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• W1991974261 abstract "CD40 belongs to the tumor necrosis factor (TNF) receptor family. CD40 signaling involves the recruitment of TNF receptor-associated factors (TRAFs) to its cytoplasmic domain. We have identified a novel intracellular CD40-binding protein termedTRAF and TNFreceptor-associated protein (TTRAP) that also interacts with TNF-R75 and CD30. The region of the CD40 cytoplasmic domain that is required for TTRAP association overlaps with the TRAF6 recognition motif. Association of TTRAP with CD40 increases profoundly in response to treatment of cells with CD40L. Interestingly, TTRAP also associates with TRAFs, with the highest affinity for TRAF6. In transfected cells, TTRAP inhibits in a dose-dependent manner the transcriptional activation of a nuclear factor-κB (NF-κB)-dependent reporter mediated by CD40, TNF-R75 or Phorbol 12-myristate 13-acetate (PMA) and to a lesser extent by TRAF2, TRAF6, TNF-α, or interleukin-1β (IL-1β). TTRAP does not affect stimulation of NF-κB induced by overexpression of the NF-κB-inducing kinase (NIK), the IκB kinase α (IKKα), or the NF-κB subunit P65/RelA, suggesting it acts upstream of the latter proteins. Our results indicate that we have isolated a novel regulatory factor that is involved in signal transduction by distinct members of the TNF receptor family. CD40 belongs to the tumor necrosis factor (TNF) receptor family. CD40 signaling involves the recruitment of TNF receptor-associated factors (TRAFs) to its cytoplasmic domain. We have identified a novel intracellular CD40-binding protein termedTRAF and TNFreceptor-associated protein (TTRAP) that also interacts with TNF-R75 and CD30. The region of the CD40 cytoplasmic domain that is required for TTRAP association overlaps with the TRAF6 recognition motif. Association of TTRAP with CD40 increases profoundly in response to treatment of cells with CD40L. Interestingly, TTRAP also associates with TRAFs, with the highest affinity for TRAF6. In transfected cells, TTRAP inhibits in a dose-dependent manner the transcriptional activation of a nuclear factor-κB (NF-κB)-dependent reporter mediated by CD40, TNF-R75 or Phorbol 12-myristate 13-acetate (PMA) and to a lesser extent by TRAF2, TRAF6, TNF-α, or interleukin-1β (IL-1β). TTRAP does not affect stimulation of NF-κB induced by overexpression of the NF-κB-inducing kinase (NIK), the IκB kinase α (IKKα), or the NF-κB subunit P65/RelA, suggesting it acts upstream of the latter proteins. Our results indicate that we have isolated a novel regulatory factor that is involved in signal transduction by distinct members of the TNF receptor family. tumor necrosis factor carbon catabolite repressor protein hemagglutinin (tag) human umbilical vein endothelial cell IκB-kinase interleukin TNF receptor-associated factor TRAF-interacting protein Janus kinase latent membrane protein 1 mitogen-activated kinase MAPK/extracellular response kinase nuclear factor κB NF-κB-inducing kinase phorbol 12-myristate 13-acetate receptor activator of NF-κB signal transducer and activator of transcription transforming growth factor-β-activated kinase TRAF-associated NF-κB activator TNF receptor TNF-R1-associated death domain protein TRAF-interacting protein TRAF and TNF receptor-associated protein expressed sequence tag N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine CD40 is a member of the tumor necrosis factor (TNF)1 receptor family that plays a critical role in many immunological processes (1.Grewal I.S. Flavell R.A. Annu. Rev. Immunol. 