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Synergistic Transcriptional Activation by hGABP and Select Members of the Activation Transcription Factor/cAMP Response Element-binding Protein Family

1999; Elsevier BV; Volume: 274; Issue: 50 Linguagem: Inglês

10.1074/jbc.274.50.35475

1083-351X

Jun‐ichi Sawada, Noriaki Simizu, Fumihiko Suzuki, Chika Sawa, Masahide Goto, Makoto Hasegawa, Takeshi Imai, Hajime Watanabe, Hiroshi Handa,

interferon and immune responses

The Ets-related DNA-binding protein human GA-binding protein (hGABP) α interacts with the four ankyrin-type repeats of hGABPβ to form an hGABP tetrameric complex that stimulates transcription through the adenovirus early 4 (E4) promoter. Using co-transfection assays, this study demonstrated that the hGABP complex mediated efficient activation of transcription from E4 promoter synergistically with activating transcription factor (ATF) 1 or cAMP response element-binding protein (CREB), but not ATF2/CRE-BP1. This synergy also partially occurred when hGABPα was used alone in place of the combination of hGABPα and hGABPβ. hGABP activated an artificial promoter containing only ATF/CREB-binding sites under coexistence of ATF1 or CREB. Consistent with these results, physical interactions of hGABPα with ATF1 or CREB were observed in vitro. Functional domain analyses of the physical interactions revealed that the amino-terminal region of hGABPα bound to the DNA-binding domain of ATF1, which resulted in the formation of ternary complexes composed of ATF1, hGABPα, and hGABPβ. In contrast to hGABPα, hGABPβ did not significantly interact with ATF1 and CREB. Taken together, these results indicate that hGABP functionally interacts with selective members of the ATF/CREB family, and also suggest that synergy results from multiple interactions which mediate stabilization of large complexes within the regulatory elements of the promoter region, including DNA-binding and non-DNA-binding factors. The Ets-related DNA-binding protein human GA-binding protein (hGABP) α interacts with the four ankyrin-type repeats of hGABPβ to form an hGABP tetrameric complex that stimulates transcription through the adenovirus early 4 (E4) promoter. Using co-transfection assays, this study demonstrated that the hGABP complex mediated efficient activation of transcription from E4 promoter synergistically with activating transcription factor (ATF) 1 or cAMP response element-binding protein (CREB), but not ATF2/CRE-BP1. This synergy also partially occurred when hGABPα was used alone in place of the combination of hGABPα and hGABPβ. hGABP activated an artificial promoter containing only ATF/CREB-binding sites under coexistence of ATF1 or CREB. Consistent with these results, physical interactions of hGABPα with ATF1 or CREB were observed in vitro. Functional domain analyses of the physical interactions revealed that the amino-terminal region of hGABPα bound to the DNA-binding domain of ATF1, which resulted in the formation of ternary complexes composed of ATF1, hGABPα, and hGABPβ. In contrast to hGABPα, hGABPβ did not significantly interact with ATF1 and CREB. Taken together, these results indicate that hGABP functionally interacts with selective members of the ATF/CREB family, and also suggest that synergy results from multiple interactions which mediate stabilization of large complexes within the regulatory elements of the promoter region, including DNA-binding and non-DNA-binding factors. human GA-binding protein activating transcription factor cAMP response element-binding protein cAMP response element-binding protein 1 cAMP response element specificity protein 1 surface plasmon resonance hemagglutinin polyacrylamide gel electrophoresis glutathioneS-transferase adeno early 4 promoter