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• W2045806492 abstract "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-κB, 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-κB. 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-κB, but not AP-1. 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-κB, 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-κB. 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-κB, but not AP-1. A family of tumor necrosis factor receptor-associated factors (TRAFs 1-7) 1The abbreviations used are: TRAF, tumor necrosis factor receptor-associated factors; TNF, tumor necrosis factor; AP-1, activation protein 1; JNK, Jun N-terminal kinase; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; ZnF, zinc finger; FRET, fluorescence resonance energy transfer. functions as adaptor molecules for TNF receptor superfamily members by associating with the intracellular domain of these proteins and subsequently mediating downstream signaling events such as NF-κB and AP-1 (1Grammer A.C. Lipsky P.E. Adv. Immunol. 2000; 76: 61-178Crossref PubMed Google Scholar, 2Bouwmeester T.A. Bauch H. Ruffner P.O. Angrand G. Bergamini K. Croughton C. Cruciat D. Eberhard J. Gagneur S. Ghidelli C. Hopf B. Huhse R. Mangano A.M. Michon M. Schirle J. Schlegl M. Schwab M.A. Stein A. Bauer G. Casari G. Drewes A.C. Gavin D. Jackson B. Joberty G. Neubauer G. Rick J. Kuster B. Superti-Furga G Nat. Cell Biol. 2004; 6: 97-105Crossref PubMed Scopus (873) Google Scholar). Biochemical approaches have revealed that TRAFs form homotypic multimers (3Ni C.Z. Welsh K. Zheng J. Havert M. Reed J.C. Ely K.R. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 1340-1342Crossref PubMed Scopus (10) Google Scholar, 4Ely K.R. Li C. J. Mol. Recognit. 2002; 15: 286-290Crossref PubMed Scopus (15) Google Scholar, 5Tsao D.H. McDonagh T. Telliez J.B. Hsu S. Malakian K. Xu G.Y. Lin L.L. Mol. Cell. 2000; 5: 1051-1057Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 6Baud V. Liu Z.G. Bennett B. Suzuki N. Xia Y. Karin M. Genes Dev. 1999; 13: 1297-1308Crossref PubMed Scopus (409) Google Scholar) as well as certain heterotypic multimers, such as those between TRAF1 and TRAF2 and between TRAF3 and TRAF5 (7Rothe M. Wong S.C. Henzel W.J. Goeddel D.V. Cell. 1994; 78: 681-692Abstract Full Text PDF PubMed Scopus (932) Google Scholar, 8Leo E. Welsh K. Matsuzawa S. Zapata J.M. Kitada S. Mitchell R.S. Ely K.R. Reed J.C. J. Biol. Chem. 1999; 274: 22414-22422Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 9Pullen S.S. Miller H.G. Everdeen D.S. Dang T.T. Crute J.J. Kehry M.R. Biochemistry. 1998; 37: 11836-11845Crossref PubMed Scopus (206) Google Scholar, 10Pullen S.S. Labadia M.E. Ingraham R.H. McWhirter S.M. Everdeen D.S. Alber T. Crute J.J. Kehry M.R. Biochemistry. 1999; 38: 10168-10177Crossref PubMed Scopus (131) Google Scholar). Previous reports have demonstrated that TRAF2 and -3 play an important role in cellular activation and differentiation following engagement of a variety of TNF receptor superfamily members such as CD40/TNFRSF5 (1Grammer A.C. Lipsky P.E. Adv. Immunol. 2000; 76: 61-178Crossref PubMed Google Scholar), OX40/TNFRSF4 (11Arch R.H. Thompson C.B. Mol. Cell. Biol. 1998; 18: 558-565Crossref PubMed Google Scholar), LTβR (12Chang Y.H. Hsieh S.L. Chen M.C. Lin W.W. Exp. Cell Res. 2002; 278: 166-174Crossref PubMed Scopus (64) Google Scholar, 13Force W.R. Glass A.A. Benedict C.A. Cheung T.C. Lama J. Ware C.F. J. Biol. Chem. 2000; 275: 11121-11129Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 14Li C. Norris P.S. Ni C.Z. Havert M.L. Chiong E.M. Tran B.R. Cabezas E. Reed J.C. Satterthwait A.C. Ware C.F. Ely K.R. J. Biol. Chem. 2003; 278: 50523-50529Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), XEDAR (15Sinha S.K. Zachariah S. Quinones H.I. Shindo M. Chaudhary P.M. J. Biol. Chem. 2002; 277: 44953-44961Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), BCMA/TNFRSF17 (16Hatzoglou A. Roussel J. Bourgeade M.F. Rogier E. Madry C. Inoue J. Devergne O. Tsapis A. J. Immunol. 2000; 165: 1322-1330Crossref PubMed Scopus (204) Google Scholar), and Fn14/TWEAKR/TNFRSF12A (17Saitoh T. Nakayama M. Nakano H. Yagita H. Yamamoto N. Yamaoka S. J. Biol. Chem. 2003; 278: 36005-36012Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 18Han S. Yoon K. Lee K. Kim K. Jang H. Lee N.K. Hwang K. Young Lee S. Biochem. Biophys. Res. Commun. 2003; 305: 789-796Crossref PubMed Scopus (97) Google Scholar). The functional significance of TRAF2 and -3 in immune responses mediated by one or more of these TNF receptor superfamily molecules was revealed by experiments with mice that were genetically altered in expression of TRAF2 or -3. Experiments using mice transgenic for only the TRAF domain of TRAF2 (amino acids 245-501; TRAF2.dominant negative; Ref. 19Cannons J.L. Bertram E.M. Watts T.H. J. Immunol. 2002; 169: 2828-2831Crossref PubMed Scopus (24) Google Scholar) or mice genetically deficient in TRAF3 expression (20Xu Y. Cheng G. Baltimore D. Immunity. 1996; 5: 407-415Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar) revealed a role for both adaptor proteins in T cell-dependent humoral immune responses. Of note, mice expressing a dominant negative form of TRAF2 exhibited an expanded B cell compartment that was evidenced by splenomegaly and lymphadenopathy (21Lee S.Y. Reichlin A. Santana A. Sokol K.A. Nussenzweig M.C. Choi Y. Immunity. 1997; 7: 703-713Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar), whereas TRAF3-/- mice exhibited decreased numbers of B cell precursors (20Xu Y. Cheng G. Baltimore D. Immunity. 1996; 5: 407-415Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Examination of signaling mechanisms mediated by TRAF2 or -3 revealed that both adaptor proteins induce activation of the mitogen-activated protein kinase Jun N-terminal kinase (JNK) as well as playing a role in the regulation of NF-κB activation (21Lee S.Y. Reichlin A. Santana A. Sokol K.A. Nussenzweig M.C. Choi Y. Immunity. 1997; 7: 703-713Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 22Yeh W.C. Shahinian A. Speiser D. Kraunus J. Billia F. Wakeham A. de la Pompa J.L. Ferrick D. Hum B. Iscove N. Ohashi P. Rothe M. Goeddel D.V. Mak T.W. Immunity. 1997; 7: 715-725Abstract Full Text Full Text PDF PubMed Scopus (712) Google Scholar, 23Nguyen L.T. Duncan G.S. Mirtsos C. Ng M. Speiser D.E. Shahinian A. Marino M.W. Mak T.W. Ohashi P.S. Yeh W.C. Immunity. 1999; 11: 379-389Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Importantly, a number of reports have indicated that TRAF3 inhibits NF-κB activation induced by TRAF2 following engagement of TNF receptor superfamily members such as CD40/TNFRSF5 (29Rothe M. Sarma V. Dixit V.M. Goeddel D.V. Science. 1995; 269: 1424-1427Crossref PubMed Scopus (978) Google Scholar) and OX40/TNFRSF4 (30Takaori-Kondo A. Hori T. Fukunaga K. Morita R. Kawamata S. Uchiyama T. Biochem. Biophys. Res. Commun. 2000; 272: 856-863Crossref PubMed Scopus (31) Google Scholar, 31Prell R.A. Evans D.E. Thalhofer C. Shi T. Funatake C. Weinberg A.D. J. Immunol. 2003; 171: 5997-6005Crossref PubMed Scopus (68) Google Scholar), but the precise mechanism of this observation has not been delineated. However, overexpression of wild-type TRAF3 has been shown to inhibit TRAF2-induced NF-κB activation (30Takaori-Kondo A. Hori T. Fukunaga K. Morita R. Kawamata S. Uchiyama T. Biochem. Biophys. Res. Commun. 2000; 272: 856-863Crossref PubMed Scopus (31) Google Scholar, 31Prell R.A. Evans D.E. Thalhofer C. Shi T. Funatake C. Weinberg A.D. J. Immunol. 2003; 171: 5997-6005Crossref PubMed Scopus (68) Google Scholar). Furthermore, proteolysis of TRAF3 by a pepstatin A inhibitable mechanism enhanced CD40-mediated NF-κB activation (32Annunziata C.M. Safiran Y.J. Irving S.G. Kasid U.N. Cossman J. Blood. 2000; 96: 2841-2848Crossref PubMed Google Scholar), whereas TRAF2-induced degradation of TRAF3 enhanced NF-κB activation (33Hostager B.S. Haxhinasto S.A. Rowland S.L. Bishop G.A. J. Biol. Chem. 2003; 278: 45382-45390Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). By contrast, expression of an alternatively spliced form of TRAF3 has been shown to activate NF-κB (34van Eyndhoven W.G. Gamper C.J. Cho E. Mackus W.J. Lederman S. Mol. Immunol. 1999; 36: 647-658Crossref PubMed Scopus (30) Google Scholar, 35Gamper C. Omene C.O. van Eyndhoven W.G. Glassman G.D. Lederman S. Hum. Immunol. 2001; 62: 1167-1177Crossref PubMed Scopus (8) Google Scholar). These findings suggest that a complex role for TRAF2 and TRAF3 in the regulation of NF-κB, but the precise molecular mechanism has not been delineated. Importantly, no direct physical interaction between TRAF2 and TRAF3 has been documented to date. The purpose of the current study was to examine whether functional inhibition of TRAF2-induced NF-κB activation was mediated by a direct interaction between TRAF2 and -3. Experiments utilizing one- and two-step FRET performed by confocal microscopy or flow cytometry clearly demonstrated that TRAF3 directly associates with TRAF2 and inhibits TRAF2-induced NF-κB, but not AP-1, activation. Plasmids—Plasmids that encode CFP- or YFP-fused to TRAF3 or -5 have been described previously (36He L. Bradrick T.D. Karpova T.S. Wu X. Fox M.H. Fischer R. McNally J.G. Knutson J.R. Grammer A.C. Lipsky P.E. Cytometry. 2003; 53A: 39-54Crossref Scopus (38) Google Scholar). Where indicated, the fragments containing TRAF3 were cloned into the appropriate sites of HcRed-C1 to prepare HcRed-TRAF3. Plasmids containing human cDNAs encompassing the complete open reading frames of hTRAF1 and hTRAF6 have been previously described (37Karpova T.S. Baumann C.T. He L. Wu X. Grammer A. Lipsky P. Hager G.L. McNally J.G. J. Microsc. 2003; 209: 56-70Crossref PubMed Scopus (262) Google Scholar, 38He L. Olson D.P. Wu X. Karpova T.S. McNally J.G. Lipsky P.E. Cytometry. 2003; 55A: 71-85Crossref Scopus (50) Google Scholar). To prepare CFP or YFP fusion protein constructs, the relevant PCR fragment was amplified using a pair of oligonucleotides (TRAF1-EcoRI top, 5′-TCGAATTCTATGGCCTCCACCAGCTCAGGCAGC-3′ and TRAF1-BamHI bottom, 5′-ATTGGATCCCTAAGTGCTGGTCTCCACAATGC-3′; TRAF6-BglII top, 5′-CTCAGATCTCGAATGAGTCTGCTAAACTGTGAA-3′ and TRAF6-HindIII bottom, 5′-TCGAAGCTTGCTATACCCCTGCATCAGTA-3′) and then cloned into the appropriate sites of CFP-C1 or YFP-C1 vectors (Clontech, San Diego, CA). Plasmids for human TRAF2 were directly amplified from a thymus cDNA library, according to the manufacturer's instructions, using a pair of oligonucleotides (TRAF2-BamHI top, 5′-CTCGGATCCATGGCTGCAGCTAGCGTG-3′ and TRAF2-Hind III bottom, 5′-AACAAGCTTAGTTAGAGCCCTGTCAGGTC-3′). Afterward, these fragments were cut with appropriate restriction enzymes and cloned into their compatible sites in CFP-C1, YFP-C1, and HcRed-C1 (Clontech) to prepare CFP-TRAF2, YFP-TRAF2 (38He L. Olson D.P. Wu X. Karpova T.S. McNally J.G. Lipsky P.E. Cytometry. 2003; 55A: 71-85Crossref Scopus (50) Google Scholar), and HcRed, respectively. The same PCR fragments for all TRAFs were cloned into His-tagged pcDNA3 (Invitrogen) to prepare His-TRAF2, His-TRAF3, His-TRAF5, and His-TRAF6 plasmids. A plasmid that encodes a FRET-negative control (CFP-TRAF2TRAF-YFP) has been previously described (36He L. Bradrick T.D. Karpova T.S. Wu X. Fox M.H. Fischer R. McNally J.G. Knutson J.R. Grammer A.C. Lipsky P.E. Cytometry. 2003; 53A: 39-54Crossref Scopus (38) Google Scholar). All constructs were confirmed by DNA sequencing. The NF-κB and AP-1 luciferase reporter plasmids were purchased from Stratagene (La Jolla, CA). Cell Culture and Plasmid Transfection—HeLa cells were obtained from the ATCC and cultured in Dulbecco's modified Eagle's medium high glucose medium supplemented with 10% fetal bovine serum, 1 mm glutamine, and antibiotics. Transient transfection was done with the LipofectAMINE Plus reagent into log-phase growing cells (Invitrogen). Routinely, transfected cells were cultured overnight and then analyzed by confocal microscopy and flow cytometry. Any samples involving TRAF6 transfection were incubated in the presence of 30 μm lactacystin (Calbiochem, La Jolla, CA) or 25 μm MG132 (Calbiochem) and were processed for the assays at 3 h post-transfection. The EBV-negative B cell line, Ramos/R2G6, was cultured as described previously (24Grammer A.C. Swantek J.L. McFarland R.D. Miura Y. Geppert T. Lipsky P.E. J. Immunol. 1998; 161: 1183-1193PubMed Google Scholar). Immunoprecipitation and Immunoblotting—Sixteen h post-transfection, cells were lysed in hyper-salt on ice. Cell extracts were examined by immunoblotting with antibodies specific for GFP (Roche Diagnostics) or individual TRAF molecules (TRAF1, -3, -5, and -6, Santa Cruz Biotechnologies, Santa Cruz, CA; TRAF2, BD Pharmingen, La Jolla, CA). Immunoprecipitation was performed with Ramos cells to determine whether there are endogenous TRAF2/TRAF3 complexes. Briefly, affinity purified rabbit anti-TRAF2 (Santa Cruz Biotechnologies) or anti-rabbit IgG1 was cross-linked to protein A-Sepharose (Roche) beads with dimethyl pimelimidate (Pierce). Twenty-five μg of protein from whole cell extracts from Ramos B cells were mixed with beads and incubated overnight at 4 °C in buffer BC-100 (20 mm HEPES, pH 7.3, 20% glycerol, 100 mm KCl, 4 mm dithiothreitol, 0.2 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride). The beads were then washed four times with 20 mm HEPES (pH 7.9), 250 mm NaCl, 0.05% Triton X-100 and subjected to electrophoresis and immunoblot analysis. FRET Detection by Flow Cytometry—All cytometric data were collected using a FACS DiVa (Digital Vantage SE.; BD Biosciences). The optical configuration for FRET measurement (FRET1) between CFP and YFP has been described previously (36He L. Bradrick T.D. Karpova T.S. Wu X. Fox M.H. Fischer R. McNally J.G. Knutson J.R. Grammer A.C. Lipsky P.E. Cytometry. 