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Protein-Protein Interactions Are Implied in Glucocorticoid Receptor Mutant 465*-mediated Cell Death

1997; Elsevier BV; Volume: 272; Issue: 41 Linguagem: Inglês

10.1074/jbc.272.41.25873

1083-351X

Hong Chen, Ganesan Srinivasan, E. Brad Thompson,

NF-κB Signaling Pathways

Previously we have shown that ICR-27, a clone of glucocorticoid-resistant human leukemic T cells, showed rapid cell loss upon transient transfection with plasmids expressing certain fragments of the human glucocorticoid receptor lacking the ligand binding domain. An extreme example was the frameshift GR mutant 465*, mutated after amino acid 465. This generated a novel 21-amino acid “tail,” beginning within the second zinc finger of the human glucocorticoid receptor DNA binding domain, a region required for ICR-27 cell death caused by hologlucocorticoid receptor plus hormone. The cell loss mediated by 465* was faster but quantitatively equivalent to that caused by hologlucocorticoid receptor plus hormone. We are therefore investigating the mechanism of action of 465*. We overexpressed 465* with or without a glutathione S-transferase tag fused to its N terminus and tested its ability to affect glucocorticoid response element (GRE)-driven reactions in vitro. Partially purified 465* showed little binding to a consensus GRE, caused virtually no stimulation of transcription from a GRE, and failed to inhibit GR-driven transcription. However, GST-465* “trapped” several proteins from ICR-27 cell extracts, including c-Jun; recombinant c-Jun also was bound in vitro. In co-transfection assays of CV-1 cells, 465* expression reduced phorbol ester (12-O-tetradecanoylphorbol-13-acetate) transcriptional activation from a promoter containing multiple AP-1 sites. Further studies proved the repressive activity of 465* was c-Jun-specific and not due to squelching artifacts. The data suggest that interaction of 465* with other proteins, such as c-Jun, might be responsible for its cell killing function. Previously we have shown that ICR-27, a clone of glucocorticoid-resistant human leukemic T cells, showed rapid cell loss upon transient transfection with plasmids expressing certain fragments of the human glucocorticoid receptor lacking the ligand binding domain. An extreme example was the frameshift GR mutant 465*, mutated after amino acid 465. This generated a novel 21-amino acid “tail,” beginning within the second zinc finger of the human glucocorticoid receptor DNA binding domain, a region required for ICR-27 cell death caused by hologlucocorticoid receptor plus hormone. The cell loss mediated by 465* was faster but quantitatively equivalent to that caused by hologlucocorticoid receptor plus hormone. We are therefore investigating the mechanism of action of 465*. We overexpressed 465* with or without a glutathione S-transferase tag fused to its N terminus and tested its ability to affect glucocorticoid response element (GRE)-driven reactions in vitro. Partially purified 465* showed little binding to a consensus GRE, caused virtually no stimulation of transcription from a GRE, and failed to inhibit GR-driven transcription. However, GST-465* “trapped” several proteins from ICR-27 cell extracts, including c-Jun; recombinant c-Jun also was bound in vitro. In co-transfection assays of CV-1 cells, 465* expression reduced phorbol ester (12-O-tetradecanoylphorbol-13-acetate) transcriptional activation from a promoter containing multiple AP-1 sites. Further studies proved the repressive activity of 465* was c-Jun-specific and not due to squelching artifacts. The data suggest that interaction of 465* with other proteins, such as c-Jun, might be responsible for its cell killing function. Glucocorticoids exert their effects on target cells by binding to an intracellular GR 1The abbreviations used are: GR, glucocorticoid receptor; hGR, human GR; GRE, glucocorticoid response element; DBD, DNA binding domain; LBD, ligand binding domain; GST, glutathioneS-transferase; TPA, 12-O-tetradecanoylphorbol-13-acetate; MMTV, mouse mammary tumor virus; LTR, long terminal repeat; ER, estrogen receptor; hER, human ER; ERE, estrogen response element; Dex, dexamethasone; TRE, TPA response element; CAT, chloramphenicol acetyltransferase; GMSA, gel mobility shift assay; PAGE, polyacrylamide gel electrophoresis; RSV, Rous sarcoma virus; PBS, phosphate-buffered saline. 1The abbreviations used are: GR, glucocorticoid receptor; hGR, human GR; GRE, glucocorticoid response element; DBD, DNA binding domain; LBD, ligand binding domain; GST, glutathioneS-transferase; TPA, 12-O-tetradecanoylphorbol-13-acetate; MMTV, mouse mammary tumor virus; LTR, long terminal repeat; ER, estrogen receptor; hER, human ER; ERE, estrogen response element; Dex, dexamethasone; TRE, TPA response element; CAT, chloramphenicol acetyltransferase; GMSA, gel mobility shift assay; PAGE, polyacrylamide gel electrophoresis; RSV, Rous sarcoma virus; PBS, phosphate-buffered saline. (1Yamamoto K.R. Annu. Rev. Genet. 1985; 19: 209-252Crossref PubMed Scopus (1329) Google Scholar). The GR is required for glucocorticoid-evoked apoptosis of lymphoid cells (2Cohen J.J. Duke R.C. Annu. Rev. Immunol. 1992; 10: 267-293Crossref PubMed Scopus (1102) Google Scholar, 3Thompson E.B. Mol. Endocrinol. 1994; 8: 665-673Crossref PubMed Scopus (155) Google Scholar). Biochemical, immunological, and genetic analyses of the GR imply that the protein folds into three distinct functional domains and several subdomains (4Giguere V. Hollenberg S.M. Rosenfeld M.G. Evans R.M. Cell. 1986; 46: 645-652Abstract Full Text PDF PubMed Scopus (674) Google Scholar, 5Evans R.M. Science. 