Department of Cell Biology (C.J.B., C.E.P., H.M., K.E.K.) and Center for Environmental Genetics (K.E.K.), University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0521; and Laboratories of Reproductive Biology (E.M.W.) and the Department of Pediatrics and Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-7500
Address all correspondence and requests for reprints to: Karen E. Knudsen, Department of Cell Biology, University of Cincinnati College of Medicine, P.O. Box 670521, 3125 Eden Avenue, Cincinnati, Ohio 45267-0521. E-mail: Karen.Knudsen{at}uc.edu.
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ABSTRACT |
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INTRODUCTION |
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Before activation by ligand, the AR resides diffusely throughout the cell and is held inactive by heat shock protein complexes (8). In the prostate, the main ligand for the AR is dihydrotestosterone (DHT), which is converted from testosterone through the action of 5-reductase (9). The AR then rapidly translocates to the nucleus, where it binds as an antiparallel homodimer to DNA at androgen response elements found in target gene promoters (10, 11, 12). The ligand-binding domain of the AR (found in the C terminus, amino acids 647919) is highly conserved among the other steroid receptors and contains the activation function 2 (AF2) transcriptional transactivation domain (13, 14). Although AF2 is a potent modulator of transactivation potential and coactivator binding in related nuclear receptors, its action in the AR is relatively weak (13, 15, 16). Fusion of this region to the receptor DNA-binding domain fails to demonstrate transactivation potential upon ligand recruitment (17). By contrast, deletion of AF2 results in a truncated protein, the activity of which is similar to that observed with the full-length receptor in reporter assays, suggesting that the majority of AR transactivation potential resides in the N-terminal region (18, 19). In the N-terminal domain (NTD), the primary ligand-dependent transactivation domain, AF1, lies between amino acids 142337 and is thought to harbor the most potent transactivation function of the receptor (20, 21). A second N-terminal transactivation function, AF5 or
5, lies downstream between residues 360528 and is not dependent on ligand binding (18, 20). Rather, the C terminus of the receptor is hypothesized to modulate AF5 function (20), and AF5 is responsive to the effects of the Rho pathway (22). Although there is no current evidence that AF5 is reactive to comodulatory proteins, a large body of evidence has demonstrated that AF1 and AF2 are strongly influenced by association with coactivators and corepressors (11, 23, 24).
The AR recruits a series of coactivator proteins to promote and enhance transcription of target promoters, such as the p160 class of coactivators [e.g. steroid receptor coactivator (SRC)-1, transcriptional intermediary factor (TIF)2/glucocorticoid receptor-interacting protein 1, and amplified in breast cancer 1/SRC3/activator of transcription of nuclear receptors/receptor-activated coactivator 3] (17, 25). Selected members of this highly homologous protein family contain some intrinsic histone acetyltransferase (HAT) activity but can also recruit p300/cAMP response element binding protein (CREB)-binding protein (CBP)-associated factor (P/CAF) and CBP/p300 to the AR complex, thus increasing the local action of histone acetylation (11, 26). Recruitment of HATs to the AR complex assists in the formation of active transcriptional complexes by acetylating histones (thereby fostering increased promoter accessibility to the RNA polymerase II complex) and also by facilitating recruitment of chromatin remodeling complexes (e.g. SWI/SNF) (27, 28). LxxLL motifs [nuclear receptor (NR) boxes] present in the p160 proteins are capable of binding to hydrophobic grooves found in the AF2 domain of nuclear receptors (29, 30). For the AR, association of p160 coactivators with the AF2 domain is required for measurable AF2 activity (31). Strikingly, it has also been shown that SRC1 and TIF2 can associate with the AR NTD independent of the coactivator NR boxes and that binding of SRC1 occurs predominantly in this region (17, 25). Deletion of the SRC1 LxxLL motifs fails to compromise coactivator function, whereas ablation of NTD binding capability eliminates SRC1 enhancement of AR function (17, 25). From these and other studies it has been hypothesized that p160 coactivators can act on both AF1 and AF2 to form a ternary complex between the NTD and AF2 hydrophobic cleft and/or that coactivator function is influenced by interaction between these two domains of the AR (32).