2003; 53A: 39-54Crossref Scopus (38) Google Scholar, 38He L. Olson D.P. Wu X. Karpova T.S. McNally J.G. Lipsky P.E. Cytometry. 2003; 55A: 71-85Crossref Scopus (50) Google Scholar). Briefly, the argon ion 488-nm laser line at 150 milliwatts and the krypton ion UV 407-nm laser line at 50 milliwatts were employed to excite the YFP and CFP, respectively. YFP signals were collected using a 546/10 nm band-pass filter in the primary laser pathway (laser 1). CFP signals were collected using a 460/20 nm band-pass filter in the third laser pathway (laser 3). FRET1 signals directly emitted from YFP during CFP → YFP FRET were collected using a 546/10-nm band-pass filter in the third laser pathway (UV1-FL7). To study FRET (FRET2) signals between YFP and HcRed, a 630/22-nm band-pass filter (FL8) was used to detect emission signals from HcRed in the primary laser pathway (laser 1), whereas HcRed was directly excited by the 568-nm line emitted from the spectrum laser at 50 milliwatts and its emission was monitored by the signals detected in the second pathway (laser 2) using a 610 LP filter (FL6). The detector in the UV1-FL7 position of the UV-laser pathway was also used to collect either two-step FRET signals emitted from HcRed during CFP → YFP → HcRed FRET using a 630/22-nm band-pass filter. All data were analyzed using CellQuest software (BD Biosciences). FRET Detection by Laser Scanning Confocal Microscopy—The method employed has been described previously (37Karpova T.S. Baumann C.T. He L. Wu X. Grammer A. Lipsky P. Hager G.L. McNally J.G. J. Microsc. 2003; 209: 56-70Crossref PubMed Scopus (262) Google Scholar, 38He L. Olson D.P. Wu X. Karpova T.S. McNally J.G. Lipsky P.E. Cytometry. 2003; 55A: 71-85Crossref Scopus (50) Google Scholar). Briefly, at 16 h post-transfection, cells were fixed in 4% paraformaldehyde in phosphate-buffered saline and mounted on silicon-coated slides. Fixation does not change fluorescence protein localization and cellular morphology (Refs. 37Karpova T.S. Baumann C.T. He L. Wu X. Grammer A. Lipsky P. Hager G.L. McNally J.G. J. Microsc. 2003; 209: 56-70Crossref PubMed Scopus (262) Google Scholar and 38He L. Olson D.P. Wu X. Karpova T.S. McNally J.G. Lipsky P.E. Cytometry. 2003; 55A: 71-85Crossref Scopus (50) Google Scholar; and data not shown). Any samples involving TRAF6 tranfection were cultured with 30 μm lacatacystin and fixed 3 h post-transfection. HeLa cells transfected with plasmids expressing CFP and YFP fusion proteins were examined routinely using a ×100 objective. Confocal microscopic images were obtained with the Carl Zeiss laser scanning microscope with LSM 510 software. An excitation wavelength of 458 nm and an emission wavelength of 480 to 500 nm were used for CFP, whereas an excitation wavelength of 514 nm and an emission wavelength of 515 to 545 nm were used for YFP. FRET was assessed and quantitated using an acceptor photobleaching method that was developed for laser-scanning confocal microscopy (37Karpova T.S. Baumann C.T. He L. Wu X. Grammer A. Lipsky P. Hager G.L. McNally J.G. J. Microsc. 