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Molecular Endocrinology 20 (3): 503-515
Copyright © 2006 by The Endocrine Society

Stress Kinase Signaling Regulates Androgen Receptor Phosphorylation, Transcription, and Localization

Daniel Gioeli, Ben E. Black, Vicki Gordon, Adam Spencer, Cristina T. Kesler, Scott T. Eblen, Bryce M. Paschal and Michael J. Weber

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

Address all correspondence and requests for reprints to: Daniel Gioeli, Department of Microbiology, P.O. Box 800734, University of Virginia Health System, Charlottesville, Virginia 22908. E-mail: dgg3f{at}virginia.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Activation of signal transduction kinase cascades is known to alter androgen receptor (AR) activity, but the molecular mechanisms are still poorly defined. Here we show that stress kinase signaling regulates Ser 650 phosphorylation and AR nuclear export. In LNCaP prostate cancer cells, activation of either MAPK kinase (MKK) 4:c-Jun N-terminal kinase (JNK) or MKK6:p38 signaling pathways increased Ser 650 phosphorylation, whereas pharmacologic inhibition of JNK or p38 signaling led to a reduction of AR Ser 650 phosphorylation. Both p38{alpha} and JNK1 phosphorylated Ser 650 in vitro. Small interfering RNA-mediated knockdown of either MKK4 or MKK6 increased endogenous prostate-specific antigen (PSA) transcript levels, and this increase was blocked by either bicalutamide or AR small interfering RNA. Stress kinase inhibition of PSA transcription is, therefore, dependent on the AR. Similar experiments involving either activation or inhibition of MAPK/ERK kinase:ERK signaling had little effect on Ser 650 phosphorylation or PSA mRNA levels. Ser 650 is proximal to the DNA binding domain that contains a nuclear export signal. Mutation of Ser 650 to alanine reduced nuclear export of the AR, whereas mutation of Ser 650 to the phosphomimetic amino acid aspartate restored AR nuclear export. Pharmacologic inhibition of stress kinase signaling reduced wild-type AR nuclear export equivalent to the S650A mutant without affecting nuclear export of the S650D mutant. Our data suggest that stress kinase signaling and nuclear export regulate AR transcriptional activity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE ANDROGEN RECEPTOR (AR) is a ligand-activated transcription factor. In addition to regulation by steroid, the AR is also regulated by posttranslational modifications generated by signal transduction pathways. Thus, the AR may also act as a node that integrates multiple extracellular signals (1, 2). During prostate cancer progression toward an androgen- independent state, there is an increase in expression of growth factors and their cognate receptors, and it is generally assumed that this promotes enhanced paracrine and autocrine stimulation of cell growth and survival (3). Studies have also shown that growth factor receptor signals can activate the AR or sensitize it to reduced levels of ligand (4, 5, 6, 7, 8). This is significant because it is clear that the AR plays a critical role in the progression of prostate cancer. Late-stage androgen-independent prostate cancer almost always retains expression of the AR despite the near absence of circulating androgens (9). Moreover, inhibiting AR expression using antibodies, ribozymes, or antisense oligonucleotides leads to an inhibition of prostate cancer cell growth (10, 11, 12, 13). Additionally, gene expression profiles of androgen-dependent and androgen-independent prostate cancers reveal that increased AR expression was the only consistent change among all the samples examined (14). This increase in AR expression hypersensitized the AR to low levels of ligand and was both necessary and sufficient to drive prostate cancer progression to androgen independence. However, the mechanisms by which peptide growth factor signaling and/or changes in AR protein levels contribute to prostate cancer progression are still poorly understood.

In an effort to understand how cross-talk between peptide growth factor and steroid hormone signaling contribute to prostate cancer progression, we set out to characterize the major phosphorylation sites in AR (15). We identified phosphorylation at serines 16, 81, 94, 256, 308, 424, and 650. Notably, diverse agonists including activators of protein kinase A (forskolin) and protein kinase C [phorbol-12-myristate-13-acetate (PMA)] increased Ser 650 phosphorylation (15). Additionally, treatment of LNCaP cells with epidermal growth factor (EGF) modestly increased Ser 650 phosphorylation. Wilson and colleagues (16) previously demonstrated that Ser 650 phosphorylation regulates AR transactivation; when suboptimal levels of steroid were used a decrease in AR transactivation of the mouse mammary tumor virus promoter was observed with a S650A mutant. This effect was not observed using the prostate-specific antigen (PSA) promoter, suggesting that cell and promoter context might shape the effect of Ser 650 phosphorylation on AR transactivation (15). The fact that Ser 650 phosphorylation occurs by both hormone-dependent and hormone-independent mechanisms (androgen, protein kinase A, EGF, and protein kinase C) suggest that modification of this site might be used to regulate steroid receptor function in response to a variety of physiological stimuli.

Previous studies on the GR, ER, and PR have suggested that different MAPK signaling pathways can regulate steroid receptor activation and localization through receptor phosphorylation, although the molecular mechanisms seem to vary among the different receptors (17, 18, 19). In this study, we have investigated which signal transduction pathways regulate Ser 650 phosphorylation and determined the effect of these signaling pathways on AR function. We have found that stress kinase signaling can regulate AR Ser 650 phosphorylation, antagonize AR transcription, and regulate AR export through phosphorylation of AR Ser 650. These observations have important implications regarding both AR function and prostate cancer biology.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Stress Kinase Signaling Regulates S650 Phosphorylation
We developed a polyclonal antiserum specific to phospho-Ser 650 to analyze Ser 650 phosphorylation (Fig. 1Go). This antiserum specifically recognizes the AR when phosphorylated on Ser 650 and does not recognize the S650A mutant or nonphosphorylated Ser 650 AR. The low level of immunoreactivity observed in untreated cells is consistent with previous observations of low levels of Ser 650 phosphorylation under basal conditions (15). The dose- and time-dependent increase in the immunoreactivity of the Ser 650 antiserum is parallel to the increase in phosphopeptide labeling observed on two-dimensional peptide maps (Fig. 1BGo). Moreover, incubating immunoprecipitated AR from PMA-treated cells with phosphatase dramatically reduced the amount of immunoreactive phospho Ser 650 AR, further demonstrating the specificity of the antiserum to phosphorylated Ser 650 AR (Fig. 1CGo).


