Activation of the noncanonical NF-κB pathway by NIK is essential for the organization of secondary lymphoid tissue, whereas deregulation of this pathway results in multiple human diseases including cancers. Although genetic studies indicate that TRAF2, TRAF3, cIAP1 and cIAP2 are all involved in the negative regulation the noncanonical NF-κB pathway, little is known about the biochemical mechanisms responsible for regulation of NIK levels before and after receptor activation. In this study, we elucidate a simple model showing the distinct roles of TRAF2 and TRAF3 in bridging the gap between the cIAPs and NIK to form a complex to promote NIK degradation. In addition, we explored the potential of SMAC mimetic compounds to mimic BAFF-like co-stimulation of B lymphocytes. Strikingly, we found that such IAP antagonists greatly augmented B lymphocyte survival and proliferation. Collectively these data advance our mechanistic understanding of the NIK regulatory complex and reveal potential complications in therapeutic application of SMAC mimetic compounds.
At present, it is clear that basal activity of the noncanonical NF-κB pathway is suppressed by constitutive suppression of NIK protein levels through direct interaction with TRAF312,13,15
. Recent genetic and pharmacological studies further suggest that TRAF2 and the cIAPs cooperate with TRAF3 in the proteasomal targeting of NIK, but a potential mechanism has not been described 12,13,15,16,19,20,32
. We find that overexpression of either TRAF2 or TRAF3 in Traf3−/−
cells, respectively, cannot compensate for the loss of the other. These results strongly suggest that both TRAF2 and TRAF3 possess highly specific and unique functions in the basal suppression of NIK. A comparison of the binding of TRAF2 and TRAF3 to the cIAPs and NIK revealed strikingly different abilities, with TRAF2 binding both cIAP1 and cIAP2 strongly and TRAF3 binding NIK strongly. With this finding, and the knowledge that TRAF2 and TRAF3 can heterodimerize in living cells25
, we reasoned that TRAF2 and TRAF3 might function as a bridge to couple cIAPs with NIK. We have further identified a chimera 3TD, containing the zinc ring and zinc finger domains of TRAF2 and the TRAF domain of TRAF3, that promotes cIAPs interaction with NIK more efficiently than either TRAF2 or TRAF3 alone. Interestingly, this 3TD chimera is able to suppress the high basal noncanonical NF-κB activities of both Traf3−/−
cells, which strongly supports our proposed model.
In addition to similar results in previous reports that TRAF2 and TRAF3 are degraded after receptor activation, our studies have further demonstrated that SMAC mimetic compounds can strongly inhibit receptor-induced TRAF2 and TRAF3 degradation, suggesting cIAPs are required for their degradation. Based upon our model, degradation of either TRAF2 or TRAF3 is sufficient to cause the dissociation of the NIK-cIAPs complex, resulting in accumulation of NIK through new protein synthesis. This also agrees with previous reports showing that BAFF-R, CD40 or LTβR-mediated noncanonical NF-κB requires new protein synthesis.
While TRAF2 and TRAF3 play distinct roles, our studies indicate that cIAP1 and cIAP2 are redundant in the negative regulation of noncanonical NF-κB activation. We have shown that either cIAP1 or cIAP2 can be recruited to the NIK complex through TRAF2 and TRAF3. More importantly, knockdown of cIAP1 in Birc3−/−
cells, knockdown of cIAP2 in Birc2−/−
cells or knockdown of both in wild-type cells results in NIK accumulation and noncanonical NF-κB activation. Consistent with recently studies, SMAC mimetic compounds, which strongly inhibit both cIAP1 and cIAP2, leads to NIK accumulation and noncanonical NF-κB activation. However, while those studies showed that SMAC mimetic compounds can potentiate cell death in certain cancer cell lines20,33
, our study has shown that the same SMAC mimetic compounds can actually promote the survival and proliferation of primary B lymphocytes due to the induction of the noncanonical NF-κB pathway.
The present work further demonstrates that disruption of a single allele of the NIK gene is sufficient to rescue the TRAF3-null lethal phenotype, suggesting that NIK levels need to be tightly and precisely regulated. Elegant work recently15,16
found that a subgroup of multiple myeloma cells expresses elevated noncanonical NF-κB activity due to Map3k14
mutation or loss of expression of both Birc2
genes. These results fit well with our proposed model in which non-redundant TRAFs cooperate with redundant cIAPs in negative regulation of NIK and noncanonical NF-κB activity. Finally, Given the known dangers of deregulated noncanonical NF-κB activity in B lymphocytes, SM compounds, which show great promise for the treatment of cancers, could inadvertently sensitize patients towards autoimmune disease or lymphomas34
. Consequently, the pursuit of this very promising new treatment strategy should proceed cautiously, in order to better define the therapeutic window and to avoid potential negative immunological side effects.