2003; 209: 56-70Crossref PubMed Scopus (262) Google Scholar, 38He L. Olson D.P. Wu X. Karpova T.S. McNally J.G. Lipsky P.E. Cytometry. 2003; 55A: 71-85Crossref Scopus (50) Google Scholar). The method assessed the extent of FRET by measuring the donor fluorescence before (Da) and after (D) photobleaching of the acceptor. The amount of energy transfer detected by confocal microscopy (FRETc) was calculated as the ratio of donor fluorescence in the presence or absence of acceptor: FRETc = D/Da. The ratio of D/Da equals or is less than 1.0 in the absence of FRET. If D/Da is >1.0, FRET is considered to have occurred (39Sharma N. Hewett J. Ozelius L.J. Ramesh V. McLean P.J. Breakefield X.O. Hyman B.T. Am. J. Pathol. 2001; 159: 339-344Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The magnitude of the D/Da ratio >1.0 is proportional to the proximity of the fluorophore. The ratio of D/Da was compared with the null hypothesis value of 1.0 by one group t-tests. Reporter Gene Assays and Flow Cytometry—For NF-κB and AP-1 reporter gene assays, HeLa cells were transfected using the LipofectAMINE Plus reagent and a total of 4.5 μg of DNA (including 0.5 μg of the NF-κB- or the AP-1 luciferase reporter constructs) and 2 μg of pEYFP plasmid to monitor transfection efficiency and 2 μg of plasmid expressing YFP- or His-tagged TRAFs at about 60% confluence in 6-well plates in triplicate. For experiments to examine whether an interaction between TRAF2 and TRAF3 affected TRAF2-mediated NF-κB or AP-1 activation, HeLa cells were transfected with 0.5 μg of the NF-κB- or AP-1 luciferase reporter plasmids and 2 or 5 μg of plasmids expressing YFP, YFP-TRAF2, or YFP-TRAF3, or 2 μg of YFP-TRAF2 plus 2 or 5 μg of YFP-TRAF3. Sixteen h post-transfection, cells were lysed with 300 μl of Promega lysis buffer. The luciferase activity of 10 μl of each lysate was measured using the Luciferase assay kit from Promega. Luciferase activity was normalized relative to YFP levels. Verification of the Functional Integrity of TRAF Fusion Proteins—The plasmids expressing fluorescent fusion proteins of each TRAF were prepared and transiently expressed in HeLa cells. Immunoblots were carried out with antibodies specific for the fluorescent tag as well as for the specific TRAF proteins to document correct expression of each TRAF fusion protein (Fig. 1, A-E). Each of the constructs was expressed and detected at the expected molecular mass. For each of the fusion proteins, except TRAF2, smaller molecular weight fragments were also detected. This was most marked for TRAF6, which was ubiquitinated within 3 h and completely degraded by 16 h post-transfection. Even when the cells were cultured in the presence of an inhibitor of the proteasome, lactacystin, only a small amount of TRAF6 was expressed at 16 h post-transfection. It is notable that HeLa cells expressed detectable levels of only two of the TRAF family members constitutively, TRAF2 and -5. Because of the relatively large size (27 kDa) of the fluorescent CFP or YFP tag fused to each TRAF, it was important to document that the TRAF fusion proteins were functionally intact. To accomplish this, plasmids were prepared that expressed TRAF proteins fused to a small His tag. As can been seen in Fig. 1F, overexpression of His or fluorescent fusion proteins of TRAF2 or -6 induced activation of NF-κB equivalently. FRET Reveals TRAF Domain-mediated Self-association of TRAF2, -3, and -5 in Living