Association of the AR NTD with AF2 has been well documented and strongly influences AR function (17, 31, 32). The NTD of the AR contains at least three distinct regions proposed to generate amphipathic -helices that can interact with the AF2 hydrophobic groove in response to ligand binding: 23FxxLF27, 179LKDIL183, and 432WxxLF436 (17, 33). In both yeast and mammalian systems, it has been demonstrated that association of the AR NTD with AF2 can occur directly, and is required for full AR activation (16, 32, 34, 35). Association of the NTD with AF2 slows the dissociation rate of bound ligand, thus promoting stabilization of the active receptor complex (33). Mutations that disrupt NTD-AF2 interaction in cultured cells retain compromised, yet detectable, AR activity. Several mutations that disrupt NTD-AF2 interaction (but do not affect ligand binding) have also been identified in patients with androgen insensitivity syndrome, thus demonstrating the biological impact of this interaction in vivo (35). Therefore, the AF1 transactivation domain is regulated by ligand binding, coactivator association, and interaction with the AF2 domain.
We and others have previously shown that cyclin D1 binds directly to the AR and is a potent repressor of AR function (36, 37). Although first identified based upon its ability to inactivate/phosphorylate the retinoblastoma tumor suppressor and promote transition into S phase of the cell cycle, it has become increasingly apparent that cyclin D1 plays additional roles as a transcriptional modulatory protein (38, 39). Cyclin D1 is known to act as both a coactivator [e.g. for the estrogen receptor (ER)] and a corepressor of multiple transcription factors (e.g. AR, signal transducer and activator of transcription 3, v-Myb, and the thyroid hormone receptor), independent of its role in the cell cycle (36, 37, 40, 41, 42, 43). We have previously demonstrated that cyclin D1 directly binds to AR and blocks AR action on multiple target promoters (42, 44). In addition, it can repress the endogenous promoter of prostate-specific antigen in the LNCaP prostate cancer cell line at stoichiometric AR to cyclin D1 expression levels (44). The ability of cyclin D1 to repress AR-mediated transcription does not appear to be cell type specific, as the inhibitory effect of overexpression is maintained in all cell lines tested (44). Additionally, AR activity is cell cycle regulated and is lowest at the peak of cyclin D1 expression (G1-S transition) (45).
Although the effects of cyclin D1 on AR activity are well characterized, the mechanism by which it mediates repression is less understood. An AR allele that harbors constitutive, ligand-independent AF5 activity and maintains cyclin D1 binding is refractory to cyclin D1 repression, suggesting that cyclin D1 manifests its repressor function through at least one of the ligand-dependent transactivation domains (AF1 or AF2) (42). Cyclin D1-mediated repression is not competed by the HAT coactivators SRC1 and P/CAF or ARA70, a coactivator capable of binding both the NTD and AF2 (42). However, we have shown previously that cyclin D1 corepressor activity could be partially reversed through the action of trichostatin A (TSA), thus implicating the recruitment of histone deacetylase (HDAC) activity as at least one potential component of cyclin D1 action (42). Subsequently, it was shown that cyclin D1 can bind to HDAC3, further suggesting that its action may be manifested, in part, through this mechanism (41, 42). Here, we identify a second, novel mechanism of cyclin D1 action, mediated through modulation of NTD-AF2 interaction. We show that cyclin D1 binds the first N-terminal -helix and strongly inhibits ligand-induced association of the AR NTD with AF2. Cyclin D1 binding to the amino terminus requires an intact 23FxxLF27 motif, and functional studies reveal that cyclin D1 impinges specifically on the association of this NTD motif with the hydrophobic cleft. Disruption of the FxxLF motif significantly impaired cyclin D1 repressor function, thus demonstrating the importance of this interaction for cyclin D1 activity. Lastly, the data revealed an unexpected ability of cyclin D1 to enhance AF2 activity in the absence of the AR amino terminus. These studies underscore the complexity of cyclin D1 action as a corepressor and demonstrate that cyclin D1 utilizes at least two distinct mechanisms to repress AR activity.
