings indicate that TRAF1 and TRAF2 cooperate in CD40 but not LMP1 signaling and suggest that cellular levels of TRAF1 may play an important role in modulating the degradation of TRAF2 and TRAF3 in response to signals from the TNFR superfamily.

Roles of TRAF molecules in B lymphocyte function

“…Using B cell lines targeted to remove TRAF1, TRAF2, or both by homologous recombination, Xie et al found that, while TRAF1 deficiency does not itself impair most CD40-mediated functions, a dual deficiency in TRAFs 1 and 2 impairs CD40 signaling to a considerably greater degree than a loss of TRAF2 alone. This suggests that TRAF1-TRAF2 interactions cooperate to enhance CD40 signals to B cells [13].…”

“…TRAF1 associates with CD40 primarily indirectly by forming multimers with TRAF2, although a small amount binds CD40 directly if TRAF2 is absent [13]. Because TRAF1 is unique among the TRAFs in lacking a RING domain, its functions have been especially mysterious.…”

“…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.…”

“…TRAF1 is expressed at high levels in LMP1+ lymphoma cells and in EBV-infected non-neoplastic lymphocytes, and LMP1 and TRAF1 co-localize in these cells [25]. Using B cell lines deficient in TRAF1, TRAF2, or both TRAF1 and 2, it was demonstrated that these TRAFs are not required for JNK phosphorylation, NFkB1 or NFkB2 activation in response to signals through LMP1, although these TRAFs do cooperate in CD40 signals to B cells [13]. This also contrasts with studies in 293 adenocarcinoma cells and TRAF2-/-MEFs implicating TRAF2 as a critical TRAF in LMP1 mediated NFkB activation [20,26].…”

“…TRAF1 also associates with TNFR2, and while its role in signaling to B cells by TNFR superfamily members appears to be primarily cooperative with TRAF2, TRAF1 deficiency also inhibits TNF-mediated IgM production [13]. Interestingly, while initial studies in other cell types did not demonstrate TRAF3 binding to TNF receptors, TRAF3 does directly associate with CD120b in B cells [32].…”