Protein 14-3-3σ Interacts with and Favors Cytoplasmic Subcellular Localization of the Glucocorticoid Receptor, Acting as a Negative Regulator of the Glucocorticoid Signaling Pathway

The glucocorticoid receptor (GR) α interacts with the highly conserved 14-3-3 family proteins. The latter bind phosphorylated serine/threonine residues of "partner" molecules and influence many signal transduction events by altering their subcellular localization and/or protecting them from proteolysis. To examine the physiologic role of 14-3-3 on the glucocorticoid-signaling pathway, we studied the nucleocytoplasmic shuttling and transactivation properties of GRα in a cell line replete with or devoid of 14-3-3σ. We found that endogenous 14-3-3σ helped localize green fluorescent protein-fused GRα in the cytoplasm in the absence of ligand and potentiated its nuclear export after ligand withdrawal. 14-3-3σ also suppressed the transcriptional activity of GRα on a glucocorticoid-responsive promoter. Disruption of the classic nuclear export signal of 14-3-3σ inactivated its ability to influence the nucleocytoplasmic trafficking and transactivation activity of GRα, whereas introduction of a mutation inactivating the binding activity of 14-3-3σ to some of its partner proteins did not. 14-3-3σ bound the ligand-binding domain of GRα through its COOH-terminal portion, in a partially ligand-dependent fashion, while it did not interact with "ligand-binding domain" of GRβ at all. These results suggest that 14-3-3σ functions as a negative regulator in the glucocorticoid signaling pathway, possibly by shifting the subcellular localization/circulation of this receptor toward the cytoplasm through its nuclear export signal. Since 14-3-3 proteins play significant roles in numerous cellular activities, such as cell cycle progression, growth, differentiation, and apoptosis, these actions might indirectly influence the transcriptional activity of GRα. Conversely, through its 14-3-3 protein interactions, GRα may influence these processes. The glucocorticoid receptor (GR) α interacts with the highly conserved 14-3-3 family proteins. The latter bind phosphorylated serine/threonine residues of "partner" molecules and influence many signal transduction events by altering their subcellular localization and/or protecting them from proteolysis. To examine the physiologic role of 14-3-3 on the glucocorticoid-signaling pathway, we studied the nucleocytoplasmic shuttling and transactivation properties of GRα in a cell line replete with or devoid of 14-3-3σ. We found that endogenous 14-3-3σ helped localize green fluorescent protein-fused GRα in the cytoplasm in the absence of ligand and potentiated its nuclear export after ligand withdrawal. 14-3-3σ also suppressed the transcriptional activity of GRα on a glucocorticoid-responsive promoter. Disruption of the classic nuclear export signal of 14-3-3σ inactivated its ability to influence the nucleocytoplasmic trafficking and transactivation activity of GRα, whereas introduction of a mutation inactivating the binding activity of 14-3-3σ to some of its partner proteins did not. 14-3-3σ bound the ligand-binding domain of GRα through its COOH-terminal portion, in a partially ligand-dependent fashion, while it did not interact with "ligand-binding domain" of GRβ at all. These results suggest that 14-3-3σ functions as a negative regulator in the glucocorticoid signaling pathway, possibly by shifting the subcellular localization/circulation of this receptor toward the cytoplasm through its nuclear export signal. Since 14-3-3 proteins play significant roles in numerous cellular activities, such as cell cycle progression, growth, differentiation, and apoptosis, these actions might indirectly influence the transcriptional activity of GRα. Conversely, through its 14-3-3 protein interactions, GRα may influence these processes. The glucocorticoid receptor (GR) 1The abbreviations used are: GR, glucocorticoid receptor; GRE, glucocorticoid response element; NES, nuclear export signal; DBD, DNA-binding domain; LBD, ligand-binding domain; GFP, green fluorescence protein; EGFP, enhanced GFP; MMTV, mouse mammary tumor virus;AD, activation domain; WT, wild type; KO, knock-out; C, cytoplasmic distribution; N, nuclear localization. belongs to the superfamily of steroid/thyroid/retinoic acid receptor proteins and mediates the diverse and pivotal actions of glucocorticoids in the maintenance of resting and stress homeostasis (1Kino T. Chrousos G.P. J. Endocrinol. 