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RESULTS |
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Cyclin D1 Binds the AR N-Terminal -Helix
We have previously demonstrated that cyclin D1 binds preferentially to the first 502 amino acids of AR (42). In addition, cyclin D1 action is directed only at the ligand-dependent transactivation functions within this region, as we have previously demonstrated that AF5 (ligand independent) activity is refractory to cyclin D1 (42). Therefore, we sought to delineate the effector point of cyclin D1 action on the NTD. To do so, amino-terminal AR truncations (AR1238 and AR46408) were generated for in vitro binding assays (Fig. 2A
). Both wild-type and mutant AR constructs were in vitro translated/transcribed in the presence of [35S]methionine and incubated with glutathione-S-transferase (GST)-cyclin D1 immobilized on glutathione-agarose beads. To control for nonspecific binding, all AR alleles were also incubated with GST alone immobilized on glutathione-agarose. Input (5% of reaction) and bound fractions were resolved by SDS-PAGE and visualized on a phosphor imager (Fig. 2B
). Analysis of input lanes confirmed that these constructs generate stable proteins (lanes 13), although the translation of AR1238 construct also results in a truncated protein (determined to be a degradation product through immunoprecipitation with an AR-specific antibody, data not shown). As can be observed in the right panel, wild-type AR (wtAR) bound to GST-cyclin D1 above the GST control. The presence of ligand (10 nM DHT) did not significantly alter the binding of AR to GST-cyclin D1 (105% of no ligand; compare lanes 46), consistent with previous reports (42). In addition, the presence of the AR antagonist biclutamide did not alter cyclin D1 binding to the AR (data not shown). Both AR
46408 and AR1238 retained specific binding to GST-cyclin D1 above the GST control (Fig. 2B
, compare lanes 7 and 8 and 9 and 10). These results implicate the first 46 amino acids of AR as a major binding site for cyclin D1.
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Cyclin D1 Inhibits NTD-AF2 Interactions
It has been shown that the first 34 amino acids of the AR are critical for its transcriptional transactivation function (25, 33). Encompassed within the first 34 amino acids is the 23FxxLF27 motif, which binds with high affinity to the AF2 domain; this interaction has been shown to stabilize the receptor-ligand interaction and is required for full AR activity (33). Two lower-affinity NTD motifs capable of binding AF2 reside outside the cyclin D1 binding region, at residues 179183 and 432436 (17, 33). The ability of the NTD to bind AF2 in the presence of ligand can be monitored using a well-defined mammalian two-hybrid assay (47), as depicted in Fig. 3A. This system is capable of specifically monitoring the interaction of the amino and carboxy termini of the AR (Fig. 3B
). CV1 cells were transfected with the Gal4-LUC reporter, cytomegalovirus (CMV)-ß-galactosidase, Gal4-AR ligand-binding domain (LBD) (the carboxyl terminus of AR, amino acids 614919, fused to the Gal4 DNA-binding domain), and/or VP16-ARTAD [the transcriptional activation domain (TAD) of VP16 fused with AR amino acids 1565]. After transfection, cells were stimulated with 0.1 nM DHT or ethanol vehicle for 24 h before harvest and analyzed for luciferase activity. VP16-ARTAD activity induced by interaction with Gal4-ARLBD in the presence of ligand was set to 100. Either construct alone in the presence of ligand or together in the absence of ligand failed to induce luciferase activity (Fig. 3B
). As expected, transfection of both constructs in the presence of ligand fostered NTD-AF2 interaction, thus stimulating a 99-fold activity from the Gal4-LUC reporter. In addition, we examined mammalian two-hybrid activity after cotransfection of expression plasmid encoding TIF2 (known to bind both the AR NTD and AF2), p53 (a known repressor of the NTD-AF2 interaction), cyclin D1, or empty vector control (Fig. 3C
