We used a cellular system to elucidate the molecular determinants of the large immunophilin FK506-binding proteins (FKBP)51 and -52 for their action on the glucocorticoid receptor in mammalian cells. Increasing the levels of FKBP51 reduced the transcriptional activity of the receptor, as reported. Elevated levels of FKBP52 per se showed no effect but mitigated the inhibition of the receptor induced by FKBP51. We discovered that nuclear translocation of the glucocorticoid receptor was delayed by FKBP51. This correlates with the reduced interaction of FKBP51 with the motor protein dynein compared with FKBP52. From mutational analyses, we concluded that three features of the immunophilins are required for efficient receptor signaling in mammalian cells: hsp90 interaction, dynein association, and peptidylprolyl isomerase (PPIase) enzyme activity. The relevance of dynein for receptor function was substantiated by several experiments: 1) coexpression of dynamitin, which disrupts the transport complex and reduces receptor activity; 2) coexpression of the PPIase domain fragment of FKBP52, which is known to disrupt interaction of the receptor to dynein and reduce glucocorticoid receptor function, in contrast to the corresponding fragment of FKBP51; and 3) swapping of the PPIase domains FKBP51 and FKBP52, which reverses the respective activity. We concluded from our results that the mechanisms of the regulatory system FKBP51/FKBP52 discovered in yeast also operate in mammals to modulate hormone binding of the receptor. In addition, differential regulation of dynein association and nuclear translocation contributes to the effects of the two immunophilins on the glucocorticoid receptor in mammals. We used a cellular system to elucidate the molecular determinants of the large immunophilin FK506-binding proteins (FKBP)51 and -52 for their action on the glucocorticoid receptor in mammalian cells. Increasing the levels of FKBP51 reduced the transcriptional activity of the receptor, as reported. Elevated levels of FKBP52 per se showed no effect but mitigated the inhibition of the receptor induced by FKBP51. We discovered that nuclear translocation of the glucocorticoid receptor was delayed by FKBP51. This correlates with the reduced interaction of FKBP51 with the motor protein dynein compared with FKBP52. From mutational analyses, we concluded that three features of the immunophilins are required for efficient receptor signaling in mammalian cells: hsp90 interaction, dynein association, and peptidylprolyl isomerase (PPIase) enzyme activity. The relevance of dynein for receptor function was substantiated by several experiments: 1) coexpression of dynamitin, which disrupts the transport complex and reduces receptor activity; 2) coexpression of the PPIase domain fragment of FKBP52, which is known to disrupt interaction of the receptor to dynein and reduce glucocorticoid receptor function, in contrast to the corresponding fragment of FKBP51; and 3) swapping of the PPIase domains FKBP51 and FKBP52, which reverses the respective activity. We concluded from our results that the mechanisms of the regulatory system FKBP51/FKBP52 discovered in yeast also operate in mammals to modulate hormone binding of the receptor. In addition, differential regulation of dynein association and nuclear translocation contributes to the effects of the two immunophilins on the glucocorticoid receptor in mammals. Corticosteroids regulate a variety of biological processes, including the metabolism of carbohydrates, lipids, and proteins, cell proliferation, development, and reproduction (1Kimberly R.P. Curr. Opin. Rheumatol. 1994; 6: 273-280Crossref PubMed Scopus (10) Google Scholar, 2Beato M. Klug J. Hum. Reprod. Update. 