help button home button Endocrine Society Molecular Endocrinology ENDO 08 Sessions Library
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Molecular Endocrinology, doi:10.1210/me.2007-0240
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow NURSA Molecule Pages Link
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kesler, C. T.
Right arrow Articles by Paschal, B. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kesler, C. T.
Right arrow Articles by Paschal, B. M.
Molecular Endocrinology 21 (9): 2071-2084
Copyright © 2007 by The Endocrine Society

Subcellular Localization Modulates Activation Function 1 Domain Phosphorylation in the Androgen Receptor

Cristina T. Kesler, Daniel Gioeli, Mark R. Conaway, Michael J. Weber and Bryce M. Paschal

Center for Cell Signaling (C.T.K., B.M.P.), Department of Microbiology (C.T.K., D.G., M.J.W.), Department of Public Health Sciences (M.R.C.), Department of Biochemistry and Molecular Genetics (B.M.P.), Cancer Center (C.T.K, D.G., M.R.C., M.J.W., B.M.P.), University of Virginia Health System, Charlottesville, Virginia 22908

Address all correspondence and requests for reprints to: Bryce Paschal, University of Virginia Center for Cell Signaling, Box 800577, Charlottesville, VA 22908. E-mail: bmp2h{at}virginia.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Although the steady-state distribution of the androgen receptor (AR) is predominantly nuclear in androgen-treated cells, androgen-bound AR shuttles between the nucleus and the cytoplasm. In the present study we have addressed how nucleocytoplasmic shuttling contributes to the regulation of AR. Nuclear transport signal fusions were used to force AR localization to the nucleus or cytoplasm of prostate cancer cells, and the effect of localization on shuttling, transcription, androgen binding, and phosphorylation was determined. Fusing the simian virus 40 nuclear localization signal or c-Abl nuclear export signal to AR resulted in androgen-independent localization to the nucleus or cytoplasm, respectively. AR forced to the nucleus was transcriptionally active on prostate-specific antigen and mouse mammary tumor virus promoters driving reporter genes. AR forced to the cytoplasm was largely inactive on the prostate-specific antigen promoter, but, surprisingly, AR was active on the mouse mammary tumor virus promoter and on two endogenous genes examined. Thus, highly transient nuclear localization of AR is sufficient to activate transcription. Androgen dissociation rates and the dissociation constant (KD) of AR for androgen were similar whether AR was localized to the cytoplasm or the nucleus, suggesting the ligand-binding cycle of AR is not strictly linked to its compartmentalization. Using phosphosite antibodies, we found that compartmentalization influences the phosphorylation state of AR. We show there is a bias for androgen-dependent phosphorylation of Ser81, Ser256, and Ser308 in the nucleus and androgen-independent phosphorylation of Ser94 in the cytoplasm. We propose that one function of nucleocytoplasmic shuttling is to integrate the signaling environment in the cytoplasm with AR activity in the nucleus.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE ANDROGEN RECEPTOR (AR) is a transcription factor that belongs to the steroid hormone receptor subfamily of nuclear receptors. AR mediates androgen-dependent transcription of genes that are required for the proper development of male reproductive organs and other target tissues (1). The AR knockout mouse has defects in sexual development and shows late-onset of obesity, impaired cardiac growth, and altered bone metabolism, indicating that the functions of AR extend to multiple tissues (2, 3). AR function is regulated primarily by androgen binding, but its activity can also be influenced by activation of signal transduction pathways that promote posttranslational modification of AR. These include phosphorylation, dephosphorylation, acetylation, sumoylation, and ubiquitylation (4, 5, 6, 7, 8, 9). Several growth factor-dependent signal transduction pathways that are upstream of AR have been implicated in prostate cancer disease progression (10). It has been postulated that signal transduction pathways sensitize AR to low concentrations of androgen or provide a mechanism for AR activation that is altogether androgen independent (11, 12). These pathways may influence AR localization as well, given that AR has a nuclear distribution in human androgen-independent prostate cancers grown in mice depleted of testicular androgens (13).

AR contains three major functional domains. The N-terminal activation function (AF)-1 domain (residues 1–555) contains binding sites for transcriptional regulators including coactivators of the p160 family, acetyltransferases including cAMP response element binding protein (CREB)-binding protein/p300, and corepressors including nuclear receptor corepressor and silencing mediator of retinoid and thyroid hormone receptor (11, 14). Downstream of the AF-1 lies the DNA-binding domain (DBD) (residues 556–624), which mediates sequence-specific binding to promoters and enhancers of target genes. The hinge region (residues 625–670) links the DBD to the C-terminal activation function 2 (AF-2), which resides within the AR ligand-binding domain (LBD) (residues 671–919).

Nuclear import of AR is necessary for its direct action on target genes. By analogy with other nuclear receptors including the glucocorticoid receptor (GR), cytoplasmic AR is thought to undergo an androgen-induced transformation that converts it to a form that is competent for nuclear import (15). AR contains a bipartite nuclear localization signal (NLS) that overlaps the DBD-hinge regions (16). The DBD-hinge NLS, which was defined by Wilson and co-workers (16) (617RKCYEAGMTLGARKLKK633), displays features that are typical of bipartite NLSs found in proteins such as nucleoplasmin. The LBD of GR contains a second NLS activity (17). The structural conservation of the LBD between nuclear receptors suggests that the AR might contain a second NLS that is located within its LBD.

AR also undergoes nuclear export. The recognition site for AR export is centered on the DBD, which is both necessary and sufficient for AR export (18). Deletion analysis and mutagenesis have suggested the presence of an additional NES in the AR LBD (19). The transport receptors that mediate nuclear import and export of AR have not been defined. In androgen withdrawal assays, nuclear export of AR is insensitive to the Crm1 inhibitor leptomycin B (LMB), an indication that the housekeeping export receptor exportin-1/Crm1 is not responsible for AR export (20). An alternative interpretation of the LMB-insensitive export of AR is that multiple export pathways can be used (15).

AR shares with other nuclear receptors the property of undergoing cycles of nucleocytoplasmic shuttling (21, 22, 23, 24). The physiological reason for nuclear receptor shuttling between the nucleus and the cytoplasm is unknown, but there are several plausible explanations. Redistributing nuclear receptors to the cytoplasm could reflect a transport-based mechanism that helps modulate gene expression by reducing the concentration of nuclear receptors in the nucleus. Shuttling may be relevant to the interactions that are important for the transactivation function of AR, such as cofactor recruitment and ligand binding. Additionally, nuclear export could be coupled to receptor turnover, as occurs with the progesterone receptor (PR) and estrogen receptor (ER) (25, 26). Alternatively, nuclear receptors may need to access the cytoplasm to act as scaffolds that facilitate kinase activation, a nongenomic function shown for AR and ER (27).