1988; 240: 889-895Crossref PubMed Scopus (6293) Google Scholar). The N-terminal portion of the hGR, containing the major transactivation activity, is referred to as τ1 (amino acids 77–262; Refs. 4Giguere V. Hollenberg S.M. Rosenfeld M.G. Evans R.M. Cell. 1986; 46: 645-652Abstract Full Text PDF PubMed Scopus (674) Google Scholar and 6Hollenberg S.M. Giguere V. Segui P. Evans R.M. Cell. 1987; 49: 39-46Abstract Full Text PDF PubMed Scopus (332) Google Scholar). A central 66-residue DNA binding domain (DBD, amino acids 420–486) comprises two zinc finger motifs and is followed by a “hinge region.” The binding of two zinc atoms by two clusters of four cysteines helps stabilize a system of loops and helices responsible for site-specific DNA binding and homodimerization (4Giguere V. Hollenberg S.M. Rosenfeld M.G. Evans R.M. Cell. 1986; 46: 645-652Abstract Full Text PDF PubMed Scopus (674) Google Scholar, 7Freedman L.P. Luisi B.F. Korszun Z.R. Basavappa R. Sigler P.B. Yamamoto K.R. Nature. 1988; 334: 543-545Crossref PubMed Scopus (346) Google Scholar, 8Zilliaus J. Wright A.P.H. Carlstedt-Duke J. Gustafsson J.-A. Mol. Endocrinol. 1995; 9: 389-440PubMed Google Scholar). The steroid binding domain (amino acids 556–777) lies at the C terminus. This region overlaps another transactivation domain, τ2 (9Hollenberg S.M. Evans R.M. Cell. 1988; 55: 899-906Abstract Full Text PDF PubMed Scopus (545) Google Scholar, 10Warriar N. Yu C. Govindan M.V. J. Biol. Chem. 1994; 269: 29010-29015Abstract Full Text PDF PubMed Google Scholar, 11Webster N.J.G. Green S. Jin J.R. Chambon P. Cell. 1988; 54: 199-207Abstract Full Text PDF PubMed Scopus (441) Google Scholar, 12Lanz R.B. Rusconi S. Endocrinology. 1994; 135: 2183-2195Crossref PubMed Scopus (57) Google Scholar). In the course of transfection experiments aimed at mapping the regions of the hGR required for its lethal effect, we discovered that certain fragments of the receptor could reduce the number of viable test cells independently of added steroid (13Nazareth L.V. Harbour D.V. Thompson E.B. J. Biol. Chem. 1991; 266: 12976-12980Abstract Full Text PDF PubMed Google Scholar). All of these hGR mutants lacked a large portion of their C termini, such that the entire LBD was missing. The hinge region of the hGR was removed to varying degrees in these mutants with no effect on their relative potencies in our assay. These mutants had been shown to have little or no transactivation activity of a GRE-driven reporter system, tested in co-transfection assays (14Schule R. Rangarajan P. Kliewer S. Ransone C.J. Bolado J. Yang N. Verma I.M. Evans R.M. Cell. 1990; 62: 1217-1226Abstract Full Text PDF PubMed Scopus (1035) Google Scholar, 15Yang-yen H.F. Chambard J.C. Sun Y.L. Smeal T. Schmidt T.T. Drouin J. Karin M. Cell. 1990; 62: 1205-1215Abstract Full Text PDF PubMed Scopus (1314) Google Scholar). An extreme case was mutant 465*, the product of a frameshift mutation at amino acid 465, within the second zinc finger of DBD. This results in retention of the amino acids that define the more amino-terminal zinc binding sequence but loss of part of the second “zinc finger” sequence from the wild type hGR DBD. Nevertheless, in our assay system, 465* was fully as active in reducing cell numbers as other C-terminal truncated hGR mutants that contained the entire DBD along with varying amounts of the hinge region. Therefore, we have focused our attention on the cell loss caused by transfection of 465*, further exploring possible mechanisms involved in the cell death process. ICR-27 cells were used to investigate the lethal effect of 465*. ICR-27 is a clone of cells selected for resistance to a high dose dexamethasone (Dex) from the Dex-sensitive cell line CEM-C7 (16Norman M.R. Thompson E.B. Cancer Res. 1977; 37: 3785-3791PubMed Google Scholar). CEM-C7 cells contain two alleles for the hGR, one wild type and the other containing a point mutation (17Harmon J.M. Thompson E.B. Mol. Cell. Biol. 1981; 1: 512-521Crossref PubMed Scopus (93) Google Scholar). In ICR-27 cells, a deletion of the wild type gene has left only the ineffective mutant GR (17Harmon J.M. Thompson E.B. Mol. Cell. Biol. 1981; 1: 512-521Crossref PubMed Scopus (93) Google Scholar, 18Palmer L.A. Hukku B. Harmon J.M. Cancer Res. 1992; 52: 6612-6618PubMed Google Scholar, 19Ashraf J. Thompson E.B. Mol. Endocrinol. 1993; 7: 631-642Crossref PubMed Scopus (56) Google Scholar). Whole cell competitive ligand binding assays show few glucocorticoid binding sites in ICR-27 cells. However, transfection with an expression vector containing the whole coding sequence of the hGR gene restores glucocorticoid responsiveness to ICR-27 cells (13Nazareth L.V. Harbour D.V. Thompson E.B. J. Biol. Chem. 1991; 266: 12976-12980Abstract Full Text PDF PubMed Google Scholar,20Harbour D.V. Chambon P. Thompson E.B. J. Steroid Biochem. Mol. Biol. 1990; 35: 1-9Crossref Scopus (29) Google Scholar). When plasmids expressing 465* are introduced into ICR-27 cells, a rapid reduction in viable cell numbers is seen, which does not require added steroid.Our in vitro studies showed that 465* could not bind to a consensus GRE, nor could it cause the stimulation of transcription of a GRE-driven reporter. We therefore hypothesize that protein-protein interactions are involved in the lethal effects of 465* and other GR mutants lacking their LBDs. Our results suggest that such interactions, rather than direct binding of 465* to GRE, are likely to be important in the observed biological effects of the GR fragment. As an example, we show that the 465* fragment interacts with c-Jun and interferes with its site-specific regulation of transcription.DISCUSSIONWhen plasmids expressing fragments of the hGR that lack the LBD but retain the DBD are transiently transfected into any of several lymphoid cell lines, they cause cell loss (13Nazareth L.V. Harbour D.V. Thompson E.B. J. Biol. Chem. 1991; 266: 12976-12980Abstract Full Text PDF PubMed Google Scholar), which appears to be apoptotic. The most extreme example of these truncated hGR fragments is 465*, in which a frameshift mutation interrupts the second zinc-binding region of the hGR DBD, replaces the ensuing 21 amino acids with a novel sequence, and then terminates the protein. We surveyed the lethal effect of 465* in a series of glucocorticoid-resistant cell lines from T cell leukemias, B cell myelomas, and myeloid leukemias (39Nazareth L.V. Johnson B.H. Chen H. Thompson E.B. Leukemia. 