In eukaryotes, the control of gene expression often involves regulated interactions of gene-specific transcription factors with promoters and enhancer regions. The regulatory properties of DNA-binding proteins are often modulated in a combinatorial fashion by interactions among them (1Thanos D. Maniatis T. Cell. 1995; 83: 1091-1100Abstract Full Text PDF PubMed Scopus (852) Google Scholar). hGABP1 has been identified as the transcription factor E4TF1 in HeLa cells because of its ability to activate transcription within the adenovirus early 4 (E4) promoter (2Watanabe H. Imai T. Sharp P.A. Handa H. Mol. Cell. Biol. 1988; 8: 1290-1300Crossref PubMed Scopus (39) Google Scholar,3Watanabe H. Wada T. Handa H. EMBO J. 1990; 9: 841-847Crossref PubMed Scopus (60) Google Scholar). Characterization of cDNAs of E4TF1 subunits (4Watanabe H. Sawada J. Yano K. Yamaguchi K. Goto M. Handa H. Mol. Cell. Biol. 1993; 13: 1385-1391Crossref PubMed Scopus (91) Google Scholar) revealed that the subunits are highly homologous to the respective rat transcription factor GABP (GA-binding protein) subunits. The GABP subunits bind to the cis-regulatory DNA sequence important for immediate early gene activation of herpes simplex virus type-1 (5LaMarco K.L. Thompson C.C. Byers B.P. Walton E.M. McKnight S.L. Science. 1991; 253: 789-792Crossref PubMed Scopus (259) Google Scholar, 6Thompson C.C. Brown T.A. McKnight S.L. Science. 1991; 253: 762-768Crossref PubMed Scopus (320) Google Scholar). Therefore, E4TF1 has been re-designated as hGABP (human GABP), as described in Ref. 7Sawa C. Goto M. Suzuki F. Watanabe H. Sawada J.-i. Handa H. Nucleic Acids Res. 1996; 24: 4954-4961Crossref PubMed Scopus (47) Google Scholar. The Ets-related protein hGABPα can bind by itself to the DNA sequence 5′-CGGAAGTG-3′ in the E4 promoter, but has no effect on in vitro or in vivo transcription (3Watanabe H. Wada T. Handa H. EMBO J. 1990; 9: 841-847Crossref PubMed Scopus (60) Google Scholar, 7Sawa C. Goto M. Suzuki F. Watanabe H. Sawada J.-i. Handa H. Nucleic Acids Res. 1996; 24: 4954-4961Crossref PubMed Scopus (47) Google Scholar, 8Sawada J-i. Goto M. Sawa C. Watanabe H. Handa H. EMBO J. 1994; 13: 1396-1402Crossref PubMed Scopus (46) Google Scholar). By itself, hGABPβ, which contains four notch/ankyrin-type repeats, neither binds to a specific sequence nor stimulates transcription, but has homodimerization activity via the carboxyl-terminal leucine zipper-like domain. However, the four ankyrin repeats mediate the association of hGABPβ with hGABPα, leading to the formation of an α2β2 heterotetrameric complex on the DNA, resulting in in vitro and in vivo transcriptional activation (3Watanabe H. Wada T. Handa H. EMBO J. 1990; 9: 841-847Crossref PubMed Scopus (60) Google Scholar, 7Sawa C. Goto M. Suzuki F. Watanabe H. Sawada J.-i. Handa H. Nucleic Acids Res. 1996; 24: 4954-4961Crossref PubMed Scopus (47) Google Scholar, 8Sawada J-i. Goto M. Sawa C. Watanabe H. Handa H. EMBO J. 1994; 13: 1396-1402Crossref PubMed Scopus (46) Google Scholar, 9Suzuki F. Goto M. Sawa C. Ito S. Watanabe H. Sawada J. Handa H. J. Biol. Chem. 1998; 273: 29302-29308Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Certain transcription factors, such as NRF-2, EF-1A, XrpFI, and RBF-1, which have been independently studied as transcription factors involved in cellular or viral gene expression, have been found to be immunologically related to GABP or hGABP (10Bolwig G.M. Bruder J.T. Hearing P. Nucleic Acids Res. 1992; 20: 6555-6564Crossref PubMed Scopus (20) Google Scholar, 11Marchioni M. Morabito S. Salvati A.L. Beccari E. Carnevali F. Mol. Cell. Biol. 1993; 13: 6479-6489Crossref PubMed Google Scholar, 12Savoysky E. Mizuno T. Sowa Y. Watanabe H. Sawada J-i. Nomura H. Ohsugi Y. Handa H. Sakai T. Oncogene. 1994; 9: 1839-1846PubMed Google Scholar, 13Virbasius J.V. Virbasiu C.A. Scarpulla R.C. Genes Dev. 1993; 7: 380-392Crossref PubMed Scopus (240) Google Scholar). In particular, subunits of NRF-2 are identical to those of hGABP at the level of cDNA (14Gugneja S. Virbasius J.V. Scarpulla R.C. Mol. Cell. Biol. 1995; 15: 102-111Crossref PubMed Scopus (100) Google Scholar). The genes for the hGABP subunits, hGABPα and hGABPβ, have been mapped to human chromosome 21.q 21.2-q21.3 and 7.q11.21, respectively (15Sawada J-i. Goto M. Watanabe H. Handa H. Yoshida M.C. Jpn. J. Cancer Res. 1995; 86: 10-12Crossref PubMed Scopus (6) Google Scholar, 16Goto M. Shimizu T. Sawada J-i. Sawa C. Watanabe H. Ichikawa H. Ohira M. Ohki M. Handa H. Gene (Amst.). 1995; 166: 337-338Crossref PubMed Scopus (8) Google Scholar). Members of the Ets family of DNA-binding proteins contain about an 85-amino acid region of similarity called the ETS domain, which is sufficient for direct DNA binding to a sequence containing a common 5′-GGA(A/T)-3′ core motif (17Janknecht R. Nordheim A. Biochim. Biophys. Acta. 1993; 1155: 346-356Crossref PubMed Scopus (206) Google Scholar, 18Wasylyk B. Hahn S.L. Giovane A. Eur. J. Biochem. 1993; 211: 7-18Crossref PubMed Scopus (811) Google Scholar). Recently, partnerships between the Ets-related proteins and transcription factors belonging to other structural families have been reported, and their functional protein-protein interactions were shown to be important for regulation of gene expression (19Treier M. Bohmann D. Mlodzik M. Cell. 1995; 83: 753-760Abstract Full Text PDF PubMed Scopus (103) Google Scholar, 20Sieweke M.H. Tekatte H. Frampton J. Graf T. Cell. 1996; 85: 49-60Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 21Giese K. Kingsley C. Kirshner J.R. Grosschedl R. Genes Dev. 1995; 9: 995-1008Crossref PubMed Scopus (484) Google Scholar). The promoter of the adenovirus E4 contains not only an hGABP-binding site, but also several binding sites for the ATF/CREB family which has been revealed to be important for its activity by deletion analysis of the promoter (22Ooyama S. Imai T. Hanaka S. Handa H. EMBO J. 1989; 8: 863-868Crossref PubMed Scopus (14) Google Scholar). Transcription factors belonging to the ATF/CREB family were found to be required for its efficient transcriptional activation in vitro (23Hai T. Horikoshi M. Roeder R.G. Green M.R. Cell. 1988; 54: 1043-1051Abstract Full Text PDF PubMed Scopus (102) Google Scholar). But it is unclear how hGABP and members of ATF/CREB family regulate the transcription. In order to gain further insight into the transcriptional activator complex involved with the Ets-related protein hGABP, and to better understand the molecular basis of synergistic transcriptional regulation, we used co-transfection and biochemical assays to examine the possibility that hGABP can cooperate with some members of ATF/CREB family. Based on the studies presented here, we propose that hGABP functions as a transcriptional partner of ATF1 or CREB, leading to efficient transcription activation. Our data suggest that a functional synergy between these factors results from a multitude of DNA-protein and protein-protein interactions which stabilize the large activator complex on promoter/enhancer elements. Luciferase reporter plasmid p4(hGABP-CRE)luc was constructed to insert the DNA fragment obtained by polymerase chain reaction using pTF1–4(C2AT) (3Watanabe H. Wada T. Handa H. EMBO J. 1990; 9: 841-847Crossref PubMed Scopus (60) Google Scholar) as a template into PicaGene PGV-B plasmid (Toyo-ink), the polymerase chain reaction product which contains four tandem repeats of 5′-AACGGAAGTGACGAA-3′ and TATA box sequence, derived from the adenovirus E4 promoter. Luciferase reporter