Title: Protein Phosphatase 5 Is a Major Component of Glucocorticoid Receptor·hsp90 Complexes with Properties of an FK506-binding Immunophilin
Abstract: Steroid receptors are recovered from hormone-free cells in multiprotein complexes containing hsp90, p23, an immunophilin, and often some hsp70. The immunophilin, which can be of the FK506- or cyclosporin A-binding class, binds to hsp90 via its tetratricopeptide repeat (TPR) domain, and different receptor heterocomplexes exist depending upon which immunophilin occupies the TPR-binding region of hsp90. We have recently reported that a protein serine/threonine phosphatase that is designated PP5 and contains four TPRs binds to hsp90 and is co-purified with the glucocorticoid receptor (GR) (Chen, M.-S., Silverstein, A. M., Pratt, W. B., and Chinkers, M. (1996) J. Biol. Chem. 271, 32315–32320). In this work, we show that PP5 is recovered with both GR that is nuclear and GR that is cytoplasmic in hormone-free cells. Approximately one-half of the GR·hsp90 heterocomplexes in L cell cytosol contains an immunophilin with high affinity FK506 binding activity, such as FKBP51 or FKBP52, and ∼35% contains PP5. Only a small (but undetermined) fraction of the native GR·hsp90 heterocomplexes contain the cyclosporin A-binding immunophilin CyP-40. PP5, FKBP52, and CyP-40 exist in separate heterocomplexes with hsp90, and competition binding experiments with the PP5 TPR domain suggest that the three proteins occupy a common binding site on hsp90. A 55-residue connecting region between the N-terminal TPR domain of human PP5 and its C-terminal phosphatase domain has 50% amino acid homology and 22% identity with the central portion of the peptidylprolyl isomerase domain of human FKBP52. Of the 9 residues in this portion of FKBP52 involved in high affinity interactions with FK506, 3 residues are retained and 4 have homologous substitutions in PP5. Although immunoadsorbed PP5 did not bind [3H]FK506, we found that both rabbit PP5 in reticulocyte lysate and purified rat PP5 were specifically retained by an FK506 affinity matrix. Thus, we propose that PP5 possesses properties of an immunophilin with low affinity FK506 binding activity and that it determines a major portion of the native GR heterocomplexes in L cell cytosol. Steroid receptors are recovered from hormone-free cells in multiprotein complexes containing hsp90, p23, an immunophilin, and often some hsp70. The immunophilin, which can be of the FK506- or cyclosporin A-binding class, binds to hsp90 via its tetratricopeptide repeat (TPR) domain, and different receptor heterocomplexes exist depending upon which immunophilin occupies the TPR-binding region of hsp90. We have recently reported that a protein serine/threonine phosphatase that is designated PP5 and contains four TPRs binds to hsp90 and is co-purified with the glucocorticoid receptor (GR) (Chen, M.-S., Silverstein, A. M., Pratt, W. B., and Chinkers, M. (1996) J. Biol. Chem. 271, 32315–32320). In this work, we show that PP5 is recovered with both GR that is nuclear and GR that is cytoplasmic in hormone-free cells. Approximately one-half of the GR·hsp90 heterocomplexes in L cell cytosol contains an immunophilin with high affinity FK506 binding activity, such as FKBP51 or FKBP52, and ∼35% contains PP5. Only a small (but undetermined) fraction of the native GR·hsp90 heterocomplexes contain the cyclosporin A-binding immunophilin CyP-40. PP5, FKBP52, and CyP-40 exist in separate heterocomplexes with hsp90, and competition binding experiments with the PP5 TPR domain suggest that the three proteins occupy a common binding site on hsp90. A 55-residue connecting region between the N-terminal TPR domain of human PP5 and its