2001; 169: 437-445Crossref PubMed Scopus (97) Google Scholar). The human GR consists of two highly homologous isoforms, α and β, produced by alternative use of exon 9 α or β of the GR gene. GRα represents the classic glucocorticoid receptor, which binds glucocorticoids and mediates almost all known glucocorticoid effects, while GRβ does not bind ligand, has dominant negative activity upon GRα and unclear physiologic role(s) (2Bamberger C.M. Schulte H.M. Chrousos G.P. Endocr. Rev. 1996; 17: 245-261Crossref PubMed Scopus (758) Google Scholar). GRα, in the ligand-free condition, resides primarily in the cytoplasm in the form of a hetero-oligomer with several heat shock proteins (hsps) and related molecules (2Bamberger C.M. Schulte H.M. Chrousos G.P. Endocr. Rev. 1996; 17: 245-261Crossref PubMed Scopus (758) Google Scholar). Upon hormone binding, GRα undergoes a conformational change, dissociates from the hsps and translocates into the nucleus depending on nuclear translocation signals 1 and 2 (3LaCasse E.C. Lefebvre Y.A. Nucleic Acids Res. 1995; 23: 1647-1656Crossref PubMed Scopus (191) Google Scholar, 4Tang Y. Ramakrishnan C. Thomas J. DeFranco D.B. Mol. Biol. Cell. 1997; 8: 795-809Crossref PubMed Scopus (44) Google Scholar, 5Wurtz J.M. Bourguet W. Renaud J.P. Vivat V. Chambon P. Moras D. Gronemeyer H. Nat. Struct Biol. 1996; 3: 87-94Crossref PubMed Scopus (685) Google Scholar). Nuclear translocation signal 1 catalyzes rapid transport of the GR through the nuclear pore, employing the importin-mediated pathway, while nuclear translocation signal 2 contributes to a slower traffic via as yet unknown mechanisms (6Savory J.G. Hsu B. Laquian I.R. Giffin W. Reich T. Hache R.J. Lefebvre Y.A. Mol. Cell. Biol. 1999; 19: 1025-1037Crossref PubMed Scopus (183) Google Scholar). After entering the nucleus, GRα binds as a homodimer to specific DNA enhancer elements, the glucocorticoid response element (GREs) in the promoter regions of glucocorticoid target genes (2Bamberger C.M. Schulte H.M. Chrousos G.P. Endocr. Rev. 1996; 17: 245-261Crossref PubMed Scopus (758) Google Scholar). Promoter-bound GRα, via its two transactivational domains, attracts histone acetyltranferase co-activators and chromatin remodeling complexes, which help transmit signals from the activated ligand-bound GRα to the transcription initiation complex (7McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Crossref PubMed Scopus (1658) Google Scholar). After dissociating from DNA, GRα is exported into the cytoplasm, becoming again fully competent for ligand binding and signal transmission (8Hache R.J. Tse R. Reich T. Savory J.G. Lefebvre Y.A. J. Biol. Chem. 1999; 274: 1432-1439Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Several mechanisms regulate the nuclear export of GRα. The CRM1/exportin and the classic nuclear export signal (NES)mediated nuclear export machineries were postulated to be involved in GRα nuclear export, based on evidence that leptomycin B, an inhibitor of these systems, abrogated the rapid nuclear to cytoplasmic translocation and cytoplasmic retention of GRα; however, no classic NES(s) are evident in the GRα molecule (6Savory J.G. Hsu B. Laquian I.R. Giffin W. Reich T. Hache R.J. Lefebvre Y.A. Mol. Cell. Biol. 1999; 19: 1025-1037Crossref PubMed Scopus (183) Google Scholar, 9Itoh M. Adachi M. Yasui H. Takekawa M. Tanaka H. Imai K. Mol. Endocrinol. 2002; 16: 2382-2392Crossref PubMed Scopus (197) Google Scholar). The calcium-calreticulin-mediated, classic NES-independent nuclear export system, on the other hand, was also reported to be involved in the nuclear export and cytoplasmic retention of GRα (10Holaska J.M. Black B.E. Rastinejad F. Paschal B.M. Mol. Cell. Biol. 2002; 22: 6286-6297Crossref PubMed Scopus (93) Google Scholar, 11Holaska J.M. Black B.E. Love D.C. Hanover J.A. Leszyk J. Paschal B.M. J. Cell Biol. 2001; 152: 127-140Crossref PubMed Scopus (225) Google Scholar). Reassembly of the GRα in the heterocomplex with hsps may not be sufficient to relocate GRα in the cytoplasm, since such complexes are also observed in the nucleus both before and after withdrawal of the ligand (8Hache R.J. Tse R. Reich T. Savory J.G. Lefebvre Y.A. J. Biol. Chem. 1999; 274: 1432-1439Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). All three main domains of GRα, i.e. the amino-terminal, DNA-binding (DBD), and ligand-binding (LBD) domains, seem to be involved in nuclear export of this molecule. A serine residue at position 226 of GRα located in the amino-terminal domain is necessary for phosphorylation by the c-Jun NH2-terminal kinase to facilitate the nuclear export of the GRα, while a 67-amino acid region in the DBD is sufficient to support calreticulin-mediated nuclear export (9Itoh M. Adachi M. Yasui H. Takekawa M. Tanaka H. Imai K. Mol. Endocrinol. 