The Early Growth Response Protein (EGR-1) Regulates Interleukin-2 Transcription by Synergistic Interaction with the Nuclear Factor of Activated T Cellsстатья из журнала
Аннотация: The early growth response-1 gene (EGR-1) is induced by a wide range of stimuli in diverse cell types; however, EGR-1-regulated genes display a highly restricted pattern of expression. Recently, an overlapping Sp1·EGR-1 binding site has been identified within the interleukin-2 (IL-2) gene promoter directly upstream of the binding site for the nuclear factor of activated T cells (NFAT). We used transfection assays to study how the abundantly and constitutively expressed Sp1 protein and the immediate early EGR-1 zinc finger protein regulate IL-2 gene expression. Here, we identify EGR-1 as an important activator of theIL-2 gene. In Jurkat T cells, EGR-1 but not Sp1 acts as a potent coactivator for IL-2 transcription, and in combination with NFATc, EGR-1 increases transcription of an IL-2 reporter construct 200-fold. Electrophoretic mobility shift assays reveal that recombinant EGR-1 and NFATc bind independently to their target sites within the IL-2 promoter, and the presence of both sites on the same DNA molecule is required for EGR-1·NFATc·DNA complex formation. The transcriptional synergy observed here for EGR-1 and NFATc explains how the abundant nuclear factor EGR-1 contributes to the expression of restrictively expressed genes. The early growth response-1 gene (EGR-1) is induced by a wide range of stimuli in diverse cell types; however, EGR-1-regulated genes display a highly restricted pattern of expression. Recently, an overlapping Sp1·EGR-1 binding site has been identified within the interleukin-2 (IL-2) gene promoter directly upstream of the binding site for the nuclear factor of activated T cells (NFAT). We used transfection assays to study how the abundantly and constitutively expressed Sp1 protein and the immediate early EGR-1 zinc finger protein regulate IL-2 gene expression. Here, we identify EGR-1 as an important activator of theIL-2 gene. In Jurkat T cells, EGR-1 but not Sp1 acts as a potent coactivator for IL-2 transcription, and in combination with NFATc, EGR-1 increases transcription of an IL-2 reporter construct 200-fold. Electrophoretic mobility shift assays reveal that recombinant EGR-1 and NFATc bind independently to their target sites within the IL-2 promoter, and the presence of both sites on the same DNA molecule is required for EGR-1·NFATc·DNA complex formation. The transcriptional synergy observed here for EGR-1 and NFATc explains how the abundant nuclear factor EGR-1 contributes to the expression of restrictively expressed genes. early growth response nuclear factor of activated T cells phosphate-buffered saline dithiothreitol phorbol 12-myristate 13-acetate phenylmethylsulfonyl fluoride glutathione S-transferase base pair(s). Antigenic activation of resting T cells initiates a cascade of biochemical and metabolic events that lead to cell proliferation, cytokine release, and T cell effector function (1Crabtree G.R. Clipstone N.A. Annu. Rev. Biochem. 1994; 63: 1045-1083Crossref PubMed Scopus (630) Google Scholar, 2Zipfel P.F. Irving S.G. Kelly K. Siebenlist U. Mol. Cell. Biol. 1989; 9: 1041-1048Crossref PubMed Scopus (148) Google Scholar, 3Fraser J.D. Straus D. Weiss A. Immunol. Today. 1993; 14: 357-362Abstract Full Text PDF PubMed Scopus (147) Google Scholar). T cell receptor engagement induces sequential signaling reactions that are converted to the nucleus, where transcription of novel genes is induced. Most of the directly activated immediate early genes encode transcription factors that regulate the nuclear events essential for cell proliferation, differentiation, and T cell effector function. In general terms the proteins encoded by the transiently expressed immediate early genes either inactivate immediate early gene transcription in an autoregulatory fashion or they function as transcriptional activators of early gene expression. The four related early growth response genes EGR-1 (4 - 6),EGR-2 (7Chavrier P. Zerial M. Lemaire P. Almendral J. Bravo R. Charnay P. EMBO J. 1988; 7: 29-35Crossref PubMed Scopus (353) Google Scholar, 8Joseph L.J. Le Beau M.M. Jamieson Jr., G.A. Acharya S. Shows T.B. Rowley J.D. Sukhatme V.P. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7164-7168Crossref PubMed Scopus (275) Google Scholar), EGR-3 (9Patwardhan S. Gashler A. Siegel M.G. Chang L.C. Joseph L.J. Shows T.B. Le Beau M.M. Sukhatme V.P. Oncogene. 1991; 6: 917-928PubMed Google Scholar, 10Mages H.W. Stamminger T. Rilke O. Bravo R. Kroczek R.A. Int. Immunol. 1993; 5: 63-70Crossref PubMed Scopus (36) Google Scholar), and EGR-4/pAT133 (11Crosby S.D. Puetz J.J. Simburger K.S. Fahrner T.J. 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Acta. 1997; 1354: 134-144Crossref PubMed Scopus (29) Google Scholar). The EGR-1 protein specifically interacts with the G-rich regulatory zinc finger protein binding site (ZIP) of the human IL-2 gene promoter, whereas EGR-2, EGR-3, and EGR-4/AT133 do not bind to this promoter element (19Skerka C. Decker E.L. Zipfel P.F. J. Biol. Chem. 1995; 270: 22500-22506Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). A large number of regulatory GC-rich promoter elements that represent overlapping binding sites for EGR-1 and Sp1 have been recently identified (20Khachigian L.M. Williams A.J. Collins T. J. Biol. Chem. 1995; 270: 27679-27686Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 21Khachigian L.M. Lindner V. Williams A.J. Collins T. Science. 1996; 271: 1427-1431Crossref PubMed Scopus (480) Google Scholar, 22Skerka C. Decker E.L. Zipfel P.F. Immunobiology. 1997; 198: 179-191Crossref PubMed Scopus (22) Google Scholar). As both zinc finger proteins bind alternatively to these elements, it is of interest to define which protein mediates transcriptional regulation through these overlapping binding sites. The regulation of the EGR-1 gene is well characterized; however, the functional mechanisms of this protein in gene transcription are less defined. EGR consensus or binding sites are present in promoters of a number of tissue specifically expressed genes such as cytokines, growth factors, and genes involved in cell cycle regulation, e.g. IL-2, tumor necrosis factor-α, tumor growth factor-β, insulin-like growth factor, platelet-derived growth factor-α, β chain, EGR-1, EGR-4/pAT133, hox 1.4, and nur77 (18Zipfel P.F. Decker E.L. Holst C. Skerka C. Biochim. Biophys. Acta. 1997; 1354: 134-144Crossref PubMed Scopus (29) Google Scholar, 19Skerka C. Decker E.L. Zipfel P.F. J. Biol. Chem. 1995; 270: 22500-22506Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 20Khachigian L.M. Williams A.J. 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Two factors have been identified that interact with the inhibitory domain and repress EGR-1 transcriptional activity in vitro (35Russo M.W. Sevetson B.R. Milbrandt J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6873-6877Crossref PubMed Scopus (255) Google Scholar, 36Svaren J. Sevetson B.R. Apel E.D. Zimonjic D.B. Popescu N.C. Milbrandt J. Mol. Cell. Biol. 1996; 16: 3545-3553Crossref PubMed Scopus (331) Google Scholar). Recently, a synergistic activation of EGR-1 and RelA has been reported in induction of NF-κB1 promoter activity (37Cogswell P.C. Mayo M.W. Baldwin Jr., A.S. J. Exp. Med. 1997; 185: 491-497Crossref PubMed Scopus (60) Google Scholar). Within the human IL-2 gene promoter, we have previously identified a novel regulatory element, termed ZIP, that serves as an overlapping binding region for EGR-1 and Sp1 (19Skerka C. Decker E.L. Zipfel P.F. J. Biol. Chem. 1995; 270: 22500-22506Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The ZIP element is located immediately upstream of the distal binding element for the nuclear factor of activated T cells (NFAT). Transfection experiments revealed that both the ZIP site and the distal NFAT site have activating functions (19Skerka C. Decker E.L. Zipfel P.F. J. Biol. Chem. 1995; 270: 22500-22506Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 27Dey B.R. Sukhatme V.P. Roberts A.B. Sporn M.B. Rauscher III, F.J. Kim S.J. Mol. Endocrinol. 