Functional Comparison of Human and Zebra Fish FKBP52 Confirms the Importance of the Proline-Rich Loop for Regulation of Steroid Hormone Receptor Activity

Previous studies demonstrated that the 52-kDa FK506-binding protein (FKBP52) proline-rich loop is functionally relevant in the regulation of steroid hormone receptor activity. While zebra fish (Danio rerio; Dr) FKBP52 contains all of the analogous domains and residues previously identified as critical for FKBP52 potentiation of receptor activity, it fails to potentiate activity. Thus, we used a cross-species comparative approach to assess the residues that are functionally critical for FKBP52 function. Random selection of gain-of-function DrFKBP52 mutants in Saccharomyces cerevisiae identified two critical residues, alanine 111 (A111) and threonine 157 (T157), for activation of receptor potentiation by DrFKBP52. In silico homology modeling suggests that alanine to valine substitution at position 111 in DrFKBP52 induces an open conformation of the proline-rich loop surface similar to that observed on human FKBP52, which may allow for sufficient surface area and increased hydrophobicity for interactions within the receptor–chaperone complex. A second mutation in the FKBP12-like domain 2 (FK2), threonine 157 to arginine (T157R), also enhanced potentiation, and the DrFKBP52-A111V/T157R double mutant potentiated receptor activity similar to human FKBP52. Collectively, these results confirm the functional importance of the FKBP52 proline-rich loop, suggest that an open conformation on the proline-rich loop surface is a predictor of activity, and highlight the importance of an additional residue within the FK2 domain

The FKBP52 Cochaperone Acts in Synergy with β-Catenin to Potentiate Androgen Receptor Signaling.

FKBP52 and β-catenin have emerged in recent years as attractive targets for prostate cancer treatment. β-catenin interacts directly with the androgen receptor (AR) and has been characterized as a co-activator of AR-mediated transcription. FKBP52 is a positive regulator of AR in cellular and whole animal models and is required for the development of androgen-dependent tissues. We previously characterized an AR inhibitor termed MJC13 that putatively targets the AR BF3 surface to specifically inhibit FKBP52-regulated AR signaling. Predictive modeling suggests that β-catenin interacts with the AR hormone binding domain on a surface that overlaps with BF3. Here we demonstrate that FKBP52 and β-catenin interact directly in vitro and act in concert to promote a synergistic up-regulation of both hormone-independent and -dependent AR signaling. Our data demonstrate that FKBP52 promotes β-catenin interaction with AR and is required for β-catenin co-activation of AR activity in prostate cancer cells. MJC13 effectively blocks β-catenin interaction with the AR LBD and the synergistic up-regulation of AR by FKBP52 and β-catenin. Our data suggest that co-regulation of AR by FKBP52 and β-catenin does not require FKBP52 PPIase catalytic activity, nor FKBP52 binding to Hsp90. However, the FKBP52 proline-rich loop that overhangs the PPIase pocket is critical for synergy

FKBP52 Directly Interacts with β-Catenin to Promote Interaction with AR.

<p>(A) <i>In vitro</i> GST-pull down assays were performed with purified, recombinant FKBP52 alone, GST-Tagged β-catenin alone, and both recombinant proteins together. Proteins were visualized on Western Blots with primary antibodies specific to human FKBP52 and β-catenin. (B) A mammalian two-hybrid assay assessing the DHT-dependent activity of a Gal4-mediated luciferase reporter in the presence or absence of a Gal4-AR LBD fusion, Vp16-β-catenin and/or FKBP52 demonstrating that FKBP52 potentiates VP-16-β-catenin/AR interaction in 293 cells. Asterisks (***) denote that hormone-dependent reporter expression in the presence of FKBP52, Vp16-β-catenin, and Gal4-AR LBD was significantly enhanced (p values ranging from < 0.01 to < 0.001) as compared to all other conditions. Hormone-dependent reporter expression in the presence of Gal4-AR LBD with FKBP52 or β-catenin alone did not significantly differ (p > 0.05) from the control with Gal4-AR LBD alone. C. A co-immunoprecipitation to detect β-catenin interaction with FKBP52 and AR with and without DHT and FKBP52 siRNA in LNCaP cell lysates. β-catenin was immunoprecipitated and blots probed for AR, FKBP52 or β-catenin. Inputs are shown at bottom. Note that FKBP52 knockdown results in reduced AR/β-catenin interaction despite similar levels of input.</p

The FKBP52-Specific AR Inhibitor MJC13 Blocks β-Catenin Interaction with AR and Potentiation of AR Activity.

<p>(A) A co-IP assay to detect β-catenin interaction with Gal4-AR LBD in 293 cells. Proteins were immunoprecipitated with an anti-Gal4 antibody in lysates from 293 cells treated with or without indicated concentrations of DHT and MJC13. AR LBD was precipitated and blots probed for AR or β-catenin +/- DHT and MJC13. Inputs are shown at bottom. (B) A mammalian two-hybrid assay assessing the DHT-dependent activity of a Gal4-mediated luciferase reporter in the presence of a Gal4-AR LBD fusion with and without Vp16-β-catenin and 30 μM MJC13. MJC13 significantly (***p < 0.001) inhibits hormone-dependent Vp16 β-catenin/Gal4-AR LBD interaction in 293. Both conditions in the presence of Vp16 β-catenin were significantly (p < 0.001) enhanced as compared to hormone-dependent activity in the presence of Gal$-AR LBD alone. (C) 52KO MEFs were co-transfected with FKBP52, β-catenin (S33A), wild type AR, the AR-inducible luciferase reporter plasmid, and the constitutively active β-galactosidase reporter plasmid. After a 1 hour soak with a range of concentrations of MJC13, cells were induced with 10 pM DHT or ethanol for 16 hours. Following cell lysis, AR expression was assessed by luciferase assay. The data represent the average reporter expression (luciferase activity/β-galactosidase activity +/- standard deviation) of four replicates. MJC13 significantly (p < 0.001) inhibited hormone-dependent activity at all concentrations at or above 1 μM. MJC13 significantly inhibited hormone-independent activity at the 2.5 μM (p < 0.05), 5 μM (p < 0.01), and 10 μM (p < 0.05) concentrations.</p

FKBP52 is Specifically Required for β-Catenin Potentiation of AR Activity.

<p>(A) AR-mediated luciferase assay in 52KO MEFs in the presence of the indicated transiently transfected expression plasmids with (Black bars) or without (grey bars) dihydrotestosterone (DHT). The asterisks denote a statistically significant difference (***p < 0.001; ****p < 0.0001) as compared to vector alone for each hormone condition. Hormone-dependent receptor activity in the presence FKBP52 and β-catenin also significantly differed as compared to activity in the presence of FKBP52 and β-catenin (S33A) (p < 0.001). The activity in the presence of FKBP52 and wild type or mutant β-catenin was also significantly higher in the presence of hormone than in the absence (p < 0.0001). All other conditions did not significantly differ from the vector alone control, or from each other for each hormone condition. (B) DHT-dependent activity of a Gal4-mediated luciferase reporter in the presence or absence of a Gal4-AR LBD fusion, β-catenin (S33Y) and/or FKBP52 was assessed in HeLa cells. The asterisks denote a statistically significant difference (**p < 0.01; ***p < 0.001) as compared to Gal4-AR LBD alone in the presence of DHT. Hormone-dependent Gal4-AR LBD activity in the presence of both β-catenin (S33Y) and FKBP52 was also significantly (p < 0.001) potentiated as compared to activity in the presence of either β-catenin (S33Y) or FKBP52 alone. (C) The same as in (B), except that transient, siRNA-mediated FKBP52 knockdown was assessed instead of overexpression. The asterisks denote a statistically significant difference (***p < 0.001) as compared to Gal4-AR LBD alone in the presence of DHT. Hormone-dependent Gal4-AR LBD activity in the presence of both β-catenin (S33Y) and Si-FKBP52 was also significantly (p < 0.001) reduced as comparecd to activity in the presence of β-catenin (S33Y) alone. (D) As a control for AR specificity, β-catenin (S33Y) potentiation of TCF4-mediated luciferase activity in HeLa cells was assessed in the presence or absence of FKBP52 overexpression. The asterisks denote a statistically significant (***p < 0.001) potentiation of TCF4-mediated luciferase activity as compared to all other conditions in the absence of β-catenin (S33Y). TCF4-mediated luciferase activity in the presence of β-catenin (S33Y) was not statistically (p > 0.05) different in the presence or absence of FKBP52.</p

FKBP52 is Required for β-Catenin Potentiation of AR in 22Rv1 Prostate Cancer Cells.

<p>(A) AR-mediated probasin-luciferase activity was assessed at a range of hormone concentrations in 22Rv1 prostate cancer cells stably transfected with a 19 base pair shRNA directed against FKBP52 (52KD) or wild type 22Rv1 cells. Significant differences at each hormone concentration are indicated (***p < 0.001). The upper panels show Western blots for AR (both the full length and truncated ARs are shown), FKBP52, and GAPDH as a loading control from 52KD and wild type 22Rv1 cell lysates. (B) A luciferase reporter assay using the AR-inducible probasin-luciferase reporter plasmid in wild type and 52KD 22Rv1 cells with and without overexpression of the indicated proteins in the presence or absence of 175 pM dihydrotestosterone (DHT). Reporter expression in the presence of β-catenin (S33A) and DHT is significantly different from all other conditions (p < 0.001). Wild type cells with empty vector alone in the presence of DHT also significantly differed from all other conditions with p values ranging from < 0.05 to < 0.001. No conditions in the presence of FKBP52 knockdown significantly differed from each other in pairwise comparisons (p>0.05).</p

The Predicted β-Catenin Binding Site on AR is Near the Putative FKBP52 Regulatory Surface.

<p>(A) Structure of the complex of nuclear receptor LRH-1 with β-catenin, PDB ID 3tx7. β-catenin is shown with a semi-transparent surface in yellow revealing its secondary structure elements as ribbons. LRH-1 is shown with blue ribbons for its secondary structure. The interfacial surface of these two molecules is shown in teal on the β-catenin surface. (B) Flufenamic acid from PDB ID 2PIT is shown in purple spheres as it binds to the androgen receptor shown with its secondary structure as teal colored ribbons. The Cα coordinates of androgen receptor in 2PIT were superimposed with the Cα coordinates of LRH-1 in 3TX7.</p

The FKBP52 Domain Requirements for FKBP52/β-Catenin Co-Regulation of AR Activity.

<p>(A-C) Wild type AR, the AR-inducible luciferase reporter plasmid, and the constitutively active β-galactosidase reporter plasmid were cotransfected simultaneously with each of the plasmids indicated for the different treatment groups in 52KO MEF cells. Cells were induced with 10 pM DHT or ethanol. Following cell lysis, AR expression was tested through a luciferase assay, followed by normalization to β-galactosidase activity. In all graphs, statistically significant differences as compared to the vector alone control for each hormone condition are denoted by asterisks (*p < 0.05; ***p < 0.001). (A) The assay was performed in the presence or absence of FKBP52, β-catenin (S33A), and the PPIase-deficient FKBP52 mutant FKBP52 (F130Y). The PPIase-deficient FKBP52 mutant retains the ability to synergize with β-catenin (S33A) indicating that PPIase enzymatic activity is not critical for synergy. (B) The assay was performed in the presence or absence of β-catenin (S33A), FKBP52, and the Hsp90 binding-deficient mutant FKBP52 (K354A). FKBP52 binding to Hsp90 is not required for the synergistic upregulation of AR activity by FKBP52 and β-catenin. C. The assay was performed in the presence or absence of β-catenin (S33A), FKBP52, FKBP51, and the FKBP51 (A116V/L119P) mutant. The FKBP51 gain of function mutant exhibits substantial synergism with β-catenin indicating that the FK1 domain and the proline-rich loop are important for synergy.</p