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FK506-Binding Protein 52 Phosphorylation: A Potential Mechanism for Regulating Steroid Hormone Receptor Activity

Mayo Clinic Arizona (M.B.C., D.L.R., D.F.S.), S. C. Johnson Research Building, Scottsdale, Arizona 85259; Border Biomedical Research Center and Department of Biological Sciences (M.B.C.), University of Texas at El Paso, El Paso, Texas 79968; and Institut fur Organische Chemie und Biochemie (M.H., F.S., J.B.), Technische Universitat Munchen, 85747 Garching, Germany

Address all correspondence and requests for reprints to: Marc B. Cox, University of Texas at El Paso, Border Biomedical Research Center and Department of Biological Sciences, 500 West University Avenue, El Paso, Texas 79968. E-mail: mbcox{at}utep.edu.

	ABSTRACT

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Functional maturation of steroid hormone receptors requires ordered assembly into a large multichaperone complex consisting of receptor monomer, an Hsp90 dimer, the p23 cochaperone, and an FK506-binding protein (FKBP) family member or alternate peptidylprolyl isomerase-related cochaperone. Previous cellular studies demonstrated that FKBP52 can potentiate receptor function. These results have been confirmed in fkbp4 gene knockout mice in which males are partially androgen insensitive and females display characteristics of progesterone insensitivity. Conversely, FKBP51, which has a high degree of similarity to FKBP52, antagonizes FKBP52-mediated potentiation. Both proteins consist of three domains: two FKBP12-like domains termed FK1 and FK2 and a tetratricopeptide repeat domain that targets binding to Hsp90. To help understand why the two FKBPs behave differently and to gain insight into FKBP52 potentiation activity, we have analyzed the loop structure that links FK1 and FK2. Within the FK linker of FKBP52 is the sequence TEEED, which forms a consensus casein kinase II phosphorylation site; the corresponding sequence in FKBP51 is FED. We demonstrate that the distinct FK linker sequences per se do not account for lack of potentiation activity by FKBP51. However, phosphorylation of the FK linker appears to be an important regulatory determinant of FKBP52-mediated potentiation of steroid receptor activity.

	INTRODUCTION

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HEAT-SHOCK PROTEIN 90 (Hsp90), one of the most abundant molecular chaperones in vertebrates, is essential for regulating the folding and activity of a wide variety of important cellular proteins. In addition to the classic chaperone function, Hsp90 and its cochaperones also serve to regulate client protein activity. Proteins known to be clients for Hsp90 include steroid hormone receptors, aryl hydrocarbon receptor, Src-related tyrosine kinases, various serine/threonine kinases, telomerase, and nitric oxide synthase. A comprehensive Hsp90 client list is maintained by Didier Picard at the University of Geneva and can be accessed at http://www.picard.ch/downloads/Hsp90interactors.pdf. Much of our current understanding of interactions between Hsp90, cochaperones, and client proteins is modeled on extensive characterizations of steroid receptor complexes. Steroid hormone receptor folding to a functionally mature state requires ordered assembly with multimeric chaperone complexes (reviewed in Refs. 1 and 2). The final stage of receptor maturation, at which the receptor is competent for hormone binding, involves receptor assembly with Hsp90 and the cochaperone p23; additionally, the mature complex contains any one of several cochaperones that compete for binding the Hsp90 C terminus. These latter cochaperones share a tetratricopeptide repeat (TPR) domain that mediates binding to the highly conserved MEEVD sequence that terminates cytoplasmic Hsp90.

Among the TPR cochaperones observed in steroid receptor complexes are two large members of the FK506-binding protein (FKBP) family of peptidylprolyl isomerases (PPIases), FKBP52 and FKBP51. The FKBPs are characterized by a conserved domain that is the binding site for immunosuppressive drugs and has PPIase activity (3). Our lab has previously shown that FKBP52 has a stimulating effect on receptors for glucocorticoid (GR), androgen (AR), and progesterone (PR) in yeast and mammalian cell models. Potentiation of receptor activity requires both Hsp90 binding ability and an intact PPIase domain (4, 5, 6), but the mechanism of receptor potentiation is not known. The physiological importance of FKBP52 in steroid receptor complexes is supported by the fact that male mice lacking the gene encoding FKBP52 have ambiguous external genitalia, nipples retained into adulthood, and dysgenic prostate and seminal vesicles, features consistent with androgen insensitivity (6, 7). Female mice lacking FKBP52 are infertile due to uterine defects in implantation that result from progesterone insensitivity (5, 8). Our lab and others have shown that FKBP51 can inhibit FKBP52-mediated potentiation of receptor activity (4, 9, 10, 11) and that FKBP51 gene expression is inducible by some steroid hormones (9, 12, 13, 14, 15). In support of an inhibitory role for FKBP51, constitutive overexpression of FKBP51 underlies cortisol resistance in New World primates (16, 17). Thus, FKBP52 and FKBP51 have opposing effects on steroid receptor function and regulate receptor responsiveness to hormone.