C-terminal phosphatase domain has 50% amino acid homology and 22% identity with the central portion of the peptidylprolyl isomerase domain of human FKBP52. Of the 9 residues in this portion of FKBP52 involved in high affinity interactions with FK506, 3 residues are retained and 4 have homologous substitutions in PP5. Although immunoadsorbed PP5 did not bind [3H]FK506, we found that both rabbit PP5 in reticulocyte lysate and purified rat PP5 were specifically retained by an FK506 affinity matrix. Thus, we propose that PP5 possesses properties of an immunophilin with low affinity FK506 binding activity and that it determines a major portion of the native GR heterocomplexes in L cell cytosol. In cytosols prepared from hormone-free cells, steroid receptors exist in multiprotein complexes that contain hsp90 1The abbreviations used are: hsp, heat shock protein; PPIase, peptidylprolyl isomerase; GR, glucocorticoid receptor; TPR, tetratricopeptide repeat; PR, progesterone receptor; PP5, protein phosphatase 5; FKBP, FK506-binding protein; CyP, cyclosporin A-binding protein; PAGE, polyacrylamide gel electrophoresis; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid. 1The abbreviations used are: hsp, heat shock protein; PPIase, peptidylprolyl isomerase; GR, glucocorticoid receptor; TPR, tetratricopeptide repeat; PR, progesterone receptor; PP5, protein phosphatase 5; FKBP, FK506-binding protein; CyP, cyclosporin A-binding protein; PAGE, polyacrylamide gel electrophoresis; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid. and some hsp90-associated proteins, including p23 and some high molecular weight immunophilins (for review see Refs. 1Pratt W.B. J. Biol. Chem. 1993; 268: 21455-21458Abstract Full Text PDF PubMed Google Scholar and 2Smith D.F. Toft D.O. Mol. Endocrinol. 1993; 7: 4-11Crossref PubMed Scopus (51) Google Scholar). The immunophilins are ubiquitous and conserved proteins that bind immunosuppressant drugs, such as FK506 and cyclosporin A (for review see Ref. 3Walsh C.T. Zydowsky L.D. McKeon F.D. J. Biol. Chem. 1992; 267: 13115-13118Abstract Full Text PDF PubMed Google Scholar). All members of the immunophilin family have peptidylprolyl isomerase (PPIase) activity, and there are two classes: the FKBPs that bind compounds like FK506 and rapamycin and the cyclophilins (CyPs) that bind cyclosporin A. The drugs bind to the isomerase site on the immunophilin and inhibitcis-trans isomerization in vitro (4Schmid F.X. Annu. Rev. Biomol. Struct. 1993; 22: 123-143Crossref PubMed Scopus (246) Google Scholar). The low molecular weight immunophilins, such as FKBP12 and CyP-18, are thought to be the cellular components responsible for the immunosuppression and are the most studied. Three high molecular weight immunophilins, FKBP52 (5Tai P.K. Maeda Y. Nakao K. Wakim N.G. Duhring J.L. Faber L.E. Biochemistry. 1986; 25: 5269-5275Crossref PubMed Scopus (189) Google Scholar, 6Yem A.W. Tomasselli A.G. Heinrikson R.L. Zurcher-Neely H. Ruff V.A. Johnson R.A. Deibel Jr., M.R. J. Biol. Chem. 1992; 267: 2868-2871Abstract Full Text PDF PubMed Google Scholar, 7Lebeau M.-C. Massol N. Herrick J. Faber L.E. Renoir J.-M. Radanyi C. Baulieu E.-E. J. Biol. Chem. 1992; 267: 4281-4284Abstract Full Text PDF PubMed Google Scholar, 8Tai P.K. Albers M.W. Chang H. Faber L.E. Schreiber S.L. Science. 1992; 256: 1315-1318Crossref PubMed Scopus (267) Google Scholar, 9Peattie D.A. Harding M.W. Fleming M.A. De Cenzo M.T. Lippke J.A. Livingston D.J. Benasutti M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10974-10978Crossref PubMed Scopus (228) Google Scholar) (also known as p59 and hsp56), FKBP51 (10Smith D.F. Baggenstoss B.A. Marion T.N. Rimerman R.A. J. Biol. Chem. 1993; 268: 18365-18371Abstract Full Text PDF PubMed Google Scholar, 11Smith D.F. Albers M.W. Schreiber S.L. Leach K.L. Deibel Jr., M.R. J. Biol. Chem. 1993; 268: 24270-24273Abstract Full Text PDF PubMed Google Scholar, 12Nair 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) (also known as avian p54), and CyP-40 (13Ratajczak T. Hlaing J. Brockway M.J. Hahnel R. J. Steroid Biochem. 1990; 35: 543-553Crossref PubMed Scopus (33) Google Scholar, 14Ratajczak T. Carrello A. Mark P.J. Warner B.J. Simpson R.J. Moritz R.L. House A.K. J. Biol. Chem. 1993; 268: 13187-13192Abstract Full Text PDF PubMed Google Scholar, 15Kieffer L.J. Seng T.W. Li W. Osterman D.G. Handschumacher R.E. Bayney R.M. J. Biol. Chem. 1993; 268: 12303-12310Abstract Full Text PDF PubMed Google Scholar), were discovered as components of steroid receptor heterocomplexes. In addition to the PPIase domains possessed by the low molecular weight immunophilins, these high molecular weight immunophilins possess three tetratricopeptide repeats (TPR) and a calmodulin-binding domain in their C-terminal half (14Ratajczak T. Carrello A. Mark P.J. Warner B.J. Simpson R.J. Moritz R.L. House A.K. J. Biol. Chem. 1993; 268: 13187-13192Abstract Full Text PDF PubMed Google Scholar, 15Kieffer L.J. Seng T.W. Li W. Osterman D.G. Handschumacher R.E. Bayney R.M. J. Biol. Chem. 1993; 268: 12303-12310Abstract Full Text PDF PubMed Google Scholar, 16Callebaut I. Renoir J.M. Lebeau M.C. Massol N. Burny A. Baulieu E.E. Mornon J.P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6270-6274Crossref PubMed Scopus (173) Google Scholar). TPRs are degenerate sequences of 34 amino acids that are often arranged in tandem repeats and are thought to be sites where intra- and intermolecular interactions occur (17Sikorski R.S. Boguski M.S. Goebl M. Hieter P. Cell. 1990; 60: 307-317Abstract Full Text PDF PubMed Scopus (391) Google Scholar). The high molecular weight immunophilins FKBP52 and CyP-40 bind to a site on hsp90 that binds a variety of proteins containing TPR domains (18Owens-Grillo J.K. Czar M.J. Hutchison K.A. Hoffmann K. Perdew G.H. Pratt W.B. J. Biol. Chem. 1996; 271: 13468-13475Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), and native immunophilin·hsp90 complexes exist in cytosols independent of the presence of steroid receptors. The existence of native immunophilin·hsp90 complexes is derived from the observation that immunoadsorption or affinity purification of FKBP52 or CyP-40 from cytosols yields co-retention of hsp90 (8Tai P.K. Albers M.W. Chang H. Faber L.E. Schreiber S.L. Science. 1992; 256: 1315-1318Crossref PubMed Scopus (267) Google Scholar, 18Owens-Grillo J.K. Czar M.J. Hutchison K.A. Hoffmann K. Perdew G.H. Pratt W.B. J. Biol. Chem. 1996; 271: 13468-13475Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 19Sanchez E.R. Faber L.E. Henzel W.J. Pratt W.B. Biochemistry. 1990; 29: 5145-5152Crossref PubMed Scopus (164) Google Scholar, 20Renoir J.M. Mercier-Bodard C. Hoffmann K. Le Bihan S. Ning Y.M. Sanchez E.R. Handschumacher R.E. Baulieu E.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4977-4981Crossref PubMed Scopus (88) Google Scholar), and immunopurification of hsp90 yields co-purification of FKBP52 and CyP-40 (21Perdew G.H. Whitelaw M.L. J. Biol. Chem. 1991; 266: 6708-6713Abstract Full Text PDF PubMed Google Scholar, 22Owens-Grillo J.K. Hoffmann K. Hutchison K.A. Yem A.W. Deibel Jr., M.R. Handschumacher R.E. Pratt W.B. J. Biol. Chem. 1995; 270: 20479-20484Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Purified FKBP52 and CyP-40 bind directly to purified hsp90 (22Owens-Grillo J.K. Hoffmann K. Hutchison K.A. Yem A.W. Deibel Jr., M.R. Handschumacher R.E. Pratt W.B. J. Biol. Chem. 1995; 270: 