) (17, 48, 49). As expected, p53 significantly reduced NTD-AF2 interaction (95.6% repression) (49), whereas overexpression of TIF2 increased Gal4 promoter activity (17, 48). Cyclin D1 significantly inhibited NTD-AF2 in-teraction, reducing activity to 41% of vector-transfected cells in the presence of ligand. These data suggest that cyclin D1 binding to the amino terminus reduces NH2-COOH interactions required for full transcriptional AR activity. We have shown previously that the repressing effects of cyclin D1 in reporter assays can be partially rescued by inhibiting HDACs with an optimum concentration of 50 nM TSA (42). To ensure that the inhibitory effect seen on the Gal4 reporter by cyclin D1 in this system was not due to HDAC recruitment and consequential reduced luciferase gene expression, the experiment shown in Fig. 3C
was repeated in the presence of TSA (Fig. 3D
). As shown, inhibition of HDAC had no significant effect on the ability of cyclin D1 to inhibit the mammalian two-hybrid system (37% of vector control compared with 41% in Fig. 3C
), which relies on robust VP16 activity. Thus, cyclin D1 is an effective inhibitor of NTD-AF2 association independent of it ability to recruit HDACs to AR transcriptional complexes.
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The First 34 Amino Acids of AR Are Critical for Receptor Function and Cyclin D1 Repression
The data presented demonstrate that cyclin D1 utilizes the NTD-binding site to regulate the NH2-COOH interaction. To assess the relative importance of this event for cyclin D1 action, an allele defective in the cyclin D1 NTD-binding site was generated (AR234) in a mammalian expression vector. Reporter assays were performed using the ARR2-LUC reporter as in Fig. 1A
. Briefly, CV1 cells were cotransfected with AR constructs (wild-type or AR
234), the ARR2-LUC reporter, and CMV-ß-galactosidase. Cells were stimulated with either 1 nM DHT or ethanol vehicle for 24 h before analysis. This concentration of ligand was essential to obtain measurable activity of the truncated AR allele. For comparison, basal activity (wtAR treated with vehicle) was set to 1 and relative luciferase activity is shown. As shown in Fig. 6A
, wtAR demonstrated a high level of transactivation potential (56.3-fold induction over basal activity), whereas AR
234 was significantly compromised (23.2% of wtAR activity), similar to that observed with wtAR and cyclin D1 (Fig. 1
). These data underscore the importance of the FxxLF and the NH2-COOH interaction for AR activity. To assess the relative expression of these two AR alleles, CV1 cells were transfected with each allele and H2B-green fluorescent protein (GFP) (as a marker for transfection efficiency). Lysates were subjected to SDS-PAGE and immunoblotted for AR and GFP. Expression of the AR
234 is comparable to wtAR (Fig. 6B
, upper panel) confirming that the loss of activity seen in Fig. 3C
is not attributed to variant expression levels. These data are consistent with previous observations (33, 51).
Although we have shown that loss of residues 234 ablates cyclin D1 binding to the AR NTD, some minimal binding was retained in the context of full-length receptor (Fig. 2D). Therefore, the effect of cyclin D1 on the AR
234 allele was examined on the ARR2-Luc reporter. This allele possesses only a fraction of the activity of the wild-type allele (Fig. 6A
). To assess the ability of cyclin D1 to repress AR in the absence of the NTD binding region, CV1 cells were transfected as in Fig. 1A
with both wtAR and AR
234 in the presence of 1.5 µg cyclin D1 plasmid. As shown in Fig. 6C
, wild-type cyclin D1 maintains some capability of repression on the AR
234 allele. However, its ability to repress is significantly less than that seen on wtAR (41.0% repression of ARD234 compared with 79.4% repression of wtAR; P
0.05). Combined, these data demonstrate that cyclin D1 utilizes the NTD-binding site to regulate AR NH2-COOH interaction, and that this activity is critical for cyclin D1 repressor function.
