AR-V7 exhibits non-canonical mechanisms of nuclear import and chromatin engagement in castrate-resistant prostate cancer
- Department of Medicine, Weill Cornell Medical College, United States
- Department of Pathology, Weill Cornell Medical College, United States
- Meyer Cancer Center, Weill Cornell Medical College, United States
Figures
Figure 1 with 2 supplements
AR-V7 exhibits fast nuclear import kinetics independently of microtubules or the importin-α/β pathway, unlike AR-fl or AR-v567.
(A) Experimental design. Plasmids encoding GFP-tagged AR-fl, AR-v567, or AR-V7 were microinjected into the nuclei of the AR-null PC3 cells. As soon as GFP-tagged proteins were detected in the …
Figure 1—figure supplement 1
AR-V7 exhibits fast nuclear import kinetics independently of microtubules, actin, or the importin-α/β pathway.
(A) Plasmid encoding GFP-tagged AR-V7 was microinjected into nuclei of PC3 cells and as soon as GFP was detected in the cytoplasm (~45 min post micro-injection) the kinetics of GFP-AR-V7 nuclear …
Figure 1—figure supplement 2
Dominant negative IPO11 does not abrogate the nuclear import of AR-fl or AR-V7.
(A) Plasmid encoding mCherry tagged AR-fl and/or a dominant negative mutant IPO11 was microinjected into PC3 cells.; 45 mins post microinjection, representative images were captured by live -cell …
Figure 2 with 1 supplement
Inactivation of Ran-GTP-mediated nuclear transport affects differentially the subcellular localization of AR-variants.
(A, B) Plasmids encoding GFP-tagged AR-fl, AR-v567, or AR-V7 were co-microinjected with plasmid encoding the catalytic mutant mCherry-tagged RanQ69L, into the nuclei of the AR-null PC3 cells. Cells …
Figure 2—figure supplement 1
AR-V7 nuclear import requires active transport via the nuclear pore complex is dependent on Ran-GTP activity and is impaired upon mutation of the dimerization box domain (D-box).
(A) Wheat germ agglutinin (WGA) blocks AR-V7 nuclear import: we incubated cells with WGA, an inhibitor of nucleoporin-mediated nuclear transport and monitored GFP-AR-V7 localization by live-cell …
Figure 3 with 1 supplement
AR-V7 drives nuclear translocation of AR-fl in the absence of ligand.
(A, B) Plasmids encoding mCherry-AR-fl or GFP-AR-V7 were micro-injected in PC3 cells. Representative microscopic images (scale bar, 10 μm) and % nuclear AR is shown. Data represent a box plot with n>…
Figure 3—figure supplement 1
AR variants enhance nuclear localization of AR-fl in the absence of ligand.
(A) In a separate experiment, PC3 cells were transfected with plasmids encoding mCherry-AR-fl and/or GFP-AR-V7. % Nuclear AR-fl is graphically represented (n~7–14 cells/condition). Data represent …
Figure 4
DBD mutation abrogates AR-V7 transcriptional activity (A, B) PC3 cells stably expressing ARE-mCherry reporter were transfected with the indicated plasmids for 48 hr and the expression of GFP protein with concomitant mCherry protein was analyzed by confocal imaging.
Representative images and quantitative results are shown (n>10 cells per condition). Scale bar, 10 µm. (C) RT-qPCR for the endogenous FKBP5 mRNA was quantified in PC3 cells after transfection of …
Figure 5 with 1 supplement
AR-V7 exhibits high intranuclear mobility compared with liganded AR-fl.
(A) FRAP was performed in PC3 cells transiently expressing GFP-AR-fl (in the absence or presence of 10 nM R1881) or GFP-AR-V7. FRAP was monitored at 1 s intervals. Representative images of cells at …
Figure 5—figure supplement 1
AR-V7 intranuclear mobility is not affected by co-expression of ligand-bound AR-fl.
(A). FRAP was performed in PC3 cells co-microinjected with mCherry-AR-fl and GFP-AR-V7 followed by treatment with 10 nM R1881 for 2 hr. Representative images from the same single cell co-expressing …
Figure 6 with 1 supplement
DNA-binding mutation increases the intranuclear mobility and abrogates the transcriptional activity of AR-fl and AR-V7.