We set out to obtain insight into how nucleocytoplasmic shuttling contributes to the function of AR by examining how cytoplasmic and nuclear localization influence androgen binding, phosphorylation, and transactivation function. Our approach involved fusing nuclear import and nuclear export signals (NESs) to AR to force its nuclear and cytoplasmic distribution, respectively. We found that the affinity of AR for androgen is the same in the nucleus and the cytoplasm, suggesting that shuttling is not fundamentally important for the ligand-binding cycle. A panel of phosphosite-specific antibodies was used to quantify how the cytoplasmic and nuclear localization of AR correlates with androgen-independent and androgen-dependent phosphorylation of AR. Nuclear localization resulted in a bias for androgen-induced phosphorylation on Ser81, Ser256, and Ser308, suggesting that the kinases for these sites reside in the nucleus. In contrast, phosphorylation of Ser94 was strongly biased for the cytoplasm. Our data suggest that nucleocytoplasmic shuttling provides a mechanism for integrating the signal transduction environment in the cytoplasm with the activity of AR in the nucleus. We propose the term "compartment sampling" to describe the transient exposure of AR to the cytoplasm during its nucleocytoplasmic shuttling cycle.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
AR function as a transcription factor requires nuclear import. After androgen-induced import, AR shuttles between the nucleus and cytoplasm (15, 20). We set out to address why AR undergoes nucleocytoplasmic shuttling using a panel of AR fusions with well-defined transport signals that confer a steady-state distribution to the nucleus or cytoplasm (Fig. 1AGo). By forcing the subcellular localization of AR to either the nucleus or cytoplasm, we were able to study how nucleocytoplasmic compartmentalization influences AR regulation and activity.


Figure 1
View larger version (21K):
[in this window]
[in a new window]

  		Fig. 1. Nuclear Transport Signal Fusions Used to Control AR Localization

A, Diagram of AR domains and positions of transport signal fusions. The c-Abl kinase NES (EAINKLESNLRELQICPAT; dark gray rectangle) and the SV40 Large T antigen NLS (PPKKKRKEDP; light gray rectangle) were each used to construct N- and C-terminal fusions with AR. The relative positions of serine phosphorylation sites analyzed in this study are indicated with arrows. B, Localization of AR transport signal fusions in the absence and presence of androgen. Cos7 cells were transfected with the indicated constructs and treated with vehicle or 1 nM R1881 for 2 h before processing for immunofluorescence microscopy using anti-AR antibody PG-21. C, Time course of NES-AR localization in PC3 cells after the addition of R1881.

 
The monopartite classical NLS from simian virus 40 (SV40) large T antigen (PPKKKRKEDP), which is recognized by importin {alpha} proteins (28), was used to drive nuclear localization of AR (Fig. 1AGo). The SV40 NLS has been used by DeFranco and co-workers (21) to study GR function. The hydrophobic NES from the c-Abl kinase (EAINKLESNLRELQICPAT), which is recognized by Crm1, was used to force the cytoplasmic localization of AR (Fig. 1AGo) (29). The c-Abl protein can localize to the cytoplasm or the nucleus, which we took as an indication that the export-promoting activity of this particular NES would not be so dominant as to completely exclude AR from the nucleus (29). Fusions of transport signals at either the N (NES-AR, NLS-AR) or C termini (AR-NES, AR-NLS) of AR were introduced into Cos7 and PC3 cells, and protein localization was determined in the absence and presence of the synthetic androgen R1881 (1 nM for 2 h). Under our culture conditions, wild-type (WT) AR is detected in both the nucleus and the cytoplasm of Cos7 cells, with a bias toward cytoplasmic distribution until the addition of androgen (Fig. 1BGo). As expected, NLS-AR and AR-NLS displayed androgen-independent nuclear localization, and NES-AR and AR-NES were predominantly cytoplasmic in the absence of androgen. AR-NES showed a low level of nuclear localization in the presence of androgen, potentially because ligand-induced repositioning of helix 12 reduces accessibility to the C-terminal NES. Fusing both an NLS and an NES to the N terminus of AR (NLS-NES-AR) resulted in a predominantly nuclear localization that became exclusively nuclear in the presence of androgen. Prolonged treatment of PC3 cells with R1881 (24 h) did not change the steady-state localization of NES-AR, indicating that the NES does not simply delay AR import (Fig. 1CGo).

Shuttling of AR Transport Signal Fusion Proteins
We used the heterokaryon shuttling assay (18) to characterize how the transport signal fusions influence the shuttling activity of AR. The heterokaryon is formed by the fusion of two cell types, one of which expresses nuclear-localized AR before fusion. Appearance of AR in the acceptor nucleus depends on AR export from a donor cell nucleus and subsequent AR import into an acceptor cell nucleus. Thus, the steady-state distribution of AR between donor and acceptor nuclei is the result of nucleocytoplasmic shuttling. The results were quantified by measuring the fluorescence intensity of donor and acceptor cell nuclei, and wild-type (WT) AR and green fluorescent protein (GFP)-NLS were used as positive and negative controls, respectively. Fusing the SV40 NLS to the C terminus of AR (AR-NLS) significantly reduced the shuttling activity of AR (P < 0.01), which was restored in the presence of androgen (Fig. 2AGo). This result suggests that in the context of this fusion protein, androgen binding can enhance the ability of AR to exit the nucleus. Fusing the SV40 NLS to the N terminus of AR also significantly reduced AR shuttling activity, but the activity of NLS-AR was not changed significantly by androgen (Fig. 2BGo). Although it is possible the N-terminal NLS alters AR conformation or subnuclear compartmentalization in a manner that reduces export, NLS-AR is functional by multiple criteria (see below). Fusing import and export signals to AR (NLS-NES-AR) generated a fusion that was slightly more active for shuttling than WT AR (Fig. 2CGo).


Figure 2
View larger version (45K):
[in this window]
[in a new window]

  		Fig. 2. Effects of Nuclear Transport Signal Fusions on AR Shuttling

Heterokaryon assays were conducted using Cos7 cells transfected with the indicated AR transport fusions and NIH 3T3 cells. Shuttling was quantified in the heterokaryons (25–40 per condition) using Openlab 4.0.2. GFP-NLS is included in each assay as a negative control for shuttling. The average ratio for each AR fusion or the nonshuttling GFP-NLS is shown on the bar graphs. A, Comparison of AR WT and AR-NLS. B, Comparison of WT AR and NLS-AR. C, Comparison of WT AR and NLS-NES-AR. D, NES-AR enters the nucleus and undergoes shuttling. NES-AR and WT AR were expressed in Cos7 cells and treated with LMB (60 min) before addition of 1 nM R1881 (2 h). Cells were fixed and processed for immunofluorescence microscopy using anti-AR antibody PG-21. Endogenous RanBP1, which undergoes Crm1-mediated nuclear export, is shown as a positive control for LMB efficacy. DAPI, 4',6-Diamidino-2-phenylindole; FITC, fluorescein isothiocyanate.

 
NES-AR Undergoes Nuclear Import and Rapid Export
We tested whether the steady-state cytoplasmic localization of NES-AR was due to rapid nuclear export or to inhibition of import via cytoplasmic retention. Cos7 cells expressing WT AR or NES-AR were treated with LMB to inhibit Crm1-dependent export, and the localization of AR was determined by immunofluorescence microscopy. We also stained cells for endogenous RanBP1, an NES-containing protein known to accumulate in the nucleus in response to LMB treatment. LMB caused nuclear accumulation of NES-AR in the presence of R1881 (Fig. 2DGo). This shows that NES-AR undergoes androgen-dependent nuclear import and rapid export to the cytoplasm via the receptor Crm1.

Steady-State Localization of AR and Transcription
The transcriptional activities of the AR transport signal fusions were measured using luciferase-based reporters containing the promoters for mouse mammary tumor virus (MMTV) and prostate-specific antigen (PSA) (30, 31). The assays were performed in the AR-negative prostate cancer cell line PC3, and immunoblotting established that activity differences were not due to protein expression levels (data not shown). NLS-AR was transcriptionally active, although the level of activity was less than that of WT AR (Fig. 3Go, A and B). AR-NLS displayed activity that was similar to that of WT AR (Fig. 3Go, C and D). AR fusions with the NES, however, gave surprising results. Both NES fusions of AR displayed low levels of activity when assayed using the PSA promoter (Fig. 3Go, B and D). In contrast NES-AR showed approximately 50% of WT AR activity, and AR-NES exceeded WT AR activity when assayed on the MMTV promoter.