2002; 16: 2382-2392Crossref PubMed Scopus (197) Google Scholar, 12Black B.E. Holaska J.M. Rastinejad F. Paschal B.M. Curr. Biol. 2001; 11: 1749-1758Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). In addition, we previously reported that removal of the LBD from GRα resulted in constitutive localization of this peptide in the nucleus, indicating that the LBD also contributes to nuclear to cytoplasmic translocation of GRα (13Kino T. Stauber R.H. Resau J.H. Pavlakis G.N. Chrousos G.P. J. Clin. Endocrinol. 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Binding of 14-3-3 to Cdc25C segregates the latter into the cytoplasm and eliminates its phosphatase activity from the nucleus, thus inhibiting cells from progressing through the G2/M check-point (22Peng C.Y. Graves P.R. Thoma R.S. Wu Z. Shaw A.S. Piwnica-Worms H. Science. 1997; 277: 1501-1505Crossref PubMed Scopus (1190) Google Scholar, 23Dalal S.N. Schweitzer C.M. Gan J. DeCaprio J.A. Mol. Cell. Biol. 1999; 19: 4465-4479Crossref PubMed Scopus (241) Google Scholar, 24Lopez-Girona A. Furnari B. Mondesert O. Russell P. Nature. 1999; 397: 172-175Crossref PubMed Scopus (506) Google Scholar). 14-3-3 proteins bind the phosphorylated serine or threonine residues of their partner proteins located within a specific amino acid sequence, RSXpSXP, identified as a "high affinity 14-3-3-binding motif" (25Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1353) Google Scholar). They contain nine α-helical structures and form a homo- or heterodimer through their NH2-terminal portions (25Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1353) Google Scholar, 26Rittinger K. Budman J. Xu J. Volinia S. Cantley L.C. Smerdon S.J. Gamblin S.J. Yaffe M.B. Mol. Cell. 1999; 4: 153-166Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 27Rosenquist M. Sehnke P. Ferl R.J. Sommarin M. Larsson C. J. Mol. Evol. 2000; 51: 446-458Crossref PubMed Scopus (172) Google Scholar). Their central third to fifth α-helices create a binding pocket for a phosphorylated serine/threonine residue, and the C-terminal seventh to ninth helices determine the specificity to target peptide motifs (25Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1353) Google Scholar, 26Rittinger K. Budman J. Xu J. Volinia S. Cantley L.C. Smerdon S.J. Gamblin S.J. Yaffe M.B. Mol. Cell. 1999; 4: 153-166Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). 14-3-3 proteins contain one classic NES in their ninth helix, which helps localize 14-3-3/partner protein complexes in the cytoplasm (26Rittinger K. Budman J. Xu J. Volinia S. Cantley L.C. Smerdon S.J. Gamblin S.J. Yaffe M.B. Mol. Cell. 1999; 4: 153-166Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 28Gorlich D. Mattaj I.W. Science. 1996; 271: 1513-1518Crossref PubMed Scopus (1067) Google Scholar). Recent research indicated that GRα formed complexes with 14-3-3 proteins and Raf-1 (29Widen C. Zilliacus J. Gustafsson J.A. Wikstrom A.C. J. Biol. Chem. 2000; 275: 39296-39301Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Although an early study reported that GRα LBD interacted with 14-3-3η in a ligand-dependent fashion in a yeast two-hybrid assay, a subsequent report indicated that GRα was associated with 14-3-3 proteins, both in the ligand-free and -bound conditions (29Widen C. Zilliacus J. Gustafsson J.A. Wikstrom A.C. J. Biol. Chem. 2000; 275: 39296-39301Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 30Wakui H. Wright A.P. Gustafsson J. Zilliacus J. J. Biol. Chem. 1997; 272: 8153-8156Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). To further investigate the functional contribution of 14-3-3 to the biologic activity of GRα, we examined the subcellular localization and transactivation properties of GRα in a cell line used in its wild type 14-3-3σ replete and in its mutant type 14-3-3σ-deficient forms (31Chan T.A. Hermeking H. Lengauer C. Kinzler K.W. Vogelstein B. Nature. 1999; 401: 616-620Crossref PubMed Scopus (814) Google Scholar). We found that endogenous 14-3-3σ helps localize ligand-free GRα in the cytoplasm and contributes to nuclear export of GRα after withdrawal of ligand. In addition, endogenous 14-3-3σ suppresses ligand-activated GRα-induced transactivation of a glucocorticoid-responsive promoter. These results indicate that 14-3-3σ functions as a negative regulator of the glucocorticoid signaling pathway by shifting the subcellular circulation of this receptor toward the cytoplasm. Plasmids—pF25-hGRα, which expresses the green fluorescence protein (GFP)-fused human GRα under the control of the cytomegalovirus promoter, was reported previously (13Kino T. Stauber R.H. Resau J.H. Pavlakis G.N. Chrousos G.P. J. Clin. Endocrinol. Metab. 