Structural features that distinguish FKBP52 and FKBP51 actions in steroid receptor regulation are not well resolved. Amino acid sequence comparisons reveal the two FKBPs are approximately 60% identical and share approximately 75% sequence similarity. Comparison of x-ray crystallographic structures of full-length FKBP51 (18) and overlapping fragments of FKBP52 (19) reveals similar domain structures and arrangements. Both proteins contain an N-terminal domain that includes an active PPIase/FK506 binding site (FK1), a structurally related middle domain (FK2) that has a similar fold to FK1 but lacks drug binding and PPIase activity (20), and a C-terminal TPR domain that is required for Hsp90 binding. FKBP51 and FKBP52 have similar PPIase activity toward peptide substrates and Hsp90-binding ability (21). Given their structural and functional similarities, one might expect redundancy in FKBP function, but this is clearly not the case in relation to steroid receptor activity. Presumably, amino acid sequence differences account for the unique ability of FKBP52 to potentiate steroid hormone receptor function.

One structural distinction between the two FKBPs lies in a seven- to nine-amino-acid loop that connects FK1 and FK2 (FK linker). Beginning at amino acid 141, the respective FK linker sequences, with differences underlined, are DLTEE/EDGG (FKBP52) and DLFEDGG (FKBP51). Interestingly, FKBP52 is readily phosphorylated at T143 by casein kinase II (CKII) in vitro, and other evidence suggests that this phosphorylation blocks FKBP52 binding to Hsp90 (22). Another study presented evidence that the FKBP52 FK linker contributes to cytoskeletal interactions of the GR heterocomplex (23). Because the FK linker of FKBP51 differs in sequence and lacks a phosphorylation site, we elected to analyze this region of FKBP52 in greater detail to determine whether the FK linker and/or FK linker phosphorylation are functionally important for FKBP52-mediated potentiation of steroid receptor activity.

	RESULTS

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Comparative Analysis of FK Linker Sequences
Although highly similar, FKBP51 and FKBP52 have divergent roles with respect to the regulation of steroid receptor activity. In a search for functionally relevant sequence differences, we focused on distinct sequences (boxed in Fig. 1) within the linker between FK domains. The FK linker contains the sequence 143-TEEED in FKBP52 and 143-FED in FKBP51. As seen in an x-ray crystallographic structure of the FKBP52 FK domains (Fig. 2), the FK linker is in an accessible position that could accommodate interaction with proteins or other cellular factors. To test whether the FK linker of FKBP52 influences Hsp90 interactions or steroid receptor activity, we generated site-directed mutants targeting this region (Fig. 3). In one mutant, the distinctive TEE sequence of the FKBP52 FK linker was substituted by the corresponding F found in FKBP51 (52-LS); in another, E144 and E145 were substituted with arginines (52-RR) to test the importance of negative charges within the linker. When compared with wild-type FKBP52 (52WT), these mutants are unaffected in their ability to associate with general or p23-containing Hsp90 complexes in vitro (Fig. 3A). To assess FKBP52 mutant function in cells, we employed an established yeast model for FKBP52 activity (4) in which steroid receptors drive expression of a β-galactosidase reporter gene in a hormone-dependent and FKBP52-enhanced manner. We have previously shown that FKBP52 potentiates by severalfold transactivation of reporter activity by GR (4), PR (5), or AR (6). Additionally, the AR-P723S mutant, first identified in an individual with complete androgen insensitivity syndrome (24), was shown to require FKBP52 for functional rescue (6) and thus provides a hypersensitive marker for FKBP52 activity. As seen in Fig. 3B, 52-LS functions equally well as 52WT to potentiate the activities of GR and AR and to rescue the function of AR-P723S; FKBP52-RR also was fully functional in receptor potentiation assays (results not shown). We further tested whether an FKBP51 mutant containing the TEEED linker sequence would gain any potentiation activity, but none was observed. Therefore, we conclude that differences in the FK linker do not contribute to the functional differences in FKBP52 and FKBP51. On the other hand, as we show below, the FK linker of FKBP52 is a potentially important determinant for regulating the ability of FKBP52 to potentiate receptor function.

View larger version (96K): [in this window] [in a new window]   Fig. 1. Human FKBP51 and FKBP52 Amino Acid Sequence Alignment FKBP51 and FKBP52 are approximately 60% identical. The FK1 (PPIase), FK2, and TPR (Hsp90 binding) domains are shaded. The unshaded region between FK1 and FK2 is the FK linker, and the boxed region of the FK linker highlights sequences that differ in this region.

View larger version (39K): [in this window] [in a new window]   Fig. 2. Structure of the FKBP52 FK Domains Shown is a depiction of the x-ray crystallographic structure (Protein Databank accession no. 1Q1C) of an FKBP52 fragment containing FK1 and FK2 domains plus the intervening FK linker; the position of T143 is indicated.

View larger version (51K): [in this window] [in a new window]   Fig. 3. Function of FK Linker Mutants A, 35S-Labeled FKBP52 (WT, 52-LS, or 52-RR) were synthesized in vitro and added to normal rabbit reticulocyte lysate. Protein complexes were coimmunoprecipitated from the lysate mixtures using a nonspecific monoclonal antibody (control), a monoclonal antibody targeting total Hsp90 complexes, or a monoclonal antibody specific for p23 that precipitates the subset of Hsp90 complexes containing p23. Images shown are of Coomassie-stained SDS gel separations (upper panels), an autoradiograph of the same gels (middle panel, bound), and an autoradiograph of a gel containing aliquots of the FKBP52 synthesis products (lower panel, input). The migration positions for Hsp90 and the precipitating antibody heavy chains (HC) are indicated. B, Yeast carrying a steroid hormone-responsive β-galactosidase reporter plasmid and a GR, AR, or AR-P723S expression vector were cotransformed with empty vector (Vect.) or the indicated FKBP expression vector and assayed for hormone-dependent expression of β-galactosidase. Reporter expression was induced with 200 nM deoxycorticosterone (GR), 0.5 nM dihydrotestosterone (AR), or 10 nM dihydrotestosterone (AR-P723S). Data are plotted as fold induction over the level of β-galactosidase activity seen in cells carrying empty vector.

View larger version (28K): [in this window] [in a new window]   Fig. 4. Phosphospecific Antibody and CKII-Dependent Phosphorylation of FKBP52 A, Monospecific rabbit polyclonal antibody was prepared against a synthetic peptide corresponding to the FK linker of FKBP52 and containing phosphothreonine at the position equivalent to T143. Antibody titer was measured in an ELISA against phosphopeptide (pT143) or control peptide lacking phosphorylation (T143). B, Purified recombinant FKBP52 (amounts shown) was treated in vitro with or without CKII, as indicated. Protein was separated on SDS gels and immunoblotted with phosphospecific antibody (p52–62) or monoclonal antibody recognizing total FKBP52 (Hi52D). C, HeLa cells were transiently transfected with an empty plasmid vector (Vect.), plasmid expressing FKBP52 (52), or CKII (CKII) or a mixture of plasmids expressing both FKBP52 and CKII (52+CKII). Forty-eight hours after transfection, cell extracts were prepared and Western immunostained for phospho-FKBP52 (p52–62) or total FKBP52 (Hi52D). Purified recombinant FKBP52 that was treated or not with CKII in vitro (right panels) was Western immunostained in parallel with cell extracts (left panels).

View larger version (65K): [in this window] [in a new window]   Fig. 5. Binding of T143 Mutants to Hsp90 and Assembly with Steroid Receptor [35S]-labeled FKBP52 (52WT or harboring point mutations T143A or T143E) were synthesized in vitro and added to normal rabbit reticulocyte lysate. Protein complexes were coimmunoprecipitated from the lysate mixtures using a nonspecific monoclonal antibody (Control), a monoclonal antibody targeting total Hsp90 complexes (Hsp90), or a monoclonal antibody specific for p23 that precipitates the subset of Hsp90 complexes containing p23. In an additional set of samples, PR complexes were assembled in reticulocyte lysate mixtures and recovered with a monoclonal antibody specific for PR. Images shown are of Coomassie-stained SDS gel separations (upper panels), an autoradiograph of the same gels (middle panel, Bound), and an autoradiograph of a gel containing aliquots of the FKBP52 synthesis products (lower panel, Input). The migration positions for Hsp90, PR, Hsp70, and the precipitating antibody heavy chains (HC) are indicated.