Figure 3
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 3. Effects of Nuclear Transport Signal Fusions on AR-Dependent Transcription

AR transport signal fusions were tested for transactivation of the MMTV and the PSA promoters. PC3 cells were cotransfected with the indicated AR transport fusion, pHH(MMTV)-luc or PSA-61-luc, and pCMV-RL-luc followed by treatment with vehicle (gray bars) or 1 nM R1881 (black bars) for 18 h. A and B, Analysis of N-terminal transport signal fusions. C and D, Analysis of C-terminal transport signal fusions. RLU, Relative luciferase units.

 
We hypothesized that the activity of the NES fusions on the MMTV promoter relative to the PSA promoter might reflect differences in the nuclear concentration of AR required for efficient transcription from the respective reporter gene. Support for this hypothesis was obtained by showing that an approximately 20-fold higher concentration of AR is required to transactivate the PSA promoter to the same level as the MMTV promoter (Fig. 4Go).


Figure 4
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 4. Effect of AR Concentration on Activation of the MMTV and PSA Promoters

A, Reporter gene assays were performed in PC3 with pHH(MMTV)-luc (diamonds; left y-axis) or pPSA-61-luc (circles; right y-axis) using 0, 10, 40, 75, 125, 250, or 500 ng of plasmid encoding WT AR. B, Expression levels of AR were analyzed by immunoblotting. C, Localization of AR analyzed by immunofluorescence microscopy. DAPI, 4',6-Diamidino-2-phenylindole; RLU, relative luciferase units; Tub, tubulin.

 
The NES used in our assays promotes efficient AR export and results in a very low nuclear concentration of AR (Figs. 1BGo and 2DGo). To determine whether a low nuclear concentration of AR is sufficient to transactivate endogenous genes, we expressed AR WT and C-terminal fusions of AR in PC3 cells and assayed the expression of the androgen-regulated genes FKBP51 and S100P (32, 33). Real-time RT-PCR analysis was used to show that NES and NLS fusions of AR assayed at a saturating concentration of R1881 (1 nM) activate the FKBP51 and S100P genes to levels that are comparable to the level obtained with unfused AR WT (Fig. 5Go).


Figure 5
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 5. AR Transport Signal Fusions Transcribe Endogenous AR-Responsive Genes

PC3 cells were transfected with either empty vector, AR WT, AR-NLS, or AR-NES and treated with vehicle or R1881 for 18 h. Relative levels of mRNA were assessed by real-time RT-PCR. A and B, Levels of FKBP51 and S100P mRNA induced by AR expression and R1881 addition. C, Expression levels of AR protein analyzed by immunoblotting. Tub, Tubulin.

 
To determine whether forced nuclear localization of AR affects androgen sensitivity to transcription, we analyzed the activity of AR-NLS over a wide range of synthetic androgen concentrations (Fig. 6Go). Androgen-dependent activation of AR-NLS and AR WT were virtually identical, indicating that forcing its localization to the nucleus does not result in AR hypersensitization to androgen.


Figure 6
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 6. Constitutive Nuclear Localization Does Not Sensitize AR to Androgen

Transactivation of the PSA promoter was measured in PC3 cells using AR WT (black diamonds) and AR-NLS (gray squares) and either vehicle or a range of R1881 concentrations (18 h). Expression levels of AR were analyzed by immunoblotting. Curves were generated using CurveExpert 1.3. Tub, Tubulin.

 
AR Affinity for Androgen Is Independent of Compartmentalization
Androgen induces AR translocation from the cytoplasm to the nucleus, indicating that the cytoplasm provides an appropriate milieu for androgen binding to AR. This includes chaperones that maintain the LBD in a conformation that is functional for ligand binding (34). To determine whether subcellular compartmentalization affects androgen binding, we transfected WT AR and AR transport signal fusions into PC3 cells and performed whole-cell binding assays using radiolabeled androgen ([3H]R1881). Scatchard analysis of the binding data revealed that the affinity for androgen is similar for NES-AR (0.36 nM) and WT AR (0.24–0.36 nM) (Fig. 7AGo). Moreover, targeting AR to the nucleus (NLS-AR, AR-NLS) did not change AR affinity for androgen (Fig. 7Go, B and C). The dissociation rate in the nucleus and cytoplasm was examined in pulse-chase experiments for WT AR, NLS-AR, and NES-AR. The half-times (t1/2) for ligand dissociation for transport signal fusion proteins (NLS-AR: t1/2 = 83 min; NES-AR: t1/2 = 79 min) were similar to that of unfused AR (WT AR: t1/2 = 77–80 min; Fig. 7Go, D and E). We conclude that androgen dissociation rate is similar whether AR is in the nucleus or the cytoplasm. Our data suggest that the complement of chaperones that are required for the androgen binding cycle are present at sufficient concentrations in both the nucleus and the cytoplasm.


Figure 7
View larger version (20K):
[in this window]
[in a new window]

  		Fig. 7. Androgen-Binding Affinity and Dissociation Rate Are Independent of the Subcellular Compartmentalization of AR

Whole-cell binding assays with [3H]R1881 were performed in PC3 cells transfected with the indicated constructs. PC3 cells were incubated with [3H]R1881 (0.05–2 nM) for 2 h. Linear regression was used to calculate KD, and the data are displayed as Scatchard plots. A, NES-AR [KD = 0.36 nM; 95% confidence interval (CI) 0.25, 0.46 nM] and WT AR (KD = 0.36 nM; 95% CI 0.28, 0.44 nM). B, NLS-AR (KD = 0.33; 95% CI 0.26, 0.51 nM) and WT AR (KD = 0.24 nM; 95% CI 0.21, 0.27 nM). C, AR-NLS (KD = 0.32 nM; 95% CI 0.24, 0.40 nM) and WT AR (KD = 0.34 nM 95% CI 0.29, 0.39 nM). D and E, Androgen dissociation assays. PC3 cells expressing the indicated AR transport signal fusions were pulsed with 2 nM [3H]R1881 for 60 min, washed, and chased with 2 µM unlabeled R1881 for 15–180 min to allow androgen dissociation. Panel D shows the plots for WT AR (t1/2 = 77 min; R2 = 0.95) and NES-AR (t1/2 = 79 min; R2 = 0.97), and panel E shows WT AR (t1/2 = 80 min; R2 = 0.96) and NLS-AR (t1/2 = 83 min; R2 = 0.98).

 
Compartmental Regulation of AR Phosphorylation
AR contains both androgen-dependent and androgen-independent phosphorylation sites, but information regarding the identities and subcellular locations of the kinases that phosphorylate AR is limited (4). To investigate whether AR phosphorylation is influenced by subcellular compartmentalization, we used a panel of phosphosite-specific antibodies to analyze the phosphorylation state of AR localized to the nucleus or cytoplasm. The level of phosphorylation at each site on AR was quantified using infrared excitation and two-color antibody imaging, an approach that enabled normalization to the signal obtained by pan-AR detection on the same immunoblot. A comparison between WT AR and the N-terminal fusions revealed that the phosphorylation of certain sites is compartment sensitive, whereas other sites appear to be compartment insensitive (Fig. 8Go). This allows an inference as to the location of AR kinases, although the results can also be interpreted with regard to the location of AR phosphatases. The reduced level of NES-AR phosphorylation at Ser81, Ser256, and Ser308 suggests that these sites are phosphorylated in the nucleus (Fig. 8Go, B, D, and E). Ser94 phosphorylation on the nonshuttling NLS-AR was dramatically lower than on WT AR and NES-AR, indicating that this site is likely phosphorylated in the cytoplasm (Fig. 8CGo). Ser16 and Ser424 do not show a compartment bias for phosphorylation in this assay (Fig. 8Go, A and F).