Cells—By confocal microscopy, TRAF fusion proteins were primarily expressed as aggregates in the cytosol (Fig. 2A, data not shown). Importantly, CFP- and YFP-tagged TRAFs localized comparably in cells. Homotypic association of TRAF2, -3, and -5, but not TRAF6, was detected in the cytosol of transfected cells by confocal FRET (Fig. 2, B and C; data not shown). Flow cytometric FRET also demonstrated homotypic association of TRAF2, -3, and -5 as well as weak, but reproducible homotypic association of TRAF6 (Fig. 2D). The ability of flow cytometry to detect homomers of TRAF6 is likely related to the enhanced sensitivity of flow cytometric FRET compared with confocal microscopic FRET (36He L. Bradrick T.D. Karpova T.S. Wu X. Fox M.H. Fischer R. McNally J.G. Knutson J.R. Grammer A.C. Lipsky P.E. Cytometry. 2003; 53A: 39-54Crossref Scopus (38) Google Scholar, 37Karpova T.S. Baumann C.T. He L. Wu X. Grammer A. Lipsky P. Hager G.L. McNally J.G. J. Microsc. 2003; 209: 56-70Crossref PubMed Scopus (262) Google Scholar, 38He L. Olson D.P. Wu X. Karpova T.S. McNally J.G. Lipsky P.E. Cytometry. 2003; 55A: 71-85Crossref Scopus (50) Google Scholar). Absence of FRET was observed in cells transfected with a plasmid expressing CFP-TRAF2TRAF-YFP in which a structurally restricted linker of nearly 100 Å (TRAF domain from TRAF2) was inserted between CFP and YFP (Fig. 2D, row 4). Importantly, self-association of TRAFs was mediated by their respective TRAF domains as comparable FRET was detected between full-length YFP-TRAFs and CFP fusion proteins of the respective TRAF domains (Fig. 3). The FRET signal for self-association of full-length TRAFs or TRAF domains of each TRAF were not statistically significant implying that the TRAF domains account for all homotypic associations detected between these family members.Fig. 3The TRAF domain mediates homotypic association of TRAF2, -3, and -5. HeLa cells were cotransfected with YFP-TRAF2 and CFP-TRAF2TRAF, or YFP-TRAF3 and CFP-TRAF3TRAF, or YFP-TRAF5 and CFP-TRAF5TRAF and analyzed by confocal microscopic FRET. Data from multiple analysis (n) are shown with the statistical significance (p value).View Large Image Figure ViewerDownload Hi-res image Download (PPT) FRET Reveals TRAF1-TRAF2, TRAF3-TRAF5, and TRAF2-TRAF3 Heterotypic Interactions in Living Cells—Heterotypic interactions between TRAF1 and TRAF2 as well as TRAF3 and TRAF5 have been previously reported (7Rothe M. Wong S.C. Henzel W.J. Goeddel D.V. Cell. 1994; 78: 681-692Abstract Full Text PDF PubMed Scopus (932) Google Scholar, 8Leo E. Welsh K. Matsuzawa S. Zapata J.M. Kitada S. Mitchell R.S. Ely K.R. Reed J.C. J. Biol. Chem. 1999; 274: 22414-22422Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 9Pullen S.S. Miller H.G. Everdeen D.S. Dang T.T. Crute J.J. Kehry M.R. Biochemistry. 1998; 37: 11836-11845Crossref PubMed Scopus (206) Google Scholar, 10Pullen S.S. Labadia M.E. Ingraham R.H. McWhirter S.M. Everdeen D.S. Alber T. Crute J.J. Kehry M.R. Biochemistry. 