1996; 10: 1789-1795PubMed Google Scholar). Transfection of 465* caused cell loss only in the T cell and B cell lines and not in the myeloid cell lines tested. This raises the possibility of therapeutic applications against glucocorticoid-resistant lymphoid malignancies for agents based on 465*. In the work presented here, we have pursued several possible mechanisms for the activity of 465*.The cell kill mediated by 465* occurs rapidly compared with that following parallel transfections of holo-GR and treatment with agonist hormone (13Nazareth L.V. Harbour D.V. Thompson E.B. J. Biol. Chem. 1991; 266: 12976-12980Abstract Full Text PDF PubMed Google Scholar). Loss of cells after 465* transfection starts 6–12 h later and usually peaks by 18–36 h. When holo-GR is transfected, no apoptosis occurs unless ligand is added, in which case cell loss begins about 24 h later and continues for 2–3 days, recapitulating the time course of apoptosis in glucocorticoid-sensitive CEM cells containing native hGR. The rapid cell death kinetics following 465* transfection suggested that the mechanism was unlikely to be through the classic GRE-driven glucocorticoid response pathway of these cells. However, in natural thymocytes, GR-steroid complexes evoke apoptosis much more swiftly (40Wyllie A.H. Nature. 1980; 284: 555-556Crossref PubMed Scopus (4138) Google Scholar), so it was incumbent to consider the classic GRE-driven pathways as a possible mechanism. Further studies showed that 465* peptide had little affinity for a consensus GRE, as assayed by GMSA. In addition, recombinant 465* peptide did not activate a GRE-driven transcription in the cell-free system, and even in excess, 465* peptide added to the in vitro transcription system did not alter the activation of the target gene caused by hGR alone. Furthermore, in CV-1 cells, 465* did not activate the MMTV-CAT, a GRE-dependent reporter gene construct, and did not interfere with holo-GR induction. All these data strongly suggested that the classic GRE-driven transcriptional activation was not involved in the death of cells caused by 465*. This led us to investigate the possibility that a repressive function of 465* might cause cell lysis.It has been shown that holo-GR interacts with various transcription factors and regulatory proteins (41Ray A. Prefontaine K.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 752-756Crossref PubMed Scopus (916) Google Scholar, 42Caldenhoven E. Liden J. Wissink S. Van de Stople A. Raaijmakers J. Koenderman L. Okret S. Gustafsson J.-A. Van der Saag P.T. Mol. Endocrinol. 1995; 9: 401-412Crossref PubMed Google Scholar, 43Scheinman R.I. Gualberto A. Jewell C.M. Cidlowski J.A. Baldwin A.S.J. Mol. Cell. Biol. 1995; 15: 943-953Crossref PubMed Google Scholar, 44Auphan N. Didonata J.A. Rosette C. Helmberg A. Karin M. Science. 1995; 270: 283-286Crossref PubMed Scopus (2143) Google Scholar, 45Kutoh E. Stromstedt P.-E. Poellinger L. Mol. Cell. Biol. 1992; 12: 4960-4969Crossref PubMed Scopus (89) Google Scholar, 46Guathier J.M. Bourachot B. Doucas V. Yaniv M. Moreau-Gachelin F. EMBO J. 1993; 12: 5089-5096Crossref PubMed Scopus (55) Google Scholar, 47Strahle U. Schmid W. Schutz G. EMBO J. 1988; 7: 3389-3395Crossref PubMed Scopus (298) Google Scholar, 48Imai E. Miller M. Otsuka-Murakami H. Renkawitz R. Nature. 1988; 332: 87-90Crossref PubMed Scopus (235) Google Scholar, 49Roux J. Pictet R. Grange T. DNA Cell Biol. 1995; 14: 385-396Crossref PubMed Scopus (111) Google Scholar) through imprecisely defined domains. The 465* peptide may recognize certain transcription factors in a novel way or factors that are normally recognized by the holo-GR. Of the transcription factors known to interact with the holo-GR, c-Jun has been implicated in GR-caused apoptosis, specifically in CEM cells (34Zhou F. Thompson E.B. Mol. Endocrinol. 1996; 10: 308-316Google Scholar). We therefore used c-Jun as a model system to test the hypothesis that the 465* peptide could interfere with the function of important cellular proteins. The results from several groups suggested that the GR DBD was indispensable for GR-c-Jun interaction (24Heck S. Kullmann M. Gast A. Ponta H. Rahmsdorf H.J. Herrlich P. Cato A.C.B. EMBO J. 1994; 13: 4087-4095Crossref PubMed Scopus (462) Google Scholar, 50Pearce D. Yamamoto K.R. Science. 1993; 259: 1161-1165Crossref PubMed Scopus (397) Google Scholar, 51Radler-Pohl A. Gebel S. Sachsenmaier C. Konig H. Kramer M. Oehler T. Streile M. Ponta H. Rapp U. Rahmsdorf H.J. Ann. N. Y. Acad. Sci. 1993; 684: 127-148Crossref PubMed Scopus (47) Google Scholar), and, in some cases at least, direct DNA binding by the GR was not necessary for the GR to repress AP-1 activity. Further study suggested that repression of AP-1 activity and transactivation functions of the GR were separable entities and that the repression could be a function of GR monomers (24Heck S. Kullmann M. Gast A. Ponta H. Rahmsdorf H.J. Herrlich P. Cato A.C.B. EMBO J. 1994; 13: 4087-4095Crossref PubMed Scopus (462) Google Scholar).Although 465* has lost part of the second zinc finger of the DBD, it retains the ability to interact with c-Jun in vitro and to inhibit AP-1 activity from an AP-1-dependent reporter gene. The data showed less inhibitory effect for a given amount of transfected expression vector carrying the 465* gene than for one carrying the holo-GR, but this result does not measure the amount of each expressed protein. To better compare the inhibitory potency between 465* and holo-GR, we examined the relative level of immunoreactive 465* or hGR found in equivalent whole cell extracts from CV-1 cells transfected with equal amount of each expression plasmid. The results demonstrated that approximately twice as much hGR protein as 465* had been expressed. Although 465* and hGR were expressed from the same promoter, the stability of these two proteins may not be the same. The lower amount of expressed 465* protein in CV-1 cells may explain the lesser inhibitory effect of transfection with pRSh465*. Nevertheless, our data clearly demonstrate that transfection of the 465* construct altered AP-1-dependent transcription. The interference was AP-1-specific, since increased expression of c-Jun counteracted the repressive effect caused by 465*. Also, 465* did not alter either GRE- or ERE-driven reporter gene activity. This indicated that 465* did not block the activity of general transcription machinery.The similar kinetics of cell death caused by 465* and several other GR mutants that lack the LBD but possess the DBD intact suggest that protein-protein interactions are more likely to be involved in the cell death process they evoke than is their direct binding to GRE sites in the genome. All of these mutants have little or no transactivation activity in co-transfection assays (14Schule R. Rangarajan P. Kliewer S. Ransone C.J. Bolado J. Yang N. Verma I.M. Evans R.M. Cell. 1990; 62: 1217-1226Abstract Full Text PDF PubMed Scopus (1035) Google Scholar, 15Yang-yen H.F. Chambard J.C. Sun Y.L. Smeal T. Schmidt T.T. Drouin J. Karin M. Cell. 1990; 62: 1205-1215Abstract Full Text PDF PubMed Scopus (1314) Google Scholar). We chose mutant 500* as an example of such mutants for comparison with 465*. Mutant 500* is as potent as 465* in affecting cell lysis, but 500* codes for a fully intact DBD. The 500* peptide bound a GRE, while 465* did not. On the other hand, by using the GST “trap” system, we found that both 465* and 500* selectively interacted with certain factors from the ICR-27 cells. It is possible that 465* and 500* interacted with the same proteins. We further demonstrated that both 465* and 500* interacted with recombinant c-Jun in vitro. By using c-Jun as a model system, we have shown that 465* not only physically interacted with c-Jun but also altered c-Jun's function. Thus, our data are consistent with the hypothesis that 465* and, by extension, other GR mutants lacking their LBDs, exert their lethal effect through binding to and altering the activity of certain transcription factors or other proteins essential for cell viability. For example, repression of potential “survival genes” could be mediated through transcriptional interference between 465* and transcription factors such as AP-1. Alternatively, 465* and similar constructs could directly interfere with some “vitality factors” or could activate lethal caspase cascades directly or indirectly (52Henkart P.A. Immunity. 1996; 4: 195-201Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar, 53Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4537) Google Scholar). These possibilities are being investigated.From the evidence described in this paper, we propose that protein-protein interactions rather than direct binding to GRE are important in the lethal effects of 465*. The observed biological cell kill by 465* could be mediated by interference with certain transcription factors or other regulatory proteins required for cell survival. Glucocorticoids exert their effects on target cells by binding to an intracellular GR 1The abbreviations used are: GR, glucocorticoid receptor; hGR, human GR; GRE, glucocorticoid response element; DBD, DNA binding domain; LBD, ligand binding domain; GST, glutathioneS-transferase; TPA, 12-O-tetradecanoylphorbol-13-acetate; MMTV, mouse mammary tumor virus; LTR, long terminal repeat; ER, estrogen receptor; hER, human ER; ERE, estrogen response element; Dex, dexamethasone; TRE, TPA response element; CAT, chloramphenicol acetyltransferase; GMSA, gel mobility shift assay; PAGE, polyacrylamide gel electrophoresis; RSV, Rous sarcoma virus; PBS, phosphate-buffered saline. 1The abbreviations used are: GR, glucocorticoid receptor; hGR, human GR; GRE, glucocorticoid response element; DBD, DNA binding domain; LBD, ligand binding domain; GST, glutathioneS-transferase; TPA, 12-O-tetradecanoylphorbol-13-acetate; MMTV, mouse mammary tumor virus; LTR, long terminal repeat; ER, estrogen receptor; hER, human ER; ERE, estrogen response element; Dex, dexamethasone; TRE, TPA response element; CAT, chloramphenicol acetyltransferase; GMSA, gel mobility shift assay; PAGE, polyacrylamide gel electrophoresis; RSV, Rous sarcoma virus; PBS, phosphate-buffered saline. (1Yamamoto K.R. Annu. Rev. Genet. 1985; 19: 209-252Crossref PubMed Scopus (1329) Google Scholar). The GR is required for glucocorticoid-evoked apoptosis of lymphoid cells (2Cohen J.J. Duke R.C. Annu. Rev. Immunol. 1992; 10: 267-293Crossref PubMed Scopus (1102) Google Scholar, 3Thompson E.B. Mol. Endocrinol. 1994; 8: 665-673Crossref PubMed Scopus (155) Google Scholar). Biochemical, immunological, and genetic analyses of the GR imply that the protein folds into three distinct functional domains and several subdomains (4Giguere V. Hollenberg S.M. Rosenfeld M.G. Evans R.M. Cell. 1986; 46: 645-652Abstract Full Text PDF PubMed Scopus (674) Google Scholar, 5Evans R.M. Science. 1988; 240: 889-895Crossref PubMed Scopus (6293) Google Scholar). The N-terminal portion of the hGR, containing the major transactivation activity, is referred to as τ1 (amino acids 77–262; Refs. 4Giguere V. Hollenberg S.M. Rosenfeld M.G. Evans R.M. Cell. 1986; 46: 645-652Abstract Full Text PDF PubMed Scopus (674) Google Scholar and 6Hollenberg S.M. Giguere V. Segui P. Evans R.M. Cell. 1987; 49: 39-46Abstract Full Text PDF PubMed Scopus (332) Google Scholar). A central 66-residue DNA binding domain (DBD, amino acids 420–486) comprises two zinc finger motifs and is followed by a “hinge region.” The binding of two zinc atoms by two clusters of four cysteines helps stabilize a system of loops and helices responsible for site-specific DNA binding and homodimerization (4Giguere V. Hollenberg S.M. Rosenfeld M.G. Evans R.M. Cell. 1986; 46: 645-652Abstract Full Text PDF PubMed Scopus (674) Google Scholar, 7Freedman L.P. Luisi B.F. Korszun Z.R. Basavappa R. Sigler P.B. Yamamoto K.R. Nature. 1988; 334: 543-545Crossref PubMed Scopus (346) Google Scholar, 8Zilliaus J. Wright A.P.H. Carlstedt-Duke J. Gustafsson J.-A. Mol. Endocrinol. 