1998; 16: 111-135Crossref PubMed Scopus (1324) Google Scholar). The receptor is present on many cell types, and its function has been studied most extensively in B cells, dendritic cells, monocytes, and endothelial cells. Characterization of mice deficient for CD40 or its ligand CD40L (also named CD154) highlights the importance of CD40-mediated signaling in the thymus-dependent humoral immune response and in isotype switching (2.Kawabe T. Naka T. Yoshida K. Tanaka T. Fujiwara H. Suematsu S. Yoshida N. Kishimoto T. Kikutani H. Immunity. 1994; 1: 167-178Abstract Full Text PDF PubMed Scopus (974) Google Scholar, 3.Renshaw B.R. Fanslow 3rd, W.C. Armitage R.J. Campbell K.A. Liggitt D. Wright B. Davison B.L. Maliszewski C.R. J. Exp. Med. 1994; 180: 1889-1900Crossref PubMed Scopus (493) Google Scholar, 4.Xu J. Foy T.M. Laman J.D. Elliott E.A. Dunn J.J. Waldschmidt T.J. Elsemore J. Noelle R.J. Flavell R.A. Immunity. 1994; 1: 423-431Abstract Full Text PDF PubMed Scopus (692) Google Scholar). CD40-mediated signal transduction induces the transcription of a large number of genes implicated in host defense against pathogens. This is accomplished by the activation of multiple transcription factors, including NF-κB (5.Berberich I. Shu G.L. Clark E.A. J. Immunol. 1994; 153: 4357-4366PubMed Google Scholar), c-Jun (6.Berberich I. Shu G. Siebelt F. Woodgett J.R. Kyriakis J.M. Clark E.A. EMBO J. 1996; 15: 92-101Crossref PubMed Scopus (174) Google Scholar), and STAT3 (7.Hanissian S.H. Geha R.S. Immunity. 1997; 6: 379-387Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). In the past 5 years we have come to understand in significant detail the cascade that leads from stimulation of TNF receptors to the activation of transcription factors. The signal transduction is triggered by binding of trimeric ligands of the TNF family to their cognate receptors, which induces oligomerization of the latter at the cell surface. This brings the intracellular domains of these receptors in close proximity whereby they serve as a high affinity binding platform for many cytoplasmic proteins involved in signal transduction. Members of the TNF receptor family, such as CD30, CD40, TNF-R75, OX40, RANK, and 4–1BB, have been implicated primarily in gene activation rather than apoptosis and transmit their signal through the direct recruitment of TRAFs (8.Arch R.H. Gedrich R.W. Thompson C.B. Genes Dev. 1998; 12: 2821-2830Crossref PubMed Scopus (512) Google Scholar). TRAFs 1–6 display similar structural features, i.e. they have an N-terminal RING finger (which is absent in TRAF1), followed by 5–7 zinc fingers, and a C-terminal TRAF domain that mediates receptor binding. CD40 associates with TRAFs 2, 3, 5, and 6 (9.Cheng G. Cleary A.M. Ye Z.S. Hong D.I. Lederman S. Baltimore D. Science. 1995; 267: 1494-1498Crossref PubMed Scopus (442) Google Scholar, 10.Rothe M. Sarma V. Dixit V.M. Goeddel D.V. Science. 1995; 269: 1424-1427Crossref PubMed Scopus (975) Google Scholar, 11.Ishida T. Mizushima S. Azuma S. Kobayashi N. Tojo T. Suzuki K. Aizawa S. Watanabe T. Mosialos G. Kieff E. Yamamoto T. Inoue J. J. Biol. Chem. 1996; 271: 28745-28748Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 12.Ishida T.K. Tojo T. Aoki T. Kobayashi N. Ohishi T. Watanabe T. Yamamoto T. Inoue J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9437-9442Crossref PubMed Scopus (311) Google Scholar). The importance of the latter for signaling by CD40 and other receptors has become clear from the characterization of TRAF6-deficient mice. Experiments performed with cells derived from these mice demonstrated that TRAF6 is crucial for CD40L, IL-1, and lipopolysaccharide-dependent activation of NF-κB (13.Lomaga M.A. Yeh W.C. Sarosi I. Duncan G.S. Furlonger C. Ho A. Morony S. Capparelli C. Van G. Kaufman S. van der Heiden A. Itie A. Wakeham A. Khoo W. Sasaki T. Cao Z. Penninger J.M. Paige C.J. Lacey D.L. Dunstan C.R. Boyle W.J. Goeddel D.V. Mak T.W. Genes Dev. 1999; 13: 1015-1024Crossref PubMed Scopus (1072) Google Scholar). These results confirmed earlier observations that TRAF6 is involved in gene activation through members of the TNF receptor and IL-1 receptor families (11.Ishida T. Mizushima S. Azuma S. Kobayashi N. Tojo T. Suzuki K. Aizawa S. Watanabe T. Mosialos G. Kieff E. Yamamoto T. Inoue J. J. Biol. Chem. 1996; 271: 28745-28748Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 14.Muzio M. Natoli G. Saccani S. Levrero M. Mantovani A. J. Exp. Med. 1998; 187: 2097-2101Crossref PubMed Scopus (525) Google Scholar, 15.Kirschning C.J. Wesche H. Merrill Ayres T. Rothe M. J. Exp. Med. 1998; 188: 2091-2097Crossref PubMed Scopus (655) Google Scholar, 16.Cao Z. Xiong J. Takeuchi M. Kurama T. Goeddel D.V. Nature. 1996; 383: 443-446Crossref PubMed Scopus (1117) Google Scholar). Recently, TRAF2 was also shown to be essential for CD40-mediated responses in mice (17.Nguyen 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). The molecular mechanisms by which TRAFs activate downstream effector proteins remain largely unknown. However, the current data suggest that this involves the interaction of TRAFs with different types of kinase. Some of these are involved in pathways leading to NF-κB activation, e.g. NIK (18.Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Crossref PubMed Scopus (1160) Google Scholar), MEKK1 (19.Baud V. Liu Z.G. Bennett B. Suzuki N. Xia Y. Karin M. Genes Dev. 1999; 13: 1297-1308Crossref PubMed Scopus (408) Google Scholar), and TAK1 (20.Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1014) Google Scholar). These kinases have the potential to activate the IκB kinases (IKKα and IKKβ) (21.Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1575) Google Scholar, 22.Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1065) Google Scholar, 23.Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1841) Google Scholar), which phosphorylate IκB. This phosphorylation triggers ubiquitination and subsequent degradation of IκB, resulting in the release of NF-κB subunits that translocate into the nucleus, where they act as transcription activators (reviewed in Ref. 24.Stancovski I. Baltimore D. Cell. 1997; 91: 299-302Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar). Signal transduction by members of the TNF receptor family also involves several regulatory factors. Most of these proteins have been identified as TRAF-binding proteins, e.g. A20 (25.Song H.Y. Rothe M. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6721-6725Crossref PubMed Scopus (369) Google Scholar), I-TRAF/TANK (26.Cheng G. Baltimore D. Genes Dev. 1996; 10: 963-973Crossref PubMed Scopus (263) Google Scholar,27.Rothe M. Xiong J. Shu H.B. Williamson K. Goddard A. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8241-8246Crossref PubMed Scopus (189) Google Scholar), and TRIP (28.Lee S.Y. Choi Y. J. Exp. Med. 1997; 185: 1275-1285Crossref PubMed Scopus (170) Google Scholar). Although their precise role in the signal transduction process remains elusive, overproduction of these factors either inhibits (in the case of A20, I-TRAF, TRIP) or synergistically activates (in the case of TANK) TRAF-mediated activation of NF-κB. As a result of a search for novel effector proteins involved in CD40 signaling, this study describes the identification of a novel regulatory protein that binds receptors and TRAFs and that inhibits activation of NF-κB. Anti-FLAG M2 monoclonal antibody was purchased from Sigma and anti-CD40 polyclonal antibody C20, that was used for Western blot, was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-hemagglutinin (HA) tag monoclonal antibody was a gift from Innogenetics S. A. (Zwijnaarde, Belgium), and anti-hCD40 5D12 monoclonal antibody, used for immunoprecipitation, was from Tanox Pharma B. V. (Amsterdam, The Netherlands). The anti-hTNF-R75 mouse monoclonal antibody utr4 was a gift of M. Brockhaus and W. Lesslauer (Roche, Basel, Switzerland). The anti-hTNF-R75 polyclonal antibodies were from W. Buurman (University Maastricht, The Netherlands). The following expression vectors for production of human and murine TRAFs were a gift from D. Goeddel (Tularik Inc., South San Francisco, CA): FLAG-hTRAF2-pRK5, FLAG-ΔTRAF2-pRK5 (insert encodes amino acids 87–501 of mouse TRAF2 (10.Rothe M. Sarma V. Dixit V.M. Goeddel D.V. Science. 1995; 269: 1424-1427Crossref PubMed Scopus (975) Google Scholar)), FLAG-hTRAF6-pRK5, and FLAG-Δ289TRAF6-pRK5 (insert encodes amino acids 289–511 of human TRAF6 (16.Cao Z. Xiong J. Takeuchi M. Kurama T. Goeddel D.V. Nature. 1996; 383: 443-446Crossref PubMed Scopus (1117) Google Scholar)). FLAG-Δ317TRAF6-pcDNA3 was constructed by PCR amplification on FLAG-hTRAF6-pRK5, engineering an EcoRI site at the 5′-end of the partial cDNA and cloning theEcoRI-XhoI fragments into FLAG-pcDNA3. HA-hTRAF3-pcDNA3 was a gift from V. Dixit (University of Michigan, Ann Arbor, MI). HA-IKKα-pcDNA and Xpress-NIK-pcDNA3 were a gift from M. Karin (University of California San Diego, La Jolla, CA). FLAG-hTRAF5-pME was provided by J. Inoue (Tokyo University, Tokyo, Japan) and P65/RelA-pRc/cytomegalovirus by S. Plaisance (University of Gent, Gent, Belgium). CD40 cDNA was amplified by PCR from a human umbilical vein endothelial cell (HUVEC) cDNA library and cloned into pcDNA3. The cDNAs encoding the cytoplasmic part of human CD40 (amino acids 216–277 (11.Ishida T. Mizushima S. Azuma S. Kobayashi N. Tojo T. Suzuki K. Aizawa S. Watanabe T. Mosialos G. Kieff E. Yamamoto T. Inoue J. J. Biol. Chem. 1996; 271: 28745-28748Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar)), human TNF-R75 (262–437 (29.Vandenabeele P. Declercq W. Vanhaesebroeck B. Grooten J. Fiers W. J. Immunol. 1995; 154: 2904-2913PubMed Google Scholar)), and human CD30 (408–595 (30.Gedrich R.W. Gilfillan M.C. Duckett C.S. Van Dongen J.L. Thompson C.B. J. Biol. Chem. 1996; 271: 12852-12858Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar)), were generated by PCR and inserted into the pEG202 vector (Gyuris et al. (32.Gyuris J. Golemis E. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1321) Google Scholar)) in-frame with the sequence encoding the LexA DNA-binding domain. The cDNA for the C-terminal cytoplasmic domain of LMP1 (amino acids 192–386 (31.Franken M. Devergne O. Rosenzweig M. Annis B. Kieff E. Wang F. J. Virol. 1996; 70: 7819-7826Crossref PubMed Google Scholar)) was obtained from M. Rowe (University of Wales, Cardiff, United Kingdom (UK)). Our CD40 deletion and point mutants were constructed by PCR, as described by Ishida and co-workers (11.Ishida T. Mizushima S. Azuma S. Kobayashi N. Tojo T. Suzuki K. Aizawa S. Watanabe T. Mosialos G. Kieff E. Yamamoto T. Inoue J. J. Biol. Chem. 1996; 271: 28745-28748Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar), and cloned into pEG202. Plasmid hTNF-R75-pcDNA6 was described previously (29.Vandenabeele P. Declercq W. Vanhaesebroeck B. Grooten J. Fiers W. J. Immunol. 1995; 154: 2904-2913PubMed Google Scholar), and human TRADD was cloned in frame with an N-terminal E tag, into pcDNA3. 4F2 and TRAF3 partial cDNAs that were picked in our two-hybrid screening were excised from pJG4–5, using EcoRI, and subcloned into the similarly digested vectors pEG202, FLAG-pcDNA3 and HA- pcDNA3. Full-length TTRAP cDNA was cloned in two ways. First, cloning into HA-pcDNA3 was done starting directly from the cDNA picked from the HUVEC library, via digestion withEcoRI and ligation into HA-pcDNA3. In doing so, 34 nucleotides from the library vector and 20 from the 5′-untranslated region of TTRAP cDNA are present between the sequence encoding the HA tag and the translation initiation codon of TTRAP. Second, for cloning of TTRAP cDNA in pJG4–5, a PCR-based approach was used. AnEcoRI site was engineered directly adjacent to the 5′-end of the TTRAP cDNA by amplification of TTRAP cDNA using the primer combination 5′-GACGAATTCAGAGGCGGCAGGAAGATGGAGTTGG and 5′-GCCTCACATCCTGAATGCAGGA. The amplified fragment was then digested with EcoRI and BglII and ligated together with a BglII-NcoI TTRAP cDNA fragment into pJG4–5. FLAG-TTRAP-pcDNA3 and TTRAP-pCS2 were obtained by ligation of the EcoRI fragment from TTRAP-pJG4–5 into FLAG-pcDNA3 and pCS2, respectively. Two-hybrid screening in yeast was performed by the interaction trap cloning method, which is often referred to as the LexA two-hybrid system (32.Gyuris J. Golemis E. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1321) Google Scholar). The cytoplasmic part of human CD40 was cloned in-frame with the LexA DNA-binding domain (the bait plasmid). Screening was done using a HeLa cell cDNA library in pJG4–5 (the prey plasmid), which was obtained from R. Brent (Harvard Medical School, Boston, MA). EGY48 (MAT α, his3, trp1, ura3–52, leu2::pLEU2-LexAop) yeast cells were transformed with the prey plasmid, the bait plasmid, and the lacZreporter plasmid pSH18–34 by the lithium acetate transformation method (33.Gietz R.D. Schiestl R.H. Willems A.R. Woods R.A. Yeast. 1995; 11: 355-360Crossref PubMed Scopus (1685) Google Scholar). Yeast cells containing bait plasmid and lacZ reporter plasmid were transformed with 20 μg of library plasmid and plated on glucose medium lacking tryptophan, histidine and uracil, to select for the presence of all three plasmids. In total, approximately 2 × 106 colonies were obtained. These transformants were harvested and frozen at −80 °C in a glycerol solution (65% glycerol (v/v), 100 mm MgSO4, 25 mmTris/HCl pH 7.4). To screen for protein-protein interaction, 20 × 106 colony-forming units of an amplified stock of original transformants were tested for a positive interaction phenotype, as described (32.Gyuris J. Golemis E. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1321) Google Scholar). When using yeast two-hybrid as test for interaction, we performed mating assays (34.Finley Jr., R.L. Brent R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12980-12984Crossref PubMed Scopus (240) Google Scholar). Bait and prey constructs were transformed in yeast strain EGY48 (mating type α) and EGY42 (mating type a), respectively. Northern analysis of human and murine mRNA blots (CLONTECH, Palo Alto, CA) was carried out with human 4F2 and the entire cDNA of mouse TTRAP (EST clone 876634) as probes, respectively. Blots were hybridized at 65 °C in QUICKHYB hybridization solution (Stratagene, La Jolla, CA). The 2-kb-long probe used for in situ hybridization was the same as used for Northern analysis of mouse TTRAP . In vitrotranscription with T3 RNA Polymerase yielded [35S]-uracil (NEN Life Science Products) labeled single-stranded riboprobe. In situ hybridization in sections of mouse embryos was done as described previously (35.Dewulf N. Verschueren K. Lonnoy O. Moren A. Grimsby S. Vande Spiegle K. Miyazono K. Huylebroeck D. Ten Dijke P. Endocrinology. 1995; 136: 2652-2663Crossref PubMed Google Scholar). Full-length human TTRAP cDNA was obtained by screening a HUVEC cDNA plasmid library with human 4F2 as probe; colony lifting and hybridization was as described previously (36.Sambrook J. Fritsch E.F. Maniatis F. Molecular Cloning: A Laboratory Manual. Second Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The mouse TTRAP homologue was obtained by screening with BLAST (37.Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59416) Google Scholar) the EST data base for sequences homologous to human TTRAP. EST clone 1262914 (GenBankTM accession number AI465781) was requested from the IMAGE consortium (Cambridge, UK) and sequenced completely to obtain the mouse TTRAP cDNA sequence. The coiled coil prediction was obtained by running the program COILS (38.Lupas A. Curr. Opin. Struct. Biol. 1997; 7: 388-393Crossref PubMed Scopus (203) Google Scholar). 293T human embryonic kidney cells were grown in Dulbecco's modified Eagle's medium supplemented with glucose (4.5 g/liter) and 10% (v/v) fetal bovine serum. Transient transfection of plasmids for luciferase reporter assay or co-immunoprecipitation analysis was done with Fugene 6 (Roche Molecular Biochemicals), using 2 μl of Fugene per μg of plasmid DNA. For luciferase reporter assays, transfections were done in duplicate using 3 × 105 293T cells per well of a 24-well plate. Each well was transfected with 15 ng of reporter plasmid NFconluc, encoding the luciferase reporter gene driven by a minimal NF-κB-responsive promoter (gift of A. Israel, Institut Pasteur, Paris, France) or 50 ng of AP-1-luc (Stratagene, La Jolla, CA). To normalize the transfection efficiency, we co-transfected 75 ng of alacZ reporter construct that contains the Rous sarcoma virus promoter inserted upstream of Escherichia coli lacZ. If the amount of TTRAP plasmid used in transfections was varied, we kept the total amount of DNA constant by adding a Myc-TTRAPmutant-pCS3 construct that does not produce TTRAP protein, because the TTRAP cDNA was cloned out-of-frame of the sequence encoding the N-terminal Myc tag. Cell extracts were prepared and assayed for luciferase activity and β-galactosidase activity according to the manufacturers' protocols (Promega (Madison, WI) andCLONTECH (Palo Alto, CA), respectively). Data were normalized by calculating the ratio of luciferase and β-galactosidase activities. The average normalized luciferase activity is presented relative to the activity in nonstimulated samples as x-fold activation. For co-immunoprecipitation, 1–2 × 106 293T cells were transfected with 2 μg of each expression vector. In the TRAF-TTRAP co-immunoprecipitation experiments, we noticed that overexpression of TRAFs 2, 3, 5, and 6 resulted in different synthesis levels of TTRAP from the co-transfected TTRAP-pcDNA3 construct. This was probably because of the fact that the cytomegalovirus promoter in the pcDNA3 vector (Invitrogen BV, Groningen, The Netherlands) is sensitive to the different levels of NF-κB induced by overexpressing these TRAFs. To circumvent this problem, we co-transfected 0.1 μg of hNIK-pcDNA3, which potently stimulates NF-κB. Stimulation of cells with CD40L was done by overlaying transfected cells with mouse 3T6 fibroblasts stably transformed with an expression vector encoding hCD40L (39.Pradier O. Willems F. Abramowicz D. Schandene L. de Boer M. Thielemans K. Capel P. Goldman M. Eur. J. Immunol. 1996; 26: 3048-3054Crossref PubMed Scopus (35) Google Scholar). As a negative control we used nontransfected 3T6 cells. Transiently transfected 293T cells at subconfluence were overlaid with approximately twice the number of 3T6 cells. Cells were harvested 24–48 h after transfection in 300 μl of lysis buffer (50 mm Tris/HCl, pH 7.4, 200 mm NaCl, 10% glycerol, 0.2% Nonidet P-40, 50 mm NaF, 1 mmNa4P2O7, 5 mmNA3VO4, 1 mm phenylmethylsulfonyl fluoride, 3 μg aprotinin/ml). Cells were then lyzed by incubation for 20 min on ice or by passing five times through a 22-gauge needle. Cellular debris and nuclei were eliminated by centrifugation (Eppendorf 4517R, 14,000 rpm, 4 °C, 10 min). Five μg of antibody was added to the lysate and incubated for 3 h at 4 °C. Subsequently, 20 μl of a 50% slurry of protein G-Sepharose (Amersham Pharmacia Biotech, Gent, Belgium) was added to the samples, and the incubation was continued for 1 h. Next, the-Sepharose was washed four times in 750 μl of lysis buffer for 10 min at 4 °C. Finally, the beads were mixed with 20 μl of sample buffer, and the samples were analyzed by SDS-polyacrylamide gel electrophoresis. To verify the expression levels of the different proteins, 0.1% of the cytoplasmic extract was analyzed on Western blot. Proteins were separated on 12.5% Tris-Tricine gels and transferred onto polyvinylidene difluoride membrane (NEN Life Science Products) using a semi-dry blotting apparatus (Sigma). For Western analysis, the membrane was blocked in 3% skimmed milk in TBS-T (10 mmTris/HCl, pH 7.4, 150 mm NaCl, 0.2% Tween 20). After sequential incubation with primary and horseradish peroxidase-conjugated secondary antibody (Jackson Laboratories, West Grove, PA) for 1 h at 24 °C, proteins were visualized with the ECL chemiluminescent detection system (NEN Life Science Products). A two-hybrid screen in yeast was set up to identify novel CD40-interacting proteins. The cDNA encoding the cytoplasmic region of CD40 was cloned into the bait vector, which was transformed in yeast together with a HeLa cDNA library cloned in the prey vector. After screening approximately 2 × 106 transformants, eight different cDNAs were isolated from yeast colonies with a positive interaction phenotype. The corresponding eight polypeptides were tested for interaction with the cytoplasmic domain of other members of the TNF receptor family, i.e. human TNF-R75 and CD30. In addition, we also used as bait the C-terminal 192 amino acids of LMP1 from Epstein-Barr virus. Similar to CD30, CD40, and TNF-R75, LMP1 can signal through direct interaction with TRAFs (40.Devergne O. Hatzivassiliou E. Izumi K.M. Kaye K.M. Kleijnen M.F. Kieff E. Mosialos G. Mol. Cell. Biol. 1996; 16: 7098-7108Crossref PubMed Google Scholar, 41.Mosialos G. Birkenbach M. Yalamanchili R. VanArsdale T. Ware C. Kieff E. Cell. 1995; 80: 389-399Abstract Full Text PDF PubMed Scopus (902) Google Scholar). One of the prey plasmids that was isolated in our screen contained a cDNA sequence encoding part of TRAF3 (amino acids 381–568, comprising part of the TRAF-N domain and the complete TRAF-C domain (9.Cheng G. Cleary A.M. Ye Z.S. Hong D.I. Lederman S. Baltimore D. Science. 1995; 267: 1494-1498Crossref PubMed Scopus (442) Google Scholar)). This hybrid prey protein associated with the cytoplasmic region of CD40, CD30, and LMP1, but not TNF-R75 (Table I), which is in accordance with published results for TRAF3 (10.Rothe M. Sarma V. Dixit V.M. Goeddel D.V. Science. 1995; 269: 1424-1427Crossref PubMed Scopus (975) Google Scholar, 30.Gedrich R.W. Gilfillan M.C. Duckett C.S. Van Dongen J.L. Thompson C.B. J. Biol. Chem. 1996; 271: 12852-12858Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 41.Mosialos G. Birkenbach M. Yalamanchili R. VanArsdale T. Ware C. Kieff E. Cell. 1995; 80: 389-399Abstract Full Text PDF PubMed Scopus (902) Google Scholar). Another positive prey, coded 4F2, bound to CD40, CD30, and TNF-R75 baits but not the LMP1 bait (Table I). The interaction phenotype of 4F2 with TNF-R75 was somewhat weaker than with CD40 and CD30. Also, the interaction phenotype of the latter two receptors with 4F2 was apparently not as strong as with the N-terminally truncated TRAF3 prey. The 1.8-kb-long partial cDNA for 4F2 encoded a novel polypeptide with no homology to TRAFs or other factors known to be involved in TNF receptor signaling.Table IInteraction test of TRAF3 (amino acids 381–566), 4F2, and TTRAP with the cytoplasmic domain of different receptors, using the yeast two-hybrid assayBaitPreyTRAF3 (381–568)4F2TTRAPCD40++++CD30++++TNF-R75−++LMP1++−−The interaction phenotype was estimated by blue/white staining of yeast colonies. Staining was scored as blue, i.e. relatively strong and visible within 12 hours (++), strong and visible within 24 hours (+), or as white (−). Open table in a new tab The interaction phenotype was estimated by blue/white staining of yeast colonies. Staining was scored as blue, i.e. relatively strong and visible within 12 hours (++), strong and visible within 24 hours (+), or as white (−). To obtain a full-length cDNA of this protein, we screened a HUVEC cDNA library using 4F2 cDNA as a probe and isolated a 2-kb-long cDNA. This yielded a complete open reading frame encoding a protein of 362 amino acids, that has been named TTRAP (TRAF andTNF Receptor-associatedprotein). Recently the complete genomic sequence of human TTRAP became available in the data base as a cosmid clone that maps to chromosome 6p22.1-22.3 (EBI accession number AL031775). The sequence of mouse TTRAP was obtained by sequencing EST clone 1262914, and the candidate Caenorhabditis elegans homologue of TTRAP was retrieved from the data base as putative protein predicted from the genomic sequence. Further comparison of TTRAP with the public data bases revealed that it is related to the C-terminal 380 amino acids of the yeast transcription factor CCR4 (42.Draper M.P. Liu H.Y. Nelsbach A.H. Mosley S.P. Denis C.L. Mol. Cell. Biol. 1994; 14: 4522-4531Crossref PubMed Scopus (73) Google Scholar) and to a CCR4-like protein named nocturnin, which has been isolated from Xenopus (43.Green C.B. Besharse J.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14884-14888Crossref PubMed Scopus (118) Google Scholar) and recently also from human and mouse (44.Dupressoir A. Barbot W. Loireau M.P. Heidmann T. J. Biol. Chem. 1999; 274: 31068-31075Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). CCR4 is distinct from TTRAP and nocturnin because it is approximately twice as big. The alignment of the TTRAP-related protein sequences shows that, although the overall amino similarities are rather low, there are stretches of identical amino acids scattered throughout the C-terminal 250 residues in the alignment (Fig. 1). The data in Table II furthermore indicate that nocturnin is more related to CCR4 than to TTRAP, whereas the C. elegans protein is more similar to TTRAP than to nocturnin. Taken together, our results indicate that TTRAP, nocturnin, and CCR4 belong to an emerging gene family. Neither nocturnin nor the C-terminal part of CCR4 has been characterized functionally.Table IIAmino acid identity (percentage) for pair wise aligned protein sequenceshTTRAPmTTRAPCelTTRAPhnocturninxnocturninmTTRAP69celTTRAP3538hnocturnin323230xnocturnin32293375yCCR4-C3230323937The aligned sequences and their GenBank™/EBI accession numbers are: hTTRAP (human, AJ269473); mTT" @default.
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• W1991974261 title "TTRAP, a Novel Protein That Associates with CD40, Tumor Necrosis Factor (TNF) Receptor-75 and TNF Receptor-associated Factors (TRAFs), and That Inhibits Nuclear Factor-κB Activation" @default.
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• W1991974261 doi "https://doi.org/10.1074/jbc.m000531200" @default.
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