Figure 8
View larger version (27K):
[in this window]
[in a new window]

  		Fig. 8. Using N-Terminal Transport Signal Fusions to Determine How Subcellular Compartmentalization Regulates AR Phosphorylation

PC3 cells expressing the indicated N-terminal AR transport signal fusions were treated with vehicle (gray bars) or 1 nM R1881 (black bars) for 2 h. AR was immunoprecipitated, and phosphorylation of AR was measured by immunoblotting using phosphosite-specific antibodies to seven AR phosphorylation sites. A–G, Quantitation of the level of AR phosphorylation in N-terminal transport signal fusions of AR using the Odyssey Infrared imaging system. The phosphosignal was always normalized to the level of AR in the same sample. pSer, Phosphorylated serine.

 
The same approach was applied to C-terminal AR transport signal fusions, and similar results were obtained (Fig. 9Go). The only major difference was that a high level of Ser650 phosphorylation was observed on NES-AR (Fig. 8GGo), whereas a low level of Ser650 phosphorylation was observed on AR-NES (Fig. 9GGo). Because Ser650 phosphorylation was enhanced on both NLS-AR and AR-NLS relative to WT AR, this site is probably phosphorylated by a kinase in the nucleus.


Figure 9
View larger version (27K):
[in this window]
[in a new window]

  		Fig. 9. Using C-Terminal Transport Signal Fusions to Determine How Subcellular Compartmentalization Regulates AR Phosphorylation

Samples were processed as described in Fig. 8Go. A–G, Quantitation of the level of AR phosphorylation in C-terminal transport signal fusions of AR using the Odyssey Infrared imaging system. The phosphosignal was always normalized to the level of AR in the same sample. pSer, Phosphorylated serine.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In the current study we have examined how subcellular compartmentalization contributes to AR regulation and activity in the cell. After its synthesis in the cytoplasm, AR can undergo both androgen-dependent and androgen-independent import into the nucleus (16, 35). In prostate cells, AR regulates the transcription of more than 100 genes, the products of which control diverse aspects of cell physiology ranging from protein trafficking to cell growth (36). AR and other steroid hormone receptors are predominately nuclear in cells and tissues in the presence of hormone, but standard immunocytochemical methods fail to reveal the fact that AR undergoes multiple rounds of export to the cytoplasm and reimport into the nucleus in a process known as nucleocytoplasmic shuttling (18, 20). Nuclear export of AR and other shuttling transcription factors has been suggested to reflect a transport-based mechanism that can dampen or terminate a transcriptional response by reducing the concentration of these factors in the nucleus. In the case of AR, however, nuclear export is predicted to be an inefficient means of reducing nuclear activity because AR rapidly returns to the nucleus. We found that NES fusions that promote an extremely low nuclear concentration of AR can transactivate reporter and endogenous genes. Whether there is a simple correlation between AR dosage and the expression of AR-regulated genes, or whether AR dosage might have gene-selective effects, as suggested by our reporter gene analysis (Fig. 3Go), is a topic for future study. High AR levels have been shown to promote androgen-independent growth in model systems, but the specific mechanism underlying this phenomena has not been elucidated (11, 37).

Function of Nucleocytoplasmic Shuttling
Transient exposure of AR to the cytoplasm during its shuttling cycle could influence AR activity in the nucleus. This could impart either positive or negative regulation of AR. We addressed this hypothesis by forcing steady-state AR localization to the nucleus or cytoplasm and testing whether this alters known biochemical properties of AR, i.e. androgen binding, phosphorylation, and transactivation of reporter and endogenous genes. Our major finding was that subcellular localization is important for determining the phosphorylation state of particular androgen-independent and androgen-dependent phosphorylation sites. Thus, one function of nucleocytoplasmic shuttling is that it allows AR to receive input from signal transduction pathways that appear to operate primarily in the cytoplasm or the nucleus. We propose that compartment sampling is a transport-based mechanism that directs AR to the cytoplasm where it acquires modifications (or binding partners) that 1) reflect the signaling environment of the cytoplasm, and 2) influence AR function in the nucleus. A better understanding of how phosphorylation regulates AR activity is needed to determine how compartment sampling positively or negatively affects AR function as a transcription factor.

Forced localization of AR was achieved with transport signal fusions based on the NLS from SV40 large T antigen and the NES from c-Abl kinase. The SV40 NLS is a strong NLS, and its fusion to the N terminus or C terminus resulted in complete nuclear localization of AR as visualized by immunofluorescence microscopy (Fig. 1BGo). For reasons that are not clear, fusing the NLS to the AR N terminus reduced nuclear export in the heterokaryon assay (Fig. 2BGo). However, this feature was helpful in exploring potential links between compartmentalization, androgen binding, and transcriptional activity. Addition of the synthetic androgen R1881 increased the shuttling of the C-terminal NLS fusion with AR (Fig. 2AGo). Although androgen binding is not required for AR export, the fact that R1881 can promote export of this AR transport signal fusion suggests that export competence might be influenced by protein conformation, chaperone composition, or posttranslational modifications promoted by androgen binding. It is interesting to note that, like AR-NLS, an NLS fusion with GR fails to shuttle unless cognate ligand is added (21). Together, these findings suggest that the export competence of steroid hormone receptors could be regulated, at least in part, by ligand binding.

In the presence of R1881, AR-NLS had near WT AR levels of shuttling and transcriptional activity. This was true whether measured with PSA or MMTV reporter genes or with endogenous androgen-responsive FKBP51 and S100P genes. In contrast, the activity of NLS-AR was lower than that of WT AR in the shuttling (30% of WT) and transcription (<50% of WT) assays using reporter genes. It is formally possible that transport signal fusions exert effects related to AR conformation and reduce AR activity in certain assays.

Compartmentalization and Ligand Binding
Nuclear import of AR occurs within minutes of androgen addition to cells (t1/2 = ~20 min). Because the nuclear import rate of AR is substantially faster than the androgen dissociation rate (t1/2 = ~75 min), it can be inferred that AR translocates into the nucleus in its androgen-bound form. This indicates that the cytoplasm contains chaperones such as heat shock protein 90 and heat shock protein 70 that are required for ligand binding (34). Given that ligand binding to AR in the cytoplasm is coupled to AR translocation into the nucleus, we explored whether there is compartment bias for the ligand binding cycle. First, we considered whether shuttling might be important for AR function, possibly because ligand binding occurs in the cytoplasm, but not the nucleus. In this scenario, AR would need to undergo export to the cytoplasm to regain its competence for ligand binding. Second, we asked whether ligand dissociation in the nucleus might be a prerequisite for nuclear export, representing, in effect, a reversal of the androgen-stimulated import mechanism. This idea was based on the fact that androgen dissociation and AR export are relatively slow as compared with androgen binding and AR import. If androgen binding is restricted to the cytoplasm, or if androgen dissociation is predominantly a nuclear event, then nuclear-localized AR might be expected to display reduced androgen binding or increased androgen dissociation as compared with WT or cytoplasmic AR. Constitutive localization of AR to the nucleus or the cytoplasm did not, however, alter the affinity for androgen or change dissociation rates (Fig. 7Go). We conclude that the chaperones required for androgen binding are present in sufficient concentrations in both the cytoplasm and the nucleus. Thus, compartment sampling does not apply to androgen binding because this reaction occurs with the same efficiency in the cytoplasm and the nucleus. Our data are consistent with the observation that the process of GR recycling does not require cytoplasmic factors for hormone binding (38, 39).

