Identification of Activating Transcription Factor 4 (ATF4) as an Nrf2-interacting Protein

Nrf2 regulates expression of genes encoding enzymes with antioxidant (e.g. heme oxygenase-1 (HO-1)) or xenobiotic detoxification (e.g. NAD(P)H:quinone oxidoreductase, glutathione S-transferase) functions via the stress- or antioxidant-response elements (StRE/ARE). Nrf2 heterodimerizes with small Maf proteins, but the role of such dimers in gene induction is controversial, and other partners may exist. By using the yeast two-hybrid assay, we identified activating transcription factor (ATF) 4 as a potential Nrf2-interacting protein. Association between Nrf2 and ATF4 in mammalian cells was confirmed by co-immunoprecipitation and mammalian two-hybrid assays. Furthermore, Nrf2·ATF4 dimers bound to an StRE sequence from the ho-1 gene. CdCl2, a potent inducer of HO-1, increased expression of ATF4 in mouse hepatoma cells, and detectable induction of ATF4 protein preceded that of HO-1 (30 minversus 2 h). A dominant-negative mutant of ATF4 inhibited basal and CdCl2-stimulated expression of a StRE-dependent/luciferase fusion construct (pE1-luc) in hepatoma cells but only basal expression in mammary epithelial MCF-7 cells. A dominant mutant of Nrf2 was equally inhibitory in both cell types in the presence or absence of CdCl2. These results indicate that ATF4 regulates basal and CdCl2-induced expression of theho-1 gene in a cell-specific manner and possibly in a complex with Nrf2. Nrf2 regulates expression of genes encoding enzymes with antioxidant (e.g. heme oxygenase-1 (HO-1)) or xenobiotic detoxification (e.g. NAD(P)H:quinone oxidoreductase, glutathione S-transferase) functions via the stress- or antioxidant-response elements (StRE/ARE). Nrf2 heterodimerizes with small Maf proteins, but the role of such dimers in gene induction is controversial, and other partners may exist. By using the yeast two-hybrid assay, we identified activating transcription factor (ATF) 4 as a potential Nrf2-interacting protein. Association between Nrf2 and ATF4 in mammalian cells was confirmed by co-immunoprecipitation and mammalian two-hybrid assays. Furthermore, Nrf2·ATF4 dimers bound to an StRE sequence from the ho-1 gene. CdCl2, a potent inducer of HO-1, increased expression of ATF4 in mouse hepatoma cells, and detectable induction of ATF4 protein preceded that of HO-1 (30 minversus 2 h). A dominant-negative mutant of ATF4 inhibited basal and CdCl2-stimulated expression of a StRE-dependent/luciferase fusion construct (pE1-luc) in hepatoma cells but only basal expression in mammary epithelial MCF-7 cells. A dominant mutant of Nrf2 was equally inhibitory in both cell types in the presence or absence of CdCl2. These results indicate that ATF4 regulates basal and CdCl2-induced expression of theho-1 gene in a cell-specific manner and possibly in a complex with Nrf2. nuclear factor-κB ferriprotoporphyrin IX heme oxygenase-1 activating transcription factor/cAMP-response element-binding protein Cap 'N′ Collar/basic-leucine zipper NF-E2 related factor nuclear factor-erythroid 2 basic region/leucine zipper Gal4 DNA binding domain yeast two-hybrid mammalian two-hybrid stress-response element antioxidant-response element Maf recognition element electrophoretic mobility shift assay activation domain amino acid hepatoma Overproduction of oxygen free radicals, attenuation of antioxidant systems, or both, commonly in response to extracellular stimuli, disturbs the cellular redox status and leads to oxidative stress. Such conditions typically elicit an adaptive response aimed at reversing this imbalance and maintaining redox homeostasis. In part, this adaptive response includes the activation of specific signaling pathways and, ultimately, the coordinate induction of a select set of genes that encode proteins with distinct activities that individually and collectively manifest antioxidant and cytoprotective functions. Central to this induction process are redox-sensitive transcription factors, such as nuclear factor-κB (NF-κB)1 and activator protein-1, arguably the two most prominent regulators of this cellular response mechanism (reviewed in Refs. 1Sen C.K. Packer L. FASEB J. 1996; 10: 709-720Crossref PubMed Scopus (1781) Google Scholar and 2Morel Y. Barouki R. Biochem. J. 1999; 342: 481-496Crossref PubMed Scopus (445) Google Scholar). Recent studies from several laboratories (3Venugopal R. Jaiswal A.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14960-14965Crossref PubMed Scopus (936) Google Scholar, 4Alam J. Stewart D. Touchard C. Boinapally S. Choi A.M.K. Cook J.L. J. Biol. Chem. 1999; 274: 26071-26078Abstract Full Text Full Text PDF PubMed Scopus (1073) Google Scholar, 5Wild A.C. Moinova H.R. Mulcahy R.T. J. Biol. Chem. 1999; 274: 33627-33636Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar, 6Itoh K. Wakabayashi N. Katoh Y. Ishii T. Igarashi K. Engel J.D. Yamamoto M. Genes Dev. 1998; 13: 67-86Google Scholar, 7Ishii T. Itoh K. Takahashi S. Sato H. Yanagawa T. Katoh Y. Bannai S. Yamamoto M. J. Biol. Chem. 2000; 275: 16023-16029Abstract Full Text Full Text PDF PubMed Scopus (1245) Google Scholar, 8Huang H.C. Nguyen T. Pickett C.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12475-12480Crossref PubMed Scopus (444) Google Scholar) have implicated another transcription regulator, Nrf2, with a potentially significant role in the adaptive response to oxidative stress. Nrf2 belongs to the CNC-bZIP subfamily of basic region/leucine zipper (bZIP) transcription factors. CNC-bZIP proteins are distinguished from other bZIP subfamilies, including those composed of Jun, Fos, ATF/CREB, or Maf factors, in that they also contain a Cap'n‘Collar (CNC) structural motif homologous to a region within theDrosophila homoeotic selector protein encoded by thecap'n‘collar gene (9Mohler J. Vani K. Leung S. Epstein A. Mech. Dev. 1991; 34: 3-10Crossref PubMed Scopus (108) Google Scholar). bZIP proteins function as obligate dimers; for example, individual Jun-Jun or Jun-Fos dimers are commonly and collectively referred to as activator protein-1 transcription factors. Sequences necessary for both dimerization and DNA binding reside within the bipartite bZIP domain. Limited but consistent observations (6Itoh K. Wakabayashi N. Katoh Y. Ishii T. Igarashi K. Engel J.D. Yamamoto M. Genes Dev. 1998; 13: 67-86Google Scholar, 8Huang H.C. Nguyen T. Pickett C.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12475-12480Crossref PubMed Scopus (444) Google Scholar) suggest that under normal conditions, and as is the case for NF-κB factors, Nrf2 exists in an inactive, cytoplasm-localized state, in part or fully as a consequence of binding to the cytoskeleton-associated protein Keap1. After exposure of cells to electrophiles or oxidative stress-generating agents, the cytoplasmic retention mechanism is inactivated, and Nrf2 is transported to the nucleus by an as yet uncharacterized mechanism(s) but one that may involve protein kinase C-mediated phosphorylation of Nrf2 (8Huang H.C. Nguyen T. Pickett C.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12475-12480Crossref PubMed Scopus (444) Google Scholar). Within the nucleus, Nrf2 activates transcription of a select set of target genes by binding to distinct but very similar DNA elements, individually or alternatively referred to as the NF-E2-binding site (10Andrews N.C. Erdjument-Bromage H. Davidson M.B. Tempst P. Orkin S.H. Nature. 1993; 362: 722-728Crossref PubMed Scopus (567) Google Scholar), the Maf recognition element (MARE, 11), the stress-response element (12Choi A.M.K. Alam J. Am. J. Respir. Cell Mol. Biol. 1996; 15: 9-19Crossref PubMed Scopus (1020) Google Scholar), or the antioxidant-response element (13Rushmore T.H. Morton M.R. Pickett C.B. J. Biol. Chem. 1991; 266: 11632-11639Abstract Full Text PDF PubMed Google Scholar). Many of the Nrf2 target genes (3Venugopal R. Jaiswal A.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14960-14965Crossref PubMed Scopus (936) Google Scholar, 4Alam J. Stewart D. Touchard C. Boinapally S. Choi A.M.K. Cook J.L. J. Biol. Chem. 1999; 274: 26071-26078Abstract Full Text Full Text PDF PubMed Scopus (1073) Google Scholar, 5Wild A.C. Moinova H.R. Mulcahy R.T. J. Biol. Chem. 1999; 274: 33627-33636Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar, 7Ishii T. Itoh K. Takahashi S. Sato H. Yanagawa T. Katoh Y. Bannai S. Yamamoto M. J. Biol. Chem. 2000; 275: 16023-16029Abstract Full Text Full Text PDF PubMed Scopus (1245) Google Scholar, 8Huang H.C. Nguyen T. Pickett C.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12475-12480Crossref PubMed Scopus (444) Google Scholar) encode proteins that play a central role in the adaptive response to oxidative stress. Among others, these include heme oxygenase-1 (HO-1), an enzyme that catalyzes the rate-limiting reaction in heme degradation, a catabolic pathway that leads to the production of bilirubin, a potent antioxidant; NAD(P)H:quinone oxidoreductase (NQO), which catalyzes two-electron reduction of quinones, preventing the participation of such compounds in redox cycling and oxidative stress; γ-glutamylcysteine synthase, which catalyzes the rate-limiting reaction in glutathione biosynthesis; and glutathione S-transferase, which conjugates hydrophobic electrophiles and reactive oxygen species with glutathione. Nrf2, like other CNC/bZIP proteins and Fos family members, belongs to a sub-class of bZIP factors with leucine zipper motifs incapable of self-dimerization. Consequently, sequence-specific DNA binding and subsequent induction of target gene transcription requires association of Nrf2 with other transcription factors. In accordance with the paradigm established by NF-E2 (10Andrews N.C. Erdjument-Bromage H. Davidson M.B. Tempst P. Orkin S.H. Nature. 1993; 362: 722-728Crossref PubMed Scopus (567) Google Scholar), the first CNC-bZIP containing mammalian transcription factor isolated, the most prominent dimerization partners of Nrf2 are the small Maf proteins, MafF, MafG and MafK (also referred to as p18 (14Andrews N.C. Kotkow K.J. Ney P.A. Erdjument-Bromage H. Tempst P. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11488-11492Crossref PubMed Scopus (238) Google Scholar)). The precise function of such Nrf2·Maf dimers, however, is controversial, as they have been proposed to function as both positive (5Wild A.C. Moinova H.R. Mulcahy R.T. J. Biol. Chem. 1999; 274: 33627-33636Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar) and negative regulators (15Dhakshinamoorthy S. Jaiswal A.K. J. Biol. Chem. 2000; 275: 40134-40141Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar) of ARE-dependent gene transcription. Jun-Nrf2 complexes have also been implicated as positive effectors of ARE-dependent genes (16Venugopal R. Jaiswal A.K. Oncogene. 1998; 17: 3145-3156Crossref PubMed Scopus (487) Google Scholar). Given our incomplete understanding of Nrf2 function, the propensity of bZIP proteins to form inter- and intra-family dimers (17Hai T. Curran T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3720-3724Crossref PubMed Scopus (1119) Google Scholar,18Vinson C.R. Hai T. Boyd S.M. Genes Dev. 1993; 7: 1047-1058Crossref PubMed Scopus (294) Google Scholar), and of transcription factors in general to form complexes that tend to provide both diversity to, and discrimination of, genetic responses to extracellular stimuli, we reasoned that additional Nrf2-containing complexes exist intracellularly and that such complexes would likely regulate Nrf2 target gene expression. Accordingly, we have used the yeast two-hybrid screening procedure to identify proteins that associate with Nrf2. Herein, we report the identification of ATF4 as a Nrf2-interacting protein and explore the potential role of ATF4 in the regulation of one Nrf2 target gene, ho-1. Tissue culture media were from Life Technologies, Inc., and fetal bovine serum was obtained from Mediatech. Restriction endonucleases and other DNA-modifying enzymes were purchased from either Life Technologies, Inc., or New England Biolabs. Oligonucleotides were synthesized by IDT, Inc. Radiolabeled nucleotides were obtained from PerkinElmer Life Sciences. Reagents for luciferase assays were purchased from Sigma. Anti-mouse Nrf2 was kindly provided by Dr. M. Yamamoto. Antibodies against other transcription factors, including anti-human Nrf2, and HO-1 were obtained from Santa Cruz Biotechnology and StressGen Biotechnologies Corp., respectively. All other chemicals were reagent grade. cDNA clones for mouse Jun D, mouse Fos B, human ATF3 (I.M.A.G.E. Clone identification number 273190), and mouse ATF4 (I.M.A.G.E. Clone identification number 1401018) were obtained from American Type Culture Collection (ATCC). Expression plasmids encoding mouse Nrf2 (pEF/Nrf2), p18 (pEF/p18), and the mutant p18 (pEF/p18M) (19Kotkow K.J. Orkin S.H. Mol. Cell. Biol. 1995; 15: 4640-4647Crossref PubMed Scopus (148) Google Scholar) were kindly provided by Dr. Stuart Orkin. Mouse and rat ATF4 cDNAs were cloned into pEF/myc/mito and pcDNA3.1/myc-his (Invitrogen), respectively, to generate pEF/mATF4 and pCMV/rATF4. A dominant mutant of mouse ATF4 was generated by overlap extension using PCR resulting in a protein with 6 amino acid substitutions within the DNA-binding domain (292RYRQKKR298 to292GYLEAAA298). The amplification product was cloned into pEF/myc/mito to generate the plasmid (pEF/mATF4M). The dominant mutant of Jun D (pCMV/JunDM) was constructed by cloning the 591-base pair BssHII/BssHII (blunt-ended) fragment of the mouse Jun D cDNA into the vector pCMV-Tag2B (Stratagene). This manipulation deletes amino acid residues 1–169 resulting in a trans-activation domain-deficient mutant of Jun D similar to one described earlier (20Hirai S.I. Ryseck R.-P. Mechta F. Bravo R. Yaniv M. EMBO J. 1989; 8: 1433-1439Crossref PubMed Scopus (392) Google Scholar). Dominant mutants of c-Jun and Nrf2 have been described previously (4Alam J. Stewart D. Touchard C. Boinapally S. Choi A.M.K. Cook J.L. J. Biol. Chem. 1999; 274: 26071-26078Abstract Full Text Full Text PDF PubMed Scopus (1073) Google Scholar). The “bait” plasmid, pDBLeu-Nrf2, for Y2H was constructed in the following manner. The mouse Nrf2 cDNA sequence encoding amino acid residues 393–581 (numbering as in Ref. 21Chui D.H.K. Tang W. Orkin S.H. Biochem. Biophys. Res. Commun. 1995; 209: 40-46Crossref PubMed Scopus (61) Google Scholar) was amplified by PCR using the primer pair Nrf2-1, 5′-CACGCGTCGACTATGCGTGAATCCCAATG-3′, and Nrf2-2, 5′-TCCTCCGGATATCAGTTTTTCTTTGTAT-3′. The amplified product was digested with SalI and EcoRV restriction endonucleases (recognition sites underlined) and cloned between theSalI and StuI sites of the pDBLeu vector (Life Technologies, Inc.) in-frame with the Gal4 DNA-binding domain (Gdbd). The integrity of the mouse Nrf2 cDNA and production of the fusion protein was confirmed by DNA sequencing and Western blotting, respectively. The mammalian Gdbd vector, pEG, was constructed by cloning the Gdbd (residues 1–147) into pEF/myc/mito. Mouse Nrf2 sequences (aa residues 13–581 or 314–581) were subsequently cloned downstream of, and in-frame with, the Gdbd to generate pEG/Nrf2 plasmids. The “activation domain” vector (pAD) was constructed by cloning an 870-base pairBglII/HindIII (blunt-ended) fragment of mouse Nrf2 (aa 13–302) into pCMV-Tag2B. Full-length ATF3, p18 and rat ATF4, sequences were subsequently cloned into pAD in-frame with the Nrf2 sequence. pFRluc, containing 5 tandem copies of the Gal4-binding site, was obtained from Stratagene. The construction of pE1-luc, containing the mouse ho-1 gene distal enhancer 1, and pStREluc, containing three copies of the mouseho-1 StRE3, has been described previously (4Alam J. Stewart D. Touchard C. Boinapally S. Choi A.M.K. Cook J.L. J. Biol. Chem. 1999; 274: 26071-26078Abstract Full Text Full Text PDF PubMed Scopus (1073) Google Scholar, 22Inamdar N.M. Ahn Y.I. Alam J. Biochem. Biophys. Res. Commun. 1996; 221: 570-576Crossref PubMed Scopus (177) Google Scholar). Plasmid pCMV/β-gal, encoding the Escherichia coliβ-galactosidase gene, was kindly provided by Dr. Ping Wei. Screening was carried out using the Y2H system from Life Technologies, Inc. Briefly, plasmid pDBLeu-Nrf2 was introduced into yeast strain MaV203 (MATα, leu2-3, 112,trp1-901, his3Δ200, ade2-101,gal4Δ, gal80Δ,SPAL10::URA3,GAL1::lacZ,HIS UAS GAL1 ::HIS[email protected]LYS2,can1R, cyh2R), and transformants were selected and purified on medium lacking leucine. Subsequently, DNA representing rat brain or liver cDNAs, cloned into the Gal4 activation domain vector pPC86, was transformed into MaV203/pDBLeu-Nrf2 strain. Transformants were selected on medium containing 25 mm 3-amino-1,2,4-triazole but lacking tryptophan, leucine, uracil, and histidine. Positive colonies were assayed for activation of the lacZ reporter gene. Plasmids isolated from the positive colonies were rescued in E. coliHD10B and re-assayed for interaction activity by transformation into the MaV203/pDBLeu-Nrf2 strain and growth on selection media. The 5′ end of the rat ATF4 cDNA was isolated by PCR amplification (5′-rapid amplification from cDNA ends) from a rat brain Marathon-Ready cDNA mixture (CLONTECH) according to the manufacturer's recommendation using two different gene-specific primers ATF4-1 (5′-TAGGACTCAGGGCTCATACAGATGCCA-3′) and ATF4-2 (5′- TTGAAGTGCTTGGCCACCTCCAGATAG-3′) and the adaptor primer provided in the kit. Both amplification products were purified and cloned into the pT-Adv vector (CLONTECH Laboratories Inc). Automated DNA sequence analysis was carried out by the Howard hughes Medical Institute Biopolymer/W. M. Keck Foundation Biotechnology Resource Laboratory at Yale University. Identical 5′ sequences were obtained from clones derived from both amplification products. COS-7 (African green monkey kidney), Hepa (mouse hepatoma), and MCF-7 (human mammary epithelial) cells were cultured in Dulbecco's modified Eagle's medium, whereas HeLa (human cervical carcinoma) cells were cultured in Eagle's minimal essential medium. All media were supplemented with 0.45% glucose, 10% fetal bovine serum, 50 μg/ml gentamicin sulfate, and 10 ng/ml insulin (MCF-7 only). Transient transfection of luciferase constructs was carried out by the calcium phosphate precipitation technique as described previously (23Alam J. J. Biol. Chem. 1994; 269: 25049-25056Abstract Full Text PDF PubMed Google Scholar) or with Fugene 6 transfection reagent (Roche Molecular Biochemicals) according to the manufacturer's recommendation. Additional details are provided in the figure legends. Transfection efficiency was monitored by co-transfection with pCMV/β-gal. Preparation of cell extract and measurement of luciferase activity were carried out as described previously (24Alam J. Zhining D. J. Biol. Chem. 1992; 267: 21894-21900Abstract Full Text PDF PubMed Google Scholar). β-Galactosidase activity was measured using the Galacto-Light (Tropix, Inc.) chemiluminescent assay kit according to the manufacturer's protocol. Full-length (p18, Fos B, and ATF3) or nearly full-length (mATF4, residues 31–349) coding regions were cloned downstream of, and in-frame with, the hexa-histidine tag in the T7 RNA polymerase-based prokaryotic expression vector series pET30a–c (Novagen Inc.). Recombinant proteins were purified from inclusion bodies by nickel affinity chromatography as per the manufacturer's protocol or according to the protocol of Holzinger et al. (25Holzinger A. Phillips K.S. Weaver T.E. BioTechniques. 1996; 20: 804-808Crossref PubMed Scopus (75) Google Scholar). Nrf2M protein (residues 393–581), containing the DNA binding and leucine zipper dimerization domains, was synthesized by coupledin vitro transcription and translation reaction as described previously (26Alam J. Wicks C. Stewart D. Gong P. Touchard C. Otterbein S. Choi A.M.K. Burrow M.E. Tou J.-S. J. Biol. Chem. 2000; 275: 27694-27702Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). EMSA was carried out as described previously (23Alam J. J. Biol. Chem. 1994; 269: 25049-25056Abstract Full Text PDF PubMed Google Scholar) using a double-stranded oligonucleotide containing the sequence 5′-TTTTCTGCTGAGTCAAGGTCCG-3′ (core StRE underlined) as probe. Five μl of Nrf2M synthesis product and/or 100 ng of recombinant protein were used in EMSA reactions. COS-7 cells were transfected 24 h after plating (1 × 106/100-mm plate) with a total of 5 μg of either empty vector (pEF/myc/mito), pEF/mATF4, pEF/Nrf2, or a combination of these plasmids using Lipofectin transfection reagent (Life Technologies, Inc.) as specified by the manufacturer. Cells were harvested 36 h after transfection and resuspended in 200 μl of lysis buffer (10 mm Tris-HCl (pH 7.5) containing 0.5% (v/v) Nonidet P-40, 150 mm NaCl, and 1 mmEDTA). Cell lysates were cleared by centrifugation, and immunoprecipitation was carried out with 100 μg of cell lysate using protein G-agarose beads as described (27Yang H. Jiang D. Li W. Liang J. Gentry L.E. Brattain M.G. Oncogene. 2000; 19: 1901-1914Crossref PubMed Scopus (31) Google Scholar). Immune complexes were eluted from the beads with 2× SDS-PAGE sample buffer and subjected to denaturing polyacrylamide gel electrophoresis. Western blotting was carried out as described previously (4Alam J. Stewart D. Touchard C. Boinapally S. Choi A.M.K. Cook J.L. J. Biol. Chem. 1999; 274: 26071-26078Abstract Full Text Full Text PDF PubMed Scopus (1073) Google Scholar). All antibodies were used at dilutions recommended by the respective suppliers. To identify proteins that interact with Nrf2, a cDNA fragment encoding the C-terminal portion of mouse Nrf2 was amplified by PCR and cloned in-frame and downstream of the Gdbd. The resulting fusion protein was used as “bait” in a yeast two-hybrid screening assay as described under “Experimental Procedures.” From a total of 2 × 106 yeast transformants, harboring either rat brain or liver cDNA/Gal4 activation domain fusions, seven independent clones (3 liver and 4 brain) that encoded Nrf2-interacting polypeptides were identified after a series of selection methods. The nucleotide sequences of the inserts within the positive clones were determined, and the results indicated that five of the cDNAs were derived from the same mRNA. A sequence similarity search using “blastn” revealed significant similarities to sequences encoding mouse ATF4 and human CREB2 (ATF4) (28Hai T. Liu F. Coukos W.J. Green M.R. Genes Dev. 1989; 3: 2083-2090Crossref PubMed Scopus (760) Google Scholar, 29Karpinski B.A. Morle G.D. Huggenvik J. Uhler M.D. Leiden J.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4820-4824Crossref PubMed Scopus (211) Google Scholar) suggesting that these clones encoded the rat homolog of ATF4. Since none of the isolates contained the initiation codon, the full-length ATF4 cDNA was obtained by PCR amplification from a rat brain cDNA library, cloned, and subjected to DNA sequence analysis. The deduced amino acid sequence of rat ATF4 is aligned with those of mouse and human ATF4 in Fig. 1. Between these three species, ATF4 exhibits 84.4% sequence conservation and the rat protein displays 94.8 and 87.2% sequence identity to mouse and human ATF4 s, respectively. As expected, the highest degree of conservation is observed within the basic region (DNA binding) and the adjacent leucine zipper (dimerization) domains. The second “leucine zipper” region, to which a function has yet to be assigned, exhibits greater divergence although the repeating leucine (or corresponding) residues at every 7th position are completely conserved in the three proteins. Association between Nrf2 and ATF4 in mammalian cells was confirmed by co-immunoprecipitation experiments and mammalian two-hybrid assays. For the former, expression plasmids encoding ATF4 or Nrf2 were transfected, individually or in combination, into COS-7 cells; the cells were lysed, and the lysates subjected to immunoprecipitation with anti-ATF4 or anti-Nrf2 antibodies in the presence or absence of the corresponding blocking peptide. Immunoprecipitates were subsequently analyzed by Western blotting. As shown in Fig. 2, Nrf2 was not detected in lysates of cells transfected with an empty vector (lane 3) or the ATF4 expression plasmid (lane 6), but was readily observed in lysates of cells transfected with both ATF4 and Nrf2 expression plasmids (lane 4). More importantly, Nrf2 could be immunoprecipitated with anti-ATF4 antibodies (lane 2), albeit to a lesser extent than that observed with anti-Nrf2 antibodies (lane 1). Immunoprecipitation of Nrf2 by both antibodies was abrogated in the presence of the corresponding blocking peptides (lanes 5and 7). The results from co-immunoprecipitation experiments were corroborated by mammalian two-hybrid assays. In these experiments, nearly full-length mouse Nrf2 (aa 13–581; Fig.3 A) or the C-terminal portion of Nrf2 (aa 314–581; Fig. 3 B) was fused to the Gdbd, and these fusions served as interaction targets. Sequences encoding test proteins were fused in-frame to an N-terminal region of Nrf2 (amino acids 13–302) that contains a potent transcription activation domain (AD). Gdbd-Nrf2-(13–581) stronglytrans-activated a luciferase reporter gene under the control of Gal4-binding sites, pFRluc. Co-expression of Nrf2 AD further increased luciferase activity by 4–5-fold suggesting self-interaction between Nrf2 proteins. AD fusions containing full-length rat ATF4 or mouse ATF3 exhibited even greater trans-activation capabilities, ∼35-fold above control and 7-fold above AD alone. The MafK (p18) fusion served as a positive control and exhibited the highest interaction activity. Gdbd-Nrf2-(314–581) contains the DNA interaction and dimerization (i.e. bZIP) domains but is transcriptionally inactive. Co-expression of AD did not stimulate luciferase activity suggesting that Nrf2 self-interaction is limited to the N-terminal portion of Nrf2. ATF3, ATF4, and p18 interacted with Gdbd-Nrf2-(314–581) with the following rank order: p18 ≫ ATF4 ≫ ATF3. Presumably these interactions reflect dimerization between leucine zipper domains. Relative to p18, ATF4 exhibits greater association with Gdbd-Nrf2-(13–581) than with Gdbd-Nrf2 (314) (∼40% versus 5%), even though the latter is produced at higher levels intracellularly (data not shown). To understand the consequence of ATF4/Nrf2 interaction on gene regulation, we initially examined the binding of ATF4 and Nrf2, individually or as a mixture, to the StRE, a cis-acting element known to regulate inducer-mediated ho-1 gene activation in an Nrf2-dependent manner (4Alam J. Stewart D. Touchard C. Boinapally S. Choi A.M.K. Cook J.L. J. Biol. Chem. 1999; 274: 26071-26078Abstract Full Text Full Text PDF PubMed Scopus (1073) Google Scholar, 22Inamdar N.M. Ahn Y.I. Alam J. Biochem. Biophys. Res. Commun. 1996; 221: 570-576Crossref PubMed Scopus (177) Google Scholar, 26Alam J. Wicks C. Stewart D. Gong P. Touchard C. Otterbein S. Choi A.M.K. Burrow M.E. Tou J.-S. J. Biol. Chem. 2000; 275: 27694-27702Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). As expected, Nrf2 did not bind to the StRE (Fig.4). ATF4 alone also did not bind to the StRE but, in the presence of Nrf2, exhibited significant binding. Presumably, this DNA-protein interaction reflects the activity of ATF4·Nrf2 dimers. p18, which can form homodimers and highly stable heterodimers with Nrf2 was used as a positive control for protein dimerization and DNA binding. p18 homodimers, which are known to interact with MARE and NF-E2 binding sites, also bound to the StRE, but the strongest binding was observed with p18·Nrf2 heterodimers. The relative affinities (and/or stability) of ATF4·Nrf2 dimers and p18·Nrf2 dimers for the StRE correlated with the relative affinities of protein-protein interactions observed in the mammalian two-hybrid assays. FosB, which cannot form homodimers and would not be expected to dimerize with Nrf2, served as a negative control and did not exhibit specific binding in the absence or presence of Nrf2. Unlike ATF4, recombinant ATF3, presumably ATF3 homodimers, bound weakly to the StRE, but the binding was decreased in the presence of Nrf2. Binding of ATF4·Nrf2 dimers to the StRE suggested a role for ATF4 inho-1 gene regulation. This potential function was investigated further by examining the ability of ATF4 totrans-activate the ho-1 enhancer, E1, in the reporter construct pE1-luc. In Hepa cells, co-transfection of an ATF4 expression plasmid, up to the maximum level tested, decreased basal pE1-luc expression by 20–25% (Fig. 5). ATF4, however, had a synergistic effect on Nrf2-dependent pE1-luc expression, increasing luciferase activity up to 2-fold. In contrast, co-expression of p18 dramatically inhibited Nrf2-mediated trans-activation of E1. This inhibition may, at least in part, be attributed to p18 homodimers as overexpression of p18 alone also inhibited basal pE1-luc expression. Previous studies from our laboratory (4Alam J. Stewart D. Touchard C. Boinapally S. Choi A.M.K. Cook J.L. J. Biol. Chem. 1999; 274: 26071-26078Abstract Full Text Full Text PDF PubMed Scopus (1073) Google Scholar, 26Alam J. Wicks C. Stewart D. Gong P. Touchard C. Otterbein S. Choi A.M.K. Burrow M.E. Tou J.-S. J. Biol. Chem. 2000; 275: 27694-27702Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar) and other laboratories (7Ishii T. Itoh K. Takahashi S. Sato H. Yanagawa T. Katoh Y. Bannai S. Yamamoto M. J. Biol. Chem. 2000; 275: 16023-16029Abstract Full Text Full Text PDF PubMed Scopus (1245) Google Scholar) have demonstrated the requirement for Nrf2 in inducer-dependentho-1 gene regulation. To determine the role, if any, of ATF4 in this process, we first examined the effect of cadmium, a known HO-1 stimulant, on ATF4 expression as earlier reports had suggested that ATF4 is a stress-response protein (30Estes S.D. Stoler D.L. Anderson G.R. Exp. Cell. Res. 1995; 220: 47-54Crossref PubMed Scopus (48) Google Scholar, 31Fawcett, T. W., Martindale, J. L., Guyton, K. Z., Hai, T., and Holbr