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DISCUSSION |
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A High-Affinity Cyclin D1-Binding Site Lies in the AR Amino-Terminal -Helix and Requires an Intact 23FxxLF27 Motif
We had previously narrowed the predominant site of cyclin D1 binding within amino acids 1502 of the AR, which encompassed both the AF1 and AF5 transactivation domains (42). Functional studies revealed that AF5 function is resistant to cyclin D1 action, indicating that cyclin D1 repressor function is restricted to the ligand-dependent transactivation domains (42). Here, deletion mapping revealed that the NTD-binding site lies within amino acids 134 of the receptor. This region of the AR is highly conserved throughout evolution and is predicted to encode a long -helix containing two putative protein interaction motifs (23FxxLF27 and 30VxxVI34) (33, 53). This stretch of 34 amino acids is highly influential in the regulation of both coactivator and AF2 binding and is therefore pivotal for full AR function (25, 54). Mutation of 23FxxLF27 to 23FxxAA27 resulted in significantly decreased cyclin D1 binding to the truncated NTD (amino acids 1238), suggesting that the structure of this motif is essential for cyclin D1 association (Fig. 4
). However, the
-helix mutant, AR
234, retained residual cyclin D1 binding using in vitro assays (Fig. 2D
). Thus, an alternate binding site likely exists outside amino acids 1238. In functional studies, cyclin D1 action on AR alleles that lack the NTD-binding site (and FxxLF) require an intact cyclin D1 LxxLL motif, suggesting that secondary binding may be to the hydrophobic cleft vacated by the loss of FxxLF. In addition, a binding site for cyclin D1 has been suggested within the hinge region of AR (residues 633668) (37), although this was not observed in our previous interaction studies (42).
Cyclin D1 Binding Regulates FxxLF-AF2 Interactions
We demonstrate that cyclin D1 inhibits interaction of the NTD with the AF2 hydrophobic cleft, as mediated specifically through the 23FxxLF27 motif (Figs. 35). Although three NTD interaction motifs have been described for association with AF2 (23FQNLF27, 179LKDIL183, and 432WHTLF436), 23FxxLF27 is considered to be the predominant interaction site and has the highest affinity for AF2 (17, 33). WxxLF is less highly conserved among species and is not required in the context of the full-length receptor for NTD/AF2 binding. The function of 179LKDIL183 has not been extensively investigated, although mutation of this site impairs NTD-AF2 interaction and reduces overall AR transactivation potential (17). We show that deletion of the FxxLF motif reduced, but did not ablate, NTD-AF2 interaction, consistent with the ability of these lower affinity sites to mediate AF2 interactions. Interestingly, interaction of these weaker motifs with AF2 was not inhibited by cyclin D1 (Fig. 5A
). Thus, these data reveal the importance of the FxxLF motif for cyclin D1 function.
Consequence of Cyclin D1 Recruitment to the AR LBD
The ability of cyclin D1 to enhance AF2 activity raises interesting questions as to the functional significance of this interaction (Fig. 5). Our data demonstrate that the ability of cyclin D1 to modulate AF2 is dependent on the cyclin D1 LxxLL motif (Fig. 5B
). As such, the manner by which cyclin D1 impinges on AF2 function is similar to that observed with the ER and presumably occurs through cyclin D1-mediated SRC1 and/or P/CAF recruitment to AF2 (40, 55). However, the ability of cyclin D1 to act on AF2 is dependent on loss of the AR NTD. This conclusion is based on the observations that cyclin D1 conferred an overall inhibitory affect on AR
234 (Fig. 6C
), and the LALA allele of cyclin D1 (defective in the LxxLL motif) retains complete AR-inhibitory function (40, 42). Moreover, we have recently generated an allele of cyclin D1 that retains the LxxLL motif, yet fails to both bind to AR and inhibit NTD-AF2 interaction. This mutant allele correspondingly lacks AR-inhibitory activity (56). Combined, these data suggest that LxxLL-dependent recruitment of cyclin D1 to the AR C terminus holds little biological relevance in the context of an N-terminal-competent receptor. However, the ability of cyclin D1 to confer marginal repression of the AR
234 allele (Fig. 6C
) suggests an alternative mechanism of action by cyclin D1 through recruitment to the carboxy terminus. One explanation for this phenomenon is that the residual cyclin D1 action on this mutant is mediated through recruitment of HDAC activity. However, attempts to reverse residual repression using TSA were confounded by the low level activity of AR
234 in comparison to a corresponding general increase in transcriptional activity of the transfection control (data not shown). Experiments to address this hypothesis are the focus of future study.