20479-20484Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 23Czar M.J. Owens-Grillo J.K. Dittmar K.D. Hutchison K.A. Zacharek A.M. Leach K.L. Deibel Jr., M.R. Pratt W.B. J. Biol. Chem. 1994; 269: 11155-11161Abstract Full Text PDF PubMed Google Scholar) at what is apparently a common binding site because they compete for the binding of each other (22Owens-Grillo J.K. Hoffmann K. Hutchison K.A. Yem A.W. Deibel Jr., M.R. Handschumacher R.E. Pratt W.B. J. Biol. Chem. 1995; 270: 20479-20484Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 24Ratajczak T. Carrello A. J. Biol. Chem. 1996; 271: 2961-2965Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Studies with mutant FKBP52 and CyP-40 showed that the region containing the three TPRs was required for binding to hsp90 (24Ratajczak T. Carrello A. J. Biol. Chem. 1996; 271: 2961-2965Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 25Radanyi C. Chambraud B. Baulieu E.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11197-11201Crossref PubMed Scopus (155) Google Scholar, 26Hoffmann K. Handschumacher R.E. Biochem. J. 1995; 307: 5-8Crossref PubMed Scopus (62) Google Scholar), and the binding of both immunophilins was competed by a fragment of CyP-40 comprising its three tetratricopeptide repeats (18Owens-Grillo J.K. Czar M.J. Hutchison K.A. Hoffmann K. Perdew G.H. Pratt W.B. J. Biol. Chem. 1996; 271: 13468-13475Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Thus, TPR domains are critical for immunophilin interaction with hsp90. Because hsp90 exists as a dimer, with dimerization being required for its function in vivo (27Minami Y. Kimura Y. Kawasaki H. Suzuki K. Yahara I. Mol. Cell. Biol. 1994; 14: 1459-1464Crossref PubMed Scopus (147) Google Scholar), it is conceivable that two different immunophilins could exist in the same hsp90 complex. However, under conditions where isolation of FKBP52 or CyP-40 from cytosol yields co-isolation of hsp90, there is no co-isolation of the other immunophilin (18Owens-Grillo J.K. Czar M.J. Hutchison K.A. Hoffmann K. Perdew G.H. Pratt W.B. J. Biol. Chem. 1996; 271: 13468-13475Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 20Renoir J.M. Mercier-Bodard C. Hoffmann K. Le Bihan S. Ning Y.M. Sanchez E.R. Handschumacher R.E. Baulieu E.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4977-4981Crossref PubMed Scopus (88) Google Scholar, 22Owens-Grillo J.K. Hoffmann K. Hutchison K.A. Yem A.W. Deibel Jr., M.R. Handschumacher R.E. Pratt W.B. J. Biol. Chem. 1995; 270: 20479-20484Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 28Johnson J.L. Toft D.O. J. Biol. Chem. 1994; 269: 24989-24993Abstract Full Text PDF PubMed Google Scholar). This is important because it suggests that there are different steroid receptor·hsp90 heterocomplexes depending upon the immunophilin that is bound. This prediction has been confirmed by direct studies that showed independent heterocomplexes of GR·hsp90·FKBP52 and GR·hsp90·CyP-40 (20Renoir J.M. Mercier-Bodard C. Hoffmann K. Le Bihan S. Ning Y.M. Sanchez E.R. Handschumacher R.E. Baulieu E.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4977-4981Crossref PubMed Scopus (88) Google Scholar, 22Owens-Grillo J.K. Hoffmann K. Hutchison K.A. Yem A.W. Deibel Jr., M.R. Handschumacher R.E. Pratt W.B. J. Biol. Chem. 1995; 270: 20479-20484Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). The binding of high molecular weight immunophilins to hsp90 via their TPR domains is conserved in plants as well as in the animal kingdom, suggesting that this may be a basic protein interaction that is critical to immunophilin function (29Owens-Grillo J.K. Stancato L.F. Hoffmann K. Pratt W.B. Krishna P. Biochemistry. 