2001; 86: 5600-5608Crossref PubMed Scopus (95) Google Scholar). pRShGRα, which expresses the human GRα, was a kind gift from Dr. R. M. Evans (Salk Institute, La Jolla, CA). pMMTV-Luc, which expresses luciferase under the control of the glucocorticoid-responsive mouse mammary tumor virus (MMTV) promoter, was a generous gift from Dr. G. L. Hager (NCI, Bethesda, MD). pCDNA4-14-3-3σ and pEGFP-C1-14-3-3σ were constructed by subcloning the coding sequence of the human 14-3-3σ into pcDNA4/HisMax (Invitrogen) or pEGFP-C1 (Clontech, Palo Alto, CA) in an in-frame fashion, respectively. pCDNA4-14-3-3σNES Mut, which expresses 14-3-3σ, defective in NES due to mutations replacing leucine at positions 243, 247, and 249 to alanine, was constructed by PCR-assisted mutagenesis using pCDNA4-14-3-3σ as a template. pEGFPC1-14-3-3σNES Mut was constructed using the same procedure employing pEGFP-C1-14-3-3σ as a template. pCDNA4-14-3-3σE182K, which expresses a 14-3-3σ mutant that may have a defective binding site for a phosphorylated serine/threonine residue due to a point mutation that replaces a glutamic acid at position 182 to lysine, was also constructed by PCR-assisted mutagenesis using the same template (26Rittinger K. Budman J. Xu J. Volinia S. Cantley L.C. Smerdon S.J. Gamblin S.J. Yaffe M.B. Mol. Cell. 1999; 4: 153-166Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). pSV40-β-Gal, which expresses β-galactosidase under the control of the simian virus 40 promoter, was purchased from Promega (Madison, WI). pLexA-GRαLBD and -GRβLBD, which express the LexA DBD fusions of the human GRα LBD or GRβ LBD, were constructed by inserting the corresponding cDNA fragments of the human GRα LBD or GRβ LBD into pLexA (Clontech) in an in-frame fashion, respectively. pGAD424-14-3-3σ-(1–270) and -(106–270), and pB42AD-14-3-3η-(1–244), -(141–244), -(190–244), -(210–244), -(110–210), and -(1–110), which respectively express the GAL4 or LexA activation domain (AD) fusions of the indicated 14-3–3 fragments, were constructed by inserting cDNA fragments of the indicated regions of 14-3-3σ or 14-3-3η into pGAD424 (Clontech) or pB42AD (Clontech), respectively. p8OP-LacZ was purchased from Clontech. Cell Cultures and Transfections—Human colon cancer-derived HCT116 wild type (WT) and 14-3-3σ knock-out (KO) cells were kindly provided by Dr. B. Vogelstein (Johns Hopkins University, Baltimore, MD) (31Chan T.A. Hermeking H. Lengauer C. Kinzler K.W. Vogelstein B. Nature. 1999; 401: 616-620Crossref PubMed Scopus (814) Google Scholar). These cells are defective in functional GRα (data not shown). They were maintained in McCoy's 5A medium supplemented with 10% fetal bovine serum, 100 units/ml of penicillin, and 1 μg/ml of streptomycin. They were transfected using Lipofectin™ with 1 μg/well of pF25-hGRα and/or 0.3 μg/well of 14-3-3σ-expressing plasmids for the study of GRα subcellular localization, as described previously (32Kino T. Kopp J.B. Chrousos G.P. J. Steroid Biochem. Mol. Biol. 2000; 75: 283-290Crossref PubMed Scopus (32) Google Scholar). For reporter assays, 0.5 μg/well of pRShGRα and 0.3 μg/well of 14-3-3σ-expressing plasmids together with 1.0 μg/well of pMMTV-Luc and 0.3 μg/well of pSV40-β-Gal were used. Detection of Subcellular Localization of GFP-fused GRα and 14-3-3σ—Cells were plated on 25-mm dishes and were transfected as described above. 24 h after transfection, the medium was replaced with McCoy's 5A medium containing 10% charcoal/dextran-treated fetal bovine serum with antibiotics. 48 h after transfection, the cells were analyzed with an inverted fluorescence microscope (Leica DM IRB, Wetzlar, Germany) as described previously (13Kino T. Stauber R.H. Resau J.H. Pavlakis G.N. Chrousos G.P. J. Clin. Endocrinol. Metab. 2001; 86: 5600-5608Crossref PubMed Scopus (95) Google Scholar, 33Kino T. Chrousos G.P. J. Biol. Chem. 2003; 278: 3023-3029Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). 12-Bit black-and-white images were captured using a digital CCD camera (Hamamatsu Photonics K.K., Hamamatsu, Japan). Image analysis and presentation was performed using the Openlab software (Improvision, Boston, MA). To examine the subcellular distribution of GFP-GRα, numbers of cells exhibiting five different distribution patterns from complete cytoplasmic distribution (C) to nuclear localization (N) were counted and percentages of each fraction to the total transfected cell number were calculated. For the experiments examining the nuclear export of GFPGRα, cells were exposed to 10–6m dexamethasone 48 h after the transfection. After culturing for 1 h, the cells were washed with PBS two times and placed in McCoy's 5A medium containing 10% charcoal/dextran-treated fetal bovine serum and antibiotics. Eight hours after replacement of the medium, numbers of cells that contained GFP-GRα mainly in the cytoplasm (subgroups corresponding to "C" and "N < C") were counted, and the nuclear export of GRα was expressed as percentages of these numbers to the total transfected cell number. Reporter Assays—Cells were plated on six-well plates and transfected as described above. 