1999; 38: 10168-10177Crossref PubMed Scopus (131) Google Scholar). To determine whether TRAF1-TRAF2 and TRAF3-TRAF5 heterotrimers could be detected in living cells, FRET was assessed in HeLa cells co-transfected with plasmids expressing CFP-TRAF1 and YFP-TRAF2 or CFP-TRAF3 and YFP-TRAF5. Along with its uniform distribution in the cytoplasm, CFP-TRAF1 co-localized with YFP-TRAF2 in punctate regions (data not shown). This close association was accompanied by positive FRET (Fig. 4). Similar results were noted for interactions between TRAF3 and -5 (Fig. 4). To determine whether TRAF2 interacted with other TRAFs, HeLa cells were co-transfected with a plasmid expressing CFP-TRAF2, along with plasmids expressing YFP-tagged TRAF3, -5, or -6 (Fig. 5A). TRAF2 colocalized with TRAF3, and FRET could be detected between TRAF2 and TRAF3 tagged with CFP and YFP, respectively (data not shown). By contrast, there was no direct interaction between TRAF2 and -5 although in some spreading cells they appeared to colocalize. FRET between TRAF2 and -6 was difficult to detect by confocal microscopy. Even when analyzed after 3 h, colocalization of TRAF2 and TRAF6 was not found reproducibly. Moreover, when colocalization was found, FRET was not routinely detected between TRAF2 and -6. Even by the more sensitive approach of flow cytometric FRET, physical interactions between TRAF2 and TRAF6 could not be reproducibly detected. The direct interaction between full-length TRAF2 and TRAF3 detected by confocal microscopy was confirmed by flow cytometry (Fig. 5B). TRAF2-TRAF3 Heterotypic Interactions Are Mediated by the TRAF-C Domain of TRAF3 and TRAF-N, ZnF4, and ZnF5 Regions of TRAF2—To identify the sequence elements that direct TRAF2 to interact with TRAF3, a series of deletion mutants of TRAF2 were fused to YFP and each was tested for an interaction with CFP-TRAF3. As noted previously, markedly positive FRET signals were detected between full-length CFP-TRAF3 and YFP-TRAF2 (Fig. 6). Of interest, the intensity of FRET for TRAF2-TRAF3 heterotypic interactions was not significantly different from FRET observed for either the TRAF2 or TRAF3 homotypic interactions. Removal of the TRAF-C of TRAF2 did not affect FRET, whereas further deletion of the TRAF-N domain and the ZnFs resulted in complete loss of FRET. Expression of either ZnF1 to ZnF5 or the TRAF domain alone of TRAF2 resulted in no FRET with CFP-TRAF3. However, ZnF1 to ZnF5 and TRAF-N of TRAF2 or the RING and ZnFs of TRAF2 were sufficient to interact with TRAF3, although these interactions were less efficient than with full-length TRAF2. Furthermore, removal of ZnF4 and ZnF5 from the RING and ZnF mutant of TRAF2 eliminated interaction with TRAF3. Taken together, the data indicate that the TRAF-N domain, ZnF4, and ZnF5 of TRAF2 are sufficient to interact with TRAF3. A series of plasmids expressing C-terminal deletion mutants of TRAF3 fused with CFP were used to examine the motifs of TRAF3 involved in heterotypic interaction with YFP-TRAF2 (Fig. 7). Deletion of TRAF-C of TRAF3 abolished the ability of TRAF3 to interact with TRAF2, although TRAF3 lacking its TRAF-C domain co-localized with TRAF2. In contrast, a mutant of TRAF3 lacking the entire TRAF domain (TRAF-C and TRAF-N) and consisting of only the RING and ZnFs did associate with TRAF2 (1.15 ± 0.10, n = 11), although to a lesser degree than the full-length TRAF3. Further deletion of ZnFs 2-5 abolished the interaction of this TRAF3 mutant with TRAF2. Moreover, a TRAF3 mutant of the ZnFs and TRAF-N in the presence or absence o" @default.
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• W2045806492 title "TRAF3 Forms Heterotrimers with TRAF2 and Modulates Its Ability to Mediate NF-κB Activation" @default.
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