View larger version (37K): [in this window] [in a new window]   Fig. 6. FKBP52 Mutant Activities in a Yeast Model of Steroid Receptor Function Yeast reporter strains for GR (A), AR (B), or mutant AR-P723S (C) were cotransformed with an empty plasmid vector (Vect.) or plasmid expressing FKBP51 (51WT), FKBP52 (52WT), or FKBP52 harboring the indicated point mutation (T143A or T143E). Yeast lysates were prepared and Western immunostained (upper panels) for the appropriate steroid receptor, total FKBP52, FKBP51, or, as a loading control, the yeast ribosomal protein L3. In parallel, yeast strains were induced with hormone and assayed for β-galactosidase activity (lower graphs). In all cases, receptor signaling in cells expressing 52WT and 52-T143A was significantly higher (P value < 0.001) compared with cells lacking FKBP (Vect.), expressing FKBP51, or expressing the FKBP52 phosphomimetic mutant 52-T143E.

View larger version (17K): [in this window] [in a new window]   Fig. 7. Phosphomimetic 52-T143E Fails to Potentiate Steroid Receptor Activity in MEF A and B, Either wild-type or 52KO MEF were cotransfected with a hormone-responsive luciferase reporter plasmid, a plasmid expressing either AR-P723S or PR-B, and an empty vector (Vect.) or plasmid expressing 52WT, 52-T143A, or 52-T143E. Cells were induced with or without 10 nM dihydrotestosterone (DHT) or 1 nM progesterone (P4), and cell lysates were assayed for luciferase activity. All data bars represent the average of triplicate samples. In all cases, receptor signaling in cells expressing 52WT or 52-T143A was significantly increased (P value < 0.05) compared with cells lacking exogenous FKBP52 (Vect.) or expressing the phosphomimetic mutant 52-T143E. C, HeLa Cells constitutively expressing a stable copy of the AR gene were cotransfected with a hormone-responsive luciferase reporter plasmid and an empty vector (Vect.) or plasmid expressing CKII. Cells were induced or not with 0.5 nM dihydrotestosterone (DHT), and cell lysates were assayed for luciferase activity. All data bars are representative of four replicate samples. Receptor signaling in cells expressing CKII was significantly reduced (P value < 0.001) compared with cells lacking exogenous CKII expression. All data in A–C are expressed as fold induction over luciferase activity observed in the absence of hormone.

Although the data presented above (Figs. 6 and 7, A and B) suggest that T143 phosphorylation abrogates FKBP52 potentiation of receptor function, it is possible that the phosphomimetic T143E mutation does not fully mimic actual T143 phosphorylation. To assess phosphorylation more directly, we attempted overexpressing exogenous CKII in MEF cells coexpressing receptor and reporter. However, using p52–62 to detect T143 phosphorylation of endogenous FKBP52 in cell extracts, no phospho-FKBP52 was detected in the MEF background (results not shown). Consistent with an apparent lack of FKBP52 phosphorylation, CKII overexpression had no effect on hormone-induced reporter activity. In an alternative approach, CKII was overexpressed in HeLa cells that contain an AR-luciferase reporter system. As shown in Fig. 4C, phospho-FKBP52 can be detected in this cellular background. In cells overexpressing CKII, we consistently observed an approximately 30% reduction in androgen-induced luciferase activity (Fig. 7C), which is consistent with a phosphorylation-dependent reduction in FKBP52-mediated potentiation of AR activity.

Phosphorylation-Induced Remodeling of the FK1 Catalytic Domain
Of the various mutations introduced in the FK linker, the only change that consistently and substantially impairs FKBP52 function is the phosphomimetic T143E substitution. 52-T143E retains binding to Hsp90 and assembles normally with PR in vitro (Fig. 5) yet fails to enhance receptor function in cells (Figs. 6 and 7). This mutation is unlikely to have a major impact on FKBP52 folding because protein-protein interactions are maintained, and mutant protein accumulates in cells at levels equivalent to 52WT. Nonetheless, mutation could impose a relevant conformational change, so we employed circular dichroism (CD) measurements and molecular dynamics simulations to compare the conformational states of purified recombinant FKBP52 variants. The far-UV CD spectra suggest no dramatic structural differences (Fig. 8A), although minor structural changes are apparent. The near-UV CD spectra further indicate that the major tertiary structure remains unchanged by the introduced mutations. However, there are clear differences in certain parts of the spectrum indicating local changes in structure (Fig. 8B).

View larger version (18K): [in this window] [in a new window]   Fig. 8. Structural Analysis of FKBP52 and Point Mutants by CD Spectroscopy Purified recombinant FKBP52 forms 52WT (solid line), 52-T143A (broken line), 52-T143E (dotted line) were analyzed by CD spectroscopy as described in Materials and Methods. A, Far-UV CD spectra of 5 µM purified recombinant protein; B, near-UV CD spectra of 20 µM protein.

View larger version (51K): [in this window] [in a new window]   Fig. 9. Predicted Structure Changes in FK Domains of 52-T143E Structures of the FK domains from 52WT and 52-T143E were derived by molecular dynamic modeling and overlaid to visualize differences. G139, which in 52-T143E loses contacts to a β-sheet in FK1, is highlighted in red.

	DISCUSSION

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Whereas our data suggest that FK linker phosphorylation abrogates FKBP52-mediated potentiation of steroid receptor activity, the mechanism by which FKBP52 activity is blocked is unclear. The steady-state level of FKBP52 in cells is unaffected by phosphomimetic mutation (Figs. 6 and 7), and there appears to be no major change in FKBP52 secondary or tertiary structure (Figs. 8 and 9). Furthermore, 52-T143E and 52WT assemble equally well into Hsp90 and steroid receptor complexes (Fig. 5). The PPIase activity of the phosphomimetic mutant 52-T143E was not significantly different from either 52WT or 52-T143A (Table 1). Thus, T143 phosphorylation appears unlikely to cause overt changes in PPIase catalytic activity. Nonetheless, structural analyses of 52-T143E do indicate allosteric rearrangements in FK1 that could possibly account for loss of receptor potentiation. Importantly, we know that FK1 is required for potentiation of steroid receptor response to hormone (4). We have recently observed that FKBP52-mediated potentiation is independent of PPIase activity but is highly sensitive to amino acid sequence changes in a loop of FK1 that overhangs the PPIase/FK506 binding pocket (Riggs, D. L., M. B. Cox, H. L. Tardif, M. Hessling, J. Buchner, and D. F. Smith, submitted for publication); allosteric conformational changes to the FK1 domain induced by phosphorylation of the FK linker could alter FK1 contacts critical for receptor potentiation.

The FKBP52 crystal structure reveals a hydrogen-bonding network between the carbonyl oxygen of T143 and the backbone amide of E146, between the T143 hydroxyl and D147 amide, and between Y225 and the side-chain carboxyl of D147 (Fig. 10). Based on the predicted structure of 52-T143E, phosphorylation of T143 would appear to introduce steric hindrance and disruption of the hydrogen bond network and structural reorientations within the FK linker and FK domains. The FK linker of 52-T143E adopts a widened architecture, and G139 loses contact with β-sheet structures in the FK1 domain, possibly increasing flexibility between FK1 and FK2 domains. Another anticipated structural change in FK1 is a twist in the short -helix that forms one side of the PPIase active site relative to β-sheet structures that complete the active site.

View larger version (68K): [in this window] [in a new window]   Fig. 10. Predicted Structural Differences in FKBP52 Variants Indicated are portions of the hydrogen bond network that interconnects FK domains and the FK linker in 52WT (A and C) and 52-T143E (B and D). The upper panels show the domains FK1 (orange) and FK2 (green) linked together by the FK linker (yellow). The lower panels are enlargements of the FK linker plus FK2 domain.

Potential Cellular Role for CKII in Regulating FKBP52 and Steroid Receptor Activities
Consistent with predictive phosphorylation site algorithms, we and others (22) have shown that FKBP52 T143 can be phosphorylated both in vitro and in cells by CKII. Although CKII was at one time thought to be largely an unregulated cellular kinase, more recent evidence indicates that CKII activity is regulated (reviewed in Ref. 26). CKII is a tetrameric enzyme consisting of two CKIIβ regulatory subunits and two CKII or CKII' catalytic subunits. In the absence of the regulatory subunit, CKII is constitutively active. One study demonstrated that inositol phosphates can significantly increase CKII activity (27), and another study showed that growing cells under hypoxic conditions not only caused the up-regulation of CKIIβ and relocalization of this subunit to the plasma membrane but also resulted in nuclear translocation of CKII and increased CKII activity (28). Finally, there is evidence that heat stress increases CKII activity by redistributing the individual subunits within the nucleus (29). These precedents for the regulation of CKII activity strengthen prospects that FKBP52 might be differentially phosphorylated by CKII in physiological settings.

