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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

The FKBP52 Domain Requirements for FKBP52/β-Catenin Co-Regulation of AR Activity.(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.
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pone.0134015.g005: The FKBP52 Domain Requirements for FKBP52/β-Catenin Co-Regulation of AR Activity.(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.

Mentions: Given that FKBP52 can potentiate AR activity through interaction with Hsp90, and in the absence of β-catenin in yeast, we aimed to determine if the pre-established domain and functional requirements for FKBP52 potentiation of AR activity alone directly mirror those of FKBP52 co-regulation with β-catenin. While both Hsp90 binding and the FKBP52 proline-rich loop are known to be critical, FKBP52 PPIase catalytic activity is not required [7, 12]. We took advantage of the FKBP52 mutants generated previously, including PPIase-deficient, Hsp90-binding deficient, and proline-rich loop mutants, to characterize the FKBP52 domain requirements for co-regulation with β-catenin using AR-mediated luciferase assays in 52KO MEF cells (Fig 5). The FKBP52 mutation F130Y abrogates PPIase activity without disrupting the conformation of the proline-rich loop, and without inhibiting FKBP52 potentiation of AR activity alone [12]. We assessed FKBP52 (F130Y) for the ability to co-regulate AR activity with β-catenin. The co-expression of FKBP52 (F130Y) with β-catenin (S33A) resulted in the synergistic up-regulation of AR-mediated luciferase reporter expression comparable to that observed with wild type FKBP52 both in the absence and presence of hormone (Fig 5A). Both wild type FKBP52 and FKBP52 (F130Y) overexpression resulted in a 9-fold enhancement of receptor-mediated reporter expression as compared to the vector control in the absence of hormone. Thus, the PPIase-deficient mutant can also support hormone-independent receptor activity. In addition, wild type FKBP52 and FKBP52 (F130Y) overexpression resulted in a 22-fold and 27-fold enhancement of hormone-dependent, receptor-mediated reporter expression respectively. These data are strikingly similar to those reported in Fig 3A. The co-expression of FKBP52 (K354A), which completely lacks Hsp90 binding [7], with β-catenin (S33A) also resulted in the synergistic up-regulation of AR-mediated luciferase reporter expression comparable to that observed with wild type FKBP52 both in the presence and absence of hormone (Fig 5B). In this assay, FKBP52 (K354A) overexpression was able to enhance hormone-independent receptor activity up to 7-fold in addition to enhancing hormone-dependent activity up to 15-fold. It is important to note that in this assay wild type FKBP52 overexpression did enhance hormone-independent receptor activity, but the increase was not statistically significant as compared to vector alone. This reflects the fact that, while a trend for increased hormone-independent activity is present in all assays, this effect is not always consistently significant. In contrast to that observed with FKBP52 alone, these data suggest that both PPIase catalytic activity and FKBP52 binding to Hsp90 are not required for the synergistic up-regulation of both hormone-independent and hormone-dependent AR activity by FKBP52 and β-catenin.


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)

The FKBP52 Domain Requirements for FKBP52/β-Catenin Co-Regulation of AR Activity.(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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134015.g005: The FKBP52 Domain Requirements for FKBP52/β-Catenin Co-Regulation of AR Activity.(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.
Mentions: Given that FKBP52 can potentiate AR activity through interaction with Hsp90, and in the absence of β-catenin in yeast, we aimed to determine if the pre-established domain and functional requirements for FKBP52 potentiation of AR activity alone directly mirror those of FKBP52 co-regulation with β-catenin. While both Hsp90 binding and the FKBP52 proline-rich loop are known to be critical, FKBP52 PPIase catalytic activity is not required [7, 12]. We took advantage of the FKBP52 mutants generated previously, including PPIase-deficient, Hsp90-binding deficient, and proline-rich loop mutants, to characterize the FKBP52 domain requirements for co-regulation with β-catenin using AR-mediated luciferase assays in 52KO MEF cells (Fig 5). The FKBP52 mutation F130Y abrogates PPIase activity without disrupting the conformation of the proline-rich loop, and without inhibiting FKBP52 potentiation of AR activity alone [12]. We assessed FKBP52 (F130Y) for the ability to co-regulate AR activity with β-catenin. The co-expression of FKBP52 (F130Y) with β-catenin (S33A) resulted in the synergistic up-regulation of AR-mediated luciferase reporter expression comparable to that observed with wild type FKBP52 both in the absence and presence of hormone (Fig 5A). Both wild type FKBP52 and FKBP52 (F130Y) overexpression resulted in a 9-fold enhancement of receptor-mediated reporter expression as compared to the vector control in the absence of hormone. Thus, the PPIase-deficient mutant can also support hormone-independent receptor activity. In addition, wild type FKBP52 and FKBP52 (F130Y) overexpression resulted in a 22-fold and 27-fold enhancement of hormone-dependent, receptor-mediated reporter expression respectively. These data are strikingly similar to those reported in Fig 3A. The co-expression of FKBP52 (K354A), which completely lacks Hsp90 binding [7], with β-catenin (S33A) also resulted in the synergistic up-regulation of AR-mediated luciferase reporter expression comparable to that observed with wild type FKBP52 both in the presence and absence of hormone (Fig 5B). In this assay, FKBP52 (K354A) overexpression was able to enhance hormone-independent receptor activity up to 7-fold in addition to enhancing hormone-dependent activity up to 15-fold. It is important to note that in this assay wild type FKBP52 overexpression did enhance hormone-independent receptor activity, but the increase was not statistically significant as compared to vector alone. This reflects the fact that, while a trend for increased hormone-independent activity is present in all assays, this effect is not always consistently significant. In contrast to that observed with FKBP52 alone, these data suggest that both PPIase catalytic activity and FKBP52 binding to Hsp90 are not required for the synergistic up-regulation of both hormone-independent and hormone-dependent AR activity by FKBP52 and β-catenin.

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