Figure 1
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  		Fig. 1. Development of Antiphospho-S650 Antibody

A, COS cell lysate expressing either wild-type (Wt) or mutant (S650A) AR was immunoblotted for total and phospho-S650 AR. Cells were either untreated or treated with 20 nM or 1 µM PMA for 1 h. B, Tryptic phosphopeptide mapping of endogenous LNCaP AR; LNCaP AR was labeled metabolically with 32P for 6 h and treated with 1 µM PMA for 2 h. C, Wild-type FLAG-AR was immunoprecipitated from PMA-treated cells and incubated with or without 100 U alkaline phosphatase. AR was immunoblotted for total and phospho-S650 AR.

 
We previously observed regulation of Ser 650 phosphorylation by multiple agonists including PMA, forskolin, and EGF. Because these agonists are capable of activating one or more MAPK cascades, we sought to determine whether any of these kinase cascades directly regulates Ser 650 phosphorylation. We overexpressed different components of MAPK signaling in COS cells along with the AR (Fig. 2Go). Phosphorylation of the AR on Ser 650 was assessed by immunoprecipitation of the AR and Western blot analysis using the antibody described above. Expression of wild-type MAPK kinase (MKK) 4 and c-Jun N-terminal kinase (JNK) 1 or MKK6 and p38{alpha} led to a robust increase in Ser 650 phosphorylation, even in the absence of stimulation with an agonist such as PMA. Expression of either wild-type or mutationally active MAPK/ERK kinase (MEK) (S218,222D) with wild-type ERK only slightly increased Ser 650 phosphorylation, even though MEK/ERK expression was higher than MKK4/JNK1 and MKK6/p38{alpha}. This suggests that signaling from the stress kinases increases Ser 650 phosphorylation.


Figure 2
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  		Fig. 2. Activation of Stress Kinase Signaling Increases S650 Phosphorylation

COS cells were transfected with wild-type FLAG-AR and HA-MKK6 or HA-MKK4 or HA-MEK with and without their cognate MAPK (FLAG-p38, FLAG-JNK, or FLAG-ERK, respectively). Cells were lysed and AR was immunoprecipitated and immunoblotted for phospho-S650. Lysate was blotted for total AR, and pan- and phospho-p38, -JNK, and -ERK. wt, Wild type; MAPKK, MAPK kinase.

 
To further investigate stress kinase signaling to the AR, we examined endogenous AR Ser 650 phosphorylation levels in response to several MAPK inhibitors. LNCaP prostate cancer cells were treated with PMA (P) for 1 h with or without pretreatment with inhibitors to p38 (SB203580), JNK (SP600125), and MEK (UO126), alone or in combination (Fig. 3AGo). Treatment with either SB203580 or SP600125 dramatically reduced endogenous AR Ser 650 phosphorylation, whereas treatment with UO126 had no effect on Ser 650 phosphorylation. Cotreatment with both SB203580 and SP600125 abolished Ser 650 phosphorylation in response to PMA. Longer exposures show that basal Ser 650 phosphorylation is also inhibited by both SB203580 and SP600125 (Fig. 3BGo). Cotreatment with SB203580 and SP600125 further decreased Ser 650 phosphorylation, although some basal Ser 650 phosphorylation remained. The effectiveness of the inhibitors was confirmed by Western blot analysis of cell lysate for phospho-ATF (activating transcription factor 2) and phospho-ERK (Fig. 3CGo). Treatment of cells with either SB203580 or SP600125 decreased phospho-ATF levels, and treatment with UO126 decreased phospho-ERK levels. These results suggest that p38 and JNK signaling are the predominant regulators of Ser 650 phosphorylation levels, both basally and in response to PMA. However, other basal regulators of Ser 650 phosphorylation may be present.


Figure 3
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  		Fig. 3. Inhibition of Stress Kinase Signaling Decreases S650 Phosphorylation

A, LNCaP cells were treated with 20 nM PMA (P) for 1 h with or without a 30 min pretreatment with 50 µM SB203580 (p38 inhibitor), 25 µM SP600125 (JNK inhibitor), or 10 µM UO126 (MEK1/2 inhibitor). Cells were lysed and AR was immunoprecipitated and immunoblotted for phospho-S650. Lysate was blotted for total AR. B, LNCaP cells were treated with 50 µM SB203580 and/or 25 µM SP600125 for 30 min. The AR was then immunoprecipitated and immunoblotted as in panel A. C, Effectiveness of the inhibitors was confirmed by Western blot analysis of cell lysate for phospho-ATF and phospho-ERK (pERK). Total ERK (totERK) was used as a loading control. X, Untreated; SB, SB203580; SP, SP600125.

 
Ser 650 is a proline-directed phosphorylation site that conforms to a casein kinase II (CK2) consensus site. However, inhibition of CK2 activity with a CK2 inhibitor DRB; 5,6-dichlorobenzimidazone did not decrease induction of Ser 650 phosphorylation in response to PMA (Supplemental Data 1 published on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). Thus, it seems unlikely that CK2 regulates Ser 650 phosphorylation under the conditions described above.

We next sought to determine whether the p38 and JNK enzymes could directly phosphorylate the AR on Ser 650 in vitro. Recombinant activated p38{alpha} and JNK1 were incubated in kinase buffer containing ATP in the absence or presence of SB203580 or SP600125. Either a glutathione-S-transferase (GST)-AR fusion protein containing the AR DNA binding domain (DBD) /hinge region encompassing AR amino acids 549–671 (Fig. 4Go, A and B), affinity purified full-length wild-type AR (Fig. 4CGo), or myelin basic protein (MBP) were used as substrate (Fig. 4DGo). Both recombinant p38{alpha} and JNK1 were able to phosphorylate a GST-AR-DBD/Hinge fusion protein that contains Ser 650 (Fig. 4Go, A and B), although JNK1 appeared to phosphorylate Ser 650 more efficiently than p38{alpha}. Interestingly, JNK1 but not p38{alpha}, was able to phosphorylate full-length AR in vitro under the conditions tested (Fig. 4CGo). JNK1 phosphorylated the full-length AR 2.2-fold over untreated control, and the SP600125 inhibitor reduced AR phosphorylation by JNK1 to control levels (0.9-fold). For comparison, an equivalent amount of full-length wild-type AR used in the in vitro kinase assays was immunoprecipitated from cells either untreated or treated with PMA or Anisomycin (Fig. 4CGo, left three lanes). As previously observed, both PMA and Anisomycin treatment increased AR phosphorylation (5.2- and 7.3-fold, respectively). However, the level of AR Ser 650 phosphorylation by recombinant JNK1 was less than that observed from cells treated with PMA or Anisomycin (Fig. 4CGo, compare lanes 2 and 3 with lane 7). Recombinant p38{alpha} and JNK1 phosphorylated MBP (38- and 44-fold, respectively) and the MBP phosphorylation was inhibited to below control levels by SB203580 and SP600125 (Fig. 4DGo).


Figure 4
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  		Fig. 4. p38 and JNK Phosphorylate AR in Vitro

A, In vitro kinase assay using purified recombinant JNK1 and p38{alpha}. Purified GST-DBD hinge AR (B) was incubated in kinase assay buffer (see Materials and Methods) with increasing amounts of purified p38{alpha} or JNK1. B, Coomasie stain of purified GST-DBD hinge region of the AR. C, In the left three lanes, COS1 cells were transfected with FLAG-hAR and either left untreated or treated with 20 nM PMA for 1 h or 1 µg/ml anisomycin for 30 min. In the right five lanes, FLAG-hAR immunoprecipitated (IP) from COS cells (as in lane 1) were incubated in kinase assay buffer with 100 ng purified p38{alpha} or JNK1 without or with either 50 µM SB203580 or 25 µM SP600125 for 20 min at 30 C. Proteins were separated by SDS-PAGE and immunoblotted for phospho-S650, total AR, p38, and JNK. Phospho- and total AR levels were quantified by densitometry using an AlphaEaseFC imaging system. The relative amounts of phospho-S650 standardized to total AR are shown below the Western blot image. D, In vitro kinase assay as in panel C using MBP and 32P{gamma}ATP. Phospho-MBP (pMBP) was visualized by autoradiography. Relative levels of MBP phosphorylation determined by scintillation counting are shown below the autoradiogram.

 
Stress Kinase Signaling Negatively Regulates AR Transcription
Many studies have implicated growth factor signaling in the regulation of AR transactivation (2). Thus, we wanted to determine the effect of stress kinase signaling and Ser 650 phosphorylation on AR transcription. Previously, we have observed cooperativity between Ras and steroid signaling when examining endogenous gene activity (20). Thus, we used small interfering RNA (siRNA) to knockdown the expression of endogenous MAPK kinases in LNCaP prostate cancer cells and real-time RT-PCR to assess PSA mRNA levels (Fig. 5Go). PSA is a well-established AR target gene. ß-Glucuronidase (GUS) was used to control for nonspecific effects of MKK knockdown on transcription (21). There was minimal effect on GUS mRNA levels in response to androgens or antiandrogens (Supplemental Data 2). The amount of PSA mRNA normalized to the amount of GUS mRNA is shown. Inhibiting expression of either MKK4 or MKK6 with siRNA increased PSA mRNA levels in both the absence and presence of dihydrotestosterone (DHT), P < 0.02 using a two-sample t test. The increase in PSA mRNA levels generated by MKK siRNA was significantly reduced with either bicalutamide (Fig. 5AGo) or AR siRNA (Fig. 5BGo); P = 0.011 and P = 0.014, respectively, using a two-sample t test. This suggests that the increased production of PSA mRNA in response to inhibition of stress kinase signaling is dependent on the AR. There was no effect on PSA mRNA levels when expression of MEK1 and MEK2 was inhibited with siRNA (Fig. 5CGo). This suggests that the negative regulation of AR transcriptional activity is specific to the stress kinase pathways. Western blot analysis demonstrating the effectiveness of the siRNA knockdowns is shown in Fig. 5DGo. When taken together, these data suggest that stress kinase signaling, and specifically, MKK4/p38 and MKK6/JNK, negatively regulate AR transcriptional activity.


Figure 5
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  		Fig. 5. Inhibition of MKK Signaling Increases Endogenous PSA Transcription

LNCaP cells were transfected with NS siRNA or siRNA to MKK4, MKK6, (A); NS, MKK4, AR (B); or NS, MEK1 and 2 (C). Cells were treated with 1 nM DHT for 12 h with or without 30 min before treatment with bicalutamide (A). Relative mRNA levels were assessed by real-time RT PCR of the PSA transcript. Plots are mean starting quantity (SQ) of PSA/mean SQ GUS with SE for the ratio shown. D, Western analysis for MKK4, MKK6, MEK1, and MEK2, AR, and tubulin; cells were treated as above.

 
Ser 650 Phosphorylation Regulates AR Nuclear Export
We next wanted to determine mechanistically how stress kinase signaling negatively regulates AR transcription. Depending on the cell type, at steady state the AR is predominantly cytoplasmic in the absence of androgen and nuclear in the presence of androgen. However, even though steady-state levels show a predominant cytoplasmic or nuclear localization, the AR is shuttling between the nucleus and cytoplasm. The AR DBD can confer nuclear export activity to a reporter protein, and point mutations in the DBD inhibit nuclear export of full-length AR (22). The proximity of the Ser 650 phosphorylation site located in the Hinge region to the DBD NES suggested that Ser 650 phosphorylation might play a role in nuclear export. Moreover, there is a growing body of evidence showing that signal transduction pathways can regulate steroid receptor localization (17, 18, 19). Therefore, we hypothesized that the stress kinase-mediated inhibition of AR transcription might be due to changes in nuclear-cytoplasmic shuttling regulated by Ser 650 phosphorylation. We used a heterokaryon shuttling assay to assess nuclear-cytoplasmic shuttling of AR phosphorylation site mutants (22). In this assay, wild-type and phosphorylation site mutant AR constructs were transfected into COS7 donor cells, and NIH 3T3 acceptor cells were labeled with a CellTracker dye that converts to a membrane impermeable form after uptake. The donor and acceptor cells were then coseeded, treated with ligand to drive nuclear import of the AR, and the cells were subsequently fused with polyethylene glycol. After fusion, the cytoplasm of heterokaryon cells fluoresces red (due to CMTMR), and the nucleus fluoresces green [due to AR-GFP fusion proteins or imunofluorescence with a fluorescein isothiocyante (FITC) secondary antibody]. The wild-type AR equilibrates between the human and mouse nuclei because it undergoes nuclear-cytoplasmic shuttling, as previously shown (22). Lack of equilibration between the donor and acceptor nuclei indicates a defect in nuclear-cytoplasmic shuttling.

Wild-type AR shuttled from donor to acceptor nuclei in this assay (Fig. 6Go; see top row) as shown previously (22). In contrast, the S650A mutant showed a significant reduction in nuclear-cytoplasmic shuttling (Fig. 6Go, second row). The S650D mutant shuttled equivalent to the wild-type AR (Fig. 6Go, third row). Neither the S650A nor S650D mutation affected ligand-induced nuclear import indicating that the defect in S650A nuclear-cytoplasmic shuttling is due to a deficiency in nuclear export of the S650A mutant. To determine whether these effects on shuttling were specific to Ser 650, we also tested whether a mutation at the highest stoichiometric ligand-induced phosphosite (Ser 81) had an effect on nuclear-cytoplasmic shuttling. The S81A mutant had no effect on shuttling (Fig. 6Go, fourth row), nor did it interfere with the effects of S650 mutations on nuclear-cytoplasmic shuttling (Fig. 6Go, fifth and sixth row). As with the single phosphorylation site S650A mutant, the double phosphorylation site mutant S81,650A failed to equilibrate between the donor and acceptor nuclei. The double S81,650D mutant shuttled equivalent to both the wild-type AR and the S650D single mutant AR. These results are consistent with AR phosphorylation on Ser 650 being important for AR nuclear-cytoplasmic shuttling.


Figure 6
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  		Fig. 6. Phosphorylation of S650 Is Required for Nuclear Export

Heterokaryon shuttling assays performed using COS cells transfected with NH2-terminally FLAG-tagged human AR (wild-type or the indicated mutant) and NIH 3T3 cells labeled with the dye CellTracker CMTMR (Rhodamine). Before fusion with PEG, cells were incubated for 45 min with 10 nM R1881 and 10 µg/ml cycloheximide to induce nuclear import. Heterokaryons were incubated for 4 h after fusion in the presence of 10 nM R1881 and 10 µg/ml cycloheximide. Cells were fixed and processed for immunofluroscence for FLAG (FITC). White arrowheads distinguish NIH 3T3 nuclei. The examples are representative of 10–25 heterokaryons from two or more independent experiments. WT, Wild type; DAPI, 4'-6-diamidino-2-phenylindole.

 
Stress Kinase Signaling Regulates AR Nuclear Export
The data presented thus far suggest that stress kinase signaling regulates AR Ser 650 phosphorylation and that Ser 650 phosphorylation regulates AR export. We next sought to determine whether stress kinase signaling could regulate AR export. Quantitative AR shuttling assays, using the heterokaryon shuttling assay, were done in the presence or absence of the p38 inhibitor, SB203580, and the JNK inhibitor, SP600125 (Figs. 7Go and 8Go). To quantify nuclear-cytoplasmic shuttling, the ratio of the total fluorescence from the acceptor nucleus to the donor nucleus was determined; the total fluorescence for a nucleus was derived by multiplying the mean fluorescence by the area. Inhibition of both p38 and JNK activity dramatically reduced wild-type GFP-AR shuttling from the donor to acceptor nuclei, decreasing wild-type AR nuclear-cytoplasmic shuttling from 0.7 to 0.34 (Fig. 7Go, rows one and two; Fig. 8Go). This decrease is equivalent to the Ser 650 to Ala mutation, which decreased shuttling to 0.38. Thus, inhibition of stress kinase signaling effectively inhibits AR nuclear-cytoplasmic shuttling. Theoretically, equilibration would yield a ratio of one; however, the ratio of acceptor to donor fluorescence for FLAG-AR and GFP-AR approximated 0.7. That the ratio does not equal one may be due to the length of time allowed for equilibration or may reflect cell constraints on the maximum nuclear-cytoplasmic shuttling in the heterokaryon assay.


Figure 7
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  		Fig. 7. Inhibition of Stress Kinase Signaling Inhibits AR Nuclear Export

Heterokaryon shuttling assays performed using COS cells transfected with wild-type GFP-hAR or NH2-terminally FLAG-tagged S650D hAR and NIH 3T3 cells labeled with the dye CellTracker CMTMR (Rhodamine). Before fusion with PEG, cells were incubated for 1 h with 1 nM DHT and 10 µg/ml cyclohemamide to induce nuclear import. During this time, cells were also treated with or without 50 µM SB203580 and 25 µM SP600125. Heterokaryons were incubated for 4.5 h after fusion in the presence of 1 nM DHT and 10 µg/ml cyclohemamide, with or without SB203580 and SP600125. Cells were fixed and visualized for GFP (FITC-wild-type) or processed for immunofluroscence for FLAG (FITC-S650D). White arrowheads distinguish NIH 3T3 nuclei. The examples are representative of 19–34 heterokaryons from two or more independent experiments. WT, Wild type; DAPI, 4'-6-diamidino-2-phenylindole.

 

Figure 8
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  		Fig. 8. Quantification of AR Nuclear-Cytoplasmic Shuttling

The heterokaryon shuttling assays illustrated in part in Fig. 5Go were quantified using Openlab 4.0.2. Nuclei were traced and the mean fluorescence and area were calculated. The total fluorescence was derived by multiplying the mean fluorescence by the area. The average total fluorescence and SEM were generated from 19–34 heterokaryons from two or more independent experiments. Wt, Wild type.

 
To determine whether stress kinase regulation of AR nuclear-cytoplasmic shuttling is dependent on Ser 650 phosphorylation, we examined the ability of the p38 and JNK inhibitors to alter nuclear-cytoplasmic shuttling of the S650D mutant. Addition of the p38 and JNK inhibitors had minimal effect on the ability of the S650D mutant to equilibrate from the donor to acceptor nucleus, altering the acceptor:donor ratio from 0.77 to 0.65 (Fig. 7Go, compare rows three and four; Fig. 8Go). Thus, mutation of Ser 650 to the phosphomimetic amino acid aspartate restores nuclear-cytoplasmic shuttling in the presence of the p38 and JNK inhibitors. This suggests that stress kinase regulation of AR export occurs specifically by phosphorylation of Ser 650. Taken together, the data suggest that stress kinase signaling promotes AR movement from the nucleus to the cytoplasm. Moreover, the corresponding enhanced transcription observed when MKK4 and MKK6 protein levels are decreased suggests the dwell time of the AR in the nucleus affects AR transcriptional output.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The fundamental clinical problem for disseminated prostate cancer is the transition of androgen-dependent disease to androgen-independent disease after androgen ablation therapy. Collectively, data suggest that although advanced prostate cancer may be functionally independent of physiologic levels of androgen, it is not independent of the AR (10, 12, 13, 23). Moreover, the notion of androgen independence in advanced prostate cancers may, in fact, reflect androgen hypersensitivity (24). One possible mechanism for this AR ligand hypersensitivity is that signal transduction pathways may regulate AR localization and activity. Here we show that stress kinase signaling can up-regulate AR Ser 650 phosphorylation and antagonize AR transcription. Moreover, we demonstrate that stress kinase phosphorylation of AR on Ser 650 regulates AR export.

Regulation of Steroid Receptor Localization by Signal Transduction Pathways
Studies on the GR, ER, and PR have shown that different MAPK signaling pathways can regulate steroid receptor activation and localization through receptor phosphorylation. Data on GR suggest that activated JNK phosphorylates GR on Ser 226 in the N-terminal transactivation domain, enhancing GR nuclear export and thus decreasing GR transcriptional activity (17, 25). In these studies, UV treatment of cells resulted in enhanced GR export, and this export was dependent on both JNK activity and phosphorylation on Ser 226. Interestingly, this export was inhibited by leptomycin B, indicating that GR export under these conditions was mediated by the export receptor Crm1. However, other laboratories have shown that GR export after ligand withdrawal is not sensitive to leptomycin B (26). These observations suggest that GR may be targeted to different export pathways depending on the physiological conditions. Our results on AR and the aforementioned data on GR indicate that kinase signaling and steroid receptor phosphorylation enhance receptor export.

Our results contrast with ER{alpha}, where kinase signaling and receptor phosphorylation decrease nuclear export. Studies by Lee and Bai (18) on ER{alpha} suggest that p38 signaling increases ER transcription through phosphorylation of Thr 311, which blocks ER export and enhances interactions with p160 coactivators. Loss of phosphorylation on Thr 311 enhances the rate of nuclear export on a pathway that is leptomycin B sensitive. However, it is unclear whether Thr 311 phosphorylation results in diminished nuclear export capacity or affects nuclear import and cytoplasmic retention (discussed in Ref.27). Regardless, the data clearly implicate p38 signaling as part of a pathway that regulates nuclear-cytoplasmic shuttling of ER. Interestingly, both nuclear import and nuclear export of progesterone receptor-B can be regulated by kinase signaling and receptor phosphorylation (19). Lange and colleagues (19) demonstrated that in the absence of ligand, EGF stimulates progesterone receptor-B import through ERK phosphorylation on Ser 294, although this phosphorylation event was not required for ligand-induced import. In the presence of ligand, ERK phosphorylation on Ser 294 increased nuclear export and receptor degradation. Although the available data suggest that signal transduction could be a common mechanism for regulating the subcellular localization and activity of nuclear receptors, whether signaling enhances cytoplasmic or nuclear localization appears to be dependent on context. This includes the particular nuclear receptor, the signal transduction pathway involved, and the export pathway to which the receptor is directed.

We propose a model for stress kinase regulation of AR localization and activity (Fig. 9Go). In the presence of ligand, the AR becomes concentrated in the nucleus where it binds DNA, homodimerizes in a reaction that involves interactions between the N and C termini, and interacts with a myriad of transcriptional coregulators, transcription factors, and components of the basal transcription machinery (1, 28). Signaling from MKK4 and/or MKK6 leads to JNK and/or p38 phosphorylation of the AR on Ser 650. We know that DNA binding is not required for phosphorylation on Ser 650; a V581F AR mutant deficient for DNA binding is phosphorylated on Ser 650 (data not shown). However, it is unclear whether Ser 650 phosphorylation on wild-type AR occurs before or after DNA binding. Also, although we have not directly identified the subcellular compartment where Ser650 phosphorylation occurs, the kinetics of the phosphorylation relative to the kinetics of androgen-induced AR import suggest that Ser650 phosphorylation occurs in the nucleus. Phosphorylation, in turn, facilitates AR export to the cytoplasm and may potentially limit AR nuclear concentration in the nucleus and decrease AR-dependent gene expression.


Figure 9
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  		Fig. 9. Model of Stress Kinase Signaling Regulation of AR Transcription and Localization

See text for details.

 
Stress Kinase Phosphorylation of AR Ser 650
Three lines of evidence support our conclusion that MKK4/JNK and MKK6/p38 directly regulate Ser 650 phosphorylation. First, exogenous expression of stress kinases can increase AR Ser 650 phosphorylation. Second, pharmacologic inhibition of stress kinases inhibits endogenous AR Ser 650 phosphorylation. Third, we observed direct phosphorylation of the AR on Ser 650 in vitro by JNK1 and p38{alpha}. Interestingly, JNK1, and not p38{alpha}, was able to phosphorylate the full-length AR in vitro. When a GST-DBD/hinge region of the AR was used as a substrate, both JNK and p38{alpha} phosphorylated Ser 650. The observation that p38{alpha} can phosphorylate an AR fragment containing Ser 650 but not the full-length AR suggests that the conformation of AR may negatively regulate the p38{alpha} AR interaction or phosphorylation. It is also possible that, whereas p38{alpha} and JNK1 are capable of phosphorylating AR Ser 650 in vitro, the constellation of AR posttranslational modifications may be suboptimal for in vitro phosphorylation of Ser 650. It is also possible that Ser650 phosphorylation requires accessory factors for optimal phosphorylation by p38 and JNK. When the in vitro kinase data are considered with the observation that cotreatment with both SB203580 and SP600125 was required to inhibit endogenous LNCaP AR Ser 650 phosphorylation, it suggests that both JNK and p38 phosphorylate the AR in cells, but that p38 phosphorylation may also require a secondary regulatory event.

Stress Kinase Signaling in Prostate Cancer
Stress kinase signaling has been implicated in a variety of cellular processes including oncogenesis and tumor suppression (29). MKK4 has been reported to function as a suppressor of tumorigenesis or metastasis in a variety of cell types (30, 31). Rinker-Schaeffer and colleagues (32) demonstrated that MKK4 could suppress AT6.1 rat prostate cancer metastasis in vivo. More significantly, the same research group demonstrated a loss of MKK4 expression in advanced prostate tumors from patients (33). An immunohistochemical and loss of heterozygosity analysis of human prostate tumor material showed a consistent down-regulation of MKK4 expression in advanced prostate tumors and loss of heterozygosity within the MKK4 locus in 31% of prostate tumors examined. Loss of stress kinase signaling components in advanced prostate cancer was also observed in the TRAMP (transgenic adenocarcinoma of the mouse prostate) model; activated forms of p38 are reduced or absent in both late stage adenocarcinomas and metastatic deposits (34). We have shown that decreasing MKK4 and MKK6 expression with siRNA can increase AR transactivation of PSA. Thus, one implication of the decrease in MKK4 expression in advanced prostate cancer is that it may hypersensitize the AR to androgen and thereby promote the acquisition of androgen-independent disease. It remains to be determined whether the loss of MKK4 (or MKK6) expression correlates with the appearance of androgen-independent prostate cancer, or whether these kinases can drive progression to androgen independence.

The p38 and JNK inhibitors used in this study have been tested for growth inhibition of prostate cancer cells (35, 36). Interestingly, JNK inhibition induced apoptosis in multiple prostate cancer cell lines in vitro and slowed DU145 xenograft growth. Given the multitude of cell processes regulated by stress kinases, it is not surprising that there are data to suggest stress kinase signaling may provide both a tumor suppressor and oncogenic role in prostate cancer (29). These observations are likely due to multiple factors including cell type, strength and duration of signal, method of signal modulation, and cell signaling environment. Moreover, it is possible that the role of stress kinase signaling may change as prostate cancer progresses. Consistent with this, well-differentiated TRAMP tumors showed elevated levels of p38 activity whereas, as mentioned above, p38 activity is decreased or absent in late stage adenocarcinomas and metastatic deposits (34).

Stress Kinase Regulation of AR Transactivation
Our data suggest that MKK4 and MKK6 signaling can antagonize AR transcription by increasing AR phosphorylation on Ser 650 and facilitating AR nuclear export. However, there are other possible mechanisms by which stress kinase signaling can antagonize AR transcription. Previous studies have suggested that PMA can decrease PSA gene expression by increasing AP-1 activity, which results in mutual repression of DNA binding (37). This study also showed that the PMA dose (1 nM) used to antagonize AR transcription did not alter nuclear levels of the AR. However, we observe a decrease in AR nuclear localization at PMA doses (20 nM) that increase AR Ser 650 phosphorylation (data not shown). Furthermore, a recent study has shown that blockade of AP-1 activity does not prevent the decrease in AR transcription by PMA suggesting alternative mechanisms for PMA induced down-regulation of AR transcription (38). Other studies have suggested that c-Jun can support AR transcription (39). This indicates that the ability of AP-1 to modulate AR activity may be cell and promoter specific. Our data suggest that JNK and p38 can directly phosphorylate the AR and antagonize AR transcription by facilitating AR export.

Ser 650 Phosphorylation in Nuclear Export
Phosphorylation on Ser 650 by stress kinases may generate a signal for nuclear export or alternatively, phosphorylation on Ser 650 may relieve nuclear retention. The Crm1/exportin 1 pathway directs export of leucine-rich nuclear export sequences (NES), yet the AR lacks such a sequence, and previous studies have shown that AR export is not inhibited by the Crm1 inhibitor, leptomycin B (40). Calreticulin (CRT) has been reported to facilitate nuclear export of GR through interactions with the DBD (22). Based on studies with GR and the fact that CRT can directly bind the AR DBD (22), CRT is a candidate receptor for AR nuclear export. However, recently the condition under which CRT mediates nuclear export of steroid receptors has been questioned (41). Another possibility is that Ser 650 phosphorylation regulates the association with factors that help retain the AR in the nucleus. For example, the Hinge region has been shown to regulate AR targeting to the nuclear matrix upon ligand binding (42). Stress kinase phosphorylation on Ser 650 could release AR from the nuclear matrix, thus freeing the AR to interact with nuclear export proteins. AR coactivators have also been shown to decrease nuclear export of the AR. Expression of SNURF (small nuclear ring finger protein/ring finger protein 4), an AR coactivator, increased AR nuclear localization in the absence of hormone, retarded nuclear export upon hormone withdrawal, and increased AR association with the nuclear matrix (43). Thus, Ser 650 phosphorylation may alter association with AR cofactors and thereby contribute to nuclear export.

Concluding Remarks
In summary we have we have found that stress kinase signaling can regulate AR Ser 650 phosphorylation, reduce AR transcription, and potentiate AR export through phosphorylation of the AR on Ser 650. Defining how signal transduction pathways regulate AR activity may provide insight into the development of androgen-independent disease. This study raises the possibility that small molecules that facilitate AR export either through modifying stress kinase signaling pathways or by affecting other components of the AR export machinery may be efficacious in the treatment of prostate cancer.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Phospho-Specific AR Antibody
The antiphospho-Ser650 antibody was raised against a peptide spanning the Ser 650 phosphorylation site in human AR by standard methods. The peptide was synthesized with phospho-serine and an N-terminal cysteine, coupled to keyhole limpet hemocyanin, and used for antibody production in rabbits (Upstate, Lake Placid, NY). Antibody titers were monitored by immunoblotting against purified S650A AR, and the terminal bleeds were affinity purified using Sepharose-immobilized phospho-peptide. To test whether the antibody detected the nonphosphorylated wild-type AR, the AR was affinity purified and incubated for 1 h with 100 U alkaline phosphatase at 37 C before SDS-PAGE. Phospho-peptide mapping of endogenous AR was previously described (15).

Cell Culture, Transfections, and Reagents
LNCaP cells (a gift from Dr. L. W. K. Chung, Emory University) were grown in T-media (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 5% fetal calf serum (Invitrogen Life Technologies). COS7 cells were grown in DMEM (Invitrogen Life Technologies) supplemented with 5% fetal calf serum. All cultures were maintained in a humidified chamber at 37 C with 5% CO2. Transfections in COS7 cells were performed using Lipofectamine (GIBCO-BRL) for biochemical experiments and FuGENE 6 (Roche, Indianapolis, IN) for heterokaryon experiments according to the manufacturer’s instructions.

The FLAG-AR, GFP-AR, FLAG-ERK, wild-type hemagglutinin (HA)-MEK and S218/222D HA-MEK have been previously described (15, 44, 45, 46). JNK1 construct was kindly provided by Roger Davis (University of Massachusetts Medical School, Boston, MA) (47). GST-p38{alpha}, HA-MKK4, and EE-MKK6 constructs were provided by Dennis J. Templeton (University of Virginia) (48). pcDNA3-FLAG-JNK1, pcDNA3-FLAG-p38{alpha}, and pcDNA3-HA-MKK6 constructs were generated by amplifying JNK1, p38{alpha}, or MKK6 coding regions by PCR and subcloning the product into the pcDNA3-FLAG or pcDNA3-HA vector (Kumar, N., T. Vomastek, J. Coyle, M.-L. Hammarskjold, M. J. Weber, manuscript in preparation). The portion of the AR cDNA corresponding to the DBD/Hinge region was PCR amplified and cloned into pGST-parallel 2, which was provided by J. Thomas Parsons (University of Virginia).

Antibodies were obtained from the following sources: anti-FLAG antibody, M2, (Sigma, St. Louis, MO); anti-HA antibody (12CA5) and anti-ERK (B3B9) from the UVa hybridoma facility; antiphospho-ATF2 antibody, antiphospho-JNK, and antiphospho-p38 MAPK (Cell Signaling, Beverly, MA); anti-MKK4 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); anti-MKK6 and anti-AR (Upstate); the anti-MEK1 and anti-MEK2 (Transduction Laboratories, Lexington, KY); and the phospho-ERK antibody was previously described (49).

Immunoprecipitations and in Vitro Kinase Reactions
For inhibitor studies, LNCaP cells were preincubated with kinase inhibitor for 30 min. The p38 inhibitor, SB203580, was used at 50 µM, the JNK inhibitor, SP60012, was used at 25 µM, and the MEK inhibitor, UO126, was used at 10 µM. LNCaP cells were then treated with 20 nM PMA for 1 h or 1 µg/ml Anisomycin for 30 min. For immunoprecipitations, LNCaP or COS7 cells were lysed in RIPA buffer [1% Nonidet P-40, 1% Na-Deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris (pH 7.5), 2 mM EDTA, 150 mM NaCl, 0.01 M Na-Phosphate, 50 mM NaF] plus the following protease and phosphatase inhibitors: 1 µg/ml pepstatin, 1 µg/ml leupeptin, 0.4 TIU/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 200 µM orthovanadate, 50 mM ß-glycerophosphate, and 0.4 µM Microcystin. The AR was immunoprecipitated from LNCaP cells with 10 µg antihuman AR, PG-21 (Upstate) per 100-mm dish. Ectopically expressed FLAG-AR was immunoprecipitated from COS7 cells with the M2 anti-Flag monoclonal antibody coupled to agarose (Sigma). Immunoprecipitates were washed three times with lysis buffer. Precipitates were resuspended in SDS-PAGE sample buffer, resolved by SDS-PAGE and transferred to nitrocellulose for immunoblotting.

For in vitro kinase assays, either affinity purified FLAG-AR (15), glutathione purified GST-DBD/hinge AR, or MBP was used as substrate. The FLAG-AR was affinity purified from transfected COS-1 cells on a M2 (Sigma) affinity column (15). The GST-DBD/hinge AR consisted of AR amino acids 549–671. The GST-DBD/hinge AR was purified from Escherichia coli as previously described (44). Purified active p38 or JNK was obtained commercially (Upstate). Kinase assays were performed in a 40-µl reaction volume containing 25 mM HEPES (pH 7.4), 20 mM magnesium acetate; 1 mM dithiothreitol, 1 mM ATP for 30 min at 30 C with either 0.5 µg of GST-DBD/hinge AR, approximately 0.1 µg of FLAG-AR, or 20 µg of MBP. Control reactions with MBP included 10 µCi of [{gamma}-32P]ATP. Reactions were stopped by addition of 4x sodium dodecyl sulfate-sample buffer followed by boiling for 5 min. Twenty microliters of these reactions were analyzed on a 12.5% SDS-PAGE gel and transferred to nitrocellulose. Phosphorylated AR was visualized and quantified by densitometry using the Alpha Innotech (Genetic Technologies, Miami, FL) imaging system. Phosphorylated MBP was cut from the membrane and quantitated by Cerenkov counting.

siRNA Knockdowns and Real-Time RT-PCR Analysis
The siRNAs to MKK4, MKK6, MEK1, and MEK2 were Dharmacon SMART pool reagents (Dharmacon). The AR siRNA was designed against the following sequence in the 3'-untranslated region: 5'-GATGTCTTCTGCCTGTTAT-3'. The nonspecific (NS) siRNA was designed against 5'-ATGTATTGGCCTGTATTA-3'. LNCaP cells were switched to RPMI 5% charcoal-stripped serum (Hyclone, Logan, UT) and transfected with siRNA using Oligofectamine (Invitrogen) according to the manufacturer’s instructions. Using a Cy3-luciferase siRNA (Dharmacon, Lafayette, CO) as a control, we observe a transfection efficiency of 100%. Knockdowns were assessed by Western blot analysis; maximal knockdown of target proteins was observed on d 3 and 4. On d 3 after transfection, cells were treated with 1 nM DHT for 12 h. On d 4, real-time RT PCR analysis was performed on siRNA knockdowns on an iCycler optical system (Bio-Rad, Hercules, CA) using the IQ SYBR Green PCR master mix. Total RNA was extracted from cells using the RNeasy kit (QIAGEN, Valencia, CA). RNA quantification to determine amounts for DNase treatment was assessed with spectrophotometry at 260 and 280 nm (1 A260 nm, 40 µg/ml). DNase I treatment was performed before the reverse transcription. Ten micrograms of total RNA were incubated in 20 µl with 10 U of DNase I (Ambion, Austin, TX) in 10 mM Tris-HCl (pH 8.0), 0.5 mM MgCl2, 1 mM dithiothreitol for 30 min at 37 C, followed by repurification of the RNA using the RNeasy kit (QIAGEN). Ribogreen (Molecular Probes, Eugene, OR) was used to quantify RNA. We reverse-transcribed 100 ng of RNA in 20 µl using the iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer’s protocol. The human-specific PCR primers used were: forward PSA 5'-TGGTGCATTACCGGAAAGTGGATCA-3'; reverse PSA 5'-GCTTGAGTCTTGGCCTGGTCATTTC-3'; forward GUS 5'-CCGACTTCTCTGACAACCGACG’3'; reverse GUS 5'-AGCCGACAAAATGCCGCAGACG-3'. Non-reverse-transcribed RNA was subject to PCR as a control; no DNA contamination was observed. For PSA, a three-step PCR was performed with an annealing temperature of 66 C. For GUS, a two-step PCR was performed with an annealing and extension temperature of 68 C.

Heterokaryon Cell Fusion Assays
The heterokaryon cell fusion assays were essentially performed as previously described (22). Briefly, NIH3T3 cells were labeled in tissue culture dishes with 500 nM CellTracker dye, (CMTMR; Molecular Probes), according to the manufacturer’s directions. Unincorporated dye was removed and cells were trypsinized and coseeded on glass coverslips with COS7 cells that had been transfected with the indicated plasmids using Fugene 6 (Roche). Equal numbers of each cell type were seeded for a total of 4 x 105 cells per coverslip and grown overnight before fusion. A concentration of 1 nM R1881 was added to the cells 1 h before fusion to induce nuclear accumulation of the AR. Cells were incubated at 37 C for 4 h after fusion in the presence of 1 nM R1881 and 10 µg/ml cyclohexamide to block protein synthesis. Cells were then fixed and processed for fluorescence microscopy. The examples shown are representative of 10–25 heterokaryons from two or more independent experiments. For quantification, nuclei were traced and the mean fluorescence and area were calculated using Openlab 4.0.2. Only heterokaryons with a single donor nucleus were included in the analysis. The total fluorescence was derived by multiplying the mean fluorescence by the area. The average total fluorescence and standard error of the mean were generated from 19–34 heterokaryons from two or more independent experiments.


   ACKNOWLEDGMENTS
 
We thank Drs. Janet Cross and Carol Chrestensen and members of the M. J. Weber, J. T. Parsons, and S. J. Parsons Research Groups for many helpful discussions. Also, we thank Dr. Mark Conaway for statistical assistance and Dr. Michael Harding for design of the PSA and GUS real-time RT-PCR primers.


   FOOTNOTES
 
This work was supported by the Department of Defense (W81XWH-04-1-0112) and Commonwealth Health Research Board (to D.G.); RO1 GM058639 (to B.M.P.); and the National Institutes of Health, National Cancer Institute (P01 CA76465, R01 CA105402) (to M.J.W.).

Present address for B.E.B.: Ludwig Institute for Cancer Research and Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California 92093.

D.G., B.E.B., V.G., A.S., C.T.K., S.T.E., and B.M.P. have nothing to declare. M.J.W. is on the Upstate Biotechnology scientific advisory board.

First Published Online November 10, 2005

Abbreviations: ATF, Activating transcription factor 2; AR, androgen receptor; CRT, calreticulin; DBD, DNA binding domain; DHT, dihydrotestosterone; EGF, epidermal growth factor; FITC, fluorescence isothiocyante; GST, glutathione-S-transferase; GUS, ß-glucuronidase; HA, hemagglutinin; JNK, c-Jun N-terminal kinase; MBP, myelin basic protein; MEK, MAPK/ERK kinase; MKK, MAPK kinase; NS, nonspecific; PMA, phorbol-12-myristate-13-acetate; PSA, prostate-specific antigen; siRNA, small interfering RNA.

Received for publication August 30, 2005. Accepted for publication November 3, 2005.


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NURSA Molecule Pages Link:

Nuclear Receptors:   AR
Ligands:   Dihydrotestosterone  |  R1881



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