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 were purchased from Nrf2 was by M. other transcription factors, including Nrf2, and HO-1 were obtained from and other were for mouse mouse Fos identification and mouse ATF4 identification were obtained from encoding mouse Nrf2 p18 and the mutant p18 K.J. Orkin S.H. Mol. Biol. 15: PubMed Scopus Google Scholar) were by and ATF4 were and to and A dominant mutant of mouse ATF4 was by using in a protein with amino acid within the domain The was to the The dominant mutant of was by the of the mouse the amino acid in a mutant of similar to one R. M. J. PubMed Scopus Google Scholar). of and Nrf2 have been 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 for was in the The mouse Nrf2 sequence encoding amino acid as in Orkin S.H. Biochem. PubMed Scopus Google Scholar) was by using the and The was with and endonucleases and between and of the Technologies, with the Gal4 domain The of the mouse Nrf2 and production of the fusion protein was confirmed by DNA and The mammalian was by the Nrf2 or were of, and the to The was by an of mouse Nrf2 p18 and were with the Nrf2 containing of the was obtained from The of containing the mouse ho-1 gene and containing of the been 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, Alam J. Biochem. 1996; PubMed Scopus Google Scholar). encoding the gene, was by was using the from Life Technologies, Inc. was yeast and were and DNA or the Gal4 activation domain was were containing but and were for activation of the gene. from the positive were in and for by the and The of the ATF4 was by from from a to the using two and and the in the were and the DNA sequence was by the M. at were obtained from from both and MCF-7 mammary cells were in cells were in media were with fetal bovine and of was by the as J. J. Biol. Chem. Full Text PDF PubMed Google Scholar) or with to the are in the was by with of cell and of were as J. D. J. Biol. Chem. Full Text PDF PubMed Google Scholar). was using the assay to the Fos and or were of, and the in the expression proteins were from by as the or to the of A. 1996; PubMed Scopus Google Scholar). protein containing the DNA binding and leucine zipper dimerization was synthesized by transcription and reaction as J. C. Stewart D. P. Touchard C. S. Choi A.M.K. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). was as J. J. Biol. Chem. Full Text PDF PubMed Google Scholar) using a containing the sequence StRE as of of protein were used in cells were with a of of either or a of these using Technologies, as by the were and in of containing and Cell were by and was with of cell using protein as H. D. J. Oncogene. 2000; PubMed Scopus Google Scholar). complexes were from the with and to was as 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). were used at by the identify proteins that with Nrf2, a encoding the of mouse Nrf2 was by and and of the The fusion protein was used as in a yeast two-hybrid screening assay as under a of 2 yeast either or activation domain and 4 that encoded Nrf2-interacting were identified a of The of the within the positive were and the results that of the were from the A sequence using significant to encoding mouse ATF4 and T. M.R. Genes Dev. PubMed Scopus Google Scholar, J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) that these encoded the of of the the the ATF4 was obtained by from a and to DNA sequence The amino acid sequence of ATF4 is with those of mouse and ATF4 in these ATF4 sequence and the protein and sequence to mouse and ATF4 the of is within the basic region and the leucine zipper The to which a function yet to the leucine at are in the Association between Nrf2 and ATF4 in mammalian cells was confirmed by co-immunoprecipitation and mammalian two-hybrid assays. the expression encoding ATF4 or Nrf2 were individually or in the cells were and the to with or in the presence or absence of the were by in Nrf2 was in of cells with an or the ATF4 expression but was in of cells with both ATF4 and Nrf2 expression Nrf2 with to a that with of Nrf2 by both was in the presence of the The results from co-immunoprecipitation were by mammalian two-hybrid assays. In these mouse Nrf2 or the of Nrf2 was to the and these as Sequences encoding proteins were to an region of Nrf2 that a potent transcription activation domain a gene under the of of Nrf2 increased by between Nrf2 containing ATF4 or mouse and The MafK fusion as a positive and the the DNA and dimerization but is of that Nrf2 is to the of Nrf2. and p18 with with the p18 ATF4 these dimerization between leucine zipper to ATF4 association with with the is at intracellularly the consequence of gene we the binding of ATF4 and Nrf2, individually or as a to the a element to regulate ho-1 gene activation in an manner 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, Alam J. Biochem. 1996; PubMed Scopus Google Scholar, J. C. Stewart D. P. Touchard C. S. Choi A.M.K. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). Nrf2 to the StRE ATF4 also to the StRE in the presence of Nrf2, significant this the of which form and with Nrf2 was used as a positive for protein dimerization and DNA p18 which are to with and NF-E2 binding also bound to the but the binding was with The of dimers and dimers for the StRE with the of in the mammalian two-hybrid assays. which form and would to with Nrf2, as a negative and specific binding in the absence or presence of Nrf2. bound to the but the binding was in the presence of Nrf2. of dimers to the StRE a role for ATF4 gene potential function was by the of ATF4 the ho-1 in the construct In cells, of an ATF4 expression to the basal expression by however, a to In of p18 inhibited of at in part, to p18 as of p18 also inhibited basal expression. studies from our 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, J. C. Stewart D. P. Touchard C. S. Choi A.M.K. 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Full Text PDF PubMed Google Scholar). the role of ATF4 in ho-1 gene we a dominant-negative mutant of ATF4 and expression. of the mutant ATF4 inhibited both basal and in a manner was and similar to that with an Nrf2 dominant an mutant of p18 inhibited but basal of or either or expression. have that is a potent activator of theho-1 gene in MCF-7 mammary epithelial cells, ho-1 by and that Nrf2 is an of this response J. C. Stewart D. P. Touchard C. S. Choi A.M.K. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). with our the Nrf2 mutant inhibited expression in the presence or absence of The p18 mutant also inhibited both basal and activities to similar The ATF4 however, inhibited only basal in the mechanism of ho-1 gene activation by and the role of ATF4 in this In this we have identified ATF4 as an Nrf2-interacting protein and have ATF4 in basal and regulation of the ho-1 gene, the in with Nrf2. association between Nrf2 and family been such an is as both of factors to the bZIP In this is that bZIP ATF4 ATF4 with and proteins under conditions and only with and form detectable at (17Hai T. Curran T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3720-3724Crossref PubMed Scopus (1119) Google Scholar). In ATF4 also with proteins M. D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: PubMed Scopus Google Scholar), a subfamily of site the or Fos within the bZIP is to that Nrf2 and ATF4 form bZIP dimers via leucine zipper in is this of that most elicit the of the basic necessary for sequence-specific DNA binding as is by the that the of Nrf2 containing the bZIP domain was used in the and the observations that ATF4 positive with a Nrf2 in and in the in the of ATF4 for Nrf2 protein and additional we the that ATF4 with additional within the Nrf2 In this is that very with the Nrf2 but is as as ATF4 in binding to the Nrf2 protein. the precise of of the Nrf2 or is The of zipper is also we that ATF4 is to associate with factors, including the protein of the H. Oncogene. PubMed Scopus Google Scholar, A. S. J. C. J. 1998; PubMed Google Scholar) and the acid A. A. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). in both the site within ATF4 was to the bZIP domain and, in the case of the association leads to for the similar to that for Nrf2. The ho-1 gene is by a of including the and of the mouse gene by most is by two and containing The are and necessary for gene activation (reviewed in A.M.K. Alam J. Am. J. Respir. Cell Mol. Biol. 1996; 15: 9-19Crossref PubMed Scopus (1020) Google Scholar). 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, J. C. Stewart D. P. Touchard C. S. Choi A.M.K. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar) have implicated Nrf2 in the mechanism of ho-1 gene activation by several and in by expression of a dominant-negative mutant of Nrf2, but of ho-1 by in and MCF-7 cells. in MCF-7 cells, of the Nrf2 mutant induction of an gene by and of that are by Nrf2 are also to of ATF4 as an Nrf2-interacting protein to that possibly in with Nrf2, gene expression. The the inhibitory of the ATF4 dominant-negative mutant basal and in cells for this is to that studies of this by the role of a specific transcription factor in gene of the of bZIP proteins to with Consequently, of gene activation with a specific dominant mutant only to the protein of but also to of dimerization one or of which may the under such is to for the role of a factor in gene such includes the observations that dimers to the that ATF4 expression is by and that ATF4 a Nrf2 of The in ATF4 from which an inhibitory similar to that of MafG of the (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, S. Jaiswal A.K. J. Biol. Chem. 2000; 275: 40134-40141Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). In the case of Nrf2, a role in ho-1 gene regulation been with the of K. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar) and cells 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). A similar for ATF4 and p18 would of very that ATF4 in part regulates ho-1 gene expression is consistent with that indicate ATF4 is a stress-response protein and, would function as a of the adaptive response to such stress. example, which HO-1 the expression and DNA binding of ATF4 in PubMed Scopus Google Scholar). another HO-1 also ATF4 DNA binding in cells T. J. K. and N. J. Biochem. J. Scholar). In ATF4 are in K. H. T. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, P.A. S. J. 1999; PubMed Google Scholar) and A. H. T. Sato T. K. J. 1991; PubMed Google Scholar) cells in response to and the two to stress. ATF4 expression is increased in cell to 2000; Google Scholar). The between and MCF-7 cells with to the of the mutant to is but to cell-specific in the induction or between ATF4 and Nrf2 requires an additional which may in a cell-specific is similar to one by and Jaiswal (16Venugopal R. Jaiswal A.K. Oncogene. 1998; 17: 3145-3156Crossref PubMed Scopus (487) Google Scholar) have proposed that of complexes is one or uncharacterized cytoplasmic consequence of this is that a role for complexes in ho-1 gene activation in MCF-7 cells and, our studies requires the of other Nrf2-containing Given the for bZIP proteins to form and distinct this is are under to the role of with to cell and inducer in the regulation of ho-1 and other Nrf2 target for in of the