The physiological significance of FKBP52 phosphorylation and conditions under which FKBP52 is phosphorylated in vivo are unknown. FKBP52 phosphorylation can be detected in HeLa cells (Fig. 4C) and COS-7 cells (22) that overexpress exogenous CKII, although in preliminary studies, we have not detected native FKBP52 phosphorylation using the phosphospecific antibody p52–62. More extensive analyses of the exact physiological conditions that favor FKBP52 T143 phosphorylation are merited given the attractive opportunity for regulating FKBP52 and steroid receptor activity. FKBP52 T143 phosphorylation may require specific stimuli and occur in a cell- and/or tissue-specific manner, which would make identification of physiological phosphorylation events more challenging.

In summary, we have identified differential phosphorylation of FKBP52 in the FK linker region as a potentially novel mechanism for regulating cellular responses to steroid hormones. The necessity for FKBP52 in both male and female reproductive processes of the mouse and the likely involvement of FKBP52 in pathological states sensitive to glucocorticoids, androgens, or progesterone encourages further study of the mechanisms by which FKBP52 alters steroid receptor function and mechanisms that regulate FKBP52 function.

	MATERIALS AND METHODS

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Antibody Production
A phosphopeptide corresponding to the human FKBP52 sequence ¹³⁷FKGEDLT(PO₄)EEEDGGI (150) was synthesized by the Mayo Clinic Peptide Synthesis Core Facility (Rochester, MN). A cysteine residue followed by a linker of two glycines was added to the N terminus to facilitate cross-linking. The peptide was cross-linked to keyhole limpet hemocyanin using the maleimide activated immunogen conjugation kit (Pierce, Rockford, IL), and was then used to immunize rabbits (Cocalico Biologicals, Inc., Reamstown, PA). The antibody was purified using a two-step protocol on columns containing Ultralink iodoacetyl gel (Pierce) cross-linked to either the phosphopeptide or the control peptide. The serum was applied to the control peptide column by gravity flow, and the flow-through was then applied to the phosphopeptide column. After extensive washing, antibody was eluted with 100 mM glycine (pH 2.5) and neutralized by the addition of 0.1 vol 1 M Tris (pH 7.5). Purified antibody was assayed for specificity to the phosphopeptide by an ELISA. In short, 96-well Immulon 4 HBX plates (Lab Systems, Franklin, MA) were coated overnight at 4 C in coating buffer consisting of carbonate/bicarbonate (pH 9.5) (Sigma Chemical Co., St. Louis, MO) plus either phosphopeptide or an identical peptide lacking phosphorylation (5 µg peptide per well). Coated wells were blocked in 2% BSA prepared in PBS and incubated with serum serially diluted in blocking buffer. Wells were washed three times in PBS supplemented with 0.05% Tween 20 and incubated with a horseradish peroxidase-conjugated secondary antirabbit IgG antibody (Pierce) diluted 1:10,000 in blocking buffer. After extensive washing, the wells were developed with 1-Step Ultra TMB-ELISA substrate (Pierce), and absorbance was measured at 450 nm.

Mutant cDNA Construction and Plasmids
An in vitro expression vector (pSPUTK; Stratagene, La Jolla, CA) containing the cDNA for human FKBP52 served as template for site-directed mutagenesis (QuikChange kit; Stratagene) to create all mutations in this study. For the loop swap mutation (52-LS) the TEEED sequence within the FK linker was replaced with FED, thereby making the linker identical to that of FKBP51. For another mutant (52-RR) glutamic acids 144 and 145 were replaced with arginines. The ability of the FKBP52 mutants to bind Hsp90 and to assemble in vitro with PR complexes were assessed by coimmunoprecipitation as described previously (30, 31).