2000; 6: 225-236Crossref PubMed Scopus (475) Google Scholar, 3Schmid W. Cole T.J. Blendy J.A. Schütz G. J. Steroid Biochem. Mol. Biol. 1995; 53: 33-35Crossref PubMed Scopus (55) Google Scholar). The mineralocorticoid receptor and the glucocorticoid receptor (GR) 1The abbreviations used are: GR, glucocorticoid receptor; TPR, tetratricopeptide repeat; FKBP, FK506-binding protein; PPIase, peptidylprolyl isomerase; CMV, cytomegalovirus; RT, reverse transcription; TAT, tyrosine aminotransferase; MT, metallothionein; GFP, green fluorescent protein; WT, wild type; DEX, dexamethasone.1The abbreviations used are: GR, glucocorticoid receptor; TPR, tetratricopeptide repeat; FKBP, FK506-binding protein; PPIase, peptidylprolyl isomerase; CMV, cytomegalovirus; RT, reverse transcription; TAT, tyrosine aminotransferase; MT, metallothionein; GFP, green fluorescent protein; WT, wild type; DEX, dexamethasone. are the two corticosteroid receptors that mediate the effects of corticosteroids. They belong to the family of ligand-dependent transcription factors (4Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schütz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6002) Google Scholar, 5Whitfield G.K. Jurutka P.W. Haussler C.A. Haussler M.R. J. Cell. Biochem. 1999; 75: 110-122Crossref Google Scholar). In the classic paradigm, GR resides in the cytoplasm in the absence of ligand. Several molecular chaperones assemble the aporeceptor in a stepwise and ATP-dependent fashion to a conformational state capable of binding to hormone with high affinity (6Pratt W.B. Toft D.O. Endocr. Rev. 1997; 18: 306-360Crossref PubMed Scopus (1514) Google Scholar). The mature GR complex consists of an hsp90 dimer, p23, possibly hsp70, and one of the tetratricopeptide repeat (TPR)-containing proteins, such as the immunophilin FK506-binding proteins (FKBP) 51 and 52 (7Tai P.K. Albers M.W. Chang H. Faber L.E. Schreiber S.L. Science. 1992; 256: 1315-1318Crossref PubMed Scopus (267) Google Scholar). Upon hormone binding, GR is translocated into the nucleus. It either activates transcription by binding to its cognate DNA elements or decreases transcription by interaction with other transcription factors (8Beato M. Herrlich P. Schütz G. Cell. 1995; 83: 851-857Abstract Full Text PDF PubMed Scopus (1624) Google Scholar, 9Karin M. Cell. 1998; 93: 487-490Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). Chaperones are not only required for proper folding of GR but may also be involved in the nuclear translocation of GR (10Pratt W.B. Silverstein A.M. Galigniana M.D. Cell. Signal. 1999; 11: 839-851Crossref PubMed Scopus (149) Google Scholar, 11Galigniana M.D. Scruggs J.L. Herrington J. Welsh M.J. Carter-Su C. Housley P.R. Pratt W.B. Mol. Endocrinol. 1998; 12: 1903-1913Crossref PubMed Scopus (155) Google Scholar, 12Murphy P.J. Kanelakis K.C. Galigniana M.D. Morishima Y. Pratt W.B. J. Biol. Chem. 2001; 276: 30092-30098Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). For example, pharmacological inhibition of hsp90 (13Whitesell L. Mimnaugh E.G. De Costa B. Myers C.E. Neckers L.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8324-8328Crossref PubMed Scopus (1310) Google Scholar) delays nuclear translocation of hormone-bound GR (14Czar M.J. Galigniana M.D. Silverstein A.M. Pratt W.B. Biochemistry. 1997; 36: 7776-7785Crossref PubMed Scopus (134) Google Scholar). Moreover, FKBP52 has been shown to interact not only with hsp90 and GR but also with the motor protein dynein (15Silverstein A.M. Galigniana M.D. Kanelakis K.C. Radanyi C. Renoir J.M. Pratt W.B. J. Biol. Chem. 1999; 274: 36980-36986Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar), apparently via dynamitin (16Galigniana M.D. Harrell J.M. O'Hagen H.M. Ljungman M. Pratt W.B. J. Biol. Chem. 2004; 279: 22483-22489Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). These findings led to the formulation of the concept of a transportosome (10Pratt W.B. Silverstein A.M. Galigniana M.D. Cell. Signal. 1999; 11: 839-851Crossref PubMed Scopus (149) Google Scholar), i.e. a protein heterocomplex that guides GR along cytoskeletal tracts to the nucleus. Without transportosome activity, GR reaches the nucleus with slower kinetics (11Galigniana M.D. Scruggs J.L. Herrington J. Welsh M.J. Carter-Su C. Housley P.R. Pratt W.B. Mol. Endocrinol. 1998; 12: 1903-1913Crossref PubMed Scopus (155) Google Scholar). This model is further supported by the observation that the inhibitory effect of geldanamycin on nuclear translocation depends on an intact cytoskeleton (11Galigniana M.D. Scruggs J.L. Herrington J. Welsh M.J. Carter-Su C. Housley P.R. Pratt W.B. Mol. Endocrinol. 1998; 12: 1903-1913Crossref PubMed Scopus (155) Google Scholar). Hsp90 and FKBP52 have recently been shown to be involved in nuclear transport of p53 (16Galigniana M.D. Harrell J.M. O'Hagen H.M. Ljungman M. Pratt W.B. J. Biol. Chem. 2004; 279: 22483-22489Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). The important question has been raised as to whether the highly homologous FKBP51 also interacts with dynein (17Pratt W.B. Galigniana M.D. Harrell J.M. DeFranco D.B. Cell. Signal. 2004; 16: 857-872Crossref PubMed Scopus (235) Google Scholar). The suggested role of immunophilins in GR signal transduction also implies novel potential ways to explain occasional GR malfunction associated with many diseases (18Kino T. Chrousos G.P. J. Endocrinol. 2001; 169: 437-445Crossref PubMed Scopus (97) Google Scholar, 19Holsboer F. Neuropsychopharmacology. 2000; 23: 477-501Crossref PubMed Scopus (1794) Google Scholar, 20Zennaro M.C. Eur. J. Endocrinol. 1998; 139: 127-138Crossref PubMed Scopus (19) Google Scholar). It has been known for several years that malfunction of GR due to low affinity binding to hormone can result from mutations of the GR receptor (21BrönnegårdStierna P. Marcus C. J. Neuroendocrinol. 1996; 8: 405-415Crossref PubMed Scopus (41) Google Scholar) or from limited hsp90 function (22Picard D. Khursheed B. Garabedian M.J. Fortin M.G. Lindquist S. Yamamoto K.R. Nature. 1990; 348: 166-168Crossref PubMed Scopus (640) Google Scholar, 23Bohen S.P. J. Biol. Chem. 1995; 270: 29433-29438Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 24Bohen S.P. Yamamoto K.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11424-11428Crossref PubMed Scopus (192) Google Scholar). More recently, it has been shown that cochaperones of hsp90 have the ability to modulate the binding affinity of GR to hormone. For example, increased levels of FKBP51 have been reported to be the common cause of glucocorticoid resistance in three New World primates (25Scammell J.G. Denny W.B. Valentine D.L. Smith D.F. Gen. Comp. Endocrinol. 2001; 124: 152-165Crossref PubMed Scopus (182) Google Scholar). Although the exact mechanistic contribution of the large immunophilins for GR function remains to be elucidated, important insight into the functional role has been provided (26Davies T.H. Ning Y.M. Sanchez E.R. J. Biol. Chem. 2002; 277: 4597-4600Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 27Riggs D.L. Roberts P.J. Chirillo S.C. Cheung-Flynn J. Prapapanich V. Ratajczak T. Gaber R. Picard D. Smith D.F. EMBO J. 2003; 22: 1158-1167Crossref PubMed Scopus (271) Google Scholar). Overexpression of FKBP51 in COS cells reduces the binding affinity of GR and, therefore, decreases the transcriptional activity of GR after hormone exposure (28Denny W.B. Valentine D.L. Reynolds P.D. Smith D.F. Scammell J.G. Endocrinology. 2000; 141: 4107-4113Crossref PubMed Scopus (181) Google Scholar). In yeast, overexpression of FKBP51 has no effect on GR, but FKBP52 enhances GR-dependent transcription (27Riggs D.L. Roberts P.J. Chirillo S.C. Cheung-Flynn J. Prapapanich V. Ratajczak T. Gaber R. Picard D. Smith D.F. EMBO J. 2003; 22: 1158-1167Crossref PubMed Scopus (271) Google Scholar). This enhancement is dependent on interaction with hsp90 and the peptidylprolyl isomerase (PPIase) activity of FKBP52 (27Riggs D.L. Roberts P.J. Chirillo S.C. Cheung-Flynn J. Prapapanich V. Ratajczak T. Gaber R. Picard D. Smith D.F. EMBO J. 2003; 22: 1158-1167Crossref PubMed Scopus (271) Google Scholar). The observation that FKBP52 displayed a similar enhancement of GR in dynein-deficient yeast (27Riggs D.L. Roberts P.J. Chirillo S.C. Cheung-Flynn J. Prapapanich V. Ratajczak T. Gaber R. Picard D. Smith D.F. EMBO J. 2003; 22: 1158-1167Crossref PubMed Scopus (271) Google Scholar) calls into question the relevance of the described interaction of FKBP52 and other TPR proteins with dynein for GR signaling. Alternatively, it is possible that yeast, which lacks steroid receptors and FKBPs, does not reflect all of the aspects of GR signaling that have developed in higher organisms to ensure optimal steroid signaling. In this study, we investigated the requirements and mechanisms of the differential regulation of GR activity by FKBP51 and FKBP52 in mammalian cells. We show that virtually all features of this regulatory system found in yeast (27Riggs D.L. Roberts P.J. Chirillo S.C. Cheung-Flynn J. Prapapanich V. Ratajczak T. Gaber R. Picard D. Smith D.F. EMBO J. 2003; 22: 1158-1167Crossref PubMed Scopus (271) Google Scholar) are also functional in mammals, e.g. hsp90 binding of the immunophilins is essential, and the PPIase activity of FKBP52 is required for modulating GR hormone binding and, thus, transcriptional activity. Moreover, we discovered that in mammalian cells nuclear translocation of GR is also regulated by the immunophilins. We detected differential binding of FKBP52 and FKBP51 to dynein, which corresponds to their differential effect on nuclear translocation. Thus, in mammals, FKBP51 and FKBP52 differentially regulate GR on two levels: hormone binding (28Denny W.B. Valentine D.L. Reynolds P.D. Smith D.F. Scammell J.G. Endocrinology. 2000; 141: 4107-4113Crossref PubMed Scopus (181) Google Scholar) and nuclear translocation. Cell Culture, Transfection, and Assays for Luciferase and β-Galactosidase—Cultivation and transfection of human neuroblastoma SK-N-MC cells (American Type Culture Collection catalog number HTB-10), HeLa cells, HEK, and H4-II-E-C3 cells were as described previously (29Schmidt U. Wochnik G.M. Rosenhagen M.C. Hartl F.U. Holsboer F. Rein T. J. Biol. Chem. 2003; 278: 4926-4931Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 30Abel A. Wochnik G. Rüegg J. Rouyer A. Holsboer F. Rein T. Mol. Endocrinol. 2002; 16: 1352-1366Crossref PubMed Scopus (31) Google Scholar). Two days before transfection, cells were seeded in medium containing charcoal-stripped serum. Unless indicated otherwise, the amounts of transfected plasmids/∼107 cells were 1.5 μg of steroid-responsive luciferase reporter plasmid MTVLuc, 3 μgof β-galactosidase expression vector pCMVβ-Gal (Stratagene) as control plasmid, various amounts of FKBP expression vector as indicated, and 0.75 μg of pRK7GR that expresses human GR from the CMV promoter of the vector pRK7 (31Spengler D. Waeber C. Pantaloni C. Holsboer F. Bockaert J. Seeburg P.H. Journot L. Nature. 