plasmids p4(hGABPmt-CRE)luc, p4(hGABP-CREmt)luc, and p4(hGABPmt-CREmt)luc were generated to substitute the tandem repeats of p4(hGABP-CRE)luc for four tandem repeats of 5′-AACGCTAGTGACGAA-3′, 5′-AACGGAAGTGTGGAA-3′, and 5′-AACGCTAGTGTGGAA-3′, respectively. The pGEX/hGABPα plasmid, which encodes hGABPα fused with glutathione S-transferase (GST) to its amino terminus (designated GST/hGABPα), was created by the insertion of aBamHI digested DNA fragment from hGABPα cDNA into pGEX/prehGABPα that was constructed by insertion of Klenow polymerase-treated NcoI DNA fragment from pET60 (4Watanabe H. Sawada J. Yano K. Yamaguchi K. Goto M. Handa H. Mol. Cell. Biol. 1993; 13: 1385-1391Crossref PubMed Scopus (91) Google Scholar) into theSmaI site of the GST fusion vector pGEX-2T. The pGEX/hGABPα mutant plasmids, which encode various GST/hGABPα mutants were constructed using available restriction sites or polymerase chain reaction-mediated strategies. The pGEX/hGABPβ plasmids, which encode hGABPβ fused with GST to their amino terminus (designated GST/hGABPβ), were constructed by the insertion of Klenow polymerase-treated NcoI-BamHI DNA fragments from pET53 (4Watanabe H. Sawada J. Yano K. Yamaguchi K. Goto M. Handa H. Mol. Cell. Biol. 1993; 13: 1385-1391Crossref PubMed Scopus (91) Google Scholar) into the SmaI site of pGEX-2T. Restriction enzyme-digested DNA fragments encoding the full-length of hGABPα and hGABPβ were cloned into NdeI-BamHI-digested pKA, a plasmid designed for the expression of a histidine-stretch and the phosphorylation target sequence of a catalytic subunit of cAMP-dependent protein kinase-fused proteins to the amino terminus in Escherichia coli. The GST-ATF1 expression plasmid pGEX-ATF1 was described previously (24Wada T. Takagi T. Yamaguchi Y. Kawase H. Hiramoto M. Ferudous A. Takayama M. Lee K.A. Hurst H.C. Handa H. Nucleic Acids Res. 1996; 24: 876-884Crossref PubMed Scopus (28) Google Scholar) and a series of GST-ATF1 mutant expression plasmids were constructed using available restriction sites or polymerase chain reaction-mediated strategies. Expression plasmids for full-length hGABP subunits in Drosophila melanogaster Schneider line 2 (SL2) cells (25Schneider I. J. Embryol. Exp. Morphol. 1972; 27: 353-365PubMed Google Scholar) were constructed as described previously (7Sawa C. Goto M. Suzuki F. Watanabe H. Sawada J.-i. Handa H. Nucleic Acids Res. 1996; 24: 4954-4961Crossref PubMed Scopus (47) Google Scholar). To facilitate the construction of expression plasmids for full-length ATF1, pBS/ATF1 was constructed by insertion of a EcoRI-HindIII DNA fragment containing ATF1 cDNA isolated from pGEM-ATF1 (gift of H. C. Hurst) (26Hurst H.C. Totty N.F. Jones C. Nucleic Acids Res. 1991; 19: 4601-4609Crossref PubMed Scopus (92) Google Scholar) into the sites of EcoRI and HindIII of pBlueScriptII SK+. The DNA fragment encoding ATF1 was isolated from pBS/ATF1 by digestion with KpnI and EcoRI, and subcloned into the sites of KpnI and EcoRI of pA5CΔP to create an ATF1 expression plasmid, pA5CΔPATF1. The coding region for CREB was obtained by polymerase chain reaction using pT7βCREB (gift of H. C. Hurst) (26Hurst H.C. Totty N.F. Jones C. Nucleic Acids Res. 1991; 19: 4601-4609Crossref PubMed Scopus (92) Google Scholar) as template, and the polymerase chain reaction product was then ligated into the site of SmaI of pUC119 to create pUC119/CREB. The CREB expression plasmid, pA5CΔP/CREB was constructed by insertion of an EcoRI-BamHI DNA fragment containing CREB cDNA isolated from pUC119/CREB into the site of BamHI and EcoRI of pA5CΔP. Nuclear extracts of HeLa cells were prepared according to Dignam et al. (27Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9153) Google Scholar). For the gel shift assay, hGABP and ATF/CREB family were purified from HeLa nuclear extract using the corresponding binding site-immobilized latex beads (28Inomota Y. Kawaguti H. Hiramoto M. Wada T. Handa H. Anal. Biochem. 