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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 Directly Interacts with β-Catenin to Promote Interaction with AR.(A) In vitro 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.
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pone.0134015.g002: FKBP52 Directly Interacts with β-Catenin to Promote Interaction with AR.(A) In vitro 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.

Mentions: Given that FKBP52 and β-catenin are predicted to co-regulate AR activity through overlapping surfaces we not only assessed the ability of FKBP52 to interact directly with β-catenin in vitro in the absence of other proteins (Fig 2A), but also the ability of FKBP52 to influence β-catenin interaction with AR (Fig 2B and 2C). Recombinant human FKBP52 co-precipitated with recombinant GST-tagged β-catenin in a cell-free system, but failed to precipitate on glutathione resin in the absence of GST-tagged β-catenin indicating a direct interaction between FKBP52 and β-catenin in the absence of other proteins (Fig 2A). In addition, the overexpression of FKBP52 in 293T cells synergistically enhanced (4.5-fold) Vp16-β-catenin interaction with Gal4-AR LBD in a mammalian two-hybrid assay as measured by hormone-dependent Gal4-mediated luciferase reporter gene expression, whereas overexpression of Vp16-β-catenin or FKBP52 alone did not significantly enhance hormone-dependent Gal4-responsive reporter expression in the presence of Gal4-AR LBD (Fig 2B). Given that this assay can only assess interactions in the presence of hormone-activated AR LBD, no conclusions regarding the ligand-dependence of the interactions can be gleaned from these data. To further validate these observations, we co-immunoprecipitated β-catenin with AR and FKBP52 from LNCaP prostate cancer cell lysates in the presence or absence of a transiently transfected siRNA targeting FKBP52. While the presence or absence of hormone had no effect on the ability of FKBP52, β-catenin and AR to form a complex, the knockdown of FKBP52 protein levels significantly abrogated β-catenin interaction with AR (Fig 2C). Thus, FKBP52 interacts directly with β-catenin in the absence of other factors and promotes β-catenin interaction with AR independent of ligand.


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 Directly Interacts with β-Catenin to Promote Interaction with AR.(A) In vitro 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134015.g002: FKBP52 Directly Interacts with β-Catenin to Promote Interaction with AR.(A) In vitro 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.
Mentions: Given that FKBP52 and β-catenin are predicted to co-regulate AR activity through overlapping surfaces we not only assessed the ability of FKBP52 to interact directly with β-catenin in vitro in the absence of other proteins (Fig 2A), but also the ability of FKBP52 to influence β-catenin interaction with AR (Fig 2B and 2C). Recombinant human FKBP52 co-precipitated with recombinant GST-tagged β-catenin in a cell-free system, but failed to precipitate on glutathione resin in the absence of GST-tagged β-catenin indicating a direct interaction between FKBP52 and β-catenin in the absence of other proteins (Fig 2A). In addition, the overexpression of FKBP52 in 293T cells synergistically enhanced (4.5-fold) Vp16-β-catenin interaction with Gal4-AR LBD in a mammalian two-hybrid assay as measured by hormone-dependent Gal4-mediated luciferase reporter gene expression, whereas overexpression of Vp16-β-catenin or FKBP52 alone did not significantly enhance hormone-dependent Gal4-responsive reporter expression in the presence of Gal4-AR LBD (Fig 2B). Given that this assay can only assess interactions in the presence of hormone-activated AR LBD, no conclusions regarding the ligand-dependence of the interactions can be gleaned from these data. To further validate these observations, we co-immunoprecipitated β-catenin with AR and FKBP52 from LNCaP prostate cancer cell lysates in the presence or absence of a transiently transfected siRNA targeting FKBP52. While the presence or absence of hormone had no effect on the ability of FKBP52, β-catenin and AR to form a complex, the knockdown of FKBP52 protein levels significantly abrogated β-catenin interaction with AR (Fig 2C). Thus, FKBP52 interacts directly with β-catenin in the absence of other factors and promotes β-catenin interaction with AR independent of ligand.

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