1993; 365: 170-175Crossref PubMed Scopus (1109) Google Scholar). In all transfections, the total amount of plasmid was equaled for each condition by supplementing “empty” expression vector. After electroporation, the cells were seeded again in medium containing charcoal-stripped serum complemented with either hormone or the respective solvent. Luciferase and β-galactosidase assays were performed as described by Schmidt et al. (29Schmidt U. Wochnik G.M. Rosenhagen M.C. Hartl F.U. Holsboer F. Rein T. J. Biol. Chem. 2003; 278: 4926-4931Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Luciferase data are presented as % stimulation (i.e. after correction of the data by galactosidase activities, fold stimulation was calculated by comparing cells without hormone to cells with hormone). The stimulation in the absence of cotransfected FKBPs was set to 100%. Tyrosine Aminotransferase (TAT) Assays and Reverse Transcription (RT)/PCR—For the measurement of native GR target genes in glucocorticoid-responsive cell lines, H4-II-E-C3 and HeLa cells were used. First, the cortisol responsivity of the TAT promoter in H4-II-E-C3 cells and of the metallothionein (MT) promoter in HeLa cells was determined. The stimulation of MT was ∼2-fold at 30 nm cortisol and the stimulation of TAT, ∼5-fold at 20 nm cortisol. To evaluate the effect of the immunophilins on the stimulation of these promoters, cells were seeded in 6-well plates and transfected the next day with 1 μg/well “empty” vector or the FKBP-expressing vectors using ExGen (Fermentas) according to the manufacturer's instructions. For TAT assays, H4-II-E-C3 cells were treated with 20 μg/ml puromycin and 20 nm cortisol or solvent the day after transfection for another 48 h. Given that the transfection efficiency was not 100%, treatment with puromycin was necessary to reduce the fraction of non-transfected cells (these cells do not express the puromycin resistance that is encoded in our expression vectors). The cells were harvested in 100 μl of lysis buffer and assays performed as described by Abel et al. (30Abel A. Wochnik G. Rüegg J. Rouyer A. Holsboer F. Rein T. Mol. Endocrinol. 2002; 16: 1352-1366Crossref PubMed Scopus (31) Google Scholar). For determination of the inducibility of MT by RT/PCR, HeLa cells were treated with 1 μg/ml puromycin and 30 nm cortisol or solvent the day after transfection for another 24 h. Total RNA was isolated using TRIzol® as recommended by the manufacturer and RT/PCR performed as described by Rüegg et al. (32Rüegg J. Holsboer F. Turck C. Rein T. Mol. Cell. Biol. 2004; 24: 9371-9382Crossref PubMed Scopus (34) Google Scholar). MT data were normalized by the coamplified GAPDH (32Rüegg J. Holsboer F. Turck C. Rein T. Mol. Cell. Biol. 2004; 24: 9371-9382Crossref PubMed Scopus (34) Google Scholar), and TAT activities were normalized to protein content (30Abel A. Wochnik G. Rüegg J. Rouyer A. Holsboer F. Rein T. Mol. Endocrinol. 2002; 16: 1352-1366Crossref PubMed Scopus (31) Google Scholar). The data were calculated as % stimulation (i.e. the stimulation in the absence of transfected immunophilin was set to 100%). Plasmids—The plasmids MTVLuc, green fluorescence protein (GFP)-GR, and pCMVβ-Gal have been described previously (33Wochnik G.M. Young J.C. Schmidt U. Holsboer F. Hartl F.U. Rein T. FEBS Lett. 2004; 560: 35-38Crossref PubMed Scopus (35) Google Scholar). All immunophilins and dynamitin were cloned downstream of the CMV promoter of the vector pRK5MCS. To create FLAG-tagged versions of the immunophilins, the reverse primer contained the sequence for the tag. The following are plasmids encoding chimeric proteins: Ch1 is a FKBP52 containing the first 138 amino acids of FKBP51 and Ch2 