1996; 35: 15249-15255Crossref PubMed Scopus (73) Google Scholar). However, the biological function(s) of the high molecular weight immunophilins and the role they play as components of steroid receptor heterocomplexes has not been defined. It has been suggested that FKBP52 is involved in targeting the cytoplasmic nuclear trafficking of receptors (18Owens-Grillo J.K. Czar M.J. Hutchison K.A. Hoffmann K. Perdew G.H. Pratt W.B. J. Biol. Chem. 1996; 271: 13468-13475Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 30Pratt W.B. Czar M.J. Stancato L.F. Owens-Grillo J.K. J. Steroid Biochem. Mol. Biol. 1993; 46: 269-279Crossref PubMed Scopus (64) Google Scholar), and there is some evidence in support of this notion for the GR (31Czar M.J. Lyons R.H. Welsh M.J. Renoir J.M. Pratt W.B. Mol. Endocrinol. 1995; 9: 1549-1560PubMed Google Scholar). Some TPR proteins are components of the mitochondrial and peroxisomal import receptor complexes (32Lamb J.R. Tugendreich S. Hieter P. Trends Biochem. Sci. 1995; 20: 257-259Abstract Full Text PDF PubMed Scopus (545) Google Scholar), which may suggest a general usefulness for TPR domains in other aspects of protein trafficking. In 1994, three laboratories isolated cDNA clones encoding a protein serine/threonine phosphatase designated PP5 (protein phosphatase 5), which is distinctive in that it contains four TPRs in an N-terminal domain (33Becker W. Kentrup H. Klumpp S. Schultz J.E. Joost H.G. J. Biol. Chem. 1994; 269: 22586-22592Abstract Full Text PDF PubMed Google Scholar, 34Chen M.X. McPartlin A.E. Brown L. Chen Y.H. Barker H.M. Cohen P.T.W. EMBO J. 1994; 13: 4278-4290Crossref PubMed Scopus (250) Google Scholar, 35Chinkers M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11075-11079Crossref PubMed Scopus (135) Google Scholar). We have recently shown that immunoadsorption of PP5 from cytosols yields co-immunoadsorption of hsp90 and that the expressed TPR domain of PP5 is sufficient for binding to hsp90 (36Chen M.-S. Silverstein A.M. Pratt W.B. Chinkers M. J. Biol. Chem. 1996; 271: 32315-32320Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Like FKBP52 and CyP-40, PP5 was co-adsorbed with the glucocorticoid receptor from L cell cytosol, suggesting that it is also a component of receptor heterocomplexes (36Chen M.-S. Silverstein A.M. Pratt W.B. Chinkers M. J. Biol. Chem. 1996; 271: 32315-32320Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Because the TPR domain of PP5 is related to those of the hsp90-binding immunophilins (35Chinkers M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11075-11079Crossref PubMed Scopus (135) Google Scholar), we predicted that it might utilize the same binding site on hsp90 (36Chen M.-S. Silverstein A.M. Pratt W.B. Chinkers M. J. Biol. Chem. 1996; 271: 32315-32320Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). That TPR domainsper se are important for GR action is suggested by the fact that expression of the PP5 TPR domain in CV-1 cells has a dominant negative effect on GR-mediated activation of transcription from a reporter plasmid (36Chen M.-S. Silverstein A.M. Pratt W.B. Chinkers M. J. Biol. Chem. 1996; 271: 32315-32320Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). It is not known whether PP5 is a major component or a trace component of the GR heterocomplex profile or whether PP5 is related in any way to the immunophilins, other than by the possession of TPR domains. Previously, it has been determined that about half the PR heterocomplexes immunoadsorbed from chick oviduct cytosol (37Smith D.F. Faber L.E. Toft D.O. J. Biol. Chem. 1990; 265: 3996-4003Abstract Full Text PDF PubMed Google Scholar) or GR heterocomplexes immunoadsorbed from L cell cytosol (38Hutchison K.A. Scherrer L.C. Czar M.J. Ning Y. Sanchez E.R. Leach K.L. Deibel M.R. Pratt W.B. Biochemistry. 