Consequence of Cyclin D1-Regulated NTD-AF2 Interaction
We showed that the ability of cyclin D1 to disrupt NTD-AF2 interaction is essential for full repressor activity. The importance of this FxxLF-AF2 interaction in the context of AR activity and prostatic epithelial proliferation is becoming increasingly apparent. It has been recently demonstrated that overexpressing peptide representing the first 34 amino acids of AR can inhibit androgen-dependent prostate growth, presumably through competition for the hydrophobic cleft and inhibition of NTD-AF2 interactions (51). In addition, abrogation of this association can result in androgen insensitivity syndrome, thus underscoring the importance of this interaction for overall AR function in vivo (35, 57). With regard to mechanism, it is clear that FxxLF-AF2 interaction slows ligand dissociation and therefore stabilizes the active AR complex (33). The AF2 hydrophobic cleft harbors a 5-fold higher affinity for FxxLF than p160 NR boxes, and it has been shown that FxxLF can exclude coactivator binding to AF2 (54). This event precludes AF2 activity (for which coactivators are essential) and thus maintains AF1 as the predominant transactivation domain. If true, cyclin D1 action on the FxxLF motif would act predominantly through abrogation of a stable AR-ligand complex and thus reduce overall AF1 function.
In this same model, p160 overexpression (such as has been observed in advanced prostate cancer) effectively competes for the AF2 binding site and induces AF2 activity (58, 59). Although both the FxxLF and WxxLF motif act to repress p160 recruitment to AF2 through occupancy of the binding pocket, the overall action on AR activity of these two motifs is quite different. Whereas the FxxLF motif confers activity to AF1 and modulates strong AR transactivation potential, the WxxLF motif lacks the ability to activate strong transactivation potential (33). In fact, this motif acts mainly to repress AF2 activity (58). It is surprising that cyclin D1, through activation of AF2, acts to reverse the negative regulatory domain of WxxLF. However, the activation of AF2 is most likely inconsequential when compared with the loss of AF1 activity.
Alternate models for p160 function (although not mutually exclusive) suggest that NTD-AF2 interaction influences the sequence of coactivator binding, provides a novel platform for coactivator association, and/or is facilitated by the ability of selected p160 coactivators to simultaneously bind the NTD and LBD. These hypotheses are generally based on the observation that p160s can bind the AR NTD and LBD and that overexpression of TIF2 or SRC1A increased activity in the mammalian two-hybrid assay (17, 25, 32). Under these models, the ability of p160 coactivators to promote AR activity through two distinct mechanisms (promotion of NTD-AF2 association and acetylation of histones) would be effectively foiled by the dual repressor actions of cyclin D1 (abrogation of NTD-AF2 association and recruitment of HDACs). The potency of these dual mechanisms likely underscores our previous observations that cyclin D1 repressor function cannot be rescued by ectopic expression of coactivators [SRC1, P/CAF, cAMP response element binding protein (CREB)-binding protein, or ARA70] (42) and that cyclin D1 is an effective repressor of AR function at stoichiometric levels with the receptor (44). Dual mechanisms have also been identified for the Dax1 orphan receptor and SMRT, which both act as AR corepressors. These repressors have been recently reported to block AR NTD-AF2 interaction and can also recruit other corepressors (HDACs, nuclear receptor corepressor, and Alien) (60, 61, 62).
In summary, we establish a second, unexpected function of cyclin D1 in transcriptional regulation. We demonstrate that cyclin D1 inhibits NTD-AF2 interactions of the AR, thus revealing a novel aspect of cyclin D1 function. We also delineate the basis and importance of this regulatory event. First, we show that cyclin D1 action is manifested through a discrete AR motif (FxxLF). This motif is critical for receptor function and is unique to AR. Therefore, these observations also draw distinctions between the disparate mechanisms by which cyclin D1 regulates AR vs. other nuclear receptors. Furthermore, we demonstrate that the ability of cyclin D1 to modulate NTD-AF2 interaction is critical for mediating repressor function. Abrogation of the FxxLF motif significantly impaired the ability of cyclin D1 to modulate AR, thus demonstrating the biological significance of this interaction. Lastly, we demonstrate that although secondary cyclin D1 binding sites were found to be present on AR, these sites fail to regulate NTD-AF2 interaction. These studies culminate in the hypothesis that the ability of cyclin D1 to modulate NTD-AF2 interaction represents a crucial mechanism of cyclin D1 function that is likely complemented by the established ability of cyclin D1 to attract HDACs. It is our belief that in-depth knowledge of receptor-specific corepressor function will lead to the design of novel therapeutic strategies for AR-dependent cancer.
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MATERIALS AND METHODS |
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Plasmids
The plasmids ARR2-LUC, pRC/CMV-cyclin D1, pRC/CMV-cyclin D1-LALA, CMV-ß-galactosidase, pGEX 3X-cyclin D1, H2B-GFP, pCMV-hAR-LFAA, pCMV-hAR, PGEX-KG, and WTAR-pGEM have been previously described (33, 42, 44). The Gal4-LUC reporter, Gal4-ARLBD, Gal4-AR628646 LBD, and VP16-AR-TAD constructs were gifts of Dr. E. Yong (47, 50). pCRAR1238 and pCR1238LFAA were generated by PCR amplification of AR amino acids 1238 using pCMV-hAR and pCMV-hAR-LFAA as templates, respectively. The following primer pairs were used for amplification: 5'-GAGCAAGAGAAGGGGAGCC3-' (sense) and 5'-TCACCACTCCTTGGCGTTGTC-3' (antisense). The resulting fragment was inserted into pCR2.1 as directed by the manufacturer (Invitrogen, San Diego, CA). pCRAR 34238 was cloned similarly by PCR from the pCMV-hAR template using the primer 5'-CGCGAAGTGATGCAGAACCCG-3' in place of the sense strand primer described above. All constructs generated were verified by sequencing and shown to be free of error. To generate the full-length allele, pcDNA-AR
234 was cloned by excising the XmaI/BamHI fragment of pAR0 and inserting it into the XmaI/BamHI sites of pCR-AR34238. The fragment and the full-length alleles were subsequently removed from the pCR vector and inserted into pcDNA3.1(-) (Invitrogen) using a NotI/BamHI digest. VP16-AR
234TAD was generated by cleaving pcDNA-AR
234 with XhoI and HindIII and inserting these fragments into the SalI and HindIII sites of pVP-16 (CLONTECH, Palo Alto, CA).
Immunoblotting
CV1 cells were transfected using the N,N-bis(2-hydro-xyethyl)-2-amino-ethane sulfonic acid/calcium phosphate method with 2.5 µg pcDNA vector, 1.0 µg H2B-GFP, and 0.5 µg of CMV-hAR or pcDNA AR234. Transfected cells were harvested via trypsinization and lysed in radioimmune precipitation assay buffer containing proteinase inhibitors (1 mM phenylmethylsulfonylfluoride, 10 µM benzamidine-HCl, 50 µM 1,10 phenanthroline-HCl, 15 µM aprotinin, 20 µM leupeptin, 15 µM pepstatin). Cell lysates were subjected to SDS-PAGE and immunoblotted with antibodies specific for AR (SC-815; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and GFP (SC-9996; Santa Cruz).
Immunoprecipitation
LNCaP cells were cultured in improved MEM containing 5% CDT serum for 48 h. Cells were then switched to incomplete MEM containing 5% heat-inactivated fetal bovine serum for 16 h, at which time cells were harvested via trypsinization. Cell pellets were lysed and sonicated in NETN (20 mM Tris-Cl, pH 8.0; 100 mM NaCl; 1 mM EDTA, pH 8.0; 0.5% Nonidet P-40) containing proteinase inhibitors. After centrifugation, supernatants were immunoprecipitated with 1 µg of antibody directed against AR (N-20; Santa Cruz), cyclin D1 (HC-295; Santa Cruz), or DBF4 (H-300; Santa Cruz) and pulled down with protein A-sepharose beads (CL-4B; Amersham Biosciences, Arlington Heights, IL). Bead complexes were washed four times with NETN. Samples were loaded on 12% SDS-PAGE, transferred to a polyvinylidine difluoride membrane, and immunoblotted with the designated antibodies.
In Vitro Binding Assay
AR and CD44 constructs were in vitro transcribed/translated using the Promega T7 rabbit reticulate lysate kit as per manufacturers instructions (Promega Corp., Madison WI) in the presence of [35S]methionine (Easy Tag Express [35S]Protein Labeling Mix; PerkinElmer Corp., Norwalk, CT). Recombinant protein was then incubated with GST alone or GST-cyclin D1 immobilized on glutathione-agarose beads (Sigma Chemical Co., St. Louis, MO) in NETN plus protease inhibitors for 3 h at 4 C with rotation. Preparation of GST and GST-cyclin D1 was performed as previously described (42). Total volume for each reaction was 500 µl. After incubation, samples were washed five times with 750 µl NETN. Samples (bound and 5% input) were then denatured in SDS-PAGE loading buffer and run on a 12% SDS-polyacrylamide gel. Signal was enhanced by incubation with Fluoro-hance (Research Products International) as indicated by the manufacturer and results were visualized and quantified on a Storm 860 PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).
Mammalian Two-Hybrid and Reporter Assays
For all reporter assays, six-well dishes of CV1 cells were cultured for 24 h in phenol-red free DMEM supplemented with 10% CDT. Cells were then transfected using the N,N-bis(2-hydroxyethyl)-2-amino-ethane sulfonic acid/calcium phosphate method with a total of 4 µg DNA per well (42). For mammalian two-hybrid assays, 0.5 µg of Gal4-LUC reporter, VP16-ARTAD, and Gal4-ARLBD were used in the presence of 1.5 µg of either empty vector or coregulator (cyclin D1, p53, TIF2). In reporter assays, 1.0 µg of ARR2-LUC, 0.5 µg of AR, and a total 1.5 µg of either cyclin D1 or pcDNA vector were used. All transfections also contained 0.25 µg of ß-galactosidase reporter and empty vector to achieve 4 µg of total DNA. After transfection, cells were washed with PBS, media were replaced, and cells were treated with the indicated concentration of DHT or ethanol vehicle (not to exceed 0.1%) for 24 h. After stimulation, cells were harvested by trypsinization and monitored for luciferase and ß-galactosidase activity using the Promega Luciferase kit and Tropix Galactar-Star systems (Tropix, Inc., Bedford, MA) respectively, as per manufacturers instructions. Transfections were performed at least twice with three independent samples. Averages and standard deviations are shown.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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First Published Online November 11, 2004
Abbreviations: AF2, Activation function 2; AR, androgen receptor; CDT serum, charcoal dextran-treated serum; CMV, cytomegalovirus; DHT, dihydrotestosterone; ER, estrogen receptor; GFP, green fluorescent protein; GST, glutathione-S-transferase; HAT, histone acetyltransferase; HDAC, histone deacetylase; LBD, ligand-binding domain; NR, nuclear receptor; NTD, N-terminal domain; P/CAF, p300/CBP-associated factor; SRC, steroid receptor coactivator; TAD, transcriptional activation domain; TIF, transcriptional intermediary factor; TSA, trichostatin A; wtAR, wild-type AR.
Received for publication July 1, 2004. Accepted for publication November 3, 2004.
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REFERENCES |
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