CAF)-Dependent Manner (original) (raw)

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1The Albert Einstein Comprehensive Cancer Center (A.T.R., M.F., C.W., C.A., R.G.P.) Division of Hormone-Dependent Tumor Biology Department of Developmental and Molecular Biology Albert Einstein College of Medicine Bronx, New York 10461

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2Department of Internal Medicine (M.J.M.) University of Texas Southwestern Medical Center, Dallas Dallas, Texas 75235-8857

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3Department of Surgery and Genetics (Z.S.) Stanford University School of Medicine Stanford, California 943054

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4Hematology-Oncology Division (S.P.B.) Department of Medicine Beth Israel Hospital Boston, Massachusetts 02215

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5Department of Physiology (O.A.J., J.J.P.) Institute of Biomedicine and Department of Clinical Chemistry University of Helsinki FIN-00014 Helsinki, Finland

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5Department of Physiology (O.A.J., J.J.P.) Institute of Biomedicine and Department of Clinical Chemistry University of Helsinki FIN-00014 Helsinki, Finland

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*Address requests for reprints to: Richard G. Pestell, The Albert Einstein Cancer Center, Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Chanin 302, 1300 Morris Park Ave, Bronx, New York 10461.

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Received:

11 September 2000

Revision received:

09 January 2001

Accepted:

30 January 2001

Cite

Anne T. Reutens, Maofu Fu, Chenguang Wang, Chris Albanese, Michael J. McPhaul, Zijie Sun, Steven P. Balk, Olli A. Jänne, Jorma J. Palvimo, Richard G. Pestell, Cyclin D1 Binds the Androgen Receptor and Regulates Hormone-Dependent Signaling in a p300/CBP-Associated Factor (P/CAF)-Dependent Manner, Molecular Endocrinology, Volume 15, Issue 5, 1 May 2001, Pages 797–811, https://doi.org/10.1210/mend.15.5.0641
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Abstract

The androgen receptor (AR) is a ligand-regulated member of the nuclear receptor superfamily. The cyclin D1 gene product, which encodes the regulatory subunit of holoenzymes that phosphorylate the retinoblastoma protein (pRB), promotes cellular proliferation and inhibits cellular differentiation in several different cell types. Herein the cyclin D1 gene product inhibited ligand-induced AR- enhancer function through a pRB-independent mechanism requiring the cyclin D1 carboxyl terminus. The histone acetyltransferase activity of P/CAF (p300/CBP associated factor) rescued cyclin D1-mediated AR _trans_-repression. Cyclin D1 and the AR both bound to similar domains of P/CAF, and cyclin D1 displaced binding of the AR to P/CAF in vitro. These studies suggest cyclin D1 binding to the AR may repress ligand-dependent AR activity by directly competing for P/CAF binding.

INTRODUCTION

The androgen receptor (AR) is a member of a nuclear receptor superfamily that regulates ligand-dependent gene transcription through binding DNA and interacting with other transcription factors (1, 2). In response to the ligand, nuclear receptors coordinate diverse physiological roles in the regulation of metabolism and development. Androgens induce differentiation of male reproductive organs (3). Inherited mutations of the AR result in defective induction of male secondary sexual characteristics (3), and somatic mutations of the AR have been linked to therapy resistance in patients with prostate cancers (4). The AR contains distinct functional domains (termed A–F), which are conserved with other members of the “classical” receptor subclass, comprising receptors for estrogens (ER), progestins (PR), glucocorticoids (GR), and mineralocorticoids (MR). Ligand-bound activated nuclear receptors direct the assembly and stabilization of a preinitiation complex at a target gene promoter through forming direct associations with the basal transcription apparatus and with hormone-dependent cofactors (5). In vitro analysis identified interactions between nuclear receptors and components of the basal transcription apparatus, including TATA-binding protein (TBP) and TAFII30 (6). These interactions are necessary but not sufficient for efficient transactivation, which requires additional coactivator proteins (7).

Coactivator proteins for the steroid receptors can be classified into structurally related groups and include the cointegrators[ CREB-binding protein (CBP) and the related functional homolog p300 (8)], the steroid receptor coactivators (SRC-1), (also referred to as p160/NCoA-1 or ERAP-160) and related family members (9), and the steroid receptor RNA activator (SRA) (10). SRC family proteins and several other nuclear receptor coregulators contain a conserved leucine-rich domain (LXXLL) and an adjacent basic region that together serve as a nuclear receptor interaction motif (9, 11). Coactivator recruitment to a nuclear receptor involves sequential docking through the basic region to charged residues on the receptor, and then docking of the LXXLL motif (11). The cointegrator proteins, p300/CBP and the SRC family, share the capacity to acetylate histones, which often correlates in part with their transcriptional coactivator function (12). The enhancement of transcriptional activity by p300/CBP requires a bridging function to associate transcription factors with the basal transcription apparatus (13), and both intrinsic and associated histone acetyltransferase (HAT) activity, which are separable functions. Acetylation facilitates binding of transcription factors to specific target DNA sequences by destabilizing nucleosomes bound to the promoter region of a target gene (14–17). The mammalian P/CAF (p300/CBP-associated factor), which contributes the p300/CBP-associated HAT activity, is homologous to the yeast HAT and transcriptional adaptor GCN5 (18). P/CAF inhibited cell cycle progression (18) and promoted myocyte differentiation (19), suggesting P/CAF may also regulate components of the cell cycle.

The eukaryotic cell cycle contains critical genetically conserved components that subserve distinct functions (20, 21). The G1 cyclins are of two classes: the D type cyclins (D1, D2, and D3) and cyclin E. The D type cyclins share structurally conserved motifs that interact with cyclin-dependent kinases 4 and 6 (cdk4/cdk6) and pRB (retinoblastoma protein). The cyclin D1 gene has been implicated in a subset of human tumors including breast and prostate cancer, and overexpression of cyclin D1 has been linked to tumor therapy resistance. Cyclin D1 collaborates with other oncogenes in cellular transformation (22), and cyclin D1 abundance is induced by oncogenic protein including mutants of Ras (23), Rac1 (24, 25), pp60src (26), STAT3 (signal transducer and activator of transcription 3) (27), and β-catenin/APC mutants (28). The proliferative and oncogenic properties of cyclin D1 may relate to its function as the regulatory subunit of the holoenzyme that phosphorylates and inactivates the tumor suppressor pRB (20, 21). However, cyclin D1 conveys holoenzyme-independent function, inhibiting myocyte differentiation (29) through partially pRB-independent means (30) and binding the estrogen receptor (ER) to induce activity in a ligand-independent manner (31–33).

In the current studies, we show that cyclin D1 selectively inhibits ligand-dependent AR function in prostate cancer cells through a pRB-independent mechanism. The ligand dependency of this interaction contrasts fundamentally with the previously described ligand-independent interaction of cyclin D1 with the ERα. Cyclin D1 inhibition of ligand-dependent AR function required a carboxyl terminal acidic-rich region that is structurally divergent from cyclin D2 and D3. Cyclin D1 binding to the AR also required the cyclin D1 acidic-rich carboxyl terminus. The correlation between cyclin D1 binding to the AR in vitro and the repression of hormone-dependent AR activity in vivo suggests either a physical interaction between cyclin D1 and the AR may form a repressor complex or that the AR and cyclin D1 may compete for a common coactivator. p300 antagonized the cyclin D1 repressor function, which occurs independently of p300’s intrinsic HAT activity, and required the associated HAT activity of P/CAF. Cyclin D1 associated directly with P/CAF. Cyclin D1 and the AR bound to the same domains of P/CAF. Cyclin D1 repression of liganded AR activity may involve competition with the AR for binding to limiting cellular P/CAF.

RESULTS

Cyclin D1 Inhibits Ligand-Dependent AR Transactivation

To examine cyclin D1 regulation of an androgen- responsive reporter gene, the AR-deficient prostate cancer cell line DU145 was transfected with the wild-type human AR and the androgen-responsive mouse mammary tumor virus (MMTV)-LUC reporter (Fig. 1A). Dihydrotestosterone (DHT) induced reporter activity 5-fold, and coexpression of cyclin D1 inhibited this activity by 60% (Fig. 1A). Cyclin D1-mediated repression of DHT-induced MMTV-LUC reporter activity was observed in DU145 cells at several different concentrations of DHT (0.1 nm to 100 nm) (Fig. 1B). To examine further the promoter specificity of cyclin D1-mediated repression, several different control reporter constructions were examined in DU145 cells in the presence of the AR and DHT. The activity of the viral promoters[ cytomegalovirus (CMV)-LUC, rous sarcoma virus (RSV)-LUC] and the luciferase reporter vectors were not regulated by cyclin D1 (Fig. 1C). Neither cyclin D2 nor cyclin D3 inhibited AR activity (Fig. 1D).

Figure 1.

Cyclin D1 Inhibits Ligand-Dependent AR Activity A, The MMTV luciferase reporter (2.4 μg) was transfected into DU145 cells with the expression vector encoding the human AR either in the presence or absence of cyclin D1 (pCMV-cyclin D1). Comparison was made with the effect of the expression of equal amounts of empty expression vector cassette (pRC CMV). DHT was added as indicated. The results are shown as mean ± sem throughout. B, Cyclin D1-mediated repression of DHT-induced MMTV-LUC reporter activity was observed in DU145 cells and occurred at several different concentrations of DHT (0.1 nm to 100 nm). C, The effect of cyclin D1 on activity of several other reporter plasmids was determined in DU145 cells. Luciferase activity of the promoters, other than MMTV, was not affected by cyclin D1 in the presence of ligand and AR. D, The effect of cyclins D2 and D3 on MMTV-LUC activity in DU145 cells treated with DHT (10−7m). E, The effect of cyclin D1 on the activity of the MMTV-LUC reporter and several other reporter genes (F) was assessed in the PC3 cell line in the presence of the AR expression plasmid and treated with DHT (10−7m).

To determine whether cyclin D1-mediated repression of ligand-induced AR-dependent reporter activity was observed in other prostate cancer cell lines, PC3 cells were transfected with the MMTV-LUC reporter. DHT-induced the MMTV-LUC reporter 7-fold, and the ligand-induced, but not the basal, activity of the reporter was inhibited by cyclin D1 (Fig. 1E). Additional reporter genes were examined to determine the specificity of cyclin D1-mediated repression of DHT- induced AR activity. Reporter constructions were used encoding immediate early gene promoters (c-jun LUC, _jun_B LUC), synthetic response elements (SRE LUC, E2F LUC), and the promoters of cell cycle control genes (cyclin D1 LUC, cyclin E LUC, p21Cip1/WAF1 LUC). In contrast with the MMTV-LUC reporter, cyclin D1 failed to repress the additional promoter reporter constructions (Fig. 1F). Repression of AR activity was also observed in 293T cell lines (data not shown). These studies suggest that cyclin D1 repression of DHT-induced AR transcriptional activity is promoter specific.

A Structurally Divergent Acid-Rich Motif in the Carboxyl Terminus of Cyclin D1 Is Required for Repression of AR Activity

To determine the domains of cyclin D1 involved in repression of AR activity, expression plasmids encoding mutant cyclin D1 were constructed and characterized in cultured mammalian cells. The carboxyl terminus of cyclin D1 diverges structurally from cyclin D2 and D3 (Fig. 2A) due to the presence of an acid-rich region (residues 272–280). The expression of the cyclin D1 mutants was assessed in cultured cells by Western blotting. Sequential deletion of the extreme carboxyl terminus did not reduce cyclin D1 protein abundance expressed in cultured cells (Fig. 2B). The abundance of the cyclin D1 mutants was also similar to wild type cyclin D1 in the presence of DHT (Fig. 2B, lower panel). Abundance of the carboxyl-terminal deletion mutant (CD1Δ C241) was increased 2-fold in abundance compared with wild type. To identify the domains of cyclin D1 required for repression of AR activity, cotransfection experiments were conducted with either the wild-type or the mutant cyclin D1 expression vectors. Deletion of the C-terminal 21 amino acids (CD1 ΔC274) reduced AR repression by 50% (Fig. 2C). Deletion of an additional three additional residues (CD1Δ C271) or point mutation of the cyclin D1 carboxyl-terminal LLXXXL motif (leucines 254/255) abolished AR repression (Fig. 2C). The repression of DHT-induced AR activity was increased 25% by the pRB-binding defective mutant (GH) (Fig. 2C). The cdk-binding defective cyclin D1 mutant (KE) was partially defective in AR repression (Fig. 2C). Therefore, the carboxyl terminus of cyclin D1 is required for full repression of AR activity. The finding that the pRB-binding domain of cyclin D1 is not required for repression is consistent with the observation that cyclin D1 inhibits DHT-induced AR activity in DU145 cells that are pRB deficient.

Figure 2.

The Cyclin D1 Carboxyl Terminus Is Required for Inhibition of Liganded AR Activity A, Alignment of carboxyl termini amino acid sequence for cyclin D1 (D1), cyclin D2, and cyclin D3 and a schematic representation of the cyclin D1 mutant expression plasmids: cdk-binding defective (CD1 KE), pRB-binding defective (CD1 GH), deleted of the carboxyl terminus, or point mutated within the cyclin D1 LLXXXL motif (36 ). B, The cyclin D1 expression plasmids were transfected into cyclin D1-deficient 293T cells. After 24 h the cells were harvested and Western blotting was performed using the cyclin D1 antibody as described in Materials and Methods. The abundance of the cyclin D1 mutant proteins was assessed by Western blotting either without (upper panel) or with DHT (10−6m) for 24 h (lower panel). C, The inhibition of DHT (10−7m)-induced MMTV-LUC reporter activity by the wild-type or mutant cyclin D1 expression plasmids is shown with the repression by wild-type cyclin D1, shown as 100%, in DU145 cells. The data are shown as mean ± sem for n = 9.

Cyclin D1 Binds the AR Hinge Region

To determine whether AR regulation by cyclin D1 may involve a direct protein-protein interaction, we examined cyclin D1 binding to the AR using either immunoprecipitation (IP) followed by Western blotting (Fig. 3A) or glutathione-_S_-transferase (GST) pull-down assay. 293T cells were used as they have high transfection efficiency and are AR deficient. Cells were transfected with expression vectors encoding either wild-type or mutant AR and cyclin D1 (Fig. 3A) and treated with DHT or vehicle for 24 h, after which IP was performed on equal amounts of protein extracts using a cyclin D1-specific antibody (DCS-11). Western blotting was then performed for the AR and cyclin D1 using specific antibodies. Wild-type AR immunoreactivity was observed in the cyclin D1 IP (Fig. 3A, lanes 1 and 2) but not in the IP from cells transfected with the empty expression vector cassette (Fig. 3A, lanes 9 and 10) or from IPs performed with control IgG (data not shown). AR immunoreactivity was observed in the cyclin D1 IP from cells transfected with the AR mutants with deleted amino acids 80–93, 96–483, and 707–902 (Fig. 3A, lanes 3–8). The C-terminal mutant AR 1–707 bound cyclin D1 with no alteration in the amount of binding in response to DHT treatment, consistent with the known deletion of most of the ligand-binding domain in AR 1–707. These results suggest that the AR between residues 483 and 707 can bind cyclin D1.

Figure 3.

Cyclin D1 Binds Wild-Type AR in Cultured Cells and in Vitro A, Schematic representation of the AR mutant expression vectors indicating the DNA binding domain (DBD) and the carboxyl terminal ligand-binding domain (LBD). Wild-type or mutant AR and the wild-type cyclin D1 expression vectors were transfected into 293T cells. IP was performed with the cyclin D1 antibody, and precipitates were analyzed by Western blotting with either the AR antibody (above) or the cyclin D1 antibody (lower panel). B, Schematic representation of GST-AR fusion proteins. GST-AR fusion products were incubated with equal amounts of in vitro expressed cyclin D1. GST pull downs were performed using the in vitro translated product from the full-length human cyclin D1 cDNA and equal amounts of GST-AR protein, and the products were separated by SDS-PAGE. The relative amount of cyclin D1 bound to the GST-AR fragment was determined by densitometry, and percent binding is shown adjacent to the GST-AR fusion proteins. Data are the mean of three separate experiments. An example of GST-AR pull-down interaction with in vitro translated cyclin D1. The[ 35S]-labeled cyclin D1 band is indicated. The GST-AR region AR-633–668 is sufficient for specific binding to cyclin D1. C, GST pull downs were performed with GST-AR/633–668 and in vitro expressed cyclin D2 or cyclin D3. Equal amounts of each of the D type cyclins were used as input. The binding of GST-AR to either cyclin D2 or cyclin D3 was similar to GST alone.

The binding of GST-AR fusion proteins to in vitro translated cyclin D1 was next assessed (Fig. 3B). The abundance of cyclin D1 in the pull down was determined by densitometry of the[ 35S]-labeled cyclin D1 product. The binding affinity for each AR fragment was expressed relative to the nonspecific binding of GST. The GST-AR fragment AR/505–676 bound with high affinity to cyclin D1 (Fig. 3B). The GST-AR/633–668 bound cyclin D1, but neither the GST-AR/505–559 nor GST-AR/552–635 bound to cyclin D1, indicating that AR residues 633–668 are sufficient for cyclin D1 binding.

Experiments were conducted to determine whether cyclin D2 or cyclin D3 was capable of binding to the AR hinge region. GST pull-down analyses were performed using equal amounts of GST-AR fusion protein and equal amounts of in vitro translated D type cyclin (Fig. 3C). In contrast with cyclin D1, neither cyclin D2 nor cyclin D3 bound to GST-AR when compared with the nonspecific binding of GST.

The Cyclin D1 Acid-Rich Carboxyl Terminus Binds the AR

To identify the region of cyclin D1 required for binding to the AR, GST pull-down experiments were performed in which in vitro translated cyclin D1 mutants were incubated with the GST-AR fragment AR/505–676. Comparison was made to the binding of wild- type cyclin D1 set as 100%. The mean binding data of three separate experiments are shown in Fig. 4. Mutation of the cdk-binding domain did not reduce AR binding. Mutation of leucines 254 and 255 in cyclin D1 diminished AR binding by only 20%, and deletion of 21 amino acids from the cyclin D1 C terminus decreased binding by 64%. The cyclin D1 mutant ΔC267 and other deletions of the C terminus gave negligible binding to the AR.

Figure 4.

The Carboxyl Terminus of Cyclin D1 Is Required for AR Binding [35S]-labeled cyclin D1 proteins, shown schematically, were incubated with equal amounts of GST-AR fusion protein (AR 633–668), and GST pull-down experiments were conducted. The results of a representative pull-down experiment are shown. The relative amount of cyclin D1 bound to the GST-AR fragment was determined by densitometry. The percent binding is shown adjacent to the cyclin D1 mutant and graphically for the mean of at least two separate experiments.

The P/CAF Histone Acetylase Domain Relieves Cyclin D1-Mediated AR Repression

We examined the mechanisms by which cyclin D1 repressed AR activity, hypothesizing that cyclin D1 may either bind the AR to form a repressor complex or interfere with coactivators regulating AR activity. We investigated the role of p300 in cyclin D1-mediated AR repression (Fig. 5A). In previous studies, the abundance of the mutant p300 proteins was shown to be similar to the wild type when transfected into mammalian cells. Overexpression of p300 decreased cyclin D1 repression of MMTV-LUC activity from 75% repression to 25% (Fig. 5A). To examine candidate functional domains of p300 required for antagonism of cyclin D1-mediated AR repression, mutants of the p300 bromo-domain (Δ1032–1138), the HAT domain (Δ1419–1721), and the CH3 region (Δ1737–1809) were assessed (Fig. 5A). The p300 CH3 regionΔ 1737–1809 mutant failed to rescue cyclin D1-mediated AR transcriptional repression (Fig. 5A). Deletion of the p300 HAT domain induced activity further than wild-type p300, suggesting the p300 HAT domain was not required for rescue.

Figure 5.

Cyclin D1-Mediated AR Repression Is Relieved by either p300 or P/CAF A, The MMTV-LUC reporter was transfected into DU145 cells and cotransfected with the human AR, cyclin D1, and p300 as indicated. Shown on the top is the schematic representation of the p300 expression vectors. The bromo, HAT, and CH3-binding domains are shown. The data are shown as the mean ± sem. DHT treatment (10−7m) was for 24 h. The repression of DHT-dependent induction of MMTV-LUC activity by cyclin D1 is shown. B, Schematic representation of the P/CAF expression vector showing the HAT domain, and the regions homologous to the Ada2 and bromo domains. Western blotting is shown of extracts from DU145 cells transfected with the Flag-tagged P/CAF expression vectors using the anti-Flag (M2) antibody. The membrane was probed with the GDI antibody as a protein loading control. The P/CAF wild-type or mutant expression vectors were cotransfected with the MMTV-LUC reporter in the presence of AR. DHT treatment was for 24 h. The repression of DHT-dependent induction of MMTV-LUC activity by cyclin D1 is shown as the mean± sem of five separate transfections. The carboxyl-terminal region of the P/CAF HAT domain is required for rescue of cyclin D1-mediated AR repression.

The CH3 region of p300 is known to interact with P/CAF and thereby contribute to the HAT activity of p300/CBP. We examined the possibility that P/CAF was the p300-associated factor required for relief of cyclin D1-mediated AR repression. P/CAF has intrinsic HAT activity (18) located within a domain adjacent to the Ada2 homology region (Fig. 5B) (34). The abundance of wild-type and mutant P/CAF expressed in cultured cells was assessed by Western blotting. DU145 cells were transfected with the Flag-tagged P/CAF expression plasmids, and Western blotting was performed with the anti-Flag antibody. The P/CAF mutant protein P/CAF Δ609–624 was expressed equally to the wild- type protein (Fig. 5B). Cyclin D1-mediated AR repression was antagonized by P/CAF overexpression (Fig. 5B). The P/CAF HAT domain deletion mutant (P/CAFΔ 609–624), however, failed to rescue and further inhibited DHT-induced AR activity (Fig. 5B). These studies indicate that P/CAF HAT activity is required for the reversal of cyclin D1-mediated AR repression.

Cyclin D1 and P/CAF Binding in Cultured Cells and in Vitro

We investigated whether cyclin D1 could directly interact with P/CAF in cultured cells. To determine whether cyclin D1 formed a complex with P/CAF, 293T cells were transfected with cyclin D1, P/CAF, and either with or without the AR. IP-Western blotting was then performed to assess complexes binding to cyclin D1 (Fig. 6). The cyclin D1 IP was immunoreactive for cyclin D1, AR, and P/CAF (Fig. 6A lanes 1 and 5). The supernatant (S) was free of cyclin D1 and contained approximately 50% of the remaining P/CAF (Fig. 6A, lane 2). IP with a control IgG contained neither cyclin D1 nor P/CAF (Fig. 6A, lane 3). AR Western blotting demonstrated AR in the cyclin D1 IP and in the supernatant (Fig. 6A, lanes 1 vs. 2). Approximately 35% of the total AR was bound to cyclin D1 and approximately 50% of the cellular P/CAF was bound to cyclin D1.

Figure 6.

Cyclin D1 Binds P/CAF and AR in Cultured Cells Expression vectors encoding cyclin D1 and P/CAF with or without the AR were transfected into 293T cells. Cells were either untreated (A) or treated with DHT (10−7m) for 24 h (B). Cellular extracts were assessed by cyclin D1 IP with comparison to equal amounts of control IgG. The precipitate (IP) and the supernatant (S) were subjected to Western blotting with the indicated antibodies for AR, P/CAF, or cyclin D1. In panel C, murine liver extracts were subjected to direct Western blotting (lane 1) or IP with the AR antibody and subsequent Western blotting of the IP (lane 2). The AR-IP contains AR, P/CAF, and cyclin D1 by Western blot.

We examined the possibility that DHT may change the relative distribution of AR as either bound or unbound to cyclin D1. Cells were transfected with expression plasmids for cyclin D1, AR, and P/CAF and treated with DHT for 24 h. Cyclin D1-IP Western blot analysis showed that the cyclin D1 IP was saturating (Fig, 6B, compare cyclin D1 Western blot, lanes 1 vs. 2). The relative proportion of AR not bound to cyclin D1 was increased by DHT, and the proportion of P/CAF associated with cyclin D1 was reduced. The relative abundance of P/CAF in the supernatant compared with the cyclin D1-bound fraction was approximately 5:1 assessed by Western blotting. Cyclin D1-IP with subsequent Western blotting was also performed on cells transfected with cyclin D1 and P/CAF. In this circumstance the addition of DHT did not increase the proportion of P/CAF unbound to cyclin D1 (Fig. 6B lanes 5 vs. 6). This finding suggests the decrease in P/CAF binding to cyclin D1 upon the addition of DHT is likely due to the presence of the AR.

To determine whether the AR binds to cyclin D1 or P/CAF in vivo, IP-Western blotting was performed using extracts from murine liver with comparison made to direct Western blotting of the hepatocellular extracts (Fig. 6C, lane 2 vs. 1). The AR antibody immunoprecipitated the AR (Fig. 6C, lane 2) and no immunoreactive AR band was observed in control IgG (not shown). The AR IP was also immunoreactive for cyclin D1 and for P/CAF. These studies demonstrate that the cyclin D1-AR interaction observed in cultured cells is also observed in tissues.

As these studies showed P/CAF can be immunoprecipitated in association with cyclin D1, it remained to be determined whether cyclin D1 could interact directly with specific domains of P/CAF. Several transcriptional activators, viral proteins, and coactivators have been shown to interact directly with P/CAF. GST pull-down experiments were therefore conducted with affinity-purified P/CAF and GST cyclin D1 or GST alone as a control (Fig. 7). The amount of P/CAF protein was confirmed by Western blotting using the Flag epitope (Fig. 7, upper panel). Incubation of affinity-purified P/CAF with GST alone showed no interaction (Fig. 7, middle panel). Incubation of affinity-purified P/CAF with GST-cyclin D1 showed strong binding (>20% of the input). The P/CAF internal deletion mutants of the HAT (Δ511–656), Ada2 homology region (Δ655–701), and bromo domain (Δ729–832) were defective in binding to cyclin D1. In addition, the internal deletion mutantΔ 5–122 was defective in binding to cyclin D1. The domains of P/CAF required for binding to cyclin D1 resemble the domains required for binding to the AR (35), raising the possibility that cyclin D1 may inhibit AR function by binding to the same sites on P/CAF.

Figure 7.

P/CAF Binds to Cyclin D1 and the AR through Overlapping Domains Affinity-purified Flag-P/CAF proteins (shown schematically) were incubated with equal amounts of full-length GST cyclin D1. Western blotting of the P/CAF mutant proteins using the Flag antibody (upper panel) confirmed that equal amounts of wild-type and mutant P/CAF proteins were incubated in the pull-down experiment. GST alone did not pull down P/CAF (middle panel). Cyclin D1-bound P/CAF protein was confirmed by Western blotting (lower panel). Domains of interaction between P/CAF and cyclin D1 identified by pull down are scored as + or −. The domains of P/CAF that bind the AR are also shown [Data reproduced from Ref. 35].

Cyclin D1 Inhibits P/CAF Binding to the AR in Vitro

As cyclin D1 bound to similar domains of P/CAF, and P/CAF enhanced AR activity, we considered the possibility that cyclin D1 may inhibit AR activity by competing with the AR for P/CAF binding. To examine this possibility, we assessed the effect of cyclin D1 on the association between P/CAF and AR in vitro. Purified Flag-tagged P/CAF produced in baculovirus was incubated with GST-AR505−676 and IP performed with the M2 Flag antibody (Fig. 8). Western blotting of the P/CAF IP using a GST antibody demonstrated the presence of the GST-AR505−676 fusion protein (Fig. 8, lower panel) indicating P/CAF and AR are capable of interacting in a pull-down assay. The preaddition of excess GST-cyclin D1 to the mix of Flag-tagged P/CAF and GST-AR505−676 fusion protein abolished the presence of immunodetectable GST-AR505−676 in the P/CAF IP (Fig. 8, lane 4, lower panel). The preaddition of equal molar amounts of GST protein did not interfere with the binding of P/CAF to GST-AR (Fig. 8 lane 2, lower panel). GST-cyclin D1 was observed in the supernatant by Western blotting in conjunction with AR505−676 (Fig. 8, lane 3). These studies suggest that cyclin D1 has the capacity to inhibit the association between P/CAF and the AR in vitro.

Figure 8.

Cyclin D1 Inhibits AR Binding to P/CAF in Vitro The in vitro association between AR and P/CAF was determined in pull-down studies in the absence of ligand. Flag-tagged P/CAF was incubated with GST-AR, and IP was performed with saturating amounts of the M2 anti-Flag antibody. The IP and supernatant (S) were subjected to Western blotting with antibodies to the AR and GST for cyclin D1. The presence of GST-AR in the P/CAF (M2) IP was confirmed through Western blotting for the AR. The addition of GST-cyclin D1 in excess (lane 4) abolished GST-AR binding to P/CAF as evidenced by the loss of AR immunoreactivity in the M2 IP. The presence of cyclin D1 in the supernatant with the AR (lane 3) is consistent with the binding of AR to cyclin D1 (Fig. 3B).

DISCUSSION

In the current studies, cyclin D1 repressed ligand-dependent AR transactivation. Cyclin D1-mediated repression of liganded AR activity was observed in pRB-deficient cells, and mutation of the pRB-binding domain of cyclin D1 did not abrogate repression. These findings suggest that the ability of cyclin D1 to inhibit liganded AR activity is distinct from its pRB phosphorylation function. Cyclin D1 regulation of liganded AR activity required the carboxyl-terminal acid-rich region of cyclin D1 and a carboxyl- terminal LXXXLL motif. The domains of cyclin D1 that were required for repression of liganded AR activity were also required for binding to the AR. These findings suggest that either binding of cyclin D1 to the AR is required to form a repressor complex or that the domain of cyclin D1 that binds to the AR may compete with the AR for a common limiting factor. The current studies are fundamentally different from the previously described interaction between cyclin D1 and the estrogen receptor α (ERα). In contrast with the current studies in which cyclin D1 selectively inhibited liganded AR activity, it is the activity of the unliganded ERα that is induced by cyclin D1 (31, 36). Although cyclin D1 binds to ERα in cultured cells, it is not known which residues of either ERα or of cyclin D1 are required for direct interaction (37). The carboxyl-terminal LXXXLL motif of cyclin D1 plays a role in cyclin D1-mediated induction of basal ERα activity through recruiting SRC-1 (36). Although the ERα is capable of binding to cyclin D1 (31, 36) and P/CAF (38), the domains of P/CAF binding to the ERα are distinct from the regions binding to the AR (data not shown). Thus, the domains of P/CAF bound by the nuclear receptor may determine functional interactions with cyclin D1.

The association between cyclin D1 and the AR demonstrated by IP-Western blotting of cultured cells and using in vitro analysis suggests the basic region of the AR interacts with the carboxy-terminal region of cyclin D1. The domains of cyclin D1 required for binding to the AR correlated well with the regions required for transcriptional repression of the ligand-bound AR. Cyclin D2 and D3 neither repressed ligand-induced AR activity nor bound to this region of the AR in GST pull-down experiments. This finding suggests that the structural divergence among the D type cyclins in this region may abrogate critical determinants for AR binding. The ΔC274 mutant was 50% defective in repression of DHT-induced MMTV-LUC activity and bound AR with 35% of wild-type binding affinity on GST pull-down analyses (Fig. 2C). The region of cyclin D1 from 267 to 274 was absolutely required for both binding to the AR and for repression of liganded AR activity. Alignment of cyclin D1 and structural comparison with cyclin A suggests the acid-rich region (267 PKAAEEEE 271) and leucine-containing motif (LLXXXL), required for AR binding, align at the C terminus of helix 5. The structure of cyclins A and H deduced through crystallization reveals a core of two repeats containing five α-helical bundles that form the cyclin fold (39). The likely amphipathic α-helical structure of cyclin D1 within the AR interaction domain may serve as an interaction domain with other proteins. The region of the AR involved in direct binding to cyclin D1 (residues 633–668) contains predominantly basic residues, suggesting an ionic basis for binding to cyclin D1.

In the current studies deletion of the p300 CH3 domain abolished relief of cyclin D1-mediated AR transrepression. Both p300 and P/CAF have intrinsic HAT function. Deletion of the P/CAF HAT domain, but not the p300 HAT domain, abolished its ability to relieve cyclin D1-mediated AR repression. As the p300 CH3 region is capable of binding P/CAF, it is likely that the p300 CH3 mutant is defective through failed recruitment of P/CAF. The finding that P/CAF HAT activity, but not p300 HAT activity, was required for relief of cyclin D1-mediated AR transrepression is consistent with recent observations in which the acetyltransferase activities and substrate specificities of p300 and P/CAF were shown to be distinguishable (40). These findings are also consistent with previous observations that the functional activities of p300 and P/CAF are restricted to specific activators or promoters (17, 41). In transfection assays, ligand-induced AR activity was inhibited by cyclin D1 and enhanced by P/CAF. The mutual antagonism between cyclin D1 and the AR may be due to competition for binding to a common intermediary protein or a limiting cofactor such as P/CAF. In support of a model in which cyclin D1 and AR may compete for a common intermediary protein are the in vitro findings that cyclin D1 and the AR bound to similar regions of P/CAF (Fig. 7) and that the preaddition of GST cyclin D1 fusion protein inhibited the binding of P/CAF to the AR (Fig. 8). Support for the biological relevance of the association between cyclin D1 and the AR include our findings that the AR was associated with cyclin D1 and P/CAF in vivo, and in transfected cells using IP-Western blot analysis (Fig. 6). Furthermore, the associations between cyclin D1 and the AR appear to be hormone regulated. Thus, in cultured cells, the amount of P/CAF associated with cyclin D1 was reduced by the presence of liganded AR. Together, these studies suggest that cyclin D1 inhibition of liganded AR activity may be due to mutually exclusive binding of cyclin D1 and AR for P/CAF. As P/CAF forms a multiprotein complex, it is likely that additional components regulating this interaction in vivo remain to be determined. As the domain of P/CAF that binds to cyclin D1 overlaps the Twist binding domain (42), it will be of interest to determine whether the differentiation function of Twist or other basic helix-loop-helix proteins are also regulated by cyclin D1.

The current studies, however, provide evidence that cyclin D1 can selectively and specifically inhibit liganded AR activity. Cyclin D1 can inhibit differentiation and either promote or inhibit cellular proliferation in a cell type-specific manner (reviewed in Ref. (43). The AR also has both differentiation and proliferation functions. For example, the DHT-liganded AR plays an important role in the induction of male secondary sexual characteristics (3). Thus, AR gene mutations described in complete androgen insensitivity syndrome causes XY genotypic males to develop as phenotypic females due to defective AR function (44). In cultured prostatic cells, DHT can induce differentiation, as evidenced by the cell cycle arrest and increased expression of enzymatic differentiation markers (45) or proliferation in cell lines with mutant AR (46). Because of the functional redundancy between components of the cell cycle-regulatory apparatus, it may be difficult to dissect the independent contribution of cyclin D1 to liganded AR activity in vivo. The AR is located in a broad array of tissues and contributes to diverse signal transduction pathways in breast, brain, and prostate, and the role of cyclin D1 in regulating signal transduction in these tissues remains to be determined.

Cyclin D1 binding to P/CAF in vitro required the HAT domain of P/CAF. Mutational analysis of the Gcn5p HAT domain identified four subdomains with catalytic activity mediated through the carboxyl-terminal subdomains (47). Mutation within subdomain IV of Gcn5p abolished HAT activity in vitro (47). In the current studies an expression plasmid encoding a deletion within the HAT domain abolished rescue of cyclin D1 repression of liganded AR activity. The carboxyl-terminal bromodomain of P/CAF, which forms a four-helix bundle that interacts specifically with acetylated lysine (48), remains intact in this mutant and may therefore reduce AR activity by competing with endogenous P/CAF. Although speculative at this time, our studies predict that the binding of cyclin D1 to the HAT domain of P/CAF may regulate other functions of P/CAF. P/CAF HAT activity contributes to regulation of transcription, inhibition of cell cycle progression, and the induction of differentiation (18, 41). The cyclin D1 gene product, in contrast, can inhibit differentiation and promote cell cycle progression in several cell types. P/CAF is associated with at least 20 polypeptides in cultured cells including the TBP-associated factors (TAFs), which are subunits of transcription factor IID (TFIID), and other TAFs (49). The association of P/CAF with these polypeptides and the direct HAT activity conveyed by P/CAF may both contribute to the regulation of AR function. P/CAF has the capacity to augment gene expression both by acetylating core histones and also by directly acetylating a subset of transcription factors including p53 (50) and the AR (35) reviewed previously (41). The contribution of cyclin D1 to other functions of P/CAF remains to be explored.

MATERIALS AND METHODS

Plasmids, Transfections, and Reporter Assays

The expression vectors pCMV-cyclin D1, CMV-cyclin D1-KE, and CMV-cyclin D1 GH (51), pCMVHA p300, p300Δ1737–1809, Δ1419–1721 (15), pCMVHAΔbromop300, which deletes residues 1,032–1,138 of p300, rP/CAF, P/CAF Δ609–624 (52), were previously described. The reporter plasmids c-fos LUC, c-Jun LUC, _Jun_B LUC, SRELUC, E2F LUC, cyclin D1 LUC, cyclin E LUC, p21Cip1/WAF1 LUC, CMV LUC, and RSV LUC were previously described (23, 53–55). The human cyclin D1 mutants were derived by PCR-directed amplification using sequence-specific primers and cloned into pRc/CMV, and the integrity of all constructs was confirmed by sequence analysis. The AR expression vectors, ARWt, ARΔ80–93, AR96–483, and AR 1–707 (56), and fusion proteins, GST-AR/676–844, GST-AR/676–919, GST-AR/505–919, GST-AR/505–676, GST-AR/505–559, GST-AR/552–635, and GST-AR/633–668 (57), the reporter MMTV-LUC (from Dr. R. Evans), and the Flag-tagged P/CAF mutants (42) were previously described. GST-cyclin D2 and GST-cyclin D3 were gifts from Dr. M. Pagano.

Cell culture, DNA transfection, and luciferase assays were performed as previously described (53, 54). A CMV-Renilla luciferase plasmid, pRL-CMV (Promega Corp., Madison, WI), was included to control for transfection efficiency as indicated. Renilla luciferase activity was assayed according to the manufacturer’s instructions for the Dual-Luciferase Reporter Assay System. The prostate cancer cell lines PC3 and DU145 (ATCC, Manassas, VA) and the 293T cell line were cultured in DMEM with 10% charcoal-stripped FCS, 1% penicillin, and 1% streptomycin. Cells were transfected by calcium phosphate precipitation or lipofection using lipofectamine PLUS (Life Technologies, Inc., Gaithersburg, MD), the medium was changed after 6 h or 3 h, respectively, and luciferase activity was determined after 48 h. At least two different plasmid preparations of each construct were used. In cotransfection experiments, a dose-response was determined in each experiment with 300 ng and 600 ng of expression vector and the promoter reporter plasmids (2.4 μg). Luciferase activity was normalized for transfection using the renilla luciferase as internal control as previously described. Luciferase assays were performed at room temperature using an Autolumat LB 953 (EG&G Berthold) as previously described (53). The -fold effect was determined for 300–600 ng of expression vector with comparison made to the effect of the empty expression vector cassette, and statistical analyses were performed using the Mann-Whitney U test.

Western Blots

The antibodies used in Western blot analysis were a monoclonal cyclin D1 antibody DCS-6 (NeoMarkers Lab Vision Corp., Fremont, CA), a rabbit anti-P/CAF antibody (18), an α-guanine nucleotide dissociation inhibitor (GDI) (58) (a gift from Dr. Perry Bickel, Washington University St. Louis, MO, used as internal control for protein abundance), anti-M2 Flag antibody (Sigma, St. Louis, MO), and antibodies from Santa Cruz Biotechnology, Inc., (Santa Cruz, CA): cyclin D1 antibody (HD11), GST (B-14), and AR (N-20). For detection of cyclin D1, the membrane was incubated with antimouse cyclin D1 antibody DCS-6 (NeoMarkers Lab Vision Corp.), washed with 0.05% TPBS three times, and then incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and washed again. An antirabbit horseradish peroxidase-conjugated secondary antibody was used for AR (1:3,000), and an antimouse horseradish peroxidase-conjugated secondary antibody was used for the Flag epitope (1:2,000). Proteins were visualized by the enhanced chemiluminescence system (Amersham Pharmacia Biotech, Arlington Heights, IL). The abundance of immunoreactive protein was quantified by phosphoimaging using a computing densitometer (Image Quant version 1.11, Molecular Dynamics, Inc., Sunnyvale, CA).

Protein Interaction Assays in Vitro and in Cultured Cells and in Vivo

_In vitro_[ 35S]methionine-labeled proteins were prepared by coupled transcription-translation with a TNT coupled reticulocyte lysate kit (Promega Corp., Madison, WI), using 1.0 μg of plasmid DNA in a total of 50 μl. Flag-tagged P/CAF proteins were expressed in Sf9 cells by infecting with recombinant baculovirus and were purified as described (15). GST fusion proteins were prepared from Escherichia coli cells as described previously (42). GST fusion proteins were induced for 4 h at 30 C with 0.1 m isopropyl-β-d-thiogalactopyranoside, and crude lysates were prepared at 4 C. Cell pellets were spun down and resuspended in 15 ml PBS containing 2 mm dithiothreitol (DTT), 1 mm phenylmethylsulfonylfluoride (PMSF), 1 μg/ml pepstatin, 1 μg/ml leupeptin, and 1 μg/ml aprotinin. Lysozyme (2 mg/sample) was added and incubation performed on ice for 10 min. Samples were sonicated using 20-sec pulses until lysed. One milliliter of 15% Triton X-100 in PBS was added, and the samples were then centrifuged at 40,000 rpm for 15 min at 4 C. The supernatant was added to 500 μl of glutathione sepharose beads (Amersham Pharmacia Biotech), and the mixture was rotated at 4 C overnight. The supernatant was discarded and the pellet washed three times in PBS containing 100 mm DTT and 1% Tween 20, and three times in PBS containing 50 mm Tris-HCl and 5 mm DTT (pH 8.0). The pellet was resuspended in 400 μl of this buffer and 50 μl of glutathione (100 mm in Tris HCl, pH 8.0) were added. Elution of proteins was performed for 1 h, and beads were separated by centrifugation. Glutathione was removed by dialysis at 4 C in 20 mm Tris, 25 mm NaCl, and 2 mm EDTA.

In vitro protein-protein interactions were performed as described (31). The in vitro translated protein (15 μl of cyclin D1) was added to 5 μg of GST or equal amounts of GST-AR, in 225 μl of binding buffer (50 mm Tris-HCl, 120 mm NaCl, 1 mm DTT, 0.5% NP-40, 1 mm EDTA, 2 μg/ml leupeptin, 2 μg/ml aprotinin, 1 mm PMSF, 2 μg/ml pepstatin) and rotated for 2 h at 4 C. Fifty microliters of glutathione-sepharose bead slurry were added, and the mixture was rotated for a further 30 min at 4 C. Beads were washed five times with 1 ml of binding buffer and 30 μl of binding buffer were added after the final wash. Baculovirus- expressed Flag-tagged P/CAF (1 μg) was used in pull-down experiments as previously described (35). In competition experiments baculovirus P/CAF (1 μg) was incubated with GST-AR505−676 (1 μg), and IP was performed with the M2 (Flag) antibody. Competition was performed through preincubation with GST-cyclin D1 (1 μg) or GST (1μ g) alone.

IP Western blot analysis was performed with murine hepatic tissue (as previously described) (59) or 293T cells. 293T cells transfected with pSV-AR, pCMV-cyclin D1, pCI-P/CAF, or expression vector control were treated for 36 h with 10−7m DHT or vehicle as control. Cells were rinsed with PBS, harvested by scraping, pelleted, and lysed in buffer (50 mm HEPES, pH 7.2, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm DTT, 0.1% Tween 20, 0.1 mm PMSF, 2.5 μg/ml leupeptin, 0.1 mm sodium orthovanadate (Sigma). Extracts were cleared by centrifugation and further precleared by rocking at 4 C with washed protein A Sepharose (Roche Molecular Biochemicals, Indianapolis, IN). Five hundred micrograms of precleared extract were immunoprecipitated with 1μ g of cyclin D1 antibody (DCS-11, NeoMarkers Lab Vision Corp.) or AR antibody (N-20, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or equivalent amounts of appropriate control IgG (Santa Cruz Biotechnology, Inc.) and 50 μl of protein A sepharose for 8–12 h at 4 C. Beads were washed five times with lysis buffer and boiled in SDS sample buffer, and released proteins were resolved by 10% SDS-PAGE. The gel was transferred to nitrocellulose and Western blotting was performed.

Acknowledgments

We thank Drs. M. Avantagiatti, D. Baltimore, S. Bhattacharya, R. Echner, P. Hinds, D. Livingston, A. Minden, and Y. Nakatani for reagents and helpful discussion.

This work was supported by an award from the Pfeiffer Foundation, RO1CA70897 and RO1CA75503 (to R.G.P.), NIH Grants CA-70297 (Z.J.S.), and American Cancer Society Grant RPG98213 (Z.J.S.). Work conducted at the Albert Einstein College of Medicine was supported by Cancer Center Core NIH Grant 5-P30-CA13330–26.

These authors contributed equally to the manuscript.

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November 2017	3
December 2017	11
January 2018	9
February 2018	8
March 2018	10
April 2018	10
May 2018	25
June 2018	19
July 2018	22
August 2018	16
September 2018	11
October 2018	15
November 2018	12
December 2018	14
January 2019	14
February 2019	18
March 2019	29
April 2019	19
May 2019	11
June 2019	17
July 2019	13
August 2019	11
September 2019	20
October 2019	21
November 2019	13
December 2019	20
January 2020	18
February 2020	8
March 2020	20
April 2020	14
May 2020	15
June 2020	27
July 2020	23
August 2020	15
September 2020	20
October 2020	17
November 2020	22
December 2020	13
January 2021	19
February 2021	9
March 2021	26
April 2021	24
May 2021	20
June 2021	27
July 2021	13
August 2021	17
September 2021	17
October 2021	11
November 2021	23
December 2021	21
January 2022	11
February 2022	13
March 2022	6
April 2022	21
May 2022	15
June 2022	9
July 2022	17
August 2022	18
September 2022	28
October 2022	25
November 2022	14
December 2022	22
January 2023	18
February 2023	21
March 2023	47
April 2023	20
May 2023	40
June 2023	7
July 2023	20
August 2023	18
September 2023	25
October 2023	42
November 2023	25
December 2023	33
January 2024	44
February 2024	34
March 2024	36
April 2024	16
May 2024	36
June 2024	15
July 2024	25
August 2024	24
September 2024	14
October 2024	17

Cyclin D1 Binds the Androgen Receptor and Regulates Hormone-Dependent Signaling in a p300/CBP-Associated Factor (P/CAF)-Dependent Manner (original) (raw)

Cite

Abstract

INTRODUCTION

RESULTS

Cyclin D1 Inhibits Ligand-Dependent AR Transactivation

A Structurally Divergent Acid-Rich Motif in the Carboxyl Terminus of Cyclin D1 Is Required for Repression of AR Activity

Cyclin D1 Binds the AR Hinge Region

The Cyclin D1 Acid-Rich Carboxyl Terminus Binds the AR

The P/CAF Histone Acetylase Domain Relieves Cyclin D1-Mediated AR Repression

Cyclin D1 and P/CAF Binding in Cultured Cells and in Vitro

Cyclin D1 Inhibits P/CAF Binding to the AR in Vitro

DISCUSSION

MATERIALS AND METHODS

Plasmids, Transfections, and Reporter Assays

Western Blots

Protein Interaction Assays in Vitro and in Cultured Cells and in Vivo

Acknowledgments

Citations

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Cyclin D1 Binds the Androgen Receptor and Regulates Hormone-Dependent Signaling in a p300/CBP-Associated Factor (P/CAF)-Dependent Manner (original) (raw)

Cite

Abstract

INTRODUCTION

RESULTS

Cyclin D1 Inhibits Ligand-Dependent AR Transactivation

A Structurally Divergent Acid-Rich Motif in the Carboxyl Terminus of Cyclin D1 Is Required for Repression of AR Activity

Cyclin D1 Binds the AR Hinge Region

The Cyclin D1 Acid-Rich Carboxyl Terminus Binds the AR

The P/CAF Histone Acetylase Domain Relieves Cyclin D1-Mediated AR Repression

Cyclin D1 and P/CAF Binding in Cultured Cells and in Vitro

Cyclin D1 Inhibits P/CAF Binding to the AR in Vitro

DISCUSSION

MATERIALS AND METHODS

Plasmids, Transfections, and Reporter Assays

Western Blots

Protein Interaction Assays in Vitro and in Cultured Cells and in Vivo

Acknowledgments

Citations

Views

Altmetric

Email alerts

More on this topic

Citing articles via

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