is a FKBP51 containing the first 138 amino acids of FKBP52. The PPIase domain fragments expressing plasmids were constructed by amplifying the cDNA coding for the first 138 amino acids, respectively, and cloning into the pRK5MCS vector. Details of the cloning procedures are available on request. Immunoprecipitation and Western Blot—For immunoprecipitation of FKBPs, HEK cells were transfected with 10 μg of a plasmid expressing a FLAG-tagged form of the protein of interest. The cells were solubilized in 1 ml of lysis buffer (20 mm Tris/HCl, pH 7.5, 130 mm NaCl, 20 mm Na2MoO4,1mm EDTA, 10% glycerol, 0.5% Triton X-100, 1:100 protease inhibitor mixture P2714 from Sigma). The extract was incubated for 1 h on ice, centrifuged for 4 min at 13,000 revolutions/min, and then the protein concentration was determined. Lysates were incubated with the anti-FLAG M2 affinity gel from Sigma (A2220) overnight at 4 °C. Beads were treated as recommended by the manufacturer. The next day, the beads were washed 3 times with 1× Tris-buffered saline, and samples were eluted with SDS-PAGE sample buffer without any reducing agent. For immunoblot detection, either 10 μg of the total protein of the whole cell lysates used for luciferase assays or FLAG immunoprecipitates were separated by SDS-PAGE under denaturing conditions. The proteins were transferred to nitrocellulose membrane (Schleicher & Schüll, GmbH). Nonspecific binding to membrane was blocked by 5% nonfat milk in Tris-buffered saline/Tween buffer, and then the following specific primary antibodies were added: Hsp90 (H-114, Santa Cruz Biotechnology); dynein (clone 70.1, Sigma); FLAG tag (M2, Sigma); hemagglutinin tag (Roche Applied Science); FKBP51 and FKBP52 (StressGen Biotech); and GR (H-300, Santa Cruz Biotechnology). Signals were visualized by appropriate secondary antibodies conjugated to horseradish peroxidase and the ECL system (Amersham Biosciences). For detection of coimmunoprecipitated and immunoprecipitated proteins on the same blotting membrane, the membrane was treated with stripping buffer (Pierce), after detection of the coprecipitated protein and the precipitated protein was detected as described above. Fluorescence Detection—HeLa cells were seeded on glass plates covered with 0.1% gelatin in a 6-well plate (∼2 × 105 cells/well) in steroid-free medium without phenol red. After 1 day, they were transfected using ExGen (Fermentas) as recommended by the manufacturer. 0.25 μg of GFP receptor and 0.75 μg of FKBP plasmid were used for each well. The cells were stimulated 24 h after transfection and then fixed with 4% paraformaldehyde. After 20 min, the cells were washed 2 times with incubation buffer (1× Tris-buffered saline/Tween buffer, 5% fetal calf serum, 1% bovine serum albumin, 1% Triton X-100) and then embedded in ProTaqs Mount Fluor (Biocyc GmbH & Co KG, Luckenwalde, Germany). For time-resolved analysis of nuclear translocation, we followed the protocol by Galigniana et al. (34Galigniana M.D. Radanyi C. Renoir J.M. Housley P.R. Pratt W.B. J. Biol. Chem. 2001; 276: 14884-14889Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). The cells were analyzed with a fluorescence microscope (Axioplan 2 imaging, Zeiss, Jena, Germany). To score the cytoplasmic/nuclear distribution of the fluorescing receptors, >100 cells were evaluated by a non-involved investigator according to a method adapted from the literature (11Galigniana M.D. Scruggs J.L. Herrington J. Welsh M.J. Carter-Su C. Housley P.R. Pratt W.B. Mol. Endocrinol. 1998; 12: 1903-1913Crossref PubMed Scopus (155) Google Scholar). 