1995; 9: 389-440PubMed Google Scholar). The steroid binding domain (amino acids 556–777) lies at the C terminus. This region overlaps another transactivation domain, τ2 (9Hollenberg S.M. Evans R.M. Cell. 1988; 55: 899-906Abstract Full Text PDF PubMed Scopus (545) Google Scholar, 10Warriar N. Yu C. Govindan M.V. J. Biol. Chem. 1994; 269: 29010-29015Abstract Full Text PDF PubMed Google Scholar, 11Webster N.J.G. Green S. Jin J.R. Chambon P. Cell. 1988; 54: 199-207Abstract Full Text PDF PubMed Scopus (441) Google Scholar, 12Lanz R.B. Rusconi S. Endocrinology. 1994; 135: 2183-2195Crossref PubMed Scopus (57) Google Scholar). In the course of transfection experiments aimed at mapping the regions of the hGR required for its lethal effect, we discovered that certain fragments of the receptor could reduce the number of viable test cells independently of added steroid (13Nazareth L.V. Harbour D.V. Thompson E.B. J. Biol. Chem. 1991; 266: 12976-12980Abstract Full Text PDF PubMed Google Scholar). All of these hGR mutants lacked a large portion of their C termini, such that the entire LBD was missing. The hinge region of the hGR was removed to varying degrees in these mutants with no effect on their relative potencies in our assay. These mutants had been shown to have little or no transactivation activity of a GRE-driven reporter system, tested in co-transfection assays (14Schule R. Rangarajan P. Kliewer S. Ransone C.J. Bolado J. Yang N. Verma I.M. Evans R.M. Cell. 1990; 62: 1217-1226Abstract Full Text PDF PubMed Scopus (1035) Google Scholar, 15Yang-yen H.F. Chambard J.C. Sun Y.L. Smeal T. Schmidt T.T. Drouin J. Karin M. Cell. 1990; 62: 1205-1215Abstract Full Text PDF PubMed Scopus (1314) Google Scholar). An extreme case was mutant 465*, the product of a frameshift mutation at amino acid 465, within the second zinc finger of DBD. This results in retention of the amino acids that define the more amino-terminal zinc binding sequence but loss of part of the second “zinc finger” sequence from the wild type hGR DBD. Nevertheless, in our assay system, 465* was fully as active in reducing cell numbers as other C-terminal truncated hGR mutants that contained the entire DBD along with varying amounts of the hinge region. Therefore, we have focused our attention on the cell loss caused by transfection of 465*, further exploring possible mechanisms involved in the cell death process. ICR-27 cells were used to investigate the lethal effect of 465*. ICR-27 is a clone of cells selected for resistance to a high dose dexamethasone (Dex) from the Dex-sensitive cell line CEM-C7 (16Norman M.R. Thompson E.B. Cancer Res. 1977; 37: 3785-3791PubMed Google Scholar). CEM-C7 cells contain two alleles for the hGR, one wild type and the other containing a point mutation (17Harmon J.M. Thompson E.B. Mol. Cell. Biol. 1981; 1: 512-521Crossref PubMed Scopus (93) Google Scholar). In ICR-27 cells, a deletion of the wild type gene has left only the ineffective mutant GR (17Harmon J.M. Thompson E.B. Mol. Cell. Biol. 1981; 1: 512-521Crossref PubMed Scopus (93) Google Scholar, 18Palmer L.A. Hukku B. Harmon J.M. Cancer Res. 1992; 52: 6612-6618PubMed Google Scholar, 19Ashraf J. Thompson E.B. Mol. Endocrinol. 1993; 7: 631-642Crossref PubMed Scopus (56) Google Scholar). Whole cell competitive ligand binding assays show few glucocorticoid binding sites in ICR-27 cells. However, transfection with an expression vector containing the whole coding sequence of the hGR gene restores glucocorticoid responsiveness to ICR-27 cells (13Nazareth L.V. Harbour D.V. Thompson E.B. J. Biol. Chem. 1991; 266: 12976-12980Abstract Full Text PDF PubMed Google Scholar,20Harbour D.V. Chambon P. Thompson E.B. J. Steroid Biochem. Mol. Biol. 1990; 35: 1-9Crossref Scopus (29) Google Scholar). When plasmids expressing 465* are introduced into ICR-27 cells, a rapid reduction in viable cell numbers is seen, which does not require added steroid. Our in vitro studies showed that 465* could not bind to a consensus GRE, nor could it cause the stimulation of transcription of a GRE-driven reporter. We therefore hypothesize that protein-protein interactions are involved in the lethal effects of 465* and other GR mutants lacking their LBDs. Our results suggest that such interactions, rather than direct binding of 465* to GRE, are likely to be important in the observed biological effects of the GR fragment. As an example, we show that the 465* fragment interacts with c-Jun and interferes with its site-specific regulation of transcription. DISCUSSIONWhen plasmids expressing fragments of the hGR that lack the LBD but retain the DBD are transiently transfected into any of several lymphoid cell lines, they cause cell loss (13Nazareth L.V. Harbour D.V. Thompson E.B. J. Biol. Chem. 1991; 266: 12976-12980Abstract Full Text PDF PubMed Google Scholar), which appears to be apoptotic. The most extreme example of these truncated hGR fragments is 465*, in which a frameshift mutation interrupts the second zinc-binding region of the hGR DBD, replaces the ensuing 21 amino acids with a novel sequence, and then terminates the protein. We surveyed the lethal effect of 465* in a series of glucocorticoid-resistant cell lines from T cell leukemias, B cell myelomas, and myeloid leukemias (39Nazareth L.V. Johnson B.H. Chen H. Thompson E.B. Leukemia. 1996; 10: 1789-1795PubMed Google Scholar). Transfection of 465* caused cell loss only in the T cell and B cell lines and not in the myeloid cell lines tested. This raises the possibility of therapeutic applications against glucocorticoid-resistant lymphoid malignancies for agents based on 465*. In the work presented here, we have pursued several possible mechanisms for the activity of 465*.The cell kill mediated by 465* occurs rapidly compared with that following parallel transfections of holo-GR and treatment with agonist hormone (13Nazareth L.V. Harbour D.V. Thompson E.B. J. Biol. Chem. 1991; 266: 12976-12980Abstract Full Text PDF PubMed Google Scholar). Loss of cells after 465* transfection starts 6–12 h later and usually peaks by 18–36 h. When holo-GR is transfected, no apoptosis occurs unless ligand is added, in which case cell loss begins about 24 h later and continues for 2–3 days, recapitulating the time course of apoptosis in glucocorticoid-sensitive CEM cells containing native hGR. The rapid cell death kinetics following 465* transfection suggested that the mechanism was unlikely to be through the classic GRE-driven glucocorticoid response pathway of these cells. However, in natural thymocytes, GR-steroid complexes evoke apoptosis much more swiftly (40Wyllie A.H. Nature. 