Abstract
Bcl10 and MALT1 are essential mediators of NF-kappaB activation in response to the triggering of a diverse array of transmembrane receptors, including antigen receptors. Additionally, both proteins are translocation targets in MALT lymphoma. Thus, a detailed understanding of the interaction between these mediators is of considerable biological importance. Previous studies have indicated that a 13-amino acid region downstream of the Bcl10 caspase recruitment domain (CARD) is responsible for interacting with the immunoglobulin-like domains of MALT1. We now provide evidence that the death domain of MALT1 and the CARD of Bcl10 also contribute to Bcl10-MALT1 interactions. Although a direct interaction between the MALT1 death domain and Bcl10 cannot be detected via immunoprecipitation, FRET data strongly suggest that the death domain of MALT1 contributes significantly to the association between Bcl10 and MALT1 in T cells in vivo. Furthermore, analysis of point mutants of conserved residues of Bcl10 shows that the Bcl10 CARD is essential for interaction with the MALT1 N terminus. Mutations that disrupt proper folding of the Bcl10 CARD strongly impair Bcl10-MALT1 interactions. Molecular modeling and functional analyses of Bcl10 point mutants suggest that residues Asp(80) and Glu(84) of helix 5 of the Bcl10 CARD directly contact MALT1. Together, these data demonstrate that the association between Bcl10 and MALT1 involves a complex interaction between multiple protein domains. Moreover, the Bcl10-MALT1 interaction is the second reported example of interactions between a CARD and a non-CARD protein region, which suggests that many signaling cascades may utilize CARD interactions with non-CARD domains.
Highlights
Bcl10 and mucosa-associated lymphatic tissue 1 (MALT1)5 are cytosolic signaling intermediates in the pathway connecting antigen receptors to activation of the NF-B transcription factor [1, 2]
To confirm and extend these previous studies, we made several deletion mutants of MALT1 and Bcl10 (Fig. 1, A and B). Various combinations of these mutants were co-transfected into HEK293T cells, and immunoprecipitation (IP) analyses were performed, using anti-FLAG to immunoprecipitate the MALT1 constructs (Fig. 1C)
Formation of Bcl10 POLKADOTS Strongly Correlates with the Ability of Bcl10 Point Mutants to Activate NF-B—In a recent study [13], we examined the requirements for formation of punctate cytosolic structures, called POLKADOTS, which are highly enriched in Bcl10 and MALT1 and which form in response to stimulation of the T cell receptor (TCR) and in conjunction with TCR-mediated activation of NF-B
Summary
Transfections, and Luciferase Assays—D10 T cells and CH12 B cells were maintained as previously described [14]. Other MALT1 deletions and the Bcl10-CFP construct were previously described [13]. Cells were transfected by the calcium phosphate method, using 1.5 g of pcDNA3–3xHA-Bcl10-GFP and 1.5 g of pcDNA3-FLAG-construct (MALT1 deletion or CARMA1) per well. Microscopy and FRET Analysis—D10 T cells stably expressing Bcl10-GFP constructs were conjugated with conalbuminprimed CH12 B cells for 20 min at 37 °C, as previously described [14]. FRET Analysis by FACS—D10 T cells were infected with retroviral vectors and drug selected to produce stable cell lines, as described above. FRET Analysis by FLIM—D10 T cell lines were fixed in 3% paraformaldehyde, and fluorescence lifetime images were collected with a LIFA frequency domain FLIM system (Lambert Instruments, The Netherlands) attached to an Olympus IX71 epifluorescence microscope, employing a ϫ60 1.45 NA objective. P values were calculated by paired t-tests comparing FRET efficiencies from cell lines expressing each Bcl mutant versus the cell line expressing Bcl10-WT
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