GCUNC-45 is a novel regulator for the progesterone receptor/hsp90 chaperoning pathway

Abstract

The hsp90 chaperoning pathway is a multiprotein system that is required for the production or activation of many cell regulatory proteins, including the progesterone receptor (PR). We report here the identity of GCUNC-45 as a novel modulator of PR chaperoning by hsp90. GCUNC-45, previously implicated in the activities of myosins, can interact in vivo and in vitro with both PR-A and PR-B and with hsp90. Overexpression and knockdown experiments show GCUNC-45 to be a positive factor in promoting PR function in the cell. GCUNC-45 binds to the ATP-binding domain of hsp90 to prevent the activation of its ATPase activity by the cochaperone Aha1. This effect limits PR chaperoning by hsp90, but this can be reversed by FKBP52, a cochaperone that is thought to act later in the pathway. These findings reveal a new cochaperone binding site near the N terminus of hsp90, add insight on the role of FKBP52, and identify GCUNC-45 as a novel regulator of the PR signaling pathway.

FIG. 1.

Identification of a novel PR-interacting protein, GCUNC-45. A. A schematic of the GCUNC-45 protein. The locations of three TPR sequences and four putative NR box motifs are indicated, as well as the UCS domain that is conserved in all UCS proteins (residues 522 to 944). In the protein sequence, the fragment identified in the yeast two-hybrid screen is residues 551 to 944. B. Interaction between GST-GCUNC-45 and PR-A, PR-B, and GR. HeLa cell lysates containing transfected PR-A, PR-B, or GR were incubated for 1 h at 4°C with glutathione-Sepharose containing bound GST-GCUNC-45 or GST alone in the presence of R5020, RU486, or dexamethasone, as indicated. Protein-bound Sepharose samples were pelleted and washed as indicated in Materials and Methods. Samples were resolved by SDS-PAGE, and PR and GR were visualized by immunoblotting. C. GCUNC-45 binds to PR in cell lysates and to purified PR. Endogenous GCUNC-45, PR-A, and PR-B were immunoprecipitated (IP) from T47D-YA or -YB cytosol using monoclonal antibodies to GCUNC-45 (UNC) and PR (PR). Mouse immunoglobulin G was used as a control (C). Proteins were detected by Western blotting. D. In vitro binding of GCUNC-45 to PR. Avian oviduct cytosol was incubated first with antibody (Ab) PR22-protein A-Sepharose resin to isolate PR. The stripped PR was then incubated with 20 μg of GCUNC-45 at the indicated temperatures. Resin-bound complexes were isolated by SDS-PAGE and stained with Coomassie blue.

FIG. 2.

TPR motifs of GCUNC-45 are essential for hsp90 binding. A. Increasing amounts of GCUNC-45, in duplicate, were added to samples of immobilized hsp90 (2 μg) and incubated for 40 min at (30°C). The protein complexes were isolated and resolved by SDS-PAGE, and Coomassie-stained bands were quantified by densitometry. The plot of arbitrary units (AU) of bound GCUNC-45 is shown and represents the mean values for each concentration. B. Scatchard analysis of the results in panel A. C. The binding of 20 μg of wild-type GCUNC-45 (lane 2) or fragment 1-284 (lane 4) or 616-944 (lane 6) to 3 μg of hsp90 was tested. Fragment 616-944 was fused with maltose-binding protein (MBP), and 20 μg of MBP was used as a control (lanes 7 and 8). Complexes were isolated using antibody resin to hsp90 and antibody resin without hsp90 was a control (C). Proteins were resolved by SDS-PAGE and stained with Coomassie blue. Lane 9 shows 1 μg of each protein. D. Comparison of the TPR motifs of human GCUNC-45 and SMUNC-45. Mutated amino acids are shaded. The positions of helices A and B are indicated according to Das et al. (9). E. Coimmunoprecipitation of 2 μg of wild-type GCUNC-45 (lane 3) and TPR1 (lane 5) and TPR2 (lane 7) mutants with 5 μg of hsp90 bound to H9010 antibody. Lanes 2, 4, and 6 represent GCUNC-45 resin controls without hsp90. F. Coimmunoprecipitation of wild-type GCUNC-45 (lane 3) and TPR1 (lane 5) and TPR2 (lane 7) mutants with avian PR-A/PR-B bound to antibody resin. Lanes 2, 4, and 6 show the background without PR. WT, wild type; C, control.

FIG. 3.

GCUNC-45 is required for optimal biological activity of PR. A. HeLa cells were transfected with 10 ng hPR1, 1 μg PRE₂-TATA_tk-LUC, and 250 ng pCH110 in the presence of increasing quantities of GCUNC-45 DNA as indicated. Luciferase and β-galactosidase activities were measured in harvested cell lysates after 16 to 20 h treatment of the cells with 10 nM R5020 (striped bars) or vehicle (open bars). Reporter activity is shown corrected for β-galactosidase activity. B to F. Cells were treated with ethanol vehicle, DharmaFECT1 reagent (FECT1), siRNA nontargeting control (NT Control), or siRNA specific to GCUNC-45 (siRNA UNC-45) SRC-1, SRC-2, FKBP51, or FKBP52. R5020 (250 nM) was added to the cells 72 h after transfection and incubated for an additional 18 h. Cells were harvested, protein levels were assessed by Western blot (B and D), and CAT enzyme production was measured by ELISA (C, E, and F). F. Time course of hormone treatment. R5020 (250 nM) was added 72 h after transfection with siRNA, and the cells were harvested at the indicated times and assessed for CAT enzyme production. EtOH, ethanol; KD, knockdown.

FIG. 4.

GFP-GCUNC-45 is a cytoplasmic protein. GFP-GCUNC-45 was transiently transfected into T47D-YB cells expressing high levels of PR-B. The cells were treated with R5020, RU486, or vehicle as indicated. Nuclei were stained blue using 4′,6′-diamidino-2-phenylindole. EtOH, ethanol.

FIG. 5.

hsp90 contains a second TPR-binding site near the N terminus. A. Hsp90 (3 μg) was bound to antibody resin and then saturated with GCUNC-45 (30 μg). Excess GCUNC-45 was removed, and samples were incubated with increasing amounts of Hop. Complexes were isolated and analyzed by SDS-PAGE. Gels were stained with Coomassie blue and scanned. Densitometry values for bound GCUNC-45 and Hop are plotted against the amount of Hop added in the reaction mixture. B. Hop and hsp90 were incubated with Hop antibody resin (F5). Excess protein was removed by centrifugation, and the resin complex was incubated with 10 μg of GCUNC-45 and 5 mM ADP for 30 min at 37°C. The complex was isolated and resolved by SDS-PAGE (lane 3). Background binding of hsp90 and GCUNC-45 to antibody resin without Hop is shown in lane 2. Lanes 4 to 6 show 1 μg of each protein, and lanes 1 and 7 show the protein standards. C. Schematic representation of the hsp90 domain of interaction and deletion fragments used to map the TPR-binding site. The ability to bind p23, Hop, or GCUNC-45 was tested as indicated. D. Immobilized GCUNC-45 (lanes 2, 4, 6, and 8) or Hop antibody complex (lanes 10, 12, and 14) was used to pull down wild-type hsp90, hsp90 fragments (1-222 or 206-728), or TRAP1, respectively. Nonspecific binding was assessed using immobilized BSA (lanes 1, 3, 5, and 7) or antibody without Hop (lanes 9, 11, and 13). The protein loads are also shown in lanes 15 to 18 (1/10 of the total). HC indicates the heavy chain of Hop antibody (F5). C, control; WT, wild type.

FIG. 6.

GCUNC-45 inhibits ATP hydrolysis by hsp90. A. ATPase activity was measured with time for 2 μM hsp90 alone (filled circles), hsp90, and 2 μM Aha1 (filled squares) or hsp90, Aha1, and 5.5 μM GCUNC-45 (open circles). B. Comparison of the inhibitory effects of GCUNC-45, Hop, and FKBP52 on hsp90 activated with Aha1. Hsp90 (2 μM) was incubated with 2 μM of Aha1 for 1 h at 37°C in the presence of the indicated ratio of protein. The broken line indicates the level of ATPase activity of hsp90 without Aha1.

FIG. 7.

GCUNC-45 is incorporated into chaperone complexes and inhibits the hormone-binding activity of PR. PR was reconstituted as described in Materials and Methods using the five-protein system (5P). GCUNC-45 (5 to 80 μg) was added to the protein mixtures. The recovered hormone-binding activity of PR is shown in panel A. B and C. FKBP52 relieved the inhibitory effect of GCUNC-45 in a concentration-dependent manner. Shown is the hormone-binding activity (B) and the protein composition of PR complexes analyzed by SDS-PAGE (C). HC indicates the heavy chain of Hop antibody (F5).

FIG. 8.

Model for PR chaperoning showing a transient association of GCUNC-45 during the progression from the intermediate to the mature complex. FKBP52, and possibly other cochaperones that bind hsp90 through TPR domains, may displace GCUNC-45 from PR complexes as a response to specific signals.