Journal: Molecular and Cellular Biology

Article Title: FKBP51 Promotes Assembly of the Hsp90 Chaperone Complex and Regulates Androgen Receptor Signaling in Prostate Cancer Cells

doi: 10.1128/mcb.01891-08

Figure Lengend Snippet: FIG. 1. FKBP51 is overexpressed in a xenograft model of AI prostate cancer. (A) Immunoblotting for chaperones in LAPC-4 AD and AI tumors grown in intact and castrated scid mice, respec- tively. The AD and AI tumors were propagated and harvested as described in Materials and Methods. The mouse number (#) refers to the particular animal from which the tumor was harvested. (B) Im- munocytochemistry of AD and AI tumors using an FKBP51 monoclo- nal antibody. (C) Real-time PCR to determine FKBP51, PSA, and S100P transcript levels in AD and AI tumors. FKBP51 overexpression was not detected in a previous microarray analysis of the LAPC-4 xenografts (3).

Article Snippet: FKBP51 Promotes Assembly of the Hsp90 Chaperone Complex and Regulates Androgen Receptor Signaling in Prostate Cancer Cells Li Ni, Chun-Song Yang, Daniel Gioeli, Henry Frierson, David O. Toft, and Bryce M. Paschal* Center for Cell Signaling and Departments of Microbiology and Pathology, University of Virginia School of Medicine, Charlottesville, Virginia 22908; Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905; and Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia 22908

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Over Expression, Microarray

Journal: Molecular and Cellular Biology

Article Title: FKBP51 Promotes Assembly of the Hsp90 Chaperone Complex and Regulates Androgen Receptor Signaling in Prostate Cancer Cells

doi: 10.1128/mcb.01891-08

Figure Lengend Snippet: FIG. 2. FKBP51 promotes AR-dependent transcription. (A) Effect of FKBP51 on AR activity in the absence (Control) or presence of 1 nM R1881. AR activity was measured by using the PSA-Luc reporter gene in PC-3 cells. AR, FKBP51, and FKBP52 levels were examined by immunoblotting (inset). FKBP51 stimulated AR activity on promoters, including the MMTV promoter, under conditions where GR activity was inhibited, as previously reported (6). (B) Effect of FKBP51 on AR activity measured by using AREs from the probasin promoter. (C) Effect of FKBP51 on AR activity using the MMTV promoter. (D) Effect of FKBP51 on GR activity using the MMTV promoter. (E) Effect of FKBP51 on AR activity using a promoter containing three synthetic AREs. (F) Effect of FK506 on FKBP51 and FKBP52 enhancement of AR activity measured by using the PSA promoter. The P values are denoted by asterisks (*, P 0.05; **, P 0.01) for effects of FKBP51 expression (A to E) and FK506 treatment (F). F/R, firefly/Renilla.

Article Snippet: FKBP51 Promotes Assembly of the Hsp90 Chaperone Complex and Regulates Androgen Receptor Signaling in Prostate Cancer Cells Li Ni, Chun-Song Yang, Daniel Gioeli, Henry Frierson, David O. Toft, and Bryce M. Paschal* Center for Cell Signaling and Departments of Microbiology and Pathology, University of Virginia School of Medicine, Charlottesville, Virginia 22908; Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905; and Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia 22908

Techniques: Activity Assay, Control, Western Blot, Expressing

Journal: Molecular and Cellular Biology

Article Title: FKBP51 Promotes Assembly of the Hsp90 Chaperone Complex and Regulates Androgen Receptor Signaling in Prostate Cancer Cells

doi: 10.1128/mcb.01891-08

Figure Lengend Snippet: FIG. 3. Androgen-dependent transcription and cell growth in prostate cancer cells engineered for FKBP51 expression. (A) Real-time PCR measurement of endogenous PSA transcript levels in LNCaP-lacZ and LNCaP-FKBP51 cell lines treated with the indicated concentrations of synthetic androgen. (B) Androgen-stimulated LNCaP cell growth of LNCaP-lacZ and LNCaP-FKBP51 cell lines measured by the MTT assay. (C) Real-time PCR to measure transcript levels of genes regulated by androgen and AR (KLK2, PSA, S100P, TMPRSS2, FKBP51). (D) Real-time PCR to measure transcript levels in CWR22Rv1 cells. The P values are denoted by asterisks (*, P 0.05; **, P 0.01) for effects of FKBP51 overexpression (A) and knockdown (C and D) on transcript levels. (E) Immunoblotting of FKBP51 and FKBP52 in control and knockdown cell lines.