24 h after transfection, the cells were exposed to the indicated amounts of dexamethasone. 48 h after transfection, the cell lysis buffer (Promega) was placed in each well, and the resulting cell lysates were harvested. Luciferase and β-galactosidase activities were determined as described previously (32Kino T. Kopp J.B. Chrousos G.P. J. Steroid Biochem. Mol. Biol. 2000; 75: 283-290Crossref PubMed Scopus (32) Google Scholar). All measurements of reporter gene activity were conducted in triplicate and all experiments were repeated at least three times. Yeast Two-hybrid Assay—Yeast strain EGY48 (Clontech) was transformed with the lacZ reporter plasmid p8OP-LacZ, pLexA-GRαLBD or -GRβLBD, and the indicated pGAD424-14-3-3σ- or pB42AD-14-3-3η-related plasmids (34Finley R.L. Brent R. Hames B.D. Glover D.M. DNA Cloning, Expression Systems: A Practical Approach. Oxford University Press, Oxford1995: 169-203Google Scholar). The cells were grown in a selective medium to the early stationary phase, permeabilized with CHCl3-SDS treatment, and β-galactosidase activity was measured in the cell suspension using Galactolight™ PLUS (Tropix, Bedford, MA), as described previously (33Kino T. Chrousos G.P. J. Biol. Chem. 2003; 278: 3023-3029Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Statistical Analyses—Statistical analysis was carried out by analysis of variance, followed by Student t test with Bonferroni correction for multiple comparisons. Endogenous 14-3-3σ Helps Non-ligand-bound GRα Remain in the Cytoplasm and Facilitates the Nuclear Export of GRα after Withdrawal of Dexamethasone—To investigate a role of 14-3-3 family proteins on the subcellular localization and transactivation activity of GRα, we employed wild type versus mutant HCT116 cells, in which both alleles of the 14-3-3σ gene were destroyed by homologous recombination (31Chan T.A. Hermeking H. Lengauer C. Kinzler K.W. Vogelstein B. Nature. 1999; 401: 616-620Crossref PubMed Scopus (814) Google Scholar). We first tested the subcellular localization of GFP-GRα in the absence of ligand. GFP-GRα was mainly located in the cytoplasm in the HCT116 WT cells, while substantial amounts of GFP-GRα were found in the nucleus in the 14-3-3σ KO cells (Fig. 1A). To determine this, we examined multiple cells and graded them into five distribution patterns from complete cytoplasmic distribution (C) to complete nuclear localization (N) (Fig. 1B). Using this analysis, HCT116 WT cells had unliganded GFPGRα mainly in the cytoplasm, while KO cells had more in the nucleus, indicating that endogenous 14-3-3σ retained GFPGRα in the cytoplasm in the absence of ligand. We also tested nuclear translocation of GFP-GRα in WT cells and KO cells in which 14-3-3σ was supplemented by the transfected expressing plasmid. GFP-GRα entered the nucleus in 10–20 min in response to 10–6m dexamethasone in both cell types, indicating that the mechanism supporting the nuclear translocation of GRα was intact in these cells (data not shown). We next examined whether 14-3-3σ plays a role in the nuclear export of GRα after withdrawal of ligand. Eight hours after removing 10–6m dexamethasone from the medium, 32% of HCT116 WT cells had GFP-GRα in the cytoplasm, while almost all KO cells still retained it in the nucleus (Fig. 1C). When the wild type 14-3-3σ was transfected in KO cells, 22% of the cells had GFP-GRα in the cytoplasm, indicating that transfected 14-3-3σ helped GRα relocate into the cytoplasm after removal of dexamethasone. Endogenous 14-3-3σ Suppresses the Transcriptional Activity of GRα—We then examined the transactivation activity of GRα stimulated with increasing concentrations of dexamethasone in WT and KO cells (Fig. 2). GRα stimulated the MMTV promoter in response to 10–6m dexamethasone by about 80- and 300-fold in WT cells and KO cells, respectively. The dexamethasone titration curve was shifted upward in the latter cells. Transfection of wild type 14-3-3σ partially reversed this change in KO cells. The EC50 (mean ± S.E.: in mm) was 4.01 ± 0.33 and 6.46 ± 1.04 in WT and KO cells, respectively (p > 0.10), whereas the B max (mean ± S.E.: × 10–2 relative luminescence unit) was 9.11 ± 0.89 and 28.01 ± 1.19, respectively (p < 0.001). Transfection of 14-3-3σ partially reversed this change in KO cells. EC50 values were similar in transfected and KO cells (p > 0.30), while the B max in the transfected cells was significantly lower than in KO cells (p < 0.01). These results indicated that endogenous 14-3-3σ functions as a negative regulator of GRα-induced transactivation. The C-terminal