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.
He
Liusheng
L
Flow Cytometry Section in the Office of Science and Technology, National Institute of Arthritis and Musculoskeletal and Skin Diseases/NIH, 9000 Rockville Pike, Building 10, Bethesda, MD 20892, USA.
Grammer
Amrie C
AC
Wu
Xiaoli
X
Lipsky
Peter E
PE
D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D019013:CD40 Antigens / Q000096:biosynthesis / Q000737:chemistry; D023201:CD40 Ligand / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004789:Enzyme Activation; D005434:Flow Cytometry; D017353:Gene Deletion; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D015398:Signal Transduction; D047992:TNF Receptor-Associated Factor 2 / Q000737:chemistry* / Q000378:metabolism; D048008:TNF Receptor-Associated Factor 3 / Q000737:chemistry / Q000502:physiology*; D048015:TNF Receptor-Associated Factor 5 / Q000378:metabolism; D013997:Time Factors; D018808:Transcription Factor AP-1 / Q000378:metabolism
D001426:Bacterial Proteins; D019013:CD40 Antigens; C485182:Cyan Fluorescent Protein; D018076:DNA, Complementary; D008164:Luminescent Proteins; D016328:NF-kappa B; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D047992:TNF Receptor-Associated Factor 2; D048008:TNF Receptor-Associated Factor 3; D048015:TNF Receptor-Associated Factor 5; D018808:Transcription Factor AP-1; C054562:yellow fluorescent protein, Bacteria; D023201:CD40 Ligand; D049452:Green Fluorescent Proteins