Article Snippet: FKBP51 Promotes Assembly of the Hsp90 Chaperone Complex and Regulates Androgen Receptor Signaling in Prostate Cancer Cells Li Ni, Chun-Song Yang, Daniel Gioeli, Henry Frierson, David O. Toft, and Bryce M. Paschal* Center for Cell Signaling and Departments of Microbiology and Pathology, University of Virginia School of Medicine, Charlottesville, Virginia 22908; Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905; and Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia 22908

Techniques: Expressing, Real-time Polymerase Chain Reaction, MTT Assay, Over Expression, Knockdown, Western Blot, Control

Journal: Molecular and Cellular Biology

Article Title: FKBP51 Promotes Assembly of the Hsp90 Chaperone Complex and Regulates Androgen Receptor Signaling in Prostate Cancer Cells

doi: 10.1128/mcb.01891-08

Figure Lengend Snippet: FIG. 4. AR activation in response to diverse ligands is enhanced by FKBP51. (A) AR activity in response to the indicated ligands is mod- ulated by FKBP51. Activity was measured in PC-3 cells by using the reporter gene PSA-Luc. (B) AR activity in the presence of R1881 (1 nM) as a function of the Cas concentration was measured by using the reporter gene PSA-Luc. CPA, cyproterone acetate; Flu, flutamide; OH-Flu, hydroxyflutamide; ASD, androstenedione; DHT, dihydrotes- tosterone; E2, estrogen.

Article Snippet: FKBP51 Promotes Assembly of the Hsp90 Chaperone Complex and Regulates Androgen Receptor Signaling in Prostate Cancer Cells Li Ni, Chun-Song Yang, Daniel Gioeli, Henry Frierson, David O. Toft, and Bryce M. Paschal* Center for Cell Signaling and Departments of Microbiology and Pathology, University of Virginia School of Medicine, Charlottesville, Virginia 22908; Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905; and Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia 22908

Techniques: Activation Assay, Activity Assay, Concentration Assay

Journal: Molecular and Cellular Biology

Article Title: FKBP51 Promotes Assembly of the Hsp90 Chaperone Complex and Regulates Androgen Receptor Signaling in Prostate Cancer Cells

doi: 10.1128/mcb.01891-08

Figure Lengend Snippet: FIG. 5. FKBP51 promotes chaperone complex assembly. (A) Chap- erone composition of AR complexes during a time course of androgen treatment. PC-3 cells were transfected with plasmids encoding AR and FKBP51 and treated with R1881 for the times indicated. AR com- plexes were isolated by IP and immunoblotted for chaperones. (B) In vitro assembly of chaperone complexes. Recombinant chaperone pro-

Article Snippet: FKBP51 Promotes Assembly of the Hsp90 Chaperone Complex and Regulates Androgen Receptor Signaling in Prostate Cancer Cells Li Ni, Chun-Song Yang, Daniel Gioeli, Henry Frierson, David O. Toft, and Bryce M. Paschal* Center for Cell Signaling and Departments of Microbiology and Pathology, University of Virginia School of Medicine, Charlottesville, Virginia 22908; Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905; and Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia 22908

Techniques: Transfection, Isolation, In Vitro, Recombinant

Journal: Molecular and Cellular Biology

Article Title: FKBP51 Promotes Assembly of the Hsp90 Chaperone Complex and Regulates Androgen Receptor Signaling in Prostate Cancer Cells

doi: 10.1128/mcb.01891-08

Figure Lengend Snippet: FIG. 6. The LBD is required for AR association with the FKBP51- p23-Hsp90 superchaperone complex. PC-3 cells were transfected with full-length AR and AR lacking the LBD (ARLBD) and FKBP51 (or empty vector). After androgen treatment, AR complexes were isolated by IP and analyzed for chaperone content by immunoblotting.

Article Snippet: FKBP51 Promotes Assembly of the Hsp90 Chaperone Complex and Regulates Androgen Receptor Signaling in Prostate Cancer Cells Li Ni, Chun-Song Yang, Daniel Gioeli, Henry Frierson, David O. Toft, and Bryce M. Paschal* Center for Cell Signaling and Departments of Microbiology and Pathology, University of Virginia School of Medicine, Charlottesville, Virginia 22908; Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905; and Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia 22908

Techniques: Transfection, Plasmid Preparation, Isolation, Western Blot

Journal: Molecular and Cellular Biology

Article Title: FKBP51 Promotes Assembly of the Hsp90 Chaperone Complex and Regulates Androgen Receptor Signaling in Prostate Cancer Cells

doi: 10.1128/mcb.01891-08

Figure Lengend Snippet: FIG. 8. Point mutations that decrease FKBP51 activity reduce its abil- ity to enhance AR-dependent transcription and superchaperone complex assembly. (A) Diagram of FKBP51 showing the three major structural domains (black shading) and the positions of the point mutations that disrupt prolyl isomerase activity (M1; F67D, D68V) and binding to Hsp90 (M2; K352A, R356A). The M1/M2 form of FKBP51 contains both sets of point mutations. (B) PSA reporter assay with wild-type and mutant forms of FKBP51. Asterisks denote P values (**, P 0.01) comparing the activities of mutant forms of FKBP51 to that of wild-type FKBP51. The mutant forms of FKBP51 show the same reductions in activity relative to wild-type FKBP51 when the data are plotted as fold activation induced by R1881 (not shown). (C) Effects of FKBP51 point mutations on enhance- ment of superchaperone complex assembly. PC-3 cells were cotransfected with Flag-Hsp90 and untagged forms of FKBP51. Hsp90 complexes were isolated by IP and immunoblotted for chaperones (transfected FKBP51, endogenous p23). (D) Analysis of mutant forms of FKBP51 by using the PP2A-AR to assess the effect on androgen binding to AR. The amount of PP2A that coimmunoprecipitates with AR was plotted as a PP2A/AR ratio. The FKBP51 enhancement of androgen binding to AR obtained with (wild-type protein) was reduced in the M1, M2, and M1/M2 mutant forms of FKBP51. V, vector.

Article Snippet: FKBP51 Promotes Assembly of the Hsp90 Chaperone Complex and Regulates Androgen Receptor Signaling in Prostate Cancer Cells Li Ni, Chun-Song Yang, Daniel Gioeli, Henry Frierson, David O. Toft, and Bryce M. Paschal* Center for Cell Signaling and Departments of Microbiology and Pathology, University of Virginia School of Medicine, Charlottesville, Virginia 22908; Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905; and Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia 22908

Techniques: Activity Assay, Binding Assay, Reporter Assay, Mutagenesis, Activation Assay, Isolation, Transfection, Plasmid Preparation

Journal: Molecular and Cellular Biology

Article Title: FKBP51 Promotes Assembly of the Hsp90 Chaperone Complex and Regulates Androgen Receptor Signaling in Prostate Cancer Cells

doi: 10.1128/mcb.01891-08

Figure Lengend Snippet: FIG. 7. FKBP51 increases the number of AR molecules that bind androgen. (A) FKBP51 regulates androgen binding to AR. Whole-cell [3H]R1881-binding assays were performed with PC-3 cells transfected with AR with or without FKBP51. (B) PP2A in- teraction assay for measuring the relative number of androgen- bound AR molecules. PP2A undergoes selective transfer from small t antigen onto AR because the PP2A A subunit recognizes features of the LBD that are unique to the agonist-bound form (35). FKBP51 increases the number of AR molecules that bind androgen, resulting in an increase in the PP2A/AR ratio. FK506 inhibits FKBP51-stimulated formation of PP2A-AR complexes and FKBP51 enhancement of AR- dependent transcription (see Fig. 2F).

Article Snippet: FKBP51 Promotes Assembly of the Hsp90 Chaperone Complex and Regulates Androgen Receptor Signaling in Prostate Cancer Cells Li Ni, Chun-Song Yang, Daniel Gioeli, Henry Frierson, David O. Toft, and Bryce M. Paschal* Center for Cell Signaling and Departments of Microbiology and Pathology, University of Virginia School of Medicine, Charlottesville, Virginia 22908; Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905; and Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia 22908

Techniques: Binding Assay, Transfection

Journal: Molecular and Cellular Biology

Article Title: FKBP51 Promotes Assembly of the Hsp90 Chaperone Complex and Regulates Androgen Receptor Signaling in Prostate Cancer Cells

doi: 10.1128/mcb.01891-08

Figure Lengend Snippet: FIG. 9. Androgen-dependent growth enhancement by FKBP51 is prevented by chaperone inhibitors. (A) LNCaP-lacZ cells were plated and grown in the absence or presence of a low androgen concentration (50 pM R1881). Inhibitors of FKBP51 (FK506, 1 M) and Hsp90

Article Snippet: FKBP51 Promotes Assembly of the Hsp90 Chaperone Complex and Regulates Androgen Receptor Signaling in Prostate Cancer Cells Li Ni, Chun-Song Yang, Daniel Gioeli, Henry Frierson, David O. Toft, and Bryce M. Paschal* Center for Cell Signaling and Departments of Microbiology and Pathology, University of Virginia School of Medicine, Charlottesville, Virginia 22908; Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905; and Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia 22908

Techniques: Concentration Assay