The Hsp90-binding peptidylprolyl isomerase FKBP52 potentiates glucocorticoid signaling in vivo

Abstract

Hsp90 is required for the normal activity of steroid receptors, and in steroid receptor complexes it is typically bound to one of the immunophilin-related co-chaperones: the peptidylprolyl isomerases FKBP51, FKBP52 or CyP40, or the protein phosphatase PP5. The physiological roles of the immunophilins in regulating steroid receptor function have not been well defined, and so we examined in vivo the influences of immunophilins on hormone-dependent gene activation in the Saccharomyces cerevisiae model for glucocorticoid receptor (GR) function. FKBP52 selectively potentiates hormone-dependent reporter gene activation by as much as 20-fold at limiting hormone concentrations, and this potentiation is readily blocked by co-expression of the closely related FKBP51. The mechanism for potentiation is an increase in GR hormone-binding affinity that requires both the Hsp90-binding ability and the prolyl isomerase activity of FKBP52.

Fig. 1. Measurement of hormone-induced reporter activity. (A) Yeast strains were transformed with a hormone receptor expression plasmid, an immunophilin expression plasmid and a plasmid carrying a corresponding reporter gene. The receptor and immunophilin genes are transcribed from the strong constitutive glycerol phosphate dehydrogenase promoter. The reporter plasmid contains the lacZ gene transcribed from a truncated yeast cytochrome C1 promoter (P_CYC1) downstream of receptor-specific hormone response elements (HRE). Receptor complexes, which contain yeast Hsp90 and other chaperones plus an immunophilin, are activated by hormone binding. The active receptor binds to HRE and recruits RNA polymerase complex (RNAP) to drive transcription from the reporter plasmid. (B) β-galactosidase was induced in the GR reporter strain by addition of deoxycorticosterone (H) at the concentrations indicated. β-galactosidase activities, measured in chemiluminescent relative light units (RLU), are plotted as a function of OD₆₀₀. (C) To generate a DOC dose–response curve for the reporter gene expression rate, the slope (ΔRLU/ΔOD₆₀₀) was calculated for the linear portion of each induction curve in (B) and plotted relative to hormone concentration.

Fig. 2. Potentiation of GR signaling by the immunophilins. (A) Reporter expression rates of the GR reporter cells transformed with an empty vector or with plasmids expressing FKBP52, FKBP51, CPR7, CyP40 or PP5. Each bar is the average expression rate (±SD) from quadruplicate cultures of a representative isolate. (B) Steady state levels of GR in the parental strain W303a and GR reporter strains in FKBP51, FKBP52 or control vector backgrounds. Extracts from each strain were analyzed by western blotting for GR (upper panel) and for the ribosomal protein L3 as a loading control (lower panel). (C) Similar to (A) except that GR reporter cells were transformed with a single vector or two vectors (52+52, 51+52 or 52+PP5) for co-expression of immunophilins. (D) For each strain shown in (C), the total cell extract was immunostained for the proteins indicated.

Fig. 3. Hormone dose–response curves. (A) GR reporter cells expressing FKBP51 (GR/51) or FKBP52 (GR/52), or transformed with empty vector (GR/vector), were treated over a range of DOC concentrations. Reporter expression rates are normalized to the rate at the highest hormone concentration. Each data point is the average expression rate (n = 4 ± SD) that is representative of two independent isolates. (B) Reporter cells expressing wild-type GR or the F620S GR mutant were transformed with an expression plasmid for FKBP51 or FKBP52. Typical hormone dose–response curves are shown.

Fig. 4. Selectivity of FKBP52-dependent potentiation. (A) Yeast report er strains were prepared that express human ER (oval features), rat GR (rectangular features) or the ER.GR or GR.ER chimera. The approximate positions for the DBD (solid fill) and the LBD (hatched fill) in each receptor construct are illustrated. (B) Reporter strains expressing FKBP51 or FKBP52 or transformed with empty vector were compared for hormone-dependent β-galactosidase activity. The background level of reporter activity is shown for uninduced vector-only cells. Other bars represent reporter activity in vector, FKBP51 and FKBP52 cells induced with the following hormone concentrations: GR strains, 25 nM DOC; ER strains, 10 pM 17-β-estradiol; GR.ER, 450 pM 17-β-estradiol; ER.GR, 400 nM DOC. These data are the average (n = 4 ± SD) of duplicate assays from two independent isolates.