1992; 206: 109-114Crossref PubMed Scopus (66) Google Scholar). Each eluate was dialyzed against 0.05 TGKEDN (50 mmTris-HCl, pH 7.9, 20% glycerol, 50 mm KCl, 1 mm EDTA, 1 mm dithiothreitol, 0.1% Nonidet P-40). hGABP was further purified by size exclusion chromatography using a Superdex 200 PC 3.2/30 column on SMART system (Amersham Pharmacia Biotech) to separate the hGABPα and hGABPβ complex from the hGABPα and hGABPγ complex. GST fusion proteins were expressed in E. coli, BL21(DE3). The cells were suspended in lysis buffer (50 mm Tris-HCl pH 7.9, 1 mm EDTA, 0.5 m NaCl, 0.5% Nonidet P-40, 5% sucrose), lysed by sonication, and subsequently centrifuged at 12,000 × g at 4 °C for 10 min to remove cell debris. The supernatants were stored at −80 °C until they were used forin vitro protein binding assays. hGABPα and hGABPβ with six histidine residues and a PKA phosphorylation site fused to their amino terminus, which were used in Figs. 5 and 6, and also expressed inE. coli BL21(DE3). These fused proteins were purified from supernatants on His-Bind Resin and dialyzed against 0.05 TGKEDN. They were stocked at −80 °C until they were used as32P-labeled proteins for GST pull-down assays. Recombinant hGABPα intact form used in Fig. 5 B was expressed inE. coli BL21(DE3) and purified from the bacteria extract using the hGABP-binding site-immobilized latex beads as described previously (9Suzuki F. Goto M. Sawa C. Ito S. Watanabe H. Sawada J. Handa H. J. Biol. Chem. 1998; 273: 29302-29308Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar).FIG. 6Mapping of the protein interaction domainsin vitro. A, schematic structures of GST-fused ATF1 variants. B, in vitro binding assay using GST-ATF1 variants. An arrowhead indicates the bound hGABPα to GST-fused ATF1 variant immobilized on glutathione-Sepharose. Binding assays and the analysis were carried out as described in the legend to Fig. 5. Coomassie Blue staining and autoradiography of the same gel are shown. C, schematic structures of GST-fused hGABPα variants. D, identification of hGABPα domain that interacts with ATF1 in vitro. Binding assays using GST-hGABPα variants were performed as inpanel B. An arrowhead indicates the bound32P-labeled ATF1 to GST-hGABPα variants immobilized on glutathione-Sepharose. Twenty percent of the total32P-labeled ATF1 protein input (IN) are shown inlane 1. Coomassie Blue staining and autoradiography of the same gel are shown.View Large Image Figure ViewerDownload (PPT) The binding reaction was performed as described previously by Watanabe et al. (3Watanabe H. Wada T. Handa H. EMBO J. 1990; 9: 841-847Crossref PubMed Scopus (60) Google Scholar). Electrophoresis was performed using a 1% agarose gel containing 2.5% glycerol and TGE buffer (50 mm Tris-HCl, pH 7.9, 380 mm glycine, 2 mm EDTA) at 4 °C and 5 V/cm for 4 h. The DNA probes derived from adenovirus early 4 promoter was prepared by digesting pUCE4-20 (3Watanabe H. Wada T. Handa H. EMBO J. 1990; 9: 841-847Crossref PubMed Scopus (60) Google Scholar) with EcoRI and HindIII. The fragments were isolated using 10% polyacrylamide gel and32P-labeled by treatment with Klenow polymerase in the presence of [α-32P]dATP, followed by purification using a Nick column (Amersham Pharmacia Biotech). About 2 ng of the DNA probe was used for the binding reactions. SL2 cells were maintained as described previously (7Sawa C. Goto M. Suzuki F. Watanabe H. Sawada J.-i. Handa H. Nucleic Acids Res. 1996; 24: 4954-4961Crossref PubMed Scopus (47) Google Scholar). Cells were plated onto 35-mm polystyrene dishes at a density of 1 × 106 cells/2 ml of medium per dish 5–10 h prior to transfection. DNA/CaPO4precipitates were formed by the dropwise addition of 100 μl of 0.25m CaCl2 containing the DNA to 100 μl of 2 × HBS (42 mm Hepes, pH 7.1, 275 mmNaCl, 1.4 mm Na2HPO4) and added to the cells 25 min later. Transfection mixture contained 0.6 μg of reporter construct, 50 ng of the β-galactosidase plasmid as an internal control for transfection efficiency and the indicated activators' expression plasmids. In each transfection assays, empty expression plasmid A5CΔP was added as necessary to achieve a constant amount of transfected DNA. After addition of DNA, cells were incubated at 27 °C and left undisturbed until the time of harvest 40 h later. Transfected SL2 cells were lysed and assayed for luciferase and β-galactosidase activity, as described previously (7Sawa C. Goto M. Suzuki F. Watanabe H. Sawada J.-i. Handa H. Nucleic Acids Res. 1996; 24: 4954-4961Crossref PubMed Scopus (47) Google Scholar). N173 cells was established by transfection of pSV2/neo and an HA-tagged hGABPα expression plasmid pCHA/E4TF1–60 which was constructed to insert HA-tagged hGABPα cDNA into mammalian expression plasmid pCAGGS (29Niwa H. Yamamura K. Miyazaki J. Gene (Amst.). 1991; 108: 193-199Crossref PubMed Scopus (4584) Google Scholar), and following two isolations of a colony in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum and 100 μg/ml G418. The cell line was maintained in tissue culture dish containing Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum and 50 μg/ml G418. N173 and HeLa monolayer cells were harvested and washed twice with ice-cold phosphate-buffered saline, and cell extracts were prepared as previous described (30Schreiber E. Matthias P. Muller M.M. Schffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3916) Google Scholar) with slight modification. The nuclear fraction and cytoplasm fraction were mixed and dialyzed against 0.1 TGKEDNP buffer (50 mm Tris-HCl, pH 7.9, 10% glycerol, 100 mm KCl, 1 mm EDTA, 1 mmdithiothreitol, 0.1% Nonidet P-40, 1 mmphenylmethylsulfonyl fluoride) for 6 h. Immunoprecipitation was performed by adding anti-HA antibody HA.11 (Babco) immobilized on protein A-Sepharose (Amersham Pharmacia Biotech) to 200 μl of the cell extract followed by rotation at room temperature for 2 h. The immunoprecipitation pellet was washed once with 200 μl of 0.1 TGKEDNP buffer, and antigens were released by five subsequent incubating for 3 min in 20 μl of 100 mm glycine, pH 2.5. Samples were loaded on SDS-PAGE and examined by immunoblotting experiment using antibodies against hGABPα, hGABPβ, CREB (Santa Cruz), and Sp1 (Santa Cruz) and the ECL Western blotting analysis system (Amersham Pharmacia Biotech). The proteins fused with both a histidine tag and a protein kinase A site and His-tagged ATF1 were phosphorylated by addition of 2 μl of [γ-32P]ATP (>7,000 Ci/mmol; Amersham Pharmacia Biotech) or 0.1 mm ATP (Amersham Pharmacia Biotech) and 5 units of PKA catalytic subunit (Promega), followed by incubation in 10 μl of kinase buffer (20 mm Tris-HCl, pH 7.9, 10 mm MgCl2, 100 mm KCl) at 30 °C for 30 min. The radiolabeled proteins were purified on His-Bind resin and subsequently loaded onto a Nick column (Amersham Pharmacia Biotech) to remove [γ-32P]ATP and imidazole. For the in vitro binding assay, equal amounts (about 5 μg) of GST fusion proteins were immobilized on 3 μl of glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) using the corresponding volumes of the supernatants containing GST fusion proteins. The GST protein-immobilized beads were equilibrated in binding buffer (50 mm Tris-HCl, pH 7.9, 10% glycerol, 50 mm KCl, 1 mm EDTA, 10 mm MgCl2, 0.1 mm CaCl2, 1 mm dithiothreitol, 0.01∼0.05% Nonidet P-40). The beads were then incubated with about 1 ng of 32P-labeled proteins at 4 °C for 2 h and packed in a 1-ml syringe as an affinity column and then washed with 300 μl of binding buffer. Bound proteins were eluted by boiling the beads in 25 μl of SDS sample buffer. Released proteins were resolved by 10% SDS-polyacrylamide gel electrophoresis. After staining with Coomassie Blue, the gels were dried and subjected to autoradiography. Protein interactions detected by surface plasmon resonance were performed using a BIACORE instrument BIACORE2000 (Biacore AB). Sensor Chip CM5 and GST Kit for fusion capture (Biacore AB) were used. For capture of GST fusion proteins, polyclonal goat anti-GST antibody was covalently coupled on the sensor chip surface using standard amine-coupling conditions. The supernatant of E. coli lysates containing GST fusion proteins were used for their immobilization on the sensor chip via anti-GST antibody. Sensor chips containing about 2,500 resonance units of GST fusion protein and running buffer 3 (50 mm Hepes, pH 7.5, 1 mm EDTA, 120 mm KCl, 10 mmMgCl2, 0.01% Tween 20) were used for experiments. Different concentrations of analyte proteins were injected over the surface in a total volume of 250 μl of running buffer 3 at 30 μl/min continuous flow. After each protein injection, the sensor chip surfaces were washed with an injection of 2 m KCl at 30 μl/min continuous flow for 1 min to dissociate proteins interacting with the surface-bound GST fusion protein. All experiments were performed at 24 °C. Kinetics constants of the interactions were calculated by the analysis software BIAevalution, adapted to the modified sensorgrams. The E4 promoter has ATF/CREB-binding sites and an hGABP-binding site. To investigate whether hGABP and ATF/CREB family proteins exist on the promoter simultaneously to regulate its activity, gel shift assays were performed with probes derived from the E4 promoter (Fig. 1). This probe DNA contains an hGABP-binding site and a major and a minor recognition site for ATF/CREB family. As shown in Fig. 1, purified hGABP, containing both α and β subunits, formed α2β2heterotetramers with the probe as described previously (lanes 1 and 8). Members of the ATF/CREB family were prepared by purification from HeLa nuclear extract using DNA affinity beads (28Inomota Y. Kawaguti H. Hiramoto M. Wada T. Handa H. Anal. Biochem. 1992; 206: 109-114Crossref PubMed Scopus (66) Google Scholar), and ATF/CREB family proteins formed DNA-protein complexes with higher mobility (lane 2). When hGABPα/hGABPβ heterotetramers were incubated with purified ATF/CREB family proteins, a new complex with the least mobility appeared (lane 3). Three retarded bands appeared when HeLa nuclear extract was used (lane 7). The mobility of the three bands were similar to those of the three bands obtained by combination use of the purified proteins of hGABP and ATF/CREB family. In either case when the purified proteins or HeLa nuclear extract were used, the DNA-protein complex with the least mobility was competed out with both cold DNA fragments containing hGABP and ATF/CREB family recognition sequences, while the two complex with faster mobility disappeared by adding either competitor containing hGABP or ATF/CREB-binding sites (lanes 4, 5, 8, and 9). This result indicates that hGABP and the ATF/CREB family can bind to the E4 promoter simultaneously with sequence-specific interaction. To further examine whether the slowest migrated DNA-protein complex by HeLa nuclear extract contains hGABP and the ATF/CREB family, antibodies were used in gel shift assay. The most slowly migrated band was obviously reacted against both anti-hGABPβ and anti-CREB antibodies (lanes 13 and 15). These results show that hGABP and some members of the ATF/CREB family simultaneously bind to the adenovirus E4 promoter in a sequence-specific manner in vitro. To study which members of the ATF/CREB family functionally interact with hGABP, we performed co-transfection assays using D. melanogaster Schneider (SL2) cells with pE4-luci, a luciferase reporter gene under control of the adenovirus E4 promoter (7Sawa C. Goto M. Suzuki F. Watanabe H. Sawada J.