Half of 14-3-3σ Interacts with the GRα LBD in a Yeast Two-hybrid Assay—We next examined the interaction of GRα and 14-3-3σ in a yeast two-hybrid assay (Fig. 3A). Administration of dexamethasone stimulated the LexA-DBDGRαLBD-induced, but not LexA-GRβLBD-induced, β-galactosidase activity by about 3-fold in the EGY48 yeast strain. Co-expression of GAL4-AD fusions of the full-length or the COOH-terminal half of 14-3-3σ enhanced β-galactosidase activity induced by LexA-DBD-GRαLBD in a partially dexamethasone-dependent fashion, whereas it did not affect the activity of LexA-DBD-GRβ LBD. These results indicated that GRα LBD interacted with the COOH-terminal half of 14-3-3σ in a partially ligand-dependent fashion, while GRβ LBD did not. No increase of β-galactosidase activity was observed in the transformed yeast cells, when they were cultured in galactose-deficient medium that did not support the expression of bait proteins (data not shown). This result indicated that expression of GAL4-AD-fused 14-3-3σs did not influence basal promoter activity. We obtained similar results using a plasmid expressing the LexA-DBD fusions of the full-length GRα (data not shown). Our results showed that 14-3-3σ interacts with GRα in the absence of dexamethasone as well as in its presence. In our system, 14-3-3η interacted with the GRα LBD in the absence of dexamethasone and the interaction was enhanced in its presence (Fig. 3B). 14-3-3η fragments, which contained the region from 210 to 240 that corresponds to the ninth α-helix, supported the binding to GRα LBD. Destruction of 14-3-3σ NES Diminishes the Ability of This Protein to Promote Cytoplasmic Retention/Nuclear Export of GRα and Suppression of GRα Transactivation—14-3-3 proteins may help translocate their partner proteins into the cytoplasm via their classic NES located in their ninth α-helix (16Fu H. Subramanian R.R. Masters S.C. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 617-647Crossref PubMed Scopus (1334) Google Scholar, 26Rittinger K. Budman J. Xu J. Volinia S. Cantley L.C. Smerdon S.J. Gamblin S.J. Yaffe M.B. Mol. Cell. 1999; 4: 153-166Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). Thus, we examined the contribution of 14-3-3σ NES on the cytoplasmic retention of GRα, by constructing a plasmid expressing a 14-3-3σ mutant (14-3-3σNES Mut), in which the NES was destroyed by clustered mutations, as a fusion with EGFP (26Rittinger K. Budman J. Xu J. Volinia S. Cantley L.C. Smerdon S.J. Gamblin S.J. Yaffe M.B. Mol. Cell. 1999; 4: 153-166Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). The EGFP-fused wild type 14-3-3σ was mainly located in the cytoplasm, while a small fraction of this fusion protein was also observed in the nucleus (Fig. 4A). The EGFP-14-3-3σNES Mut, on the other hand, was distributed more in the nucleus, indicating that the introduced mutations inactivated the NES. Co-expression of 14-3-3σNES Mut did not change the distribution of unliganded GFP-GRα, in contrast to the wild type 14-3-3σ (Fig. 4B). We next examined the effect of this mutant on the nuclear export of GFP-GRα after withdrawal of dexamethasone. Supplementation of the wild type 14-3-3σ brought GFP-GRα into the cytoplasm in 23% of KO cells, while expression of 14-3-3σNES Mut did not change the nuclear export of this receptor (Fig. 4C). In a functional reporter assay, 14-3-3σNES Mut did not suppress GRα-induced transactivation of the MMTV promoter in a dexamethasone titration curve (Fig. 4D). These results suggest that 14-3-3σ retained the ligand-free GFP-GRα in the cytoplasm and helped it through its NES to redistribute in the cytoplasm after the withdrawal of dexamethasone. Since the destruction of NES also abolished the suppressive effect of 14-3-3σ on GRα transactivation, it is possible that 14-3-3σ suppressed GRα-induced transcriptional activity by segregating GRα away from the nucleus. The Phosphopeptide Binding Activity of 14-3-3σ May Not Be Necessary for Cytoplasmic Retention of GFP-GRα and Suppression of GRα-dependent Transactivation—Since a previous publication indicated that 14-3-3 forms a complex with GRα together with its partner protein Raf-1, we employed a 14-3-3σE182K mutant, to examine whether 14-3-3 partner proteins might contribute to the action of 14-3-3σ on the activity of GRα (29Widen C. Zilliacus J. Gustafsson J.A. Wikstrom A.C. J. Biol. Chem. 2000; 275: 39296-39301Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). This mutation corresponds to the replacement of a glutamic acid at 180 by a lysine in Drosophila 14-3-3ϵ that inactivates the binding activity of this protein to Raf-1 because of destruction of the phosphopeptide-binding pocket (26Rittinger K. Budman J. Xu J. Volinia S. Cantley L.C. Smerdon S.J. Gamblin S.J. Yaffe M.B. Mol. Cell. 1999; 4: 153-166Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). 