(A, B) FRAP was performed in PC3 cells transiently expressing GFP-AR-fl or GFP-AR-V7 or their respective DBD mutants (A573D). Kinetics of protein recovery after photobleaching are graphically …
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Figure 6—source data 1
DNA-binding mutation increases the intranuclear mobility and abrogates the transcriptional activity of AR-fl and AR-V7.
- https://cdn.elifesciences.org/articles/73396/elife-73396-fig6-data1-v2.pdf
Figure 6—figure supplement 1
DBD mutation increases the intranuclear mobility of liganded-AR-fl and AR-V7.
(A) FRAP was performed in the nuclei of PC3 cells following transient expression of GFP-AR-fl or GFP-AR-fl A573D (in the presence of 10 nM R1881) or GFP-AR-V7 or GFP-AR-fl A573D. Representative …
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | PC3 | ATCC | RRID: CVCL_4885 | |
| Cell line (H. sapiens) | LNCaP | ATCC | RRID: CVCL_0395 | |
| Cell line (H. sapiens) | C4-2 | ATCC | RRID: CVCL_4782 | |
| Cell line (H. sapiens) | HEK-293T | ATCC | RRID: CVCL_0063 | |
| Cell line (H. sapiens) | 22rv1 | ATCC | RRID: CVCL_1045 | |
| Cell line (H. sapiens) | M12 | ATCC | RRID: CVCL_4860 | |
| Antibody | (Rabbit polyclonal) anti-AR-N-terminal (AR-N-21) | This paper | Custom antibody produced by the Giannakakou Lab (1:500) | |
| Antibody | (Rabbit monoclonal) anti-AR-V7 | RevMab | Cat #: 31-1109-00 RRID: AB_2716436 | (1:500) |
| Antibody | (Rabbit monoclonal) anti-AR-C-terminal | Abcam | Cat #: ab52615 RRID: AB_867653 | (1:1000) |
| Antibody | (Rabbit polyclonal) anti-actin | Sigma-Aldrich | Cat #: A2066 RRID: AB_476693 | (1:500) |
| Antibody | (Rabbit polyclonal) anti-beta tubulin | Abcam | Cat #: ab6046 RRID: AB_2210370 | (1:500) |
| Antibody | (Rabbit polyclonal) anti-Histone H3 | Abcam | Cat #: ab1791 RRID: AB_302613 | (1:1000) |
| Antibody | (Rabbit polyclonal) anti-GFP | Novus Biologicals | Cat #: NB600-303 RRID: AB_10001300 | (1:500) |
| Antibody | (Mouse monoclonal) anti-mCherry 1C51 | Novus Biologicals | Cat #: NBP1-96752 RRID: AB_11034849 | (1:500) |
| Antibody | Wheat Germ Agglutinin (WGA) - Alexa Fluor 594 Conjugate | Molecular Probes | Cat #: W11262 RRID: AB_2334867 | 5 µg/ml |
| Antibody | (Goat anti-Rabbit) IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | Thermo Fisher Scientific | Cat#: A-11008 | (1:10,000) |
| Antibody | (Goat anti-Mouse) IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 | Thermo Fisher Scientific | Cat#: A-11004 | (1:10,000) |
| Chemical compound, drug | Docetaxel (Taxotere) | Sigma-Aldrich | Cat #: 01885 | 1 µM |
| Chemical compound, drug | Importazole | Sigma-Aldrich | Cat #: SML0341 | 50 µM |
| Chemical compound, drug | Cytochalasin D | Sigma-Aldrich | Cat #: C8273 | 1 µg/ml |
| Chemical compound, drug | Hygromycin | Santa Cruz Biotechnology | Cat #: sc-29067 | (1:1000) |
| Chemical compound, drug | Doxycycline | Enzo Life Sciences | Cat #: ALX380273G005 | 1 µg/ml |
| Chemical compound, drug | R1881 | Sigma-Aldrich | Cat #: R0908 | 10 nM |
| Chemical compound, drug | Puromycin Dihydrochloride | Thermo Fisher Scientific | Cat #: A1113803 | 1 µg/ml |
| Chemical compound, drug | Hoechst 33342, Trihydrochloride, Trihydrate | Thermo Fisher Scientific | Cat #: H3570 | 10 µg/ml |
| Transfected construct | pmCherry-AR-fl (PC3) | This paper | Produced by Giannakakou Lab | |
| Transfected construct | pEGFP-C1-AR-fl (PC3, M12) | This paper, Thadani-Mulero et al., 2014 | Produced by Giannakakou Lab | |
| Transfected construct | pEGFP-C1-AR-v567 (PC3, M12) | This paper, Thadani-Mulero et al., 2014 | Produced by Giannakakou Lab | |
| Transfected construct | pEGFP-AR-V7 (PC3,M12) | This paper, Thadani-Mulero et al., 2014 | Produced by Giannakakou Lab | |
| Transfected construct | pEGFP-C1-AR-fl A573D/ (PC3) | This paper | Produced by Giannakakou Lab | |
| Transfected construct | pEGFP-C1-AR-V7 A573D/ (PC3) | This paper | Produced by Giannakakou Lab | |
| Transfected construct | pEGFP-C1-AR-fl (A596T/S597T) | This paper | Produced by Giannakakou Lab | |
| Transfected construct | pEGFP-C1-AR-V7 (A596T/S597T) | This paper | Produced by Giannakakou Lab | |
| Recombinant DNA reagent | mEos4b-C1 | Paez-Segala et al., 2015 | Obtained from Dr. Michael Davidson | |
| Transfected construct | mEos4b-AR-fl (PC3) | This paper | Produced by Giannakakou Lab | |
| Transfected construct | mEos4b-AR-v567 (PC3) | This paper | Produced by Giannakakou Lab | |
| Transfected construct | mEos4b-AR-V7 (PC3) | This paper | Produced by Giannakakou Lab | |
| Transfected construct | pCW57.1-GFP-AR-V7 (C42) | This paper | Produced by Giannakakou Lab | |
| Transfected construct | pCW57.1-GFP-AR-V7 DBox (C42) | This paper | Produced by Giannakakou Lab | |
| Transfected construct | pmCherry-C1-RanQ69L (22rv1, PC3) | Kazgan et al., 2010 | Obtained from Dr. Jay Brenman | |
| Transfected construct | CS-GS241B-mCHER-LV152 lentivirus (PC3) | Azeem et al., 2017 | Obtained from Dr. Karl-Henning Kalland | |
| Recombinant DNA reagent | pCW57.1 tet-on vector | Addgene | Cat #: 41393 RRID: Addgene_41393 | Obtained from Dr. David Root |
| Software, algorithm | Zeiss Zen Software | Zeiss | RRID:SCR_013672 | https://www.zeiss.com/ microscopy/ us/products/microscope-software/zen-lite.html |
| Software, algorithm | GraphPad Prism | GraphPad | RRID:SCR_005375 | http://www.graphpad.com/scientific-software/prism/ |
| Software, algorithm | Fiji | ImageJ | RRID:SCR_002285 | https://imagej.net/Fiji |
| Chemical compound | Fast SYBR Green Mastermix | Thermo Fischer Applied Biosystems | Cat #: 4385612 | |
| Commercial assay, kit | Macherey-Nagel Kit | Macherey-Nagel | Cat #: 740609.250 | |
| Commercial assay, kit | Nucelofector Kit R | Lonza Bioscience | Cat #: VVCA-1001 | Used LNCaP program T007 with Amaxa II |
| Commercial assay, kit | FuGENE HD Transfection Reagent | Promega Corporation | Cat #: E2691 | |
| Sequence-based reagent | Q-PCR Amplification Primer FKBP5e: Fwd: GGT TCC TGG GCA GGA GTA AG Rev: AAC GTG GAT CCC ACA CTC TC | IDT DNA | ||
| Sequence-based reagent | Q-PCR Amplification Primer PSAe: Fwd-TGG GAC AAC TTG CAA ACC TG Rev-GAT CCA GGC TTG CTT ACT GT | IDT DNA | ||
| Sequence-based reagent | Q-PCR Amplification Primer AREneg: Fwd: GCT GAT TCA ATT ACC TCC CAG AA Rev: AGT TTG GGA CAG ACG GGA AA | IDT DNA |