1993; 32: 3953-3957Crossref PubMed Scopus (59) Google Scholar) contains FKBPs. Also, we have previously reported that only a few GR·hsp90 complexes contain CyP-40, which binds rather weakly to hsp90 (18Owens-Grillo J.K. Czar M.J. Hutchison K.A. Hoffmann K. Perdew G.H. Pratt W.B. J. Biol. Chem. 1996; 271: 13468-13475Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 22Owens-Grillo J.K. Hoffmann K. Hutchison K.A. Yem A.W. Deibel Jr., M.R. Handschumacher R.E. Pratt W.B. J. Biol. Chem. 1995; 270: 20479-20484Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). In this work, we provide evidence that PP5 binds to the same site on hsp90 as the immunophilins and that complexes containing PP5 account for 35% of the GR heterocomplexes in L cell cytosol. We also show that a region in the center of PP5 has some homology with the FK506 binding site of FKBP52 and that PP5 binds in a specific manner to an FK506 affinity matrix. This leads us to suggest that PP5 has properties of an immunophilin with a low affinity FK506 binding activity and that it accounts for one of every three native GR·hsp90 heterocomplexes in L cell cytosol. Untreated rabbit reticulocyte lysate was from Green Hectares (Oregon, WI). [6,7-3H]Triamcinolone acetonide (35.4 Ci/mmol), [propyl-3H]FK506 (95 Ci/mmol), and125I-conjugated goat anti-mouse and anti-rabbit IgGs were from DuPont NEN. Iron-supplemented bovine calf serum was from HyClone Laboratories, Inc. (Logan, UT). Actigel ALD (activated aldehyde agarose) affinity support for protein immobilization was purchased from Sterogene Biochemicals (San Gabriel, CA). Powdered Dulbecco's modified Eagle's medium (high glucose), goat anti-mouse IgG-horseradish peroxidase conjugate, monoclonal nonimmune IgG and IgM, nonimmune rabbit serum, and protein A-Sepharose were from Sigma. Donkey anti-rabbit IgG-horseradish peroxidase conjugate was from Pierce. Ampholytes were from Bio-Rad. Immobilon-P was from Millipore (Bedford, MA). Cyclosporin A was provided by Sandoz Research Institute (East Hanover, NJ), and FK506 was from Alexis Biochemicals (San Diego, CA). The AC88 monoclonal IgG against hsp90 was from StressGen (Victoria, British Columbia). The BuGR2 IgG monoclonal antibody against the GR, the 3G3 monoclonal IgM against hsp90, and the anti-cyclophilin 40 (C-terminal peptide) antibody were from Affinity Bioreagents (Golden, CO). The anti-FLAG M2 monoclonal IgG was from IBI (New Haven, CT). The DS14F5 monoclonal antibody against p60 (39Smith D.F. Sullivan W.P. Marion T.N. Zaitsu K. Madden B. McCormick D.J. Toft D.O. Mol. Cell. Biol. 1993; 13: 869-876Crossref PubMed Scopus (246) Google Scholar) and Escherichia coli expressing human p60 were kindly provided by Dr. David Smith (University of Nebraska, Omaha, NE). The EC1 monoclonal antibody against hsp56 (40Nakao K. Meyers J.E. Faber L.E. Can. J. Biochem. Cell Biol. 1985; 63: 33-40Crossref PubMed Scopus (48) Google Scholar) was kindly provided by Dr. Lee Faber (Medical College of Ohio). The UPJ56 rabbit antiserum against hsp56 (41Ruff V.A. Yem A.W. Munns P.L. Adams L.D. Reardon I.M. Deibel Jr., M.R. Leach K.L. J. Biol. Chem. 1992; 267: 21285-21288Abstract Full Text PDF PubMed Google Scholar) was a kind gift from Dr. Karen Leach (The Upjohn Co., Kalamazoo, MI). Hybridoma cells producing FiGR monoclonal IgG against the GR (42Bodwell J.E. Orti E. Coull J.M. Pappin D.J.C. Smith L.I. Swift F. J. Biol. Chem. 1991; 266: 7549-7555Abstract Full Text PDF PubMed Google Scholar) were generously provided by Dr. Jack Bodwell (Dartmouth Medical School, Lebanon, NH). Rabbit antiserum to PP5, purified FLAG-PP5, and the FLAG-tagged TPR domain of rat PP5 were prepared as described previously (36Chen M.