Compartmentalization and Phosphorylation
We explored the hypothesis that compartment sampling exposes the predominantly nuclear-localized AR to a signal transduction pathway that is active predominantly in the cytoplasm. In this manner, compartment sampling could circumvent the need to deliver an activated kinase to the nucleus, thereby restricting signaling to a single compartment. We tested the basis of the hypothesis by forcing the localization of AR to the nucleus or the cytoplasm and determining the phosphorylation state of individual sites in AR. Quantitative analysis using a panel of phosphosite-specific antibodies revealed that of the seven AR phosphorylation sites examined in this study, Ser94 has a strong bias for phosphorylation in the cytoplasm. This conclusion was based on a comparison of the ratios of Ser94 phosphorylation detected on AR transport fusions and WT AR (Table 1Go). In the context of transport signal fusions to the N terminus of AR, the ratio of Ser94 phosphorylation on NES/WT (1.42) was higher than NLS/WT (0.25). The same trend was noted for Ser94 phosphorylation in the C-terminal fusions of AR, where the ratio of NES/WT (0.79) was higher than that of NLS/WT (0.50). The fact that phosphorylation of NLS fusions to AR is less than that of WT AR also suggests that AR does not achieve a maximal level of Ser94 phosphorylation between translation and the initial round of nuclear import. Rather, it suggests that Ser94 phosphorylation occurs when AR is exposed to the cytoplasm during a subsequent round of export and reimport.


View this table:
[in this window]
[in a new window]

  		Table 1. Effect of Subcellular Localization on AR Phosphorylation

 
Three sites in AR, Ser81, Ser256, and Ser308, were found to be highly phosphorylated when androgen-bound AR was localized to the nucleus, an indication that these sites are recognized by nuclear kinases. This deduction was based on a comparison of the ratios of phosphorylation detected on the transport fusions. Ser81 in AR was recently reported to be phosphorylated by Cdk1, a kinase that has multiple nuclear targets (40, 41). The data did not allow us to establish whether or not there is compartment bias for Ser16, Ser424, and Ser650. Ser650 phosphorylation is mediated by stress kinases including p38. Because activated p38 is localized to the nucleus and Ser650 phosphorylation is important for efficient AR export from the nucleus, it seems likely that phosphorylation of this site occurs in the nucleus (5, 42). The phosphorylation status of Ser213 in AR was not evaluated in this study, although it is predicted to be phosphorylated by Akt in the nucleus (43).

Nucleocytoplasmic Shuttling of Other Nuclear Receptors
Nucleocytoplasmic shuttling appears to be a general property of nuclear receptors. AR, GR, ER, progesterone receptor (PR), thyroid hormone receptor (TR), and mineralocorticoid receptor have been shown to undergo shuttling in the heterokaryon assay, which remains the system of choice for demonstrating that a protein with a steady-state nuclear localization undergoes export and reimport (21, 22, 23, 24). The functional significance of shuttling of these other nuclear receptors is not fully understood, but there are clear links to the mechanisms that regulate nuclear receptor turnover. Lange and co-workers (25) have characterized a pathway that involves MAPK phosphorylation of Ser294 in PR and have shown that it is required for efficient nuclear export and hormone-dependent degradation by the proteasome. Also, fusing a hydrophobic NES to GR accelerates turnover of the fusion protein, and this can be inhibited by treatment with LMB (44). A similar mechanism may operate with ER, given that inhibiting ER export from the nucleus results in protein stabilization rather than hormone-dependent degradation (26). These findings suggest that at least one function of nucleocytoplasmic shuttling is to deliver nuclear receptors to the protein degradation machinery in the cytoplasm. Like GR, PR, and ER, AR is degraded by the 26S proteasome (45). However, androgen binding to AR promotes protein stabilization, which contrasts with the protein turnover induced by cognate ligand binding to GR, PR, and ER. This suggests that AR localization, and possibly its export, may not be tightly coupled to protein turnover.

It seems likely that nucleocytoplasmic shuttling is important for nuclear receptors to perform nongenomic functions related to kinase activation in the cytoplasm. Progestin signaling through PR has been connected to cell cycle progression through activation of cytoplasmic signal transduction cascades (46). Additionally, ER can facilitate estrogen-dependent activation of phosphatidylinositol 3-kinase via binding to the regulatory subunit p85 (47). Also, AR and ER can attenuate apoptosis in several cell types by activating the Src/Shc/ERK signal transduction pathway (48). Whether these nongenomic activities are designed to simply regulate cytoplasmic kinase activity in a steroid hormone-dependent manner or whether they are part of a complicated scheme for amplifying cytoplasmic signals that regulate nuclear function remains to be determined. Elucidating the AR transport mechanism and developing a better understanding of the relevant signal transduction pathways should provide further insight into how nucleocytoplasmic shuttling regulates the activity of AR.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmid Construction
pcDNA3-flag-AR (pfAR) was used as WT AR and as the vector for adding N-terminal transport fusions and has been described elsewhere (4). Annealed synthetic oligonucleotides (Sigma Genosys, The Woodlands, TX) were used to construct the transport signals. For pcDNA3-flag-NLS-AR (pNLS-AR), an annealed oligonucleotide encoding the monopartite SV40 NLS (PPKKKRKEDP) was inserted into pcDNA3 using HindIII and BamHI sites to form the intermediate vector pcDNA3-flag-NLS. AR was inserted into this vector using XhoI and XbaI sites. For construction of pcDNA3-flag-NES-AR (pNESAR) and pcDNA3-flag-NLS-NES-AR, an annealed oligonucleotide encoding the c-Abl kinase NES (EAINKLESNLRELQICPAT) was inserted into pfAR and pNLSAR, respectively, using BamHI/XhoI. To generate AR C-terminal fusions, the AR LBD was PCR amplified to contain AscI and BamHI sites. This product, along with the N-terminal flag-AR (1–565) fragment excised from pfAR, was inserted into pSPORT6 via EcoR1/HindIII and HindIII/XbaI, respectively, to make pSPORT6-flag-AR (pSPAR). This construct is designated AR WT, and it encodes the sequence GAPGS immediately before the stop codon. Transport signals were inserted into pSPAR using AscI and BamHI sites to construct pAR-NLS and pAR-NES. Construction of pEGFP-Stv-NLS has been described (49). Reporter plasmids used were pHH-luc, which contains the MMTV promoter fused to luciferase and pPSA-61-luc, which contains the complete, 6-kb prostate-specific antigen promoter (30, 31). pPSA-61-luc was kindly provided by M. Brown.

Cell Culture and Transfection
PC3, Cos7, and NIH3T3 cells were purchased from American Type Culture Collection (Manassas, VA). PC3 cells were maintained and transfected in RPMI 1640 (Life Technologies, Inc., Carlsbad, CA) supplemented with 5% fetal bovine serum. Cos7 and NIH3T3 cells were maintained and transfected in DMEM (Life Technologies, Inc.) supplemented with 10% newborn calf serum. Transient transfections were performed using the Fugene 6 protocol (Roche, Indianapolis, IN). For reporter assays, PC3 cells were transfected with 225 µg reporter plasmid, 25 ng pCMV-Renilla-luc, and 200–500 ng AR plasmid (total = 750 ng/well; 12-well dish). For immunofluorescence microscopy, PC3 cells grown on glass coverslips in six-well dishes were transfected with 1.5 µg total plasmid DNA, and Cos7 cells were transfected with 1 µg total DNA. For immunoprecipitation, PC3 cells plated in 100-mm dishes were transfected with 5 µg total plasmid DNA.

Phosphosite-Specific Antibody Production
The phosphosite antibodies have been described elsewhere (50). In brief, peptides were coupled to keyhole limpet hemocyanin through an N-terminal cysteine injected into rabbits, and antibodies were purified on immobilized peptide. Antibodies to phosphoserines 81 and 650 were prepared by Upstate Biotechnology, Inc. (Lake Placid, NY). Antibodies to phosphoserines 16, 94, 256, 308, and 424 were raised by Cocalico (Reamstown, PA). Specificity of antibodies was verified by ELISA as well as immunoblotting against relevant phosphorylation mutants of AR.

Immunofluorescence Microscopy
Cos7 cells were plated at 5 x 104 cells per well on glass coverslips in six-well dishes. PC3 cells were plated at 8 x 104 cells per well on coverslips precoated with 0.5 mg poly-D-lysine (Sigma Chemical Co., St. Louis, MO) dissolved in PBS. Coverslips were fixed in 4% formaldehyde for 20 min followed by permeabilization in 0.8% Triton-X. Immunofluorescence was performed using anti-AR antibody PG-21 (Upstate Biotechnology) on transiently transfected Cos7 and PC3 cells treated with R1881 (PerkinElmer, Boston, MA) for times indicated in the legends. Heterokaryon nucleocytoplasmic shuttling assays were performed as previously described (18). CellTracker Orange CMTMR (Molecular Probes, Eugene, OR) was used to visualize heterokaryon fusions. Transfected Cos7 cells were coseeded with CMTMR-stained NIH 3T3 cells onto glass coverslips for 24 h. AR nuclear import in Cos7 was induced with 10 nM R1881 for 90 min. Cells were fused using polyethylene glycol and incubated for 4.5 h in 10% newborn calf serum DMEM containing 10 µg/ml cyclohexamide and 10 nM R1881. Indirect immunofluorescence was performed using PG-21. Cells were also stained with 4'-6-diamidino-2-phenylindole (Sigma). AR shuttling in heterokaryons was quantified from digital images using commercial software (Openlab 4.0.2). The ratio of the acceptor cell nuclear fluorescence (in NIH 3T3 cells) to donor cell nuclear fluorescence (in Cos7 cells) was calculated for 25–40 heterokaryons under each condition. All experiments in this study were performed at least three times.

Immunoprecipitation and Immunoblotting
PC3 cell immunoprecipitation was performed using extracts prepared in lysis buffer [0.5% Nonidet P-40, 100 mM NaCl, 10 mM EDTA, 20 mM Tris-HCl (pH 8.0), containing 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml pepstatin, 0.5 mM dithiothreitol, 0.2 mM sodium vanadate, 100 nM microcysteine]. The extracts were sonicated on ice and clarified by centrifugation at 18,000 x g for 15 min. AR was immunoprecipitated using the M2 antiflag monoclonal antibody (Sigma) prebound to Protein G beads. Immobilized immune complexes were washed three times, eluted in sample buffer, resolved by SDS-PAGE (8% gels), and transferred to nitrocellulose for immunoblot analysis. Membranes were blocked in 5% milk diluted in PBS for 1 h, after which they were incubated with phosphosite-specific AR primary antibodies diluted in 1% milk/PBS overnight at 4 C. Membranes were then incubated with antirabbit Alexa Fluor 680 (Molecular Probes) diluted in PBS-1% Tween for 40 min. For probing for total AR, a monoclonal anti-AR antibody (BD Biosciences, San Jose, CA) was used. The secondary antibody used was antimouse IRDye 800 (Rockland Immunochemicals, Gilbertsville, PA). Blots were scanned and analyzed using the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE) to quantify the levels of phosphorylated AR and total AR.

Reporter Gene Analysis
Luciferase reporters pHH-luc and pPSA-61-luc were cotransfected with AR expression plasmids and CMV-Renilla-luciferase into PC3 cells. After transfection, media was changed to phenol red-free RPMI supplemented with 5% charcoal-stripped (Dextran) serum containing 1 nM R1881 or vehicle and incubated at 37 C for 18 h. Luciferase assays were performed using the Promega Dual-Luciferase Assay System (Promega Corp., Madison, WI). Curves for dose-response assays were fit using CurveExpert 1.3 (HYAMS, Hixon, TN).

Real-Time RT-PCR
Real-time RT-PCR was performed as previously described (5). Briefly, RNA was extracted from PC3 cells transiently expressing various constructs using the RNeasy kit (QIAGEN, Chatsworth, CA). Assays were conducted on an iCycler optical system (Bio-Rad Laboratories, Inc., Hercules, CA) using the IQ SYBR Green PCR master mix. DNase I treatment was performed directly on the RNeasy mini column with 27 Kunitz units of DNase I (QIAGEN) for 15 min at room temperature according to the QIAGEN protocol. RNA was quantified using Ribogreen (Molecular Probes). RNA (500 ng) was reverse transcribed in 20 µl reaction volume using the iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer’s protocol. The human specific PCR primers used were: forward FKBP51, 5'-CATCAAGGCATGGGACATTGG-3'; reverse FKBP51, 5'-TCGAGGGAATTTTAGGGAGACT-3'; forward S100P, 5'-ATGACGGAACTAGAGACAGCC-3'; reverse S100P, 5'-AGGAAGCCTGGTAGCTCCTT-3'; forward ß-glucuronidase (GUS), 5'-CCGACTTCTCTGACAACCGACG-3'; reverse GUS, 5'-AGCCGACAAAATGCCGCAGACG-3'. Nonreversed transcribed RNA was subject to PCR as a control. The primer annealing temperatures were 60 C (FKBP51), 58 C (S100P), and 68 C (GUS).

Androgen Binding and Dissociation Assays
Binding assays using R1881 were performed as described (51). Briefly, PC3 cells in 12-well dishes were transfected and treated in duplicate with [3H]R1881 (PerkinElmer, Boston, MA) concentrations ranging from 0.05 nM to 2 nM. Nonspecific binding was determined using the same concentrations of [3H]R1881 in the presence of a 200-fold excess of unlabeled R1881. The dissociation constant (KD), t1/2, 95% confidence limits, and SE were determined from Scatchard analysis using linear regression (Microsoft Excel and Minitab version 14). For dissociation assays, transfected PC3 cells were pulsed with 2 nM [3H]R1881 for 60 min, washed with phenol red-free, serum free RPMI 1640, and chased with 2 nM cold R1881 for 15, 30, 60, 90, 120, 150, and 180 min.


   ACKNOWLEDGMENTS
 
We thank Myles Brown (Harvard University, Boston, MA) for providing plasmids and Adam Spencer (University of Virginia, Charlottesville, VA) for preparation of antibodies. The Paschal and Weber laboratories are acknowledged for helpful discussions throughout the course of the study.


   FOOTNOTES
 
This work was supported by National Cancer Institute Grant PO1CA104106.

Disclosure Statement: The authors have nothing to disclose.

First Published Online June 19, 2007

Abbreviations: AF, Activation function; AR, androgen receptor; DBD, DNA-binding domain; ER, estrogen receptor; GFP, green fluorescence protein; GR, glucocorticoid receptor; GUS, ß-glucuronidase; LBD, ligand-binding domain; LMB, leptomycin B; MMTV, mouse mammary tumor virus; NES, nuclear export signal; NLS, nuclear localization signal; PR, progesterone receptor; PSA, prostate-specific antigen; SV40, simian virus 40; WT, wild type.