1994; 8: 595-602Crossref PubMed Scopus (197) Google Scholar, 38Durand D.B. Shaw J.P. Bush M.R. Replogle R.E. Belagaje R. Crabtree G.R. Mol. Cell. Biol. 1988; 8: 1715-1724Crossref PubMed Scopus (431) Google Scholar, 39Emmel E.A. Verweij C.L. Durand D.B. Higgins K.M. Lacy E. Crabtree G.R. Science. 1989; 246: 1617-1620Crossref PubMed Scopus (643) Google Scholar), and a combination of the ZIP and NFAT elements contribute significantly to IL-2 gene expression (19Skerka C. Decker E.L. Zipfel P.F. J. Biol. 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Curran T. Rao A. Nature. 1993; 365: 352-355Crossref PubMed Scopus (692) Google Scholar). Besides this cooperation with AP-1 proteins, a functional interaction of NFATp with the proto-oncogene c-Maf in transcription of the murine IL-4gene has been reported (50Ho I.C. Hodge M.R. Rooney J.W. Glimcher L.H. Cell. 1996; 85: 973-983Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar). To characterize which of the two ZIP binding proteins mediatesIL-2 gene transcription, we have analyzed the regulatory role of these zinc finger proteins. Here, we report that the transcription factor Sp1 has no effect on IL-2 transcription but that EGR-1 and NFATc interact synergistically. EGR-1 and NFATc bind independently to adjacent sites within the IL-2 gene promoter to form an EGR-1·NFATc·DNA complex. Although by itself EGR-1 has little transactivating capacity, this zinc finger protein enhances NFATc transactivation 25-fold. This is the first report that describes a functional interaction between the early growth response protein EGR-1 and a member of the NFAT family of transcription factors. The reporter plasmid pZNA3-Luc contains three copies of the ZIP, NFAT, and AP-1 binding elements linked to a minimal IL-2 promoter fragment. For construction of pZNA3-Luc, three double-stranded oligonucleotides covering the ZIP·NFAT·AP-1 binding region of the human IL-2 promoter (position −302 to −258) were ligated in head to tail orientation and inserted into plasmid pMILuc4 (19Skerka C. Decker E.L. Zipfel P.F. J. Biol. Chem. 1995; 270: 22500-22506Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), which contains a minimal IL-2 promoter (position −63 to +51) linked to the firefly luciferase gene. Plasmid pNA3-Luc contains three copies of the NFAT and AP-1 binding sites (position −280 to −258) linked to the minimal IL-2 promoter and the firefly luciferase gene. The sequence of the reporter constructs was confirmed by DNA sequence analysis. Expression plasmid pSG5-EGR1 has a full-length human EGR-1 cDNA inserted (18Zipfel P.F. Decker E.L. Holst C. Skerka C. Biochim. Biophys. Acta. 1997; 1354: 134-144Crossref PubMed Scopus (29) Google Scholar). The deletion constructs of this expression vector include the DNA binding zinc finger domain and N- or C-terminal parts of the sequence conferring to amino acids 11–435 (ΔC-EGR-1) and 321–543 (ΔN-EGR-1). Plasmid pPacSp1, which has 2.1 kilobases of the human Sp1 cDNA (encoding the C-terminal 696 amino acids of Sp1) inserted, was kindly provided by Robert Tjian (University of California, Berkeley). Plasmid pSH107c, which contains the full-length human NFATc cDNA (51Northrop J.P. Ho S.N. Chen L. Thomas D.J. Timmerman L.A. Nolan G.P. Admon A. Crabtree G.R. Nature. 1994; 369: 497-502Crossref PubMed Scopus (532) Google Scholar), was a generous gift of Gerald R. Crabtree (Stanford University, Stanford, CA). The c-Fos and c-Jun expression constructs, pRSVc-fos (52Van Beveren C. van Straaten F. Curran T. Muller R. Verma I.M. Cell. 1983; 32: 1241-1255Abstract Full Text PDF PubMed Scopus (424) Google Scholar) and pRSV-cJ (53Angel P. Hattori K. Smeal T. Karin M. Cell. 1988; 55: 875-885Abstract Full Text PDF PubMed Scopus (1154) Google Scholar), were obtained from Peter Angel (Forschungszentrum, Karlsruhe). Plasmid pGEX-NFATc, which contains the full-length NFATc cDNA, was kindly provided by E. Serfling (Würzburg, Germany). All plasmids used for transfection were purified by CsCl density gradient centrifugation. The human helper T cell line Jurkat and 293 kidney cells (kindly provided by Hermann Eibel, University Hospital Freiburg, Germany) were maintained in RPMI 1640 medium (Bio Whittaker), supplemented with 10% heat-inactivated fetal calf serum, penicillin, streptomycin, and fungizone at a density between 0.5 × 105 and 8 × 105 cells per ml. For transient transfections of Jurkat cells, 1 × 107 cells were washed with PBS and resuspended in 0.8 ml of transfection buffer (10 mm glucose, 0.1 mm DTT in RPMI 1640). Cells were transfected by electroporation at 300 V and 960 μF using 5 μg of reporter construct and 7 μg of the indicated expression plasmids. In all experiments, the total amount of transfected DNA was kept constant by addition of pSG5 plasmid DNA. After transfection, cells were incubated for 48 h in culture medium. When indicated, cells were stimulated 24 h after transfection with 1 μg/ml PHA (Murex Diagnostics) and 20 ng/ml PMA (Sigma) for another 24 h. Cells were harvested by washing two times with PBS, lysed by incubation in 250 μl of lysis reagent (Promega) for 8 min at room temperature, and centrifuged for 10 s at maximum speed in an Eppendorf microfuge. Cell supernatant (50 μl) was mixed with 100 μl of luciferase assay reagent (Promega), and the light emission was measured immediately at 25 °C using a luminometer (Berthold Biolumat LB 9500C). The initial 10-s integral of light emission was recorded. All assays were performed in triplicate. Transfection of 293 cells was performed by calcium phosphate precipitation according to standard procedures (54Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). About 4 × 105 cells were transfected with 2 μg of reporter plasmid and 3 μg of the various expression plasmids, together with 0.5 μg of pRL-TK vector (Promega) to control for transfection efficiency. After 24 h, cells were washed twice with PBS and harvested. Cell lysis and measurement of luciferase activity were performed using the dual-luciferase reporter assay system (Promega). Recombinant EGR-1 protein was expressed in the baculovirus system as described (18Zipfel P.F. Decker E.L. Holst C. Skerka C. Biochim. Biophys. Acta. 1997; 1354: 134-144Crossref PubMed Scopus (29) Google Scholar). Sf9 cells were infected in Insect-Xpress medium (Bio Whittaker), with recombinant EGR-1 containing virus using a multiplicity of infection of 5. After 3 days, cells were harvested, washed twice in ice-cold PBS, and incubated on ice in hypotonic buffer (10 mm Tris-Cl, pH 7.4, 10 mm NaCl, 1.5 mm MgCl2, 10 mm NaF, 0.5 mm DTT, 0.5 mm PMSF, Sigma) for 10 min. Cells were pelleted by centrifugation at 12,000 × g for 10 s, and the pellets were incubated on ice for 45 min in lysis buffer (20 mm HEPES, pH 7.9, 420 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 10 mm NaF, 25% glycerol, 0.5 mm DTT, 0.5 mm PMSF). The extracts were cleared by centrifugation at 12,000 × g for 20 min at 4 °C and stored in aliquots at −70 °C. Recombinant NFATc protein was expressed as a GST-fusion protein using plasmid pGEX-NFATc, which contains the full-length NFATc cDNA (40Serfling E. Avots A. Neumann M. Biochim. Biophys. Acta. 1995; 1263: 181-200Crossref PubMed Scopus (206) Google Scholar). An overnight culture of pGEX-NFATc transformed cells (DH5α/BL21) was added to 100 ml of LB medium containing 50 μg/ml ampicillin, 0.1% glucose and grown for 3 h at 37 °C. After induction of protein expression by isopropyl-1-thio-β-d-galactopyranoside (0.4 mm), cells were grown at room temperature for another hour. Cells were harvested, resuspended in 2 ml of PBS containing 1% Triton X-100 (Sigma), 0.5 mm DTT, and 0.5 mm PMSF, and lysed on ice by sonication; cellular debris were pelleted by centrifugation. Upon addition of 1 ml of glutathione-agarose beads (1:2 (v/v), Sigma), the suspension was incubated for 20 min at 4 °C on a rotating platform. Beads were collected by centrifugation (1 min at 800 × g) and washed five times with PBS (containing DTT and PMSF). GST-NFATc fusion protein was eluted using 50 mm Tris-HCl, pH 8.0, containing 10 mmglutathione (Boehringer Mannheim). Eluted protein was stored in aliquots at −70 °C. Recombinant Sp1 protein was obtained from Promega. SDS-polyacrylamide gel electrophoresis was performed as described previously (54Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar, 55Heukeshoven J. Dernick R. Electrophoresis. 1986; 6: 103-112Crossref Scopus (1286) Google Scholar) using 10% separating gels and prestained molecular weight marker proteins (Life Technologies, Inc.). Proteins were transferred onto nitrocellulose membrane by semidry blotting (56Kyhse-Andersen J. J. Biochem. Biophys. Methods. 1984; 10: 203-209Crossref PubMed Scopus (2248) Google Scholar). Membranes were blocked with 5% (w/v) dried milk in PBS for 30 min and incubated overnight at 4 °C with specific polyclonal rabbit anti-human EGR-1 antiserum (Santa Cruz Biotechnologies) or monoclonal mouse anti-human NFATc antibody (ABR, Inc.). For electrophoretic mobility shift assays, 1–5 pm double-stranded oligonucleotides were end-labeled with [γ-33P]ATP (specific activity, 3000 Ci/mm; Amersham Buchler). Double-stranded oligonucleotides representing the ZIP site of the human IL-2 promoter (Z; position −302 to −280 relative to the transcription start site), the NFAT site (N; position −280 to −260), as well as the complete IL-2 ZIP·NFAT region (ZN; position −302 to −258) were used. Double-stranded oligonucleotides with the mutated binding sites included ZmN (5′ TGTAT AACCA CCAAC TTAAA GAAAG GAGGA AAAAC TGTTT CATA 3′), ZNm (5′ TGTAT CCCCA CCCCC TTAAA GAAAG GAGGC AAAAC TGTTG CATA 3′), and ZmNm (5′ TGTATAACCA CCAAC TTAAA GAAAG GAGGC AAAAC TGTTG CATA 3′); mutated nucleotides are underlined. Labeled oligonucleotides (10–30 μm) were incubated at 4 °C with recombinant proteins in 20 μl of buffer containing 20 mm HEPES, pH 7.9, 50 mm KCl, 0.5 mmDTT, 1 mm MgCl2, and 4% (v/v) Ficoll for 30 min in the presence of 0.5 μg of poly(dI-dC) (Amersham Pharmacia Biotech). In some cases, competitor DNA or specific antiserum directed against EGR-1 (Santa Cruz) or NFATc (ABR) was added to the incubation mixture as indicated. The resulting DNA-protein complexes were separated in a 5% nondenaturing polyacrylamide gel at 4 °C in 0.25 × TBE at 150 V and 20 mA. For competition experiments, the indicated oligonucleotides were used in 200-fold excess. A combination of the ZIP, NFAT, and AP-1 elements of the human IL-2 gene promoter is important for gene induction. As each element represents binding sites for several transcription factors, we were interested in identifying the factors required for maximal transcription. To this end, cotransfection experiments were performed with various expression vectors and with reporter construct pZNA3-Luc containing three copies of the ZIP·NFAT·AP-1 region linked to a minimal IL-2gene promoter and the firefly luciferase gene. The zinc finger proteins Sp1 and EGR-1 showed little effect when used as single proteins (1.1- and 1.3-fold induction), AP-1 proteins (c-Fos and c-Jun) showed a weak activating capacity (2.8-fold induction), whereas NFATc displayed a strong activating effect on reporter gene transcription (8.1-fold) (Table I and Fig. 1 A). The transcriptional activity observed with NFATc is in agreement with previous results (39Emmel E.A. Verweij C.L. Durand D.B. Higgins K.M. Lacy E. Crabtree G.R. Science. 1989; 246: 1617-1620Crossref PubMed Scopus (643) Google Scholar) and underlines the important role of this protein in IL-2gene induction. Similar results were obtained with a reporter construct that lacks the ZIP site but has three copies of the NFAT·AP-1 sites linked to the minimal IL-2 gene promoter (pNA3-Luc) (Fig. 1 B).Table ITransactivating effects of ZIP, NFAT, and AP-1 binding factorsExpression plasmidRelative light units (± S.D.)Fold inductionNone105 (± 10)1.0EGR-1141 (± 43)1.3Sp1119 (± 86)1.1AP-1 (c-Fos, c-Jun)296 (± 65)2.8NFATc854 (± 73)8.1EGR-1 + NFATc21,460 (± 2,192)204.4Sp1 + NFATc870 (± 739)8.3NFATc + AP-13,442 (± 1,562)32.8EGR-1 + NFATc + AP-119,537 (± 12,900)186.1Transactivating activity of EGR-1, Sp1, AP-1 proteins c-Fos and c-Jun, and NFATc on reporter construct pZNA3-Luc, which contains three copies of the ZIP · NFAT · AP-1 region of the humanIL-2 gene promoter linked to a minimal IL-2 gene promoter and to the firefly