A Novel TRAF6 Binding Site in MALT1 Defines Distinct Mechanisms of NF-κB Activation by API2·MALT1 Fusions

Heidi Noels,Geert Van Loo,Sofie Hagens,Vicky Broeckx,Rudi Beyaert,Peter Marynen,Mathijs Baens

doi:10.1074/jbc.m611038200

Abstract

The recurrent translocation t(11;18)(q21;q21) associated with mucosa-associated lymphoid tissue (MALT) lymphoma results in the expression of an API2.MALT1 fusion protein that constitutively activates NF-kappaB. The first baculovirus IAP repeat (BIR) domain of API2 and the C terminus of MALT1, which contains its caspase-like domain, are present in all reported fusion variants and interact with TRAF2 and TRAF6, respectively, suggesting their contribution to NF-kappaB signaling by API2.MALT1. Also, the involvement of BCL10 has been suggested via binding to BIR1 of API2 and via its interaction with the immunoglobulin domains of MALT1, present in half of the fusion variants. However, conflicting reports exist concerning their roles in API2.MALT1-induced NF-kappaB signaling. In this report, streptavidin pulldowns of biotinylated API2.MALT1 fusion variants showed that none of the fusion variants interacted with endogenous BCL10; its role in NF-kappaB signaling by API2.MALT1 was further questioned by RNA interference experiments. In contrast, TRAF6 was essential for NF-kappaB activation by all fusion variants, and we identified a novel TRAF6 binding site in the second immunoglobulin domain of MALT1, which enhanced NF-kappaB activation when present in the fusion protein. Furthermore, inclusion of both immunoglobulin domains in API2.MALT1 further enhanced NF-kappaB signaling via intramolecular TRAF6 activation. Finally, binding of TRAF2 to BIR1 contributed to NF-kappaB activation by API2.MALT1, although additional mechanisms involving BIR1-mediated raft association are also important. Taken together, these data reveal distinct mechanisms of NF-kappaB activation by the different API2.MALT1 fusion variants with an essential role for TRAF6.

Highlights

Extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT)[4] represents with 8% one of the most common types of non-Hodgkin B-cell lymphoma
NF-␬B Activation by API21⁄7MALT1 Does Not Require BCL10—The API21⁄7MALT1 fusion protein containing both Ig domains of MALT1 (A7M3 or case 2; see Fig. 1A) possessed a higher NF-␬B activating potential in 293 cells compared with the variant lacking these domains (A7M8 or case 1) (18)
In MALT1, the Ig domains mediate the interacvalues were normalized for galactosidase values to correct tion with BCL10, which is essential for MALT1 to synergistifor differences in transfection efficiency (plotted as cally enhance NF-␬B activation with BCL10 (17)

Summary

EXPERIMENTAL PROCEDURES

Constructs—A vector enabling expression of biotinylated proteins was constructed by introducing oligonucleotides encoding the biotinylation (bio) sequence (GLNDIFEAQKIEWHE) described by Beckett et al (24) downstream of the N-terminal FLAG epitope in the plasmid pcDNA3.1 (pcD-F-bio). A second vector contained the bio-tag preceded by a flexible linker (SGSSGSSG) C-terminal of the multiple cloning site (pcD-F-bioC). These vectors were used to generate bio-constructs for MALT1 and the API21⁄7MALT1 fusion variants A7M8, A7M5, and A7M3 (fusion of exon 7 of API2 with exons 8, 5, and 3 of MALT1, respectively; see Fig. 1A). The open reading frame of the Escherichia coli BirA biotin protein ligase was amplified from genomic DNA via long distance PCR and cloned in the MSCV-FLAG-puromycin vector. Monoclonal Jurkat and SSK41 cell lines were generated stably expressing BirA from a pMSCV-FLAG-puromycin vector alone or in combination with a bio-tagged version of MALT1 (Jurkat) and A7M3 or A7M8 (SSK41), respectively, via their expression from a pcDF-bio-neomycin vector.

Cell lysates and precipitated protein complexes were fractionated on

RESULTS

DISCUSSION