Mammalian Cell Lines and Assays
MEFs were isolated from d-13 wild-type and homozygous 52KO embryos and immortalized as previously described (5). MEF or HeLa cells were cultured at 5% CO₂ in MEM supplemented with 10% fetal bovine serum and essential amino acids. All plasmid transfections were performed in six-well plates at approximately 80% confluence for 3 h using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions at a DNA (µg) to Lipofectamine (µl) ratio of 1:3 in MEM lacking fetal bovine serum. For Western immunoblot analyses, the cells were lysed 48 h after transfection as described below. For the receptor activity assays, MEF lines were transfected with the following plasmids (1 µg each plasmid per well): a hormone-responsive firefly luciferase reporter, a mammalian expression vector (pCI-neo; Promega, Madison, WI) constitutively expressing steroid hormone receptor, and pCI-neo constitutively expressing an FKBP variant. To control for transfection efficiency, each well was also transfected with 50 ng of a constitutive β-galactosidase expression plasmid. Twenty-four hours after transfection, cells were treated with hormone in ethanol carrier (concentration of ethanol in media never exceeded 0.01%). Approximately 16 h after hormone addition, cells were lysed by addition of M-PER (Pierce; 200 µl/well) and incubation at room temperature for 15 min. Luciferase activity was determined by addition of 100 µl luciferase assay reagent (Promega) to 10 µl cell lysate in an opaque 96-well plate; light emission was measured immediately in a luminescence plate reader (Lumicount, model BL70000; Packard Instrument Co., Meriden, CT). β-Galactosidase activity was measured by addition of 100 µl assay reagent (Gal-Screen; Tropix, Bedford, MA) to 6 µl lysate in an opaque 96-well plate. After 2 h at room temperature, plates were assayed in a luminescence plate reader. After normalizing for transfection efficiency (relative light units/β-galactosidase activity), data were plotted as fold induction of luciferase activity over background activity observed in the absence of hormone. Statistical significance was determined by one-way ANOVA followed by pair-wise comparisons using Bonferroni’s multiple comparisons test; P values 0.05 determine significant differences.

Yeast Strains and Assays
β-Galactosidase reporter assays used as a quantitative indicator of steroid hormone receptor activity were described previously (4). The designer deletion strain BY4741 (MATa his31 leu20 ura30 met150) with an additional TRP1 gene deletion was the parental strain for GR assays; W303 (MAT leu2-112 ura3–1 trp1–1 his3–11,15 ade2–1 can1-100 GAL SUC2) with a deleted PDR5 gene was the parental strain for AR assays; and W303a (MATa leu2-112 ura3-1 trp1-1 his3-11,15 ade2-1 can1-100 GAL SUC2) was the parental strain for the AR-P723S assays. Parental strains were cotransformed with three plasmids: a constitutive receptor expression plasmid (p415GPD-GR, p425GPD-hAR, or p425GPD-hAR-P723S), a hormone-inducible β-galactosidase reporter plasmid (pUCS-26x), and a plasmid constitutively expressing an FKBP variant. Hormone-induced reporter activity was quantitated in yeast extracts as described previously (4).

Immunoblots
Yeast cells in exponential phase growth (OD at 600 nm, OD₆₀₀, approximately equal to 1) were pelleted, resuspended in extract buffer [20 mM Tris (pH 7.5), 100 mM NaCl, 5% glycerol supplemented with protease inhibitors], and vortexed vigorously in the presence of glass beads. Cell extracts were clarified at 14,000 rpm for 20 min at 4 C. To prepare mammalian cell extracts, growth medium was replaced with protein extraction reagent (M-PER; Pierce) supplemented with protease inhibitors (Complete mini EDTA-free; Roche, Indianapolis, IN) and, in samples to be probed with phosphospecific antibody, phosphatase inhibitor cocktails I and II (Calbiochem, San Diego, CA). Protein concentrations in both yeast and mammalian extracts were determined using Coomassie Plus (Pierce). Typically, 20 µg total cellular protein per lane was separated on a 10–20% Criterion gel (Bio-Rad, Hercules, CA) and transferred to polyvinylidene difluoride membranes. Mouse monoclonal antibodies Hi52D and Hi51B (32) were used to detect FKBP52 and FKBP51, respectively. Specific antibodies to detect AR (PG-21) and GR (BuGR2) were obtained commercially (Affinity Bioreagents, Golden, CO). As protein loading controls, yeast extracts were blotted with antibody recognizing the L3 ribosomal subunit (33), and mammalian samples were blotted with antibody recognizing glyceraldehyde-3-phosphate dehydrogenase (6C5; Biodesign International, Saco, MN). The secondary antibody was an alkaline phosphatase-conjugated goat antimouse antibody (Pierce), and bands were visualized with Immun-Star AP substrate (Bio-Rad) and exposed to x-ray film.

Recombinant Protein Expression and Purification
His-tagged FKBP52 variants were expressed in Escherichia coli from pET28a⁺ vectors (Statagene). The protein variants were affinity purified from bacterial extracts on a Ni-NTA High Trap column (GE Healthcare, Munich, Germany). After loading and extensive washing with buffer A [50 mM sodium phosphate (pH 7.5), 200 mM NaCl], bound protein was eluted with buffer A plus 300 mM imidazole and dialyzed against 40 mM HEPES (pH 7.5) plus 50 mM NaCl. The eluate was loaded on a ResourceQ column (GE Healthcare) and eluted with a linear gradient from 50 mM to 1 M NaCl. The FKBP eluted at 300 mM NaCl and was judged by SDS-PAGE to be at least 99% pure.

CD Spectroscopy
Purified recombinant proteins were dialyzed against 5 mM sodium phosphate (pH 7.5). Spectra were recorded on a Jasco 715 spectropolarimeter (Jasco, Gross-Umstadt Germany) with a protein concentration of 5 µM for all FKBP52 variants at a temperature of 20 C. For the far-UV CD spectra, samples were loaded in a cuvette with a path length of 1 mm, and spectra were recorded from 260–195 nm. Near-UV CD spectra were recorded from 320–250 nm in a 5-mm path-length cuvette.

Protease-Coupled PPIase Assay
FKBP PPIase activities were measured as described previously (21). The synthetic peptide substrates were N-succinyl-Ala-Ile-Pro-Phe-p-nitroanilide and N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide. Assay mixtures contained purified recombinant FKBP (10–150 nM final concentration), substrate peptide (50 µM), and chymotrypsin (1 mg/ml) in 40 mM HEPES (pH 7.5) at 10 C. Because chymotrypsin cleaves only the peptide amide bond preceding the proline moiety in its trans conformation, cis-trans proline isomerization becomes rate limiting for cis peptide cleavage. The reaction was followed by generation of the free chromophore p-nitroanilide, which absorbs at 390 nm. Kinetic traces were fitted to a single exponential equation with floating end point. The experimentally observed rate (k_obs) values were plotted against FKBP52 concentration and fitted by a linear regression to determine the specificity constants.

Molecular Modeling
The structure of the FK1 and FK2 domains of FKBP52 (Protein Databank accession no. 1Q1C) was analyzed in a yamba2 force field with the program Yasara (34). The simulation cell was centered on the protein structure, and the protein distance from cell boundaries was at least 5 Å. With the pH set to 7.0 and a temperature of 298 K, the wild-type protein structure was analyzed in the molecular dynamic simulation force field for 7 nsec. No structural differences were noted between the simulated and crystal structures. To gauge the potential effects of phosphorylation, threonine at position 143 was converted to glutamic acid, and dynamic simulations were carried out under the same experimental conditions for 5 nsec.

	ACKNOWLEDGMENTS

 
We thank Brian Freeman for the yeast β-galactosidase reporter plasmid, Khalil Ahmed for the CKII expression plasmid, Jonathan Warner for the L3 antibody, and Tammy Brehm-Gibson in the Mayo Immunology Core facility for her advice on the development of the phosphospecific antibody.

	FOOTNOTES

 
This work was supported by National Institutes of Health (NIH) F32 DK068983 (M.B.C.), NIH R01 DK48218 (D.F.S.), the Mayo Foundation, and the DFG SFB594 (J.B.).

Disclosures: No author associated with this manuscript has potential conflicts of interests that need to be disclosed.

First Published Online August 23, 2007

Abbreviations: AR, Androgen receptor; CD, circular dichroism; CKII, casein kinase II; FKBP, FK506-binding protein; Hsp90, heat-shock protein 90; GR, glucocorticoid receptor; 52KO, fkbp52 null; MEF, mouse embryonic fibroblast; PPIase, peptidylprolyl isomerase; PR, progesterone receptor; TPR, tetratricopeptide repeat; 52WT, wild-type FKBP52.

Received for publication December 21, 2006. Accepted for publication August 13, 2007.

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NURSA Molecule Pages Link:

Nuclear Receptors:   GR  |  PR  |  AR

Ligands:   Dihydrotestosterone  |  Progesterone

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