14-3-3σE182K was distributed mainly in the cytoplasm similarly to the wild type 14-3-3σ (Fig. 5A). 14-3-3σE182K preserved property of the wild type 14-3-3σ on the subcellular distribution and transactivation activity of GRα (Fig. 5, B and C), suggesting that association of 14-3-3σ to partner proteins, such as Raf-1, may not be necessary for its effect on these GRα activities. GRα forms heterocomplexes with 14-3-3 and its partner protein Raf-1 in the cytoplasmic fraction of adrenalectomized rat liver shown by immunoaffinity chromatography and co-immunoprecipitation experiments (29Widen C. Zilliacus J. Gustafsson J.A. Wikstrom A.C. J. Biol. Chem. 2000; 275: 39296-39301Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Although these complexes were observed in the absence of ligand, the interaction of constituent molecules was further strengthened by the presence of the hormone. Association of GRα with one of the 14-3-3 proteins, 14-3-3η, was also found in a yeast two-hybrid screening, using the GRα LBD as a bait (30Wakui H. Wright A.P. Gustafsson J. Zilliacus J. J. Biol. Chem. 1997; 272: 8153-8156Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In agreement with the above reports, we examined the functional effect of 14-3-3 on the activity of GRα by employing wild type versus mutant HCT116 cells, in which both alleles of the 14-3-3σ gene were destroyed by homologous recombination (31Chan T.A. Hermeking H. Lengauer C. Kinzler K.W. Vogelstein B. Nature. 1999; 401: 616-620Crossref PubMed Scopus (814) Google Scholar). We found that endogenous 14-3-3σ helped GFP-GRα remain in the cytoplasm in the absence of dexamethasone and supported the nuclear export of GFP-GRα after withdrawal of dexamethasone via its NES. 14-3-3σ, thus, appeared to function as an "attached" partner NES, a finding that might explain the results of previous reports demonstrating that the nuclear export of GRα is sensitive to leptomycin B, even though the GRα molecule does not contain a classic NES (6Savory J.G. Hsu B. Laquian I.R. Giffin W. Reich T. Hache R.J. Lefebvre Y.A. Mol. Cell. Biol. 1999; 19: 1025-1037Crossref PubMed Scopus (183) Google Scholar, 9Itoh M. Adachi M. Yasui H. Takekawa M. Tanaka H. Imai K. Mol. Endocrinol. 2002; 16: 2382-2392Crossref PubMed Scopus (197) Google Scholar). We previously reported that deletion of the GRα LBD from GRα localized this GRα fragment in the nucleus (13Kino T. Stauber R.H. Resau J.H. Pavlakis G.N. Chrousos G.P. J. Clin. Endocrinol. Metab. 2001; 86: 5600-5608Crossref PubMed Scopus (95) Google Scholar). Since 14-3-3σ interacts with the LBD, a defect in binding of this domain to 14-3-3σ may explain its particular subcellular distribution. We also demonstrated that endogenous 14-3-3σ functioned as a negative regulator of GRα-induced transactivation. This activity correlated with the ability of 14-3-3σ to localize unliganded GFP-GRα in the cytoplasm, in the experiment employing the 14-3-3σ mutants NES Mut and E182K. Therefore, it is likely that 14-3-3σ suppresses GRα-induced transactivation by shifting intracellular circulation of GRα toward the cytoplasm, possibly by reducing the chance of ligand-bound GRα to interact with GREs, steroid hormone receptor co-activators, and other related specific or general transcription factors in the nucleus. For instance, GRα dynamically interacts with GREs in living cells, binding to and dissociating from them in the order of seconds (35Fletcher T.M. Ryu B.W. Baumann C.T. Warren B.S. Fragoso G. John S. Hager G.L. Mol. Cell. Biol. 2000; 20: 6466-6475Crossref PubMed Scopus (80) Google Scholar, 36McNally J.G. Muller W.G. Walker D. Wolford R. Hager G.L. Science. 2000; 287: 1262-1265Crossref PubMed Scopus (652) Google Scholar). In this situation, the drive created by 14-3-3σ, which shifts GRα toward the cytoplasm, may reduce the probability of GRα binding to GREs and, hence, may reduce its transcriptional activity. A similar effect was also observed in a recent report, which showed that c-Jun NH2-terminal kinase suppressed GRα-induced transactivation by phosphorylating serine 216 and facilitating its nuclear export (9Itoh M. Adachi M. Yasui H. Takekawa M. Tanaka H. Imai K. Mol. Endocrinol. 2002; 16: 2382-2392Crossref PubMed Scopus (197) Google Scholar). A previous report demonstrated that overexpressed 14-3-3η enhanced GRα-induced transactivation of a synthetic GRE-containing heterologous promoter in African monkey kidney-derived COS7 cells, while we showed that endogenous 14-3-3σ suppressed GRα transactivation in HCT116 cells (30Wakui H. Wright A.P. Gustafsson J. Zilliacus J. J. Biol. Chem. 1997; 272: 8153-8156Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). This discrepancy may result from differences in the experimental systems employed, including the type of cell lines, the different 14-3-3 isoforms and overexpression versus normal expression versus knock-out of 14-3-3. Indeed, overexpression of a protein may sometimes cause artificial effects (37Frew I.J. Dickins R.A. Cuddihy A.R. Del Rosario M. Reinhard C. O'Connell M.J. Bowtell D.D. Mol. Cell. Biol. 2002; 22: 8155-8164Crossref PubMed Scopus (34) Google Scholar). We examined 14-3-3 "loss of function" by employing the KO cells and showed that the physiologic activity of 14-3-3σ is that of a negative regulator of the glucocorticoid signaling pathway. We demonstrated that the GRα LBD interacts with 14-3-3σ as well as η in a partially ligand-dependent fashion. A previous report indicated that this domain of GRα interacted with 14-3-3η in an absolutely ligand-dependent fashion in the same LexA yeast two-hybrid system (30Wakui H. Wright A.P. Gustafsson J. Zilliacus J. J. Biol. Chem. 1997; 272: 8153-8156Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Differences in the GRα fragments employed or yeast strains used in the assay might have led to the different results. Since a recent report also demonstrated partial ligand-dependent interaction between GRα and 14-3-3 in a semiquantitative coimmunoprecipitation assay, it is likely that GRα and 14-3-3 proteins associate with each other in the absence of ligand. In contrast to the GRα LBD, the GRβ "LBD" did not interact with 14-3-3σ at all. Since GRβ is constitutively located in the nucleus, the inability of GRβ to interact with 14-3-3σ might, to some extent, contribute to its constitutive nuclear localization (13Kino T. Stauber R.H. Resau J.H. Pavlakis G.N. Chrousos G.P. J. Clin. Endocrinol. Metab. 2001; 86: 5600-5608Crossref PubMed Scopus (95) Google Scholar, 38Oakley R.H. Sar M. Cidlowski J.A. J. Biol. Chem. 1996; 271: 9550-9559Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar, 39Oakley R.H. Webster J.C. Sar M. Parker Jr., C.R. Cidlowski J.A. Endocrinology. 1997; 138: 5028-5038Crossref PubMed Scopus (156) Google Scholar, 40Vottero A. Chrousos G.P. Trends Endocrinol. Metab. 1999; 10: 333-338Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Our results employing 14-3-3σE182K suggest that the association of known partner proteins with 14-3-3σ may not be necessary for this molecule to influence the subcellular localization of GRα and the suppression of its transactivation. However, an E180K mutation in Drosophila 14-3-3ϵ, which corresponds to the E182K mutation in human 14-3-3σ, abolishes the interaction of this protein with Raf-1 and BAD, but preserves that with IRS-1, indicating that the E182K mutation in 14-3-3σ might not completely exclude the interaction of this 14-3-3 subtype with all partner proteins (26Rittinger K. Budman J. Xu J. Volinia S. Cantley L.C. Smerdon S.J. Gamblin S.J. Yaffe M.B. Mol. Cell. 1999; 4: 153-166Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 41Chang H.C. Rubin G.M. Genes Dev. 1997; 11: 1132-1139Crossref PubMed Scopus (124) Google Scholar). In agreement with the above-indicated evidence, about half of the 14-3-3-partner proteins use a different phosphopeptide-binding motif to interact with 14-3-3, suggesting that a single surface of 14-3-3σ in the phosphopeptide-binding pocket, which the E182K mutation destroys, may not support its association generically with all partner proteins. Further experiments are required to address this issue. In summary, endogenous 14-3-3σ functions as a negative regulator of GRα-induced transactivation, most likely by shifting the subcellular distribution and circulation of GRα toward the cytoplasm. These results indicate that change in the intracellular concentration as well as the subcellular distribution of 14-3-3σ may contribute to the altered sensitivity of tissues to glucocorticoids seen in several physiologic and pathologic conditions (1Kino T. Chrousos G.P. J. Endocrinol. 2001; 169: 437-445Crossref PubMed Scopus (97) Google Scholar). Since 14-3-3 proteins are involved in a broad array of cellular activities, such as cell cycle progression, growth, differentiation, and apoptosis, these activities might indirectly influence the transcriptional activity of GRα, by changing the availability of 14-3-3 and/or altering partner proteins associated with 14-3-3. On the other hand, the opposite may be true. Ligand-bound GRα may influence these cellular processes by segregating and/or influencing 14-3-3 and partner molecules. We thank Drs. R. M. Evans and G. L. Hager for providing their plasmids and K. Zachman, Anton Alatsatianos, and Ly Chheng for their superb technical assistance.