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J. Biol. Chem., Vol. 279, Issue 29, 30643-30653, July 16, 2004
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¶**
From the
Laboratories for Reproductive Biology and the Departments of Pediatrics, ||Medicine, and **Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599
Received for publication, March 19, 2004 , and in revised form, April 21, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(185 amino acid residues) to the glucocorticoid receptor (GR1; 420 residues), androgen receptor (AR; 558 residues), progesterone receptor (B form; 566 residues), and mineralocorticoid receptor (602 residues). In contrast, transcription factors such as p53, NF-
B, and VP16 typically have transcriptional activation domains of <100 residues.
The importance of the NH2-terminal domain for AR functional activity is attributed largely to activation function-1 (AF1) between amino acid residues 142 and 337 (6). This region was further resolved into two noncontiguous activation segments, AF1a and AF1b (7). The functional importance of the AR NH2-terminal region is supported by the extensive post-translational modification that occurs in the region, including phosphorylation (8–10), sumoylation (11), and ubiquitylation (12, 13). In addition, a conserved region between AR residues 225 and 259 in the region of AF1 was predicted to form an -helix and to contribute to AR transactivation and was shown to be a binding site for the general transcription factor TFIIF (14). Earlier evidence also suggested that the AR NH2-terminal region contains functional domains involved in transcriptional repression (15). In contrast, activation function-2 (AF2) in the AR ligand-binding domain, although highly conserved, has little inherent transcriptional activity when assayed in mammalian cells (16).
In this study, we identify sequence 234AKELCKAVS-VSMGL247 in the human AR NH2-terminal domain, which is highly conserved in all AR sequences characterized thus far through multiple fish species. Based on the premise that strict amino acid sequence conservation indicates a site of critical function, we investigated the functional importance of human AR residues 234–247 using a yeast two-hybrid screen. The COOH terminus of the Hsp70-interacting protein (CHIP) was identified as a binding partner and was found to contribute to AR down-regulation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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TPR, expressing CHIP with a deletion of the tetratricopeptide repeat (TPR) domain at residues 27–147 (see Fig. 3A), were created by digesting pcDNA3-CHIP and pcDNA3-CHIPTPR with EcoRI and XbaI and ligating the inserts to the EcoRI and XbaI sites of pCMV5. All PCR-amplified sequences were verified for the absence of random errors.
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-galactosidase filter assay performed following the instructions of the manufacturer (Clontech, yeast protocols handbook, catalog number PT3024-1, PR13103). Large fresh colonies were transferred to Whatman No. 5 filter paper, and the filters were immersed in liquid nitrogen for 10 s and allowed to thaw at room temperature. Freshly prepared Z buffer containing 60 mM NA2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 50 mM 2-mercaptoethanol, pH 7.0, and 0.33 mg/ml 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-gal) was added to the filter and incubated at room temperature to score blue colonies after 30 min to overnight. Plasmids from the positive yeast colonies in the -galactosidase filter assay were rescued. Colonies that grew on synthetic medium lacking Leu gradually lost the bait vector, but not the library plasmids. The library vector was purified using the Clontech YEAST-MAKER yeast plasmid isolation kit (catalog number K1611-1).
In Vitro Protein Interaction Assay—GST-AR-(220–270), GST-AR-(220–270m4), GST-AR-(220–270)-A234T, and GST-AR-(220–270)-E236G were expressed from pGEX-5X-1 as GST fusion proteins. The GST fusion proteins were expressed in Escherichia coli XL1-Blue cells treated with 0.5 mM isopropyl--D-thiogalactopyranoside; extracted in 0.15 M NaCl, 5 mM EDTA, 10% glycerol, 100 µM phenylmethylsulfonyl fluoride, 10 mM dithiothreitol, and 50 mM Tris-HCl (pH 8.0); and incubated with glutathione-agarose beads (Amersham Biosciences) as described (18). Full-length CHIP and mutants CHIPTPR, CHIPU, and CHIP-K30A were expressed from pcDNA3 and in vitro translated in the presence of 25 µCi of [35S]methionine (PerkinElmer Life Sciences) using the TNT T7 quick coupled transcription/translation system (Promega). Washed beads were boiled in SDS-containing buffer. Input lanes contained 30% of the binding reactions.
Transient Transfection Assays—Human hepatocellular carcinoma HepG2 cells (American Type Culture Collection) were maintained in 5% CO2 at 37 °C in Eagle's minimal essential medium (Invitrogen) containing 10% fetal bovine serum (Gemini Laboratories), 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, and penicillin and streptomycin. Cells were plated at 2 x 105 cells/well on 12-well tissue culture plates and transfected with the indicated concentrations of expression vector DNA and 0.1 µg of 5xGal4-Luc3 reporter vector using (per well) 50 µl of EC buffer (QIAGEN Inc.), 1 µg of enhancer, and 1 µl of Effectene. 24 h after transfection, the medium was changed to serum-free medium, and when appropriate, the indicated hormones were added. Luciferase activity was determined 24 h later by harvesting cells in 0.22 ml of lysis buffer containing 1% Triton X-100, 2 mM EDTA, and 25 mM Tris phosphate (pH 7.8) (19). After a 30-min incubation at room temperature, 0.1-ml aliquots were assayed for luciferase light units using a LumiStar Galaxy multiplate reader luminometer (BMG Labtechnologies).
To measure inherent transcriptional activation by the AR NH2-terminal conserved motif, Gal4 DNA-binding domain fusion proteins were expressed in HepG2 cells with the indicated regions of the AR NH2-terminal domain and the 5xGal4-Luc3 reporter vector. The effect of the NH2-terminal conserved motif on AR transactivation was determined in HepG2 cells using full-length AR vectors with the indicated deletions or mutations and the mouse mammary tumor virus-luciferase (MMTV-Luc) reporter or the PSA-Enh-Luc reporter (provided by Michael Carey, University of California, Los Angeles). The influence of CHIP on AR transcriptional activity was determined in HepG2 cells using (per well) 50 ng of pCMV5 AR expression vector, 25 ng of pCMV5-CHIP and pCMV5-CHIPTPR vectors, and 0.25 µg of MMTV-Luc reporter. Cells were incubated for 24 h in the absence and presence of the indicated hormones and assayed for luciferase activity as described above.
In mammalian two-hybrid experiments, human epithelioid cervical carcinoma HeLa cells were maintained in Eagle's minimal essential medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and penicillin and streptomycin. HeLa cells (1.5 x 105/well of 12-well plates) were transfected with 0.1 µg of 5xGal4-Luc3 reporter and the indicated AR and CHIP vectors using Effectene as described above.
Immunoblot Analysis—Monkey kidney COS cells were cultured in Dulbecco's modified Eagle's medium containing 10% bovine calf serum (Hyclone Laboratories), 2 mM L-glutamine, 20 mM HEPES (pH 7.2), and penicillin and streptomycin in a 5% CO2 incubator at 37 °C. Cells (1.5 x 106/10-cm dish) were transfected using DEAE-dextran or the Effectene reagent kit (QIAGEN Inc.), which included (per 10-cm dish) 0.3 ml of EC buffer, 16 µl of enhancer, 10 µl of Effectene, and 1 ml of medium added to 7 ml of fresh medium. After transfection, the medium was replaced with serum-free medium with and without 50 nM dihydrotestosterone (DHT). The next day, cells were washed with cold phosphate-buffered saline and harvested in 1 ml of phosphate-buffered saline. After centrifugation at 5000 rpm at 4 °C, cells were suspended in 0.1 ml of lysis buffer containing 0.15 M NaCl, 0.5% Nonidet P-40, 50 mM NaF, 1 mM dithiothreitol, 50 mM Tris-HCl (pH 7.5), 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor mixture (Sigma) and incubated for 1 h at 0 °C. After centrifugation at 12,000 rpm for 15 min at 4 °C, the supernatant protein concentration was determined using the Bio-Rad assay. Samples (10 µg of protein/lane) were separated on 10 or 8–16% acrylamide gels containing SDS. After electrophoresis, gel proteins were electrophoretically transferred to nitrocellulose membranes. The blots were incubated with 1 µg/ml rabbit antibody AR32, raised against an AR NH2-terminal peptide, or with a 1:500 dilution of mouse anti-Gal4 DNA-binding domain IgG2a (sc-510, Santa Cruz Biotechnology). Immunoreactive bands were visualized using chemiluminescence (SuperSignal Western Dura extended duration substrate, Pierce).
Immunocytochemistry—COS cells (1.25 x 106/well) were transfected in 12-well tissue culture plates, with each well containing a 0.15-mm thick cover glass (Fisher). Cells were transfected using Effectene as described above with 0.2 µg of pCMV-hAR and pCMV5-FLAG-CHIP and treated with and without 100 nM DHT 24 and 48 h after transfection. Cells were fixed with freshly prepared 3% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked with 0.5% bovine serum albumin as described (20), and incubated for 1 h with primary antibodies: mouse anti-FLAG monoclonal antibody M2 (1:250 dilution; Sigma) and rabbit anti-AR polyclonal antibody 3510 (1:250 dilution; Abcam, Inc.). After several washes with phosphate-buffered saline, cells were incubated for 30 min with secondary antibodies: TRITC-conjugated AffiniPure donkey anti-mouse IgG (1:50 dilution; Jackson ImmunoResearch Laboratories, Inc.) and fluorescein isothiocyanate-conjugated AffiniPure donkey anti-rabbit IgG (1:50 dilution; Jackson ImmunoResearch Laboratories, Inc.). The cover glasses were mounted on slides using DakoCytomation fluorescent mounting medium (Fisher) and viewed using a Zeiss LSM 210 confocal microscope.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sequence conservation of AR NH2-terminal residues 234–247 suggests a conserved motif for