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

Storer Samaniego C, Suh JH, Chattopadhyay A, Olivares K, Guy N, Sivils JC, Dey P, Yumoto F, Fletterick RJ, Strom AM, Gustafsson JÅ, Webb P, Cox MB - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences and Border Biomedical Research Center, The University of Texas at El Paso, El Paso, Texas, United States of America; Department of Chemistry and Biochemistry, Kettering University, Flint, Michigan, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

FKBP52 is Specifically Required for β-Catenin Potentiation of AR Activity.(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.
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pone.0134015.g003: FKBP52 is Specifically Required for β-Catenin Potentiation of AR Activity.(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.

Mentions: Since the AR BF3 surface is the putative binding and/or regulatory site for FKBP52 [13] and both FKBP52 and β-catenin are known positive regulators of AR, it is possible that FKBP52 and β-Catenin work in concert at this surface. Thus, we aimed to assess the effect of FKBP52 on β-catenin potentiation of AR activity. The fkbp52-deficient mouse embryonic fibroblast (52KO MEF) cell line provides a true FKBP52-negative background in which to assess the ability of β-catenin to potentiate AR activity. Thus, we assessed the ability of β-catenin or the degradation-resistant β-catenin (S33A) mutant to potentiate hormone-dependent and hormone-independent AR-mediated luciferase reporter gene expression in the presence or absence of FKBP52 (Fig 3A). While overexpression of β-catenin, or β-catenin (S33A), alone had no significant effect on AR-mediated luciferase expression in this system, the co-expression of FKBP52 with β-catenin, or β-catenin (S33A), resulted in an impressive synergistic up-regulation of both hormone-dependent (17- and 25-fold enhancement respectively as compared to vector alone) and hormone-independent (10-fold enhancement as compared to vector alone) AR activity. Based on these data, we conclude that β-catenin potentiation of AR activity in 52KO MEF cells requires the presence of FKBP52, and that this co-regulation can promote hormone-independent receptor-mediated reporter expression in this system.


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

Storer Samaniego C, Suh JH, Chattopadhyay A, Olivares K, Guy N, Sivils JC, Dey P, Yumoto F, Fletterick RJ, Strom AM, Gustafsson JÅ, Webb P, Cox MB - PLoS ONE (2015)

FKBP52 is Specifically Required for β-Catenin Potentiation of AR Activity.(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.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4514735&req=5

pone.0134015.g003: FKBP52 is Specifically Required for β-Catenin Potentiation of AR Activity.(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.
Mentions: Since the AR BF3 surface is the putative binding and/or regulatory site for FKBP52 [13] and both FKBP52 and β-catenin are known positive regulators of AR, it is possible that FKBP52 and β-Catenin work in concert at this surface. Thus, we aimed to assess the effect of FKBP52 on β-catenin potentiation of AR activity. The fkbp52-deficient mouse embryonic fibroblast (52KO MEF) cell line provides a true FKBP52-negative background in which to assess the ability of β-catenin to potentiate AR activity. Thus, we assessed the ability of β-catenin or the degradation-resistant β-catenin (S33A) mutant to potentiate hormone-dependent and hormone-independent AR-mediated luciferase reporter gene expression in the presence or absence of FKBP52 (Fig 3A). While overexpression of β-catenin, or β-catenin (S33A), alone had no significant effect on AR-mediated luciferase expression in this system, the co-expression of FKBP52 with β-catenin, or β-catenin (S33A), resulted in an impressive synergistic up-regulation of both hormone-dependent (17- and 25-fold enhancement respectively as compared to vector alone) and hormone-independent (10-fold enhancement as compared to vector alone) AR activity. Based on these data, we conclude that β-catenin potentiation of AR activity in 52KO MEF cells requires the presence of FKBP52, and that this co-regulation can promote hormone-independent receptor-mediated reporter expression in this system.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences and Border Biomedical Research Center, The University of Texas at El Paso, El Paso, Texas, United States of America; Department of Chemistry and Biochemistry, Kettering University, Flint, Michigan, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus