Both CD40 and LMP1 stimulate B cell activation and NF-κB/JNK activation 7294143
. Previous studies have shown that the cytoplasmic domain of LMP1 is necessary and sufficient to deliver these activation signals 7264144
, and have suggested that the LMP1 cytoplasmic domain can mimic that of CD40 545
, the major difference between the two being that LMP1 signals constitutively via self-aggregation, whereas CD40 requires engagement by CD154. However, several reports have also presented evidence that important differences exist between the signaling pathways used by CD40 and LMP1. LMP1 expression in the Jurkat T cell line stimulates detectable CD54 upregulation, whereas CD40 does not 44
, and transgenic B cell expression of LMP1 in CD40−/
− mice does not restore the normal development of B cell memory 8
. Additionally, earlier studies from our laboratory showed that CD40 and LMP1 signals can cooperate in B cell activation, further supporting the concept that the two receptors use overlapping but distinct mechanisms of signaling 7
A very early event associated with signaling by both LMP1 and CD40 is the recruitment of the cytoplasmic adapter proteins TRAFs 1, 2, and 3 to cholesterol-rich, detergent-insoluble membrane microdomains, or rafts 2223
. We have recently shown that degradation of TRAF 2 rapidly follows CD40-mediated raft recruitment 23
, a finding we have confirmed in normal splenic B cells (unpublished observations). Although TRAF1 is also recruited to rafts by both CD40 and LMP1, signaling through neither molecule stimulates any detectable degradation of TRAF1 (data not shown). To determine whether LMP1 also stimulates degradation of TRAFs 2 and 3, we used an inducibly expressed LMP1 molecule that we previously found stimulates NF-κB activation and B cell effector functions 7
. Unlike CD40, self-aggregation of WT-LMP1 stimulated no detectable TRAF degradation. This was not due to a deficiency in the strength of the self-aggregation stimulus compared with normal ligand-induced aggregation of CD40, as a chimeric molecule with the membrane-spanning domains of LMP1 and the cytoplasmic domain of CD40 induced TRAF degradation as effectively as did WT CD40 ( versus ).
The failure of LMP1 to mimic CD40 in stimulating the degradation of TRAFs 2 and 3 led us to ask whether this difference is reflected in altered signal strength or duration in downstream effects on B cell activation. Although both CD40 engagement and induced LMP1 expression have been shown to stimulate increased expression of CD80 7
, it is not possible to directly compare this effect between WT CD40 and LMP1, because the modes of initiation of the stimulus are quite different. However, when signaling through the cytoplasmic domains of WT-LMP1 and LMP1-hCD40 molecules was initiated for both molecules by self-aggregation of the LMP1 membrane-spanning domains, induced WT-LMP1 expression stimulated greater upregulation of CD80 than did induced expression of the LMP1-hCD40 molecule ( B). This finding suggested that the failure of LMP1 signaling to induce TRAF degradation may contribute to enhanced B cell activation signals delivered via LMP1. However, direct comparison of signaling, particularly early signaling events, is difficult to make between molecules with LMP1 external domains. These molecules begin to signal as their B cell expression is induced, so initiation of signaling cannot be carefully regulated or synchronized between cell lines. Additionally, as their relative expression must be determined by Western blotting, a technique that is only semiquantitative, it is difficult to ensure that two different transfected cell lines are expressing the same amounts of different LMP1 molecules.
As mentioned above, we and others have shown previously that the cytoplasmic domain of LMP1 is necessary and sufficient for B cell activation signals, and shows that TRAF degradation behavior also maps to the cytoplasmic domain of CD40 versus LMP1. We thus produced a chimeric molecule consisting of the external and transmembrane domains of human CD40 and the cytoplasmic domain of LMP1. This molecule allows quantitation of cell surface expression in transfectants by flow cytometry, and, like CD40, does not signal unless and until it is engaged by CD154 or anti-CD40 mAb. B cell transfectants were generated that were expression matched for either WT-hCD40 or the hCD40-LMP1 chimera ( A). Ligation of these two receptors led to the same outcome as self-aggregation of LMP1 versus LMP1-hCD40; namely, WT-hCD40 induced TRAF 2 and 3 degradation, whereas hCD40-LMP1 did not (). These data validated the use of the hCD40-LMP1 molecule to directly compare signaling by the cytoplasmic domains of CD40 and LMP1. The ability of an inhibitor of the cellular 26S proteasome to diminish the CD40-induced TRAF degradation suggests that the process of ubiquitination may be involved in the degradation, a possibility we are currently investigating.
The inability of the LMP1 molecule to degrade TRAFs 2 and 3 cannot be solely accounted for by strength of TRAF binding. demonstrates that properties of TRAF binding in B cells confirm previous in vitro binding assays 16
. That is, although CD40 appears to bind TRAF2 more effectively, LMP1 shows stronger binding than CD40 to TRAF3. However, CD40 signaling stimulates degradation of both TRAFs, whereas LMP1 signaling stimulates degradation of neither. This suggests that there may be a specific alteration induced in TRAFs 2 and 3 by association with CD40, but not LMP1. This idea is supported by two pieces of evidence. First, although we and others have shown that certain CD40-mediated activation signals are independent of the binding of TRAFs 2 and 3 171920303446
, data presented in show that hCD40 mutants that cannot bind TRAFs 2 or 3 cannot stimulate TRAF degradation. Second, shows that when B cells receive signals from both CD40 and LMP1 cytoplasmic domains, TRAFs 2 and 3 are degraded. Thus, LMP1 signaling does not block CD40-mediated TRAF degradation, although in these transfectants the hCD40-LMP1 molecule is expressed at higher levels than the endogenous mCD40 molecule (not shown). This finding argues against the hypothesis that additional intracellular molecules that bind to LMP1 but not CD40, such as TNFR-associated death domain (TRADD), block TRAF degradation. Additionally, LMP1 mutant molecules lacking the TRADD binding site do not gain the ability to degrade TRAFs (unpublished observations). However, intracellular molecules that associate with CD40 but not LMP1 may participate in the differential ability to degrade TRAFs 2 and 3. For example, CD40 but not LMP1 binds TRAF6, and CD40 utilizes TRAF6 in several of its signaling functions 154748
. We are currently examining the potential role of TRAF6 and other intracellular molecules in mediating degradation of TRAFs 2 and 3 by CD40 signals.
The model system described in this report permitted a direct comparison of the effects of signaling via CD40 and LMP1 on early B cell activation and later B cell effector functions, and allowed us to determine whether the differential ability to degrade TRAFs 2 and 3 was reflected by differences in downstream signaling through CD40 and LMP1. Analysis in both M12.4.1 and CH12.LX transfectants indicated that the cytoplasmic domain of LMP1 was a more potent stimulus for JNK activation, a very early event after initiation of signaling through each of these molecules. Additionally, NF-κB activation was considerably sustained in cells signaled via the LMP1 cytoplasmic domain compared with those signaled via WT-hCD40 (). Both JNK and NF-κB activation have been shown to play important roles in signaling by both CD40 (for a review, see reference 49
) and LMP1 184150
. To determine whether enhancement in these early signals correlated with enhanced B cell effector functions, we compared surface molecule upregulation, IL-6 and IgM secretion induced by ligation of either WT-hCD40 or hCD40-LMP1, and found that for all three effector functions, the LMP1 cytoplasmic domain provided a markedly greater stimulus than that of CD40 ().
This study identifies a novel and important mechanism by which TRAF signaling and thereby the signaling of receptors that utilize TRAFs may be regulated. The potential importance of these results is underscored by the fact that LMP1 is a transforming protein 2
. Previous reports have concluded that the transforming effect of LMP1 on B cells is mostly due to its constitutive CD40 mimicry (see above). The data presented here suggest that the capacity of LMP1 to transform cells may also be due to the maintenance of an amplified and sustained LMP1 signal, permitted by avoiding TRAF degradation. As the TRAF binding region of LMP1 appears to play a necessary and sufficient role in cellular transformation by LMP1 5152
, stimulation through CD40 may be able to ultimately attenuate LMP1 signaling by degrading the TRAFs necessary for its transforming effect. This hypothesis is supported by experimental data indicating CD40 signals protect EBV-infected human peripheral B cells from undergoing transformation in vitro and in vivo 53
In summary, we have identified a novel mechanism for regulating CD40 signaling. This mode of regulation involves rapid proteasome-dependent degradation of TRAF adapter molecules after initiation of signaling through CD40. Strikingly, this mode of regulation is absent after initiation of signaling through LMP1, possibly contributing to the enhanced signaling potency of LMP1 and its ability to transform B cells.