A dual role for the API2 moiety in API2-MALT1-dependent NF-κB activation: heterotypic oligomerization and TRAF2 recruitment

Abstract

Mucosa-associated lymphoid tissue (MALT) lymphoma is the most common extranodal lymphoid neoplasm. Chromosomal translocation t(11;18)(q21,q21) is found in 30% of gastric MALT lymphomas and is associated with a failure to respond to standard treatment and a tendency to disseminate. This translocation generates a chimeric protein composed of N-terminal sequences of Inhibitor of Apoptosis 2 (API2, also known as BIRC3 and cIAP2) fused to C-terminal sequences of MALT1. API2-MALT1 promotes cell survival and proliferation via activation of nuclear factor-jB (NF-jB). Here, we investigate the mechanism by which the API2 moiety contributes to NF-jB stimulation. We find that the API2 moiety mediates oligomerization of API2-MALT1 as well as interaction with tumor necrosis factor receptor-associated factor 2 (TRAF2). Surprisingly, oligomerization does not occur via homotypic interaction; rather, the API2 moiety of one monomer interacts with the MALT1 moiety of another monomer. Further, the specific region of the API2 moiety responsible for mediating oligomerization is distinct from that mediating TRAF2 binding. Although deletion or mutation of the TRAF2 binding site does not inhibit oligomerization, it does lead to dramatically decreased NF-jB activation. Deletion of both TRAF2 binding and oligomerization regions results in nearcomplete loss of NF-jB activation. Thus, API2 moietymediated heterotypic oligomerization and TRAF2 binding both contribute to maximal API2-MALT1-dependent NF-jB stimulation.

TRAF2-binding BIR1 domain of c-IAP2/MALT1 fusion protein is essential for activation of NF-κB

“…In this regard, a novel TRAF6 binding site has been reported in the second immunoglobulin domain of MALT1 (Noels et al, 2007). In agreement with others (Lucas et al, 2007), we observed that DRING TRAF6 dominant-negative also suppresses NF-kB signaling by c-IAP2/MALT1 (not shown). Thus, it is likely that both TRAF2 and TRAF6 are required for c-IAP2/MALT1 activity, though further analysis is needed to confirm this hypothesis.…”

“…Thus, when these residues are mutated to alanine, the E47A/R48A double-mutant loses its ability to bind TRAF2. Recently, it was shown, and we have confirmed (data not shown) that although the double-point mutant cannot bind TRAF2 the c-IAP2/MALT1 protein still oligomerizes (Lucas et al, 2007). Using plasmids encoding three different c-IAP2/ MALT1 fusion proteins (cases 1, 2 and 3), we performed DNA transfection dose response studies to explore their ability to induce NF-kB.…”

“…A recent report suggests that TRAF2 does not play a role in the ability of c-IAP2/MALT1 to induce NF-kB, and that the binding of TRAF2 simply plays a role in oligomerization (Varfolomeev et al, 2006). In contrast, others have found TRAF2 to be necessary for effective NF-kB activation (Lucas et al, 2007). Therefore, we sought to address this issue using alternative and complementary approaches.…”

“…Our comparisons of the effects of expressing the c-IAP2 fusion region in cis versus trans with MALT1 confirm that the fusion event produced by t(11;14) translocations is required for activating this oncoprotein. One functionality previously ascribed to the fusion protein that is not found in either c-IAP2 or MALT1 individually its ability to oligomerize (Lucas et al, 2007). In this sense, the c-IAP2/MALT fusion protein may mimic physiological mechanisms for TRAF activation, which normally occurs when these E3 ligases are recruited to clustered TNF-receptor and Toll-like receptor complexes (Rothe et al, 1994;Silverman and Maniatis, 2001;Akira and Takeda, 2004).…”

“…The portions of MALT1 involved in c-IAP2/MALT1 fusions retain the TRAF6 binding site, and thus TRAF6 would be expected to participate. However, c-IAP2 also binds TRAF2 through its first BIR domain, suggesting that TRAF2 could also contribute to NF-kB activation, but conflicting results have been obtained (Varfolomeev et al, 2006;Lucas et al, 2007).…”