0 was assigned to a cell with a balanced distribution, + 1 for a cell with enhanced nuclear (–1 with enhanced cytoplasmic) fluorescence, and + 2 for a cell with exclusively nuclear (–2 with exclusively cytoplasmic) fluorescence. FKBP51 and FKBP52 Act Differentially on GR in Mammalian Cells—To set up a mammalian model system to investigate the effect of FKBP51 and FKBP52 on GR, we used the neuroblastoma cell line SK-N-MC, HEK cells, and HeLa cells in a GR-responsive reporter assay. In all of these cell lines, we observed a dose-dependent inhibition of GR-mediated transcription by FKBP51 as reported for COS cells (28Denny W.B. Valentine D.L. Reynolds P.D. Smith D.F. Scammell J.G. Endocrinology. 2000; 141: 4107-4113Crossref PubMed Scopus (181) Google Scholar), whereas FKBP52 had no effect (Fig. 1A). Overexpression of the carboxy terminus of Hsc70-interacting protein, another TPR domain protein, leads to degradation of GR through the proteasome machinery (35Connell P. Ballinger C.A. Jiang J. Wu Y. Thompson L.J. Höhfeld J. Patterson C. Nat. Cell Biol. 2001; 3: 93-96Crossref PubMed Scopus (0) Google Scholar). In yeast, overexpression of FKBP52 or FKBP51 increased the levels of GR. In our mammalian cells, overexpression of FKBP51 or FKBP52 did not change the level of GR (Fig. 1A). This excludes degradation of GR as an explanation for the inhibitory effect of FKBP51. We also observed that, in the presence of FKBP51, higher concentrations of cortisol were necessary to elicit a GR response (Fig. 1B). This reflects the change in Kd of GR hormone binding observed by others (28Denny W.B. Valentine D.L. Reynolds P.D. Smith D.F. Scammell J.G. Endocrinology. 2000; 141: 4107-4113Crossref PubMed Scopus (181) Google Scholar). We noted that, at saturating levels of cortisol, FKBP51 still slightly but significantly decreased GR activity, indicating an additional level of action of FKBP51 on GR (i.e. additive to the Kd effect). GR activities were the same for the vector control and FKBP52 coexpression (not shown). It was important to test whether the same pattern of activity of FKBP51 and FKBP52 can be observed on an endogenous GR-regulated gene as in our transient reporter system. We used the glucocorticoid-responsive cell lines HeLa and H4-II-E-C3 to measure the influence of additional FKBP51 or FKBP52 on the established GR target genes MT (metallothionein) and TAT (tyrosine aminotransferase), respectively (36Karin M. Haslinger A. Heguy A. Dietlin T. Imbra R. Experientia Suppl. 1987; 52: 401-405Crossref PubMed Scopus (15) Google Scholar, 37Nitsch D. Boshart M. Schutz G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5479-5483Crossref PubMed Scopus (161) Google Scholar). We observed that the FKBP51 abolished the enhancement of MT RNA after the addition of hormone, whereas FKBP52 had no significant effect (Fig. 1C and data not shown). Likewise, TAT activity was significantly reduced by FKBP51 but not by FKBP52 (Fig. 1C). Thus, the inhibitory action of FKBP51 is independent of the cell line used and, importantly, also independent of the promoter and its chromosomal context. Although FKBP52 enhanced GR signaling in yeast (27Riggs D.L. Roberts P.J. Chirillo S.C. Cheung-Flynn J. Prapapanich V. Ratajczak T. Gaber R. Picard D. Smith D.F. EMBO J. 2003; 22: 1158-1167Crossref PubMed Scopus (271) Google Scholar), the environment provided for GR activity is obviously different in mammals and yeast. Both WT and FLAG-tagged proteins produced indistinguishable results (not shown). The Inhibitory Activity of FKBP51 Requires Interaction with Hsp90 and Is Competed by FKBP52—Hsp90 dependence has been demonstrated for the FKBP52-induced enhancement of GR in yeast (27Riggs D.L. Roberts P.J. Chirillo S.C. Cheung-Flynn J. Prapapanich V. Ratajczak T. Gaber R. Picard D. Smith D.F. EMBO J. 2003; 22: 1158-1167Crossref PubMed Scopus (271) Google Scholar). However, GR and FKBP51/52 evolved only in higher organisms, and the immunophilins also exhibit hsp90-independent features (15Silverstein A.M. Galigniana M.D. Kanelakis K.C. Radanyi C. Renoir J.M. Pratt W.B. J. Biol. Chem. 1999; 274: 36980-36986Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 38Pirkl F. Buchner J. J. Mol. Biol. 2001; 308: 795-806Crossref PubMed Scopus (145) Google Scholar, 39Pirkl F. Fischer E. Modrow S. Buchner J. J. Biol. Chem. 2001; 276: 37034-37041Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). To test the hsp90 dependence of the FKBP51-inhibitory effect on GR in our mammalian reporter system, we replaced the positively charged amino acids lysine 352 and arginine 356 with alanines in the hsp90-interacting TPR repeat motif, (see Fig. 6 for domain structure). The corresponding mutation in the TPR domain of protein phosphatase 5 has been shown to disrupt binding to hsp90 (40Russell L.C. Whitt S.R. Chen M.S. Chinkers M. J. Biol. Chem. 1999; 274: 20060-20063Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). The TPR-defective FKBP51 did not inhibit GR-dependent transcription (Fig. 2A). The protein was expressed at the same level as WT FKBP51 and coimmunoprecipitation confirmed that there is no interaction with hsp90 (Fig. 2A, lower panels). HEK and SK-N-MC cells yielded the same results. The requirement for hsp90 interaction was also confirmed by the inability of FKBP51 to inhibit a constitutively active GR that lacks the hsp90-interacting ligand binding domain (not shown).Fig. 2Inhibition of GR by FKBP51 requires interaction with hsp90 and is mitigated by FKBP52. A, SK-N-MC cells were transfected with the reporter plasmid MTVLuc, increasing amounts of a plasmid expressing FKBP51 with a double point mutation in the TPR domain (K352A/R356A), the β-galactosidase plasmid, and the GR plasmid (pRK7GR). After transfection, the cells were cultivated for 16 h in the presence of 10 nm cortisol. Upper panel, luciferase data were normalized by the β-galactosidase activities and are presented as relative activities ± S.E. of five independent experiments performed in duplicate. Middle panel, Western blot of cell extracts probed with an anti-FLAG antibody, demonstrating increasing expression of the mutant FKBP51. Lower panel, Western blot of immunoprecipitations (IP) from wtFKBP51 and mutant FKBP51-expressing cells using anti-FLAG beads. The blot was probed with an anti-FLAG antibody to control the efficiency of the precipitation and with an hsp90 antibody. B, HEK cells were transfected with the reporter plasmid MTVLuc, the GR plasmid pRK7GR, the β-galactosidase plasmid, 3 μg of the FKBP51 plasmid, and increasing amounts of the FKBP52 plasmid. The cells were then cultivated for 16 h in the presence of 10 nm cortisol. Upper panel, luciferase data were normalized by the β-galactosidase activities and are presented as relative activities ± S.E. of three independent experiments performed in duplicate. Lower panel, Western blot detection of untagged FKBP51 and FLAG-tagged FKBP52.View Large Image Figure ViewerDownload Hi-res image Download (PPT) There is strong evidence in vitro (41Nair S.C. Rimerman R.A. Toran E.J. Chen S. Prapapanich V. Butts R.N. Smith D.F. Mol. Cell. Biol. 1997; 17: 594-603Crossref PubMed Scopus (165) Google Scholar) and in yeast (27Riggs D.L. Roberts P.J. Chirillo S.C. Cheung-Flynn J. Prapapanich V. Ratajczak T. Gaber R. Picard D. Smith D.F. EMBO J. 2003; 22: 1158-1167Crossref PubMed Scopus (271) Google Scholar) that FKBP51 and FKBP52 compete with each other for binding to the GR heterocomplex. In other words, the activity of
