TRAF3 forms heterotrimers with TRAF2 and modulates its ability to mediate NF-{kappa}B activation.
FRET experiments utilizing confocal microscopy or flow cytometry assessed homo- and heterotrimeric association of human tumor necrosis factor receptor-associated factors (TRAF) in living cells. Following transfection of HeLa cells with plasmids expressing CFP- or YFP-TRAF fusion proteins, constitutive homotypic association of TRAF2, -3, and -5 was observed, as well as ... heterotypic association of TRAF1-TRAF2 and TRAF3-TRAF5. A novel heterotypic association between TRAF2 and -3 was detected and confirmed by immunoprecipitation in Ramos B cells that constitutively express both TRAF2 and -3. Experiments employing deletion mutants of TRAF2 and TRAF3 revealed that this heterotypic interaction minimally involved the TRAF-C domain of TRAF3 as well as the TRAF-N domain and zinc fingers 4 and 5 of TRAF2. A novel flow cytometric FRET analysis utilizing a two-step approach to achieve linked FRET from CFP to YFP to HcRed established that TRAF2 and -3 constitutively form homo- and heterotrimers. The functional importance of TRAF2-TRAF3 heterotrimerization was demonstrated by the finding that TRAF3 inhibited spontaneous NF-kappaB, but not AP-1, activation induced by TRAF2. Ligation of CD40 on Ramos B cells by recombinant CD154 caused TRAF2 and TRAF3 to dissociate, whereas overexpression of TRAF3 in Ramos B cells inhibited CD154-induced TRAF2-mediated activation of NF-kappaB. Together, these results reveal a novel association between TRAF2 and TRAF3 that is mediated by unique portions of each protein and that specifically regulates activation of NF-kappaB, but not AP-1.
Mesh Terms:
Animals, Antigens, CD40, B-Lymphocytes, Bacterial Proteins, CD40 Ligand, DNA, Complementary, Dimerization, Enzyme Activation, Flow Cytometry, Gene Deletion, Genes, Reporter, Green Fluorescent Proteins, HeLa Cells, Humans, Immunoprecipitation, Luminescent Proteins, Microscopy, Confocal, Mutation, NF-kappa B, Plasmids, Protein Binding, Protein Structure, Tertiary, Recombinant Fusion Proteins, Recombinant Proteins, Signal Transduction, TNF Receptor-Associated Factor 2, TNF Receptor-Associated Factor 3, TNF Receptor-Associated Factor 5, Time Factors, Transcription Factor AP-1
Animals, Antigens, CD40, B-Lymphocytes, Bacterial Proteins, CD40 Ligand, DNA, Complementary, Dimerization, Enzyme Activation, Flow Cytometry, Gene Deletion, Genes, Reporter, Green Fluorescent Proteins, HeLa Cells, Humans, Immunoprecipitation, Luminescent Proteins, Microscopy, Confocal, Mutation, NF-kappa B, Plasmids, Protein Binding, Protein Structure, Tertiary, Recombinant Fusion Proteins, Recombinant Proteins, Signal Transduction, TNF Receptor-Associated Factor 2, TNF Receptor-Associated Factor 3, TNF Receptor-Associated Factor 5, Time Factors, Transcription Factor AP-1
J. Biol. Chem.
Date: Dec. 31, 2004
PubMed ID: 15383523
View in: Pubmed Google Scholar
Download Curated Data For This Publication
142700
Switch View:
- Interactions 6