-S. Silverstein A.M. Pratt W.B. Chinkers M. J. Biol. Chem. 1996; 271: 32315-32320Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). The WCL2 line of Chinese hamster ovary cells overexpressing the mouse GR was established by Hirst et al. (43Hirst M.A. Northrup J.P. Danielsen M. Ringold G.M. Mol. Endocrinol. 1990; 4: 162-170Crossref PubMed Scopus (49) Google Scholar). WCL2 cells and L929 mouse fibroblasts (L cells) were grown in monolayer culture in Dulbecco's modified Eagle's medium supplemented with 10% iron-supplemented calf serum. For WCL2 cells, this medium was supplemented with 3 μmmethotrexate and 40 μg/ml proline. Cells were harvested by scraping into Earle's balanced saline, washed once, suspended in 1.5 volumes of HE buffer (10 mm Hepes, 1 mm EDTA) or HEM (HE plus 20 μm sodium molybdate), and ruptured by Dounce homogenization. Homogenates were centrifuged for 1 h at 100,000 × g, with the supernatant from this step being the “cytosol” from which the GR was immunoadsorbed. The GR heterocomplex was immunoadsorbed from 400-μl aliquots of L cell cytosol or 300-μl aliquots of WCL2 cytosol by rotation for 2 h at 4 °C with 15% volume of covalently coupled FiGR-Actigel. The immune pellets were washed three times with 1 ml of TEGM buffer (10 mm TES, 50 mm NaCl, 4 mm EDTA, 10% (w/v) glycerol, 20 mm sodium molybdate, pH 7.6), and proteins were resolved by SDS-polyacrylamide gel electrophoresis. For immunoadsorption of hsp90 or immunophilins, aliquots (100 μl) of rabbit reticulocyte lysate were immunoadsorbed for 2 h at 4 °C to 7.5-μl pellets of Actigel-ALD precoupled with nonimmune mouse ascites or 3G3 anti-hsp90 IgM or to 8 μl of protein A-agarose prebound with the EC1 monoclonal IgG against FKBP52 (10%), anti-CyP-40 (5%), anti-PP5 serum (10%), nonimmune mouse IgG, or nonimmune rabbit serum. Immunopellets were washed twice by suspension in 1 ml of HEG buffer (10 mmHepes, pH 7.4, 1 mm EDTA, 10% glycerol) and centrifugation prior to gel electrophoresis. Immunopellets were boiled in SDS sample buffer, and proteins were resolved on 10 or 12% SDS-polyacrylamide gels. For two-dimensional gel electrophoresis, immunopellets were dissolved by incubating them in 9.5 murea, 2% (w/v) Nonidet P-40, 5% ampholytes (pH range 5–8), and 5% β-mercaptoethanol. Proteins were transferred to Immobilon-P membranes and probed with 1 μg/ml BuGR2 antibody for the GR, 1 μg/ml AC88 for hsp90, 0.1% UPJ56 or 1 μg/ml EC1 for hsp56, 0.1% PP5 antiserum for PP5, 1 μg/ml M2 monoclonal for the FLAG-PP5 and FLAG-TPR, 0.1% DS14F5 for p60, or 0.1% anti-cyclophilin 40 for CyP-40. The immunoblots were then incubated a second time with the appropriate125I-conjugated counter antibody to visualize the immunoreactive bands. Rabbit hsp90 was purified from brain cytosol by sequential chromatography over DE52, hydroxylapatite, and ATP-agarose exactly as described by Hutchisonet al. (44Hutchison K.A. Dittmar K.D. Czar M.J. Pratt W.B. J. Biol. Chem. 1994; 269: 5043-5049Abstract Full Text PDF PubMed Google Scholar). Aliquots (30 μl) of purified rabbit hsp90 (1 mg/ml) were immunoadsorbed to 7.5-μl pellets of Actigel precoupled with 75 μl of 3G3 ascites. Pellets were washed once with 1 ml of HE buffer and suspended in Hepes buffer, pH 7.4, plus 0.1% Nonidet P-40 in a final volume of 130 μl, including 30 μl of the hsp90-free DE52 fraction of rabbit brain cytosol containing p60, PP5, FKBP52, and CyP-40. This is DE52 fraction pool A as described by Dittmar et al. (45Dittmar K.D. Hutchison K.A. Owens-Grillo J.K. Pratt W.B. J. Biol. Chem. 1996; 271: 12833-12839Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). In experiments where binding of proteins to hsp90 was competed with the PP5 TPR domain, 7.5 μg of purified FLAG-tagged PP5 TPR in 30 μl of 20 mm Hepes, 1 mmdithiothreitol, 150 mm NaCl were added, maintaining the same final incubation volume of 130 μl. Samples were incubated on ice with occasional manual resuspension for 40 min, washed three times with 1 ml of HEG, and proteins were resolved by SDS-PAGE and Western blotting. Reconstitution of GR·hsp90 heterocomplexes was performed essentially as described previously (44Hutchison K.A. Dittmar K.D. Czar M.J. Pratt W.B. J. Biol. Chem. 1994; 269: 5043-5049Abstract Full Text PDF PubMed Google Scholar, 45Dittmar K.D. Hutchison K.A. Owens-Grillo J.K. Pratt W.B. J. Biol. Chem. 1996; 271: 12833-12839Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Immunoadsorbed GR was stripped of associated hsp90 by incubating the immunopellet for 2 h at 4 °C with 0.5m NaCl followed by two washes with 1 ml of Hepes buffer. Salt-stripped immune pellets were then incubated for 20 min at 30 °C with 50 μl of rabbit reticulocyte lysate plus 5 μl of an ATP-regenerating system (50 mm ATP, 250 mmcreatinine phosphate, 20 mm MgCl2, and 100 units/ml creatinine phosphokinase). The mixtures were resuspended every 5 min during the incubation. After the incubation, the pellets were washed three times with 1 ml of TEGM, and replicate washed pellets were assayed for steroid binding activity or GR-associated proteins. For assay of steroid binding activity, pellets were incubated with 50 nm [3H]triamcinolone acetonide in TEGM overnight on ice, washed twice with HEM buffer, and counted for radioactivity in a scintillation counter. Pellets from replicate incubations were analyzed by SDS-PAGE and Western blotting for GR, hsp90, PP5, FKBP52, and CyP-40. For bacterial lysates containing p60, cDNA for the 60-kDa human protein (IEF SSP 3521) cloned by Honoré et al. (46Honoré B. Leffers H. Madsen P. Rasmussen H.H. Vandekerckhove J. Celis J.E. J. Biol. Chem. 1992; 267: 8485-8491Abstract Full Text PDF PubMed Google Scholar), which is the homolog of the rabbit p60 (39Smith D.F. Sullivan W.P. Marion T.N. Zaitsu K. Madden B. McCormick D.J. Toft D.O. Mol. Cell. Biol. 1993; 13: 869-876Crossref PubMed Scopus (246) Google Scholar), was subcloned into a pET23C vector (Novagen) using theEcoRI and NotI sites. 2W. P. Sullivan and D. O. Toft, unpublished observations. This construct was used to transform E. coli strain BL21 (DE3), which harbors an integrated T7 polymerase gene. Control E. coli and bacteria expressing p60 were grown to an A 600 of 0.6, induced with isopropyl-β-d-thiogalactopyranoside for 3 h at 25 °C, and harvested. Bacterial lysates were prepared by sonication in phosphate-buffered saline, and aliquots were flash frozen and stored at −70 °C. The preparation of FK506 immobilized onto an Affi-Gel 10 matrix was described by Yem et al. (6Yem A.W. Tomasselli A.G. Heinrikson R.L. Zurcher-Neely H. Ruff V.A. Johnson R.A. Deibel Jr., M.R. J. Biol. Chem. 1992; 267: 2868-2871Abstract Full Text PDF PubMed Google Scholar), and the affinity matrix used here is the remainder of that original batch prepared at the Upjohn Co. Rabbit reticulocyte lysate (100 μl) or purified PP5 (100 μl, 10 μg/ml) was rotated with 100 μl of affinity resin at 4 °C for 2 h. The resin was then washed six times with 1 ml of HE buffer by rotating at 4 °C for 15 min on each wash. After washing, the resin was rotated at 4 °C for 15 min with 100 μl of vehicle (10 or 4% ethanol, 0.02% Triton X-100), cyclosporin A, or FK506 as indicated. The matrix was then stripped by incubating it with 100 μl of 0.1% trifluoroacetic acid for 1 h at 4 °C. Proteins eluted from 1-ml washes were contracted to 100 μl in a rapid vacuum apparatus. The prot