1980; 284: 555-556Crossref PubMed Scopus (4138) Google Scholar), so it was incumbent to consider the classic GRE-driven pathways as a possible mechanism. Further studies showed that 465* peptide had little affinity for a consensus GRE, as assayed by GMSA. In addition, recombinant 465* peptide did not activate a GRE-driven transcription in the cell-free system, and even in excess, 465* peptide added to the in vitro transcription system did not alter the activation of the target gene caused by hGR alone. Furthermore, in CV-1 cells, 465* did not activate the MMTV-CAT, a GRE-dependent reporter gene construct, and did not interfere with holo-GR induction. All these data strongly suggested that the classic GRE-driven transcriptional activation was not involved in the death of cells caused by 465*. This led us to investigate the possibility that a repressive function of 465* might cause cell lysis.It has been shown that holo-GR interacts with various transcription factors and regulatory proteins (41Ray A. Prefontaine K.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 752-756Crossref PubMed Scopus (916) Google Scholar, 42Caldenhoven E. Liden J. Wissink S. Van de Stople A. Raaijmakers J. Koenderman L. Okret S. Gustafsson J.-A. Van der Saag P.T. Mol. Endocrinol. 1995; 9: 401-412Crossref PubMed Google Scholar, 43Scheinman R.I. Gualberto A. Jewell C.M. Cidlowski J.A. Baldwin A.S.J. Mol. Cell. Biol. 1995; 15: 943-953Crossref PubMed Google Scholar, 44Auphan N. Didonata J.A. Rosette C. Helmberg A. Karin M. Science. 1995; 270: 283-286Crossref PubMed Scopus (2143) Google Scholar, 45Kutoh E. Stromstedt P.-E. Poellinger L. Mol. Cell. Biol. 1992; 12: 4960-4969Crossref PubMed Scopus (89) Google Scholar, 46Guathier J.M. Bourachot B. Doucas V. Yaniv M. Moreau-Gachelin F. EMBO J. 1993; 12: 5089-5096Crossref PubMed Scopus (55) Google Scholar, 47Strahle U. Schmid W. Schutz G. EMBO J. 1988; 7: 3389-3395Crossref PubMed Scopus (298) Google Scholar, 48Imai E. Miller M. Otsuka-Murakami H. Renkawitz R. Nature. 1988; 332: 87-90Crossref PubMed Scopus (235) Google Scholar, 49Roux J. Pictet R. Grange T. DNA Cell Biol. 1995; 14: 385-396Crossref PubMed Scopus (111) Google Scholar) through imprecisely defined domains. The 465* peptide may recognize certain transcription factors in a novel way or factors that are normally recognized by the holo-GR. Of the transcription factors known to interact with the holo-GR, c-Jun has been implicated in GR-caused apoptosis, specifically in CEM cells (34Zhou F. Thompson E.B. Mol. Endocrinol. 1996; 10: 308-316Google Scholar). We therefore used c-Jun as a model system to test the hypothesis that the 465* peptide could interfere with the function of important cellular proteins. The results from several groups suggested that the GR DBD was indispensable for GR-c-Jun interaction (24Heck S. Kullmann M. Gast A. Ponta H. Rahmsdorf H.J. Herrlich P. Cato A.C.B. EMBO J. 1994; 13: 4087-4095Crossref PubMed Scopus (462) Google Scholar, 50Pearce D. Yamamoto K.R. Science. 1993; 259: 1161-1165Crossref PubMed Scopus (397) Google Scholar, 51Radler-Pohl A. Gebel S. Sachsenmaier C. Konig H. Kramer M. Oehler T. Streile M. Ponta H. Rapp U. Rahmsdorf H.J. Ann. N. Y. Acad. Sci. 1993; 684: 127-148Crossref PubMed Scopus (47) Google Scholar), and, in some cases at least, direct DNA binding by the GR was not necessary for the GR to repress AP-1 activity. Further study suggested that repression of AP-1 activity and transactivation functions of the GR were separable entities and that the repression could be a function of GR monomers (24Heck S. Kullmann M. Gast A. Ponta H. Rahmsdorf H.J. Herrlich P. Cato A.C.B. EMBO J. 1994; 13: 4087-4095Crossref PubMed Scopus (462) Google Scholar).Although 465* has lost part of the second zinc finger of the DBD, it retains the ability to interact with c-Jun in vitro and to inhibit AP-1 activity from an AP-1-dependent reporter gene. The data showed less inhibitory effect for a given amount of transfected expression vector carrying the 465* gene than for one carrying the holo-GR, but this result does not measure the amount of each expressed protein. To better compare the inhibitory potency between 465* and holo-GR, we examined the relative level of immunoreactive 465* or hGR found in equivalent whole cell extracts from CV-1 cells transfected with equal amount of each expression plasmid. The results demonstrated that approximately twice as much hGR protein as 465* had been expressed. Although 465* and hGR were expressed from the same promoter, the stability of these two proteins may not be the same. The lower amount of expressed 465* protein in CV-1 cells may explain the lesser inhibitory effect of transfection with pRSh465*. Nevertheless, our data clearly demonstrate that transfection of the 465* construct altered AP-1-dependent transcription. The interference was AP-1-specific, since increased expression of c-Jun counteracted the repressive effect caused by 465*. Also, 465* did not alter either GRE- or ERE-driven reporter gene activity. This indicated that 465* did not block the activity of general transcription machinery.The similar kinetics of cell death caused by 465* and several other GR mutants that lack the LBD but possess the DBD intact suggest that protein-protein interactions are more likely to be involved in the cell death process they evoke than is their direct binding to GRE sites in the genome. All of these mutants have little or no