luciferase gene, is shown. Open table in a new tab Transactivating activity of EGR-1, Sp1, AP-1 proteins c-Fos and c-Jun, and NFATc on reporter construct pZNA3-Luc, which contains three copies of the ZIP · NFAT · AP-1 region of the humanIL-2 gene promoter linked to a minimal IL-2 gene promoter and to the firefly luciferase gene, is shown. As previous experiments demonstrated that a combination of the ZIP- and NFAT elements induces strong IL-2 promoter activity (19Skerka C. Decker E.L. Zipfel P.F. J. Biol. Chem. 1995; 270: 22500-22506Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), we asked whether factors binding to these adjacent promoter sites do interact. The transcriptional activity obtained by a combination of two proteins is shown as fold induction compared with the activity obtained with the NFATc expression vector, which was set to 1. A combination of Sp1 and NFATc showed no enhancing effect (Fig. 2and Table I); however, EGR-1 potentiated NFATc transcription and increased transactivation 25-fold (Fig. 2 A and Table I). Although both Sp1 and EGR-1 bind to the same promoter element, EGR-1 but not Sp1 cooperates with NFATc in IL-2 gene induction. To test whether this cooperation is dependent on the DNA binding sites, a reporter plasmid (pNA3-Luc) was used that contains the NFAT and AP-1 binding sites but lacks the ZIP site. The presence of the ZIP element is essential for the observed interaction, as in the absence of an EGR binding site, EGR-1 affected NFATc activity only 2.3-fold (Fig. 2 B). This reduced effect suggests that binding to DNA is a prerequisite for the functional interaction of the two nuclear factors. As AP-1 proteins are reported to interact with NFAT factors (57Jain J. Miner Z. Rao A. J. Immunol. 1993; 151: 837-848PubMed Google Scholar, 58Nolan G.P. Cell. 1994; 77: 795-798Abstract Full Text PDF PubMed Scopus (100) Google Scholar), we asked whether the AP-1 proteins c-Fos and c-Jun do influence the functional synergy between EGR-1 and NFATc. Transfection experiments with reporter plasmid pZNA3-Luc confirmed the previously described interaction of AP-1 with NFATc, as a combination of c-Fos and c-Jun increased the transcriptional activity of NFATc 4-fold (Fig. 2 C and Table I). Although c-Fos and c-Jun increased NFATc activity, the AP-1 factor failed to further enhance the synergistic interaction of EGR-1 and NFATc. In contrast, a slight decrease was observed when all four proteins (EGR-1, NFATc, c-Fos, and c-Jun) were simultaneously expressed (Fig. 2 C and Table I). The observed interaction of AP-1 proteins c-Fos and c-Jun with NFATc was also observed with reporter construct pNA3-Luc, which lacks the ZIP site. In the absence of an EGR-1 binding site, AP-1 proteins influenced the transcriptional activity of NAFTc to a similar extent (Fig. 2 D). Several activating domains and one inhibitory domain have been identified within the N-terminal part of the EGR-1 protein (33Gashler A.L. Swaminathan S. Sukhatme V.P. Mol. Cell. Biol. 1993; 13: 4556-4571Crossref PubMed Scopus (214) Google Scholar, 34Russo M.W. Matheny C. Milbrandt J. Mol. Cell. Biol. 1993; 13: 6858-6865Crossref PubMed Scopus (89) Google Scholar). Given this modular composition, we tried to localize the domain responsible for the functional interaction with NFATc. To this end, a vector encoding a truncated EGR-1 protein with the N-terminal 320 amino acids deleted (ΔN-EGR-1) was transfected together with NFATc. This truncated protein retained the activity of wild-type EGR-1 almost completely (Fig. 3). The interaction with
Год издания: 1998
Авторы: Eva L. Decker, Christine Skerka, Peter F. Zipfel
Издательство: Elsevier BV
Источник: Journal of Biological Chemistry
Ключевые слова: Signaling Pathways in Disease, Immune Cell Function and Interaction, Cytokine Signaling Pathways and Interactions
Другие ссылки: Journal of Biological Chemistry (PDF)
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PubMed (HTML)
Journal of Biological Chemistry (HTML)
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Открытый доступ: hybrid
Том: 273
Выпуск: 41
Страницы: 26923–26930