The Trk fused gene-product (Tfg) is part of a 600-700kDa CARMA1 complex

Abstract

B cell receptor (BCR) mediated activation of nuclear factor κB (NF-κB) is key to humoral immunity. CARMA1 (CARD11) is essential for BCR mediated NF-κB activation by interacting with Bcl10 and MALT1. Besides these two main players, few interaction partners of the CARMA1 complex are known. Here we identified new interaction partners of CARMA1. We generated two rabbit antisera against mouse CARMA1 to immunopurify endogenous CARMA1 from lysates of mouse B cells. Nik-binding protein (NIBP), Ras-GAP SH3 binding protein 2 (G3BP1) and Trk-fused gene (Tfg) were identified by peptide mass fingerprinting in immunopurified CARMA1 complexes. The interaction of Tfg and CARMA1 was confirmed by co-immunoprecipitation and Blue native polyacrylamide gel electrophoresis using the anti CARMA1 and newly generated anti Tfg antibodies. This analysis revealed that CARMA1 formed complexes of 600-1000 kDa. Additionally, Tfg was found in complexes of 500-600 kDa which increased in size to ∼740 kDa upon overexpresssion.