-i. Handa H. Nucleic Acids Res. 1996; 24: 4954-4961Crossref PubMed Scopus (47) Google Scholar). In this study, activating transcription factor 1 (ATF1) (31Hai T. Liu F. Coukos W.J. Green M.R. Genes Dev. 1989; 3: 2083-2090Crossref PubMed Scopus (755) Google Scholar), cAMP response element-binding protein (CREB) (32Gonzalez G.A. Yamamoto K.K. Fisher W.H. Karr D. Menzel P. Biggs W. Vale W.W. Montminy M.R. Nature. 1989; 337: 749-752Crossref PubMed Scopus (648) Google Scholar), and cAMP response element-binding protein 1 (CRE-BP1 (33Maekawa T. Sakura H. Kanei-Ishii C. Sudo T. Yoshimura T. Fujisawa J. Yoshida M. Ishii S. EMBO J. 1989; 8: 2023-2028Crossref PubMed Scopus (292) Google Scholar) which is identical to ATF2 except for two amino acids) were examined as representative members of the ATF/CREB family because these three factors belonging to ATF/CREB family were reported to be involved in the regulation of adenovirus E4 gene (26Hurst H.C. Totty N.F. Jones C. Nucleic Acids Res. 1991; 19: 4601-4609Crossref PubMed Scopus (92) Google Scholar, 33Maekawa T. Sakura H. Kanei-Ishii C. Sudo T. Yoshimura T. Fujisawa J. Yoshida M. Ishii S. EMBO J. 1989; 8: 2023-2028Crossref PubMed Scopus (292) Google Scholar). Transfected ATF1 increased luciferase activity only slightly above background levels, even when large quantities were transfected (Fig. 2 A, solid columns). In contrast, co-transfections of increasing amounts of transfected ATF1 with constant amounts of hGABPα and hGABPβ led to a marked synergistic transactivation in a dose-dependent manner (Fig. 2 A, white columns). Qualitatively similar results were obtained with the use of transfected CREB as substitute for transfected ATF1 (Fig. 2 B). But ATF2/CRE-BP1 transfected together with hGABP did not lead to a synergistic activation of transcription (Fig. 2 C). These results show that hGABP functionally interacts with ATF1 and CREB, but not ATF2/CRE-BP1, resulting in stimulation of synergistic transcription. To explore whether the synergy between hGABP and ATF1 or CREB entirely depends on the interactions to their binding sites on DNA, we used four luciferase reporter gene constructs. The p4(hGABP-CRE)luc reporter plasmid was constructed to be under control of an artificial promoter including four tandem repeats of an E4 promoter-derived DNA sequence containing both an hGABP-binding site and a CRE site (ATF/CREB family binding site). It is noted that the CRE sequence 5′-TGACGAAA-3′ is not a very good CRE site with some deference compared with the consensus one 5′-TGACGTCA-3′. The p4(hGABPmt-CRE)luc, p4(hGABP-CREmt)luc, and p4(hGABPmt-CREmt)luc reporter plasmids were also constructed to have mutations in their hGABP-binding sites, CREs, or both sites, respectively, resulting in the inability of each factor to bind to their reporter genes. Lack of the hGABP-binding motif resulted in no activation by transfected hGABPα and hGABPβ, even when large amounts of the expression plasmids were used (Fig. 3 A, compare solid columns 2–5 with white columns 2–5). But p4(hGABPmt-CRE)luc did not abrogate the ability of hGABP to synergistically activate transcription in the presence of co-transfected ATF1 or CREB (Fig. 3 A, solid columns 6–15). Importantly, the magnitude of the synergistic effect was reduced when compared with the reporter constructs p4(hGABP-CRE)luc (Fig. 3 A, compare solid columns 6–15 with white columns