transactivation activity in co-transfection assays (14Schule R. Rangarajan P. Kliewer S. Ransone C.J. Bolado J. Yang N. Verma I.M. Evans R.M. Cell. 1990; 62: 1217-1226Abstract Full Text PDF PubMed Scopus (1035) Google Scholar, 15Yang-yen H.F. Chambard J.C. Sun Y.L. Smeal T. Schmidt T.T. Drouin J. Karin M. Cell. 1990; 62: 1205-1215Abstract Full Text PDF PubMed Scopus (1314) Google Scholar). We chose mutant 500* as an example of such mutants for comparison with 465*. Mutant 500* is as potent as 465* in affecting cell lysis, but 500* codes for a fully intact DBD. The 500* peptide bound a GRE, while 465* did not. On the other hand, by using the GST “trap” system, we found that both 465* and 500* selectively interacted with certain factors from the ICR-27 cells. It is possible that 465* and 500* interacted with the same proteins. We further demonstrated that both 465* and 500* interacted with recombinant c-Jun in vitro. By using c-Jun as a model system, we have shown that 465* not only physically interacted with c-Jun but also altered c-Jun's function. Thus, our data are consistent with the hypothesis that 465* and, by extension, other GR mutants lacking their LBDs, exert their lethal effect through binding to and altering the activity of certain transcription factors or other proteins essential for cell viability. For example, repression of potential “survival genes” could be mediated through transcriptional interference between 465* and transcription factors such as AP-1. Alternatively, 465* and similar constructs could directly interfere with some “vitality factors” or could activate lethal caspase cascades directly or indirectly (52Henkart P.A. Immunity. 1996; 4: 195-201Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar, 53Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4537) Google Scholar). These possibilities are being investigated.From the evidence described in this paper, we propose that protein-protein interactions rather than direct binding to GRE are important in the lethal effects of 465*. The observed biological cell kill by 465* could be mediated by interference with certain transcription factors or other regulatory proteins required for cell survival. When plasmids expressing fragments of the hGR that lack the LBD but retain the DBD are transiently transfected into any of several lymphoid cell lines, they cause cell loss (13Nazareth L.V. Harbour D.V. Thompson E.B. J. Biol. Chem. 1991; 266: 12976-12980Abstract Full Text PDF PubMed Google Scholar), which appears to be apoptotic. The most extreme example of these truncated hGR fragments is 465*, in which a frameshift mutation interrupts the second zinc-binding region of the hGR DBD, replaces the ensuing 21 amino acids with a novel sequence, and then terminates the protein. We surveyed the lethal effect of 465* in a series of glucocorticoid-resistant cell lines from T cell leukemias, B cell myelomas, and myeloid leukemias (39Nazareth L.V. Johnson B.H. Chen H. Thompson E.B. Leukemia. 1996; 10: 1789-1795PubMed Google Scholar). Transfection of 465* caused cell loss only in the T cell and B cell lines and not in the myeloid cell lines tested. This raises the possibility of therapeutic applications against glucocorticoid-resistant lymphoid malignancies for agents based on 465*. In the work presented here, we have pursued several possible mechanisms for the activity of 465*. The cell kill mediated by 465* occurs rapidly compared with that following parallel transfections of holo-GR and treatment with agonist hormone (13Nazareth L.V. Harbour D.V. Thompson E.B. J. Biol. Chem. 1991; 266: 12976-12980Abstract Full Text PDF PubMed Google Scholar). Loss of cells after 465* transfection starts 6–12 h later and usually peaks by 18–36 h. When holo-GR is transfected, no apoptosis occurs unless ligand is added, in which case cell loss begins about 24 h later and continues for 2–3 days, recapitulating the time course of apoptosis in glucocorticoid-sensitive CEM cells containing native hGR. The rapid cell death kinetics following 465* transfection suggested that the mechanism was unlikely to be through the classic GRE-driven glucocorticoid response pathway of these cells. However, in natural thymocytes, GR-steroid complexes evoke apoptosis much more swiftly (40Wyllie A.H. Nature. 1980; 284: 555-556Crossref PubMed Scopus (4138) Google Scholar), so it was incumbent to consider the classic GRE-driven pathways as a possible mechanism. Further studies showed that 465* peptide had little affinity for a consensus GRE, as assayed by GMSA. In addition, recombinant 465* peptide did not activate a GRE-driven transcription in the cell-free system, and even in excess, 465* peptide added to the in vitro transcription system did not alter the activation of the target gene caused by hGR alone. Furthermore, in CV-1 cells, 465* did not activate the MMTV-CAT, a GRE-dependent reporter gene construct, and did not interfere with holo-GR induction. All these data strongly suggested that the classic GRE-driven transcriptional activation was not involved in the death of cells caused by 465*. This led us to investigate the possibility that a repressive function of 465* might cause cell lysis. It has been shown that holo-GR interacts with various transcription factors and regulatory proteins (41Ray A. Prefontaine K.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 752-756Crossref PubMed Scopus (916) Google Scholar, 42Caldenhoven E. Liden J. Wissink S. Van de Stople A. Raaijmakers J. Koenderman L. Okret S. Gustafsson J.-A. Van der Saag P.T. Mol. Endocrinol. 1995; 9: 401-412Crossref PubMed Google Scholar, 43Scheinman R.I. Gualberto A. Jewell C.M. Cidlowski J.A. Baldwin A.S.J. Mol. Cell. Biol. 1995; 15: 943-953Crossref PubMed Google Scholar, 44Auphan N. Didonata J.A. Rosette C. Helmberg A. Karin M. Science. 1995; 270: 283-286Crossref PubMed Scopus (2143) Google Scholar, 45Kutoh E. Stromstedt P.-E. Poellinger L. Mol. Cell. Biol. 1992; 12: 4960-4969Crossref PubMed Scopus (89) Google Scholar, 46Guathier J.M. Bourachot B. Doucas V. Yaniv M. Moreau-Gachelin F. EMBO J. 1993; 12: 5089-5096Crossref PubMed Scopus (55) Google Scholar, 47Strahle U. Schmid W. Schutz G. EMBO J. 1988; 7: 3389-3395Crossref PubMed Scopus (298) Google Scholar, 48Imai E. Miller M. Otsuka-Murakami H. Renkawitz R. Nature. 1988; 332: 87-90Crossref PubMed Scopus (235) Google Scholar, 49Roux J. Pictet R. Grange T. DNA Cell Biol. 1995; 14: 385-396Crossref PubMed Scopus (111) Google Scholar) through imprecisely defined domains. The 465* peptide may recognize certain transcription factors in a novel way or factors that are normally recognized by the holo-GR. Of the transcription factors known to interact with the holo-GR, c-Jun has been implicated in GR-caused apoptosis, specifically in CEM cells (34Zhou F. Thompson E.B. Mol. Endocrinol. 1996; 10: 308-316Google Scholar). We therefore used c-Jun as a model system to test the hypothesis that the 465* peptide could interfere with the function of important cellular proteins. The results from several groups suggested that the GR DBD was indispensable for GR-c-Jun interaction (24Heck S. Kullmann M. Gast A. Ponta H. Rahmsdorf H.J. Herrlich P. Cato A.C.B. EMBO J. 1994; 13: 4087-4095Crossref PubMed Scopus (462) Google Scholar, 50Pearce D. Yamamoto K.R. Science. 1993; 259: 1161-1165Crossref PubMed Scopus (397) Google Scholar, 51Radler-Pohl A. Gebel S. Sachsenmaier C. Konig H. Kramer M. Oehler T. Streile M. Ponta H. Rapp U. Rahmsdorf H.J. Ann. N. Y. Acad. Sci. 1993; 684: 127-148Crossref PubMed Scopus (47) Google Scholar), and, in some cases at least, direct DNA binding by the GR was not necessary for the GR to repress AP-1 activity. Further study suggested that repression of AP-1 activity and transactivation functions of the GR were separable entities and that the repression could be a function of GR monomers (24Heck S. Kullmann M. Gast A. Ponta H. Rahmsdorf H.J. Herrlich P. Cato A.C.B. EMBO J. 1994; 13: 4087-4095Crossref PubMed Scopus (462) Google Scholar). Although 465* has lost part of the second zinc finger of the DBD, it retains the ability to interact with c-Jun in vitro and to inhibit AP-1 activity from an AP-1-dependent reporter gene. The data showed less inhibitory effect for a given amount of transfected expression vector carrying the 465* gene than for one carrying the holo-GR, but this result does not measure the amount of each expressed protein. To better compare the inhibitory potency between 465* and holo-GR, we examined the relative level of immunoreactive 465* or hGR found in equivalent whole cell extracts from CV-1 cells transfected with equal amount of each expression plasmid. The results demonstrated that approximately twice as much hGR protein as 465* had been expressed. Although 465* and hGR were expressed from the same promoter, the stability of these two proteins may not be the same. The lower amount of expressed 465* protein in CV-1 cells may explain the lesser inhibitory effect of transfection with pRSh465*. Nevertheless, our data clearly demonstrate that transfection of the 465* construct altered AP-1-dependent transcription. The interference was AP-1-specific, since increased expression of c-Jun counteracted the repressive effect caused by 465*. Also, 465* did not alter either GRE- or ERE-driven reporter gene activity. This indicated that 465* did not block the activity of general transcription machinery. The similar kinetics of cell death caused by 465* and several other GR mutants that lack the LBD but possess the DBD intact suggest that protein-protein interactions are more likely to be involved in the cell death process they evoke than is their direct binding to GRE sites in the genome. All of these mutants have little or no transactivation activity in co-transfection assays (14Schule R. Rangarajan P. Kliewer S. Ransone C.J. Bolado J. Yang N. Verma I.M. Evans R.M. Cell. 1990; 62: 1217-1226Abstract Full Text PDF PubMed Scopus (1035) Google Scholar, 15Yang-yen H.F. Chambard J.C. Sun Y.L. Smeal T. Schmidt T.T. Drouin J. Karin M. Cell. 1990; 62: 1205-1215Abstract Full Text PDF PubMed Scopus (1314) Google Scholar). We chose mutant 500* as an example of such mutants for comparison with 465*. Mutant 500* is as potent as 465* in affecting cell lysis, but 500* codes for a fully intact DBD. The 500* peptide bound a GRE, while 465* did not. On the other hand, by using the GST “trap” system, we found that both 465* and 500* selectively interacted with certain factors from the ICR-27 cells. It is possible that 465* and 500* interacted with the same proteins. We further demonstrated that both 465* and 500* interacted with recombinant c-Jun in vitro. By using c-Jun as a model system, we have shown that 465* not only physically interacted with c-Jun but also altered c-Jun's function. Thus, our data are consistent with the hypothesis that 465* and, by extension, other GR mutants lacking their LBDs, exert their lethal effect through binding to and altering the activity of certain transcription factors or other proteins essential for cell viability. For example, repression of potential “survival genes” could be mediated through transcriptional interference between 465* and transcription factors such as AP-1. Alternatively, 465* and similar constructs could directly interfere with some “vitality factors” or could activate lethal caspase cascades directly or indirectly (52Henkart P.A. Immunity. 1996; 4: 195-201Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar, 53Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4537) Google Scholar). These possibilities are being investigated. From the evidence described in this paper, we propose that protein-protein interactions rather than direct binding to GRE are important in the lethal effects of 465*. The observed biological cell kill by 465* could be mediated by interference with certain transcription factors or other regulatory proteins required for cell survival. We thank Jian Zhong for help in developing the in vitro transcription system and Betty Johnson for comments on this manuscript.