Fig. 5. The role of Hsp90 binding in FKBP52-mediated potentiation. (A) Major structural domains of human FKBP51. FKBP51 contains two FKBP12-like domains, FK1 and FK2, that have predominant β-sheet structure. FK1 forms the FK506-binding site and has PPIase activity; the 80S and 40S loop regions characteristic of FKBP12 are indicated. Six adjacent amino acids in the FK1 domain (broken lines) that correspond to the 40S loop of FKBP12 are unresolved. The N- and C-terminal ends of the molecule, as indicated, are also unresolved. FK2, which is structurally similar to FK1, lacks PPIase and FK506-binding activities. The C-terminal TPR domain is predominantly α-helical and forms the Hsp90-binding site; mutation of Lys-352 within the TPR domain will abrogate Hsp90 binding. FKBP52 is likely to have an overall structure very similar to FKBP51. (B) The FKBP52 point mutant K354A (corresponding to Lys-352 in FKBP51) was compared with wild-type FKBP52 for binding to Hsp90. Radiolabeled FKBP52 and FKBP52-K354A were prepared in vitro (first two lanes); aliquots of each synthesis mixture were tested for co-immunoprecipitation with Hsp90 complexes (final two lanes). Samples were separated by SDS–PAGE and visualized by Coomassie Blue staining (upper panel) or autoradio graphy (lower panel). (C) Hormone-induced β-galactosidase activity in GR reporter cells containing vector or plasmids expressing FKBP52 or FKBP52-K354A. All data are the average (n = 4 ± SD) of duplicate assays from two independent isolates.

Fig. 6. Mapping of FKBP52 domains required for potentiation. (A) Domain structures are illustrated for full-length FKBPs, FKBP52 truncation mutants and FKBP chimeric proteins. (B) Reporter expression rates were measured in separate GR reporter strains containing empty vector or expressing the indicated immunophilin product. Expression data are the average (n = 4 ± SD) of duplicate assays from two independent isolates. Expression levels for FKBP52 and truncation mutants were compared by western immunostaining. Owing to the loss of individual epitopes in truncation mutants, two anti-FKBP52 antibodies (Hi52b and Hi52d) were used along with an L3 probe as loading control. (C) Reporter expression rates were similarly determined in GR reporter strains expressing wild-type proteins or full-length chimeras containing the FK1 domain from FKBP52 (chimera 1) or FKBP51 (chimera 2). Expression of each form was verified by western immuno staining with anti-FKBP51 antibodies FF1 or Hi51b and anti-FKBP52 Hi52c. Note that FF1, which has an epitope in FK1 of FKBP51, detects wild-type FKBP51 and chimera 2, Hi51b, whose epitope is in the C-terminal region of FKBP51, detects FKBP51 and chimera 1, and Hi52c, with an epitope in FK1 of FKBP52 detects wild-type FKBP52 and chimera 2. Immunostaining of L3 (asterisk) provided an internal loading reference for each sample.

Fig. 7. Roles of dynein binding and PPIase activity in potentiation. (A) Reporter expression in cells containing empty vector, FKBP51 or FKBP52 in a wild-type DYN1 or dyn1-null background in response to 50 nM DOC. All data are the average (n = 4 ± SD) of duplicate assays from two independent isolates. (B) Cultures of GR reporter cells expressing the indicated immunophilin were treated with FK506 (10 µg/ml) for 30 min before addition of DOC (50 nM) and measurement of reporter activity (A and B normalized to the level of expression in vector-only cells). (C) Expression rates in GR reporter cells containing empty vector, FKBP51, FKBP52 or the PPIase-deficient mutant FKBP52-FD67DV. All data are the average (n = 4 ± SD) of duplicate assays from two independent isolates. The inset shows a western immunostain for FKBP52 or L3 which compares FKBP52 expression levels in the wild-type and mutant strains.

Fig. 8. Model for FKBP-mediated changes in GR hormone-binding affinity. FKBP52 or FKBP51 assemble with GR complexes through an interaction with Hsp90. The three major FKBP domains (FK1, FK2 and TPR) are indicated, as are the major domains of GR (N-terminal domain, DBD and LBD). Hsp90 is a multidomain protein that normally functions as a dimer but, for simplicity, is illustrated as a single entity. When FKBP52 is present in the GR complex, the active PPIase domain (FK1) of FKBP52 interacts with the LBD of GR, stimulating a conformational change and increase in hormone-binding affinity. Conversely, FKBP51 in the GR complex favors an alternative lower-affinity LBD conformation. The distinction between FKBP52 and FKBP51 is perhaps not in PPIase activity per se, but in the specificity of each for sequences within the LBD.