Received for publication May 9, 2007. Accepted for publication June 14, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Avila DM, Zoppi S, McPhaul MJ 2001 The androgen receptor (AR) in syndromes of androgen insensitivity and in prostate cancer. J Steroid Biochem Mol Biol 76:135–142[CrossRef][Medline]
  2. Matsumoto T, Takeyama K, Sato T, Kato S 2005 Study of androgen receptor functions by genetic models. J Biochem 138:105–110[Abstract/Free Full Text]
  3. Ikeda Y, Aihara K, Sato T, Akaike M, Yoshizumi M, Suzaki Y, Izawa Y, Fujimura M, Hashizume S, Kato M, Yagi S, Tamaki T, Kawano H, Matsumoto T, Azuma H, Kato S, Matsumoto T 2005 Androgen receptor gene knockout male mice exhibit impaired cardiac growth and exacerbation of angiotensin II-induced cardiac fibrosis. J Biol Chem 280:29661–29666[Abstract/Free Full Text]
  4. Gioeli D, Ficarro SB, Kwiek JJ, Aaronson D, Hancock M, Catling AD, White FM, Christian RE, Settlage RE, Shabanowitz J, Hunt DF, Weber MJ 2002 Androgen receptor phosphorylation. J Biol Chem 277:29304–29314[Abstract/Free Full Text]
  5. Gioeli D, Black BE, Gordon V, Spencer A, Kesler CT, Eblen ST, Paschal BM, Weber MJ 2006 Stress kinase signaling regulates androgen receptor phosphorylation, transcription, and localization. Mol Endocrinol 20:503–515[Abstract/Free Full Text]
  6. Lin HK, Wang L, Hu YC, Altuwaijri S, Chang C 2002 Phosphorylation-dependent ubiquitylation and degradation of androgen receptor by Akt require Mdm2 E3 ligase. EMBO J 21:4037–4048[CrossRef][Medline]
  7. Poukka H, Karvonen U, Janne OA, Palvimo JJ 2000 Covalent modification of the androgen receptor by small ubiquitin-like modifier 1 (SUMO-1). Proc Natl Acad Sci USA 97:14145–14150[Abstract/Free Full Text]
  8. Burgdorf S, Leister P, Scheidtmann KH 2004 TSG101 interacts with apoptosis-antagonizing transcription factor and enhances androgen receptor-mediated transcription by promoting its monoubiquitination. J Biol Chem 279:17524–17534[Abstract/Free Full Text]
  9. Fu M, Wang C, Wang J, Zhang X, Sakamaki T, Yeung YG, Chang C, Hopp T, Fuqua SAW, Jaffray E, Hay RT, Palvimo JJ, Janne OA, Pestell RG 2002 Androgen receptor acetylation governs trans activation and MEKK1-induced apoptosis without affecting in vitro sumoylation and trans-repression function. Mol Cell Biol 22:3373–3388[Abstract/Free Full Text]
  10. Gioeli D 2005 Signal transduction in prostate cancer progression. Clin Sci 108:293–308[CrossRef][Medline]
  11. Culig Z, Bartsch G 2006 Androgen axis in prostate cancer. J Cell Biochem 99:373–381[CrossRef][Medline]
  12. Bakin RE, Gioeli D, Sikes RA, Bissonette EA, Weber MJ 2003 Constitutive activation of the ras/mitogen-activated protein kinase signaling pathway promotes androgen hypersensitivity in LNCaP prostate cancer cells. Cancer Res 63:1981–1989[Abstract/Free Full Text]
  13. Zhang L, Johnson M, Le KH, Sato M, Ilagan R, Iyer M, Gambhir SS, Wu L, Carey M 2003 Interrogating androgen receptor function in recurrent prostate cancer. Cancer Res 63:4552–4560[Abstract/Free Full Text]
  14. Wang L, Hsu CL, Chang C 2005 Androgen receptor corepressors: an overview. Prostate 63:117–130[CrossRef][Medline]
  15. Shank LC, Paschal BM 2005 Nuclear transport of steroid hormone receptors. Crit Rev Eukaryot Gene Expr 15:49–73[CrossRef][Medline]
  16. Zhou Z, Sar M, Simental JA, Lane MV, Wilson EM 1994 A ligand-dependent biopartite nuclear targeting signal in the human androgen receptor. J Biol Chem 269:13115–13123[Abstract/Free Full Text]
  17. Picard D, Yamamoto KR 1987 Two signals mediate hormone-dependent nuclear localization of the glucocorticoid receptor. EMBO J 6:3333–3340[Medline]
  18. Black BE, Holaska JM, Rastinejad F, Paschal BM 2001 DNA binding domains in diverse nuclear receptors function as nuclear export signals. Curr Biol 11:1749–1758[CrossRef][Medline]
  19. Saporita AJ, Zhang Q, Navai N, Dincer Z, Hahn J, Cai X, Wang Z 2003 Identification and characterization of a ligand-regulated nuclear export signal in androgen receptor. J Biol Chem 278:41998–42005[Abstract/Free Full Text]
  20. Tyagi RK, Lavrovsky Y, Ahn SC, Song CS, Chatterjee B, Roy AK 2000 Dynamics of intracellular movement and nucleocytoplasmic recycling of the ligand-activated androgen receptor in living cells. Mol Endocrinol 14:1162–1174[Abstract/Free Full Text]
  21. Madan AP, DeFranco DB 1993 Bidirectional transport of glucocorticoid receptors across the nuclear envelope. Proc Natl Acad Sci USA 90:3588–3592[Abstract/Free Full Text]
  22. Dauvois S, White R, Parker MG 1993 The antiestrogen ICI 182780 disrupts estrogen receptor nucleocytoplasmic shuttling. J Cell Sci 106:1377–1388[Abstract]
  23. Guichon-Mantel A, Delabre K, Lescop P, Milgrom E 1996 Intracellular traffic of steroid hormone receptors. J Steroid Biochem Mol Biol 56:3–9[CrossRef][Medline]
  24. Bunn CF, Neidig JA, Freidinger KE, Stankiewicz TA, Weaver BS, McGrew J, Allison LA 2001 Nucleocytoplasmic shuttling of the thyroid hormone receptor {alpha}. Mol Endocrinol 15:512–533[Abstract/Free Full Text]
  25. Qiu M, Olsen A, Faivre E, Horwitz KB, Lange CA 2003 Mitogen-activated protein kinase regulates nuclear association of human progesterone receptors. Mol Endocrinol 17:628–642[Abstract/Free Full Text]
  26. Callige M, Kieffer I, Richard-Foy H 2005 CSN5/Jab1 is involved in ligand-dependent degradation of estrogen receptor {alpha} by the proteasome. Mol Cell Biol 25:4349–4358[Abstract/Free Full Text]
  27. Migliaccio A, Castoria G, di Domenico M, de Falco A, Bilancio A, Lombardi M, Barone MV, Ametrano D, Zannini MS, Abbondanza C, Auricchio F 2000 Steroid-induced androgen receptor-oestradiol receptor ß-Src complex triggers prostate cancer cell proliferation. EMBO J 19:5406–5417[CrossRef][Medline]
  28. Permberton LF, Paschal BM 2005 Mechanisms of receptor-mediated nuclear import and nuclear export. Traffic 6:187–198[CrossRef][Medline]
  29. Taagepera S, McDonald D, Loeb JE, Whitaker LL, McElroy AK, Wang JYJ, Hope TJ 1998 Nuclear-cytoplasmic shuttling of c-Abl tyrosine kinase. Proc Natl Acad Sci USA 95:7457–7462[Abstract/Free Full Text]
  30. Cleutijens KBJM, van der Korput HAGM, van Eekelen CCEM, van Rooij HCJ, Faber PW, Trapman J 1997 An androgen response element in a far upstream enhancer region is essential for high, androgen-regulated activity of the prostate-specific antigen promoter. Mol Endocrinol 11:148–161[Abstract/Free Full Text]
  31. Nordeen S 1988 Luciferase reporter gene vectors for analysis of promoters and enhancers. Biotechniques 6:454–456[Medline]
  32. Magee JA, Chang L, Stormo GD, Milbrandt J 2006 Direct, androgen receptor mediated regulation of the FKBP5 gene via a distal enhancer element. Endocrinology 147:590–598[Abstract/Free Full Text]
  33. Averboukh L, Liang P, Kantoff PW, Pardee AB 1996 Regulation of S100P expression by androgen. Prostate 29:350–355[CrossRef][Medline]
  34. Fang Y, Fliss AE, Robins DM, Caplan AJ 1996 Hsp90 regulates androgen receptor hormone binding affinity in vivo. J Biol Chem 271:28697–28702[Abstract/Free Full Text]
  35. Gregory CW, Johnson RT, Mohler JL, French FS, Wilson EM 2001 Androgen receptor stabilization in recurrent prostate cancer is associated with hypersensitivity to low androgen. Cancer Res 61:2892–2898[Abstract/Free Full Text]
  36. Nelson PS, Clegg N, Arnold H, Ferguson C, Bonham M, White J, Hood L, Lin B 2002 The program of androgen-responsive genes in neoplastic prostate epithelium. Proc Natl Acad Sci USA 99:11890–11895[Abstract/Free Full Text]
  37. Chen CD, Welsbie DS, Tran C, Baek SH, Chen R, Vessella R, Rosenfeld MG, Sawyers CL 2004 Molecular determinants of resistance to antiandrogen therapy. Nat Med 10:33–39[CrossRef][Medline]
  38. Sackey FNA, Hache RJG, Reich T, Kwast-Welfeld J, Lefebvre YA 1996 Determinants of subcellular distribution of the glucocorticoid receptor. Mol Endocrinol 10:1191–1205[Abstract/Free Full Text]
  39. Liu J, DeFranco DB 1999 Chromatin recycling of glucocorticoid receptors: implication for multiple roles of heat shock protein 90. Mol Endocrinol 13:355–365[Abstract/Free Full Text]
  40. Chen S, Xu Y, Yuan X, Bubley GJ, Balk SP 2006 Androgen receptor phosphorylation and stabilization in prostate cancer by cyclin-dependent kinase 1. Proc Natl Acad Sci USA 103:15969–15974[Abstract/Free Full Text]
  41. Takizawa CG, Morgan DO 2000 Control of mitosis by changes in the subcellular location of cyclin-B1-Cdk1 and Cdc25C. Curr Opin Cell Biol 12:658–665[CrossRef][Medline]
  42. Ben-Levy R, Hooper S, Wilson R, Paterson HF, Marshall CJ 1998 Nuclear export of the stress-activated protein kinase p38 mediated by its substrate MAPKAP kinase-2. Curr Biol 8:1049–1057[CrossRef][Medline]
  43. Taneja SS, Ha S, Swenson NK, Huang HY, Lee P, Melamed J, Shapiro E, Garabedian MJ, Logan SK 2005 Cell-specific regulation of androgen receptor phosphorylation in vivo. J Biol Chem 280:40916–40924[Abstract/Free Full Text]
  44. Liu J, DeFranco DB 2000 Protracted nuclear export of glucocorticoid receptor limits its turnover and does not require the exportin 1/CRM1-directed nuclear export pathway. Mol Endocrinol 14:40–51[Abstract/Free Full Text]
  45. Sheflin L, Keegan B, Zhang W, Spaulding SW 2000 Inhibiting proteasomes in human HepG2 and LNCaP cells increases endogenous androgen receptor levels. Biochem Biophys Res Commun 276:144–150[CrossRef][Medline]
  46. Skildum A, Faivre E, Lange CA 2005 Progesterone receptors induce cell cycle progression via activation of mitogen-activated protein kinases. Mol Endocrinol 19:327–339[Abstract/Free Full Text]
  47. Simoncini T, Hafezi-Moghadam A, Brazil DP, Ley K, Chin WW, Liao JK 2000 Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH-kinase. Nature 407:538–541[CrossRef][Medline]
  48. Kousteni S, Bellido T, Plotkin LI, O’Brien CA, Bodenner DL, Han L, Han K, DiGregorio GB, Katzenellenbogen JA, Katzenellenbogen BS, Roberson PK, Weinstein RS, Jilka RL, Manolagas SC 2001 Nongenotropic, sex-nonspecific signaling through the estrogen or androgen receptors: dissociation from transcriptional activity. Cell 104:719–730[Medline]
  49. Black BE, Levesque L, Holaska JM, Wood TC, Paschal BM 1999 Identification of the NTF2-related factor that binds ran-GTP and regulates nuclear protein export. Mol Cell Biol 19:8616–8624[Abstract/Free Full Text]
  50. Yang CS, Vitto MJ, Busby SA, Garcia BA, Kesler CT, Gioeli D, Shabanowitz J, Hund DF, Rundell K, Brautigan DL, Paschal BM 2005 Simian virus 40 small t antigen mediates conformation-dependent transfer of protein phosphatase 2A onto the androgen receptor. Mol Cell Biol 25:1298–1308[Abstract/Free Full Text]
  51. Blok LJ, Ruiter PE, Brinkmann AO 1998 Forskolin-induced dephosphorylation of the androgen receptor impairs ligand binding. Biochemistry 37:3850–3857[CrossRef][Medline]

NURSA Molecule Pages Link:

Nuclear Receptors:   AR
Ligands:   R1881



This article has been cited by other articles:


Home page
 Mol. Endocrinol.Home page
S. Kaikkonen, T. Jaaskelainen, U. Karvonen, M. M. Rytinki, H. Makkonen, D. Gioeli, B. M. Paschal, and J. J. Palvimo
SUMO-Specific Protease 1 (SENP1) Reverses the Hormone-Augmented SUMOylation of Androgen Receptor and Modulates Gene Responses in Prostate Cancer Cells
Mol. Endocrinol., March 1, 2009; 23(3): 292 - 307.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
M. Li, J.-S. Ai, B.-Z. Xu, B. Xiong, S. Yin, S.-L. Lin, Y. Hou, D.-Y. Chen, H. Schatten, and Q.-Y. Sun
Testosterone Potentially Triggers Meiotic Resumption by Activation of Intra-Oocyte SRC and MAPK in Porcine Oocytes
Biol Reprod, November 1, 2008; 79(5): 897 - 905.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
L. C. Shank, J. B. Kelley, D. Gioeli, C.-S. Yang, A. Spencer, L. A. Allison, and B. M. Paschal
Activation of the DNA-dependent Protein Kinase Stimulates Nuclear Export of the Androgen Receptor in Vitro
J. Biol. Chem., April 18, 2008; 283(16): 10568 - 10580.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow NURSA Molecule Pages Link
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kesler, C. T.
Right arrow Articles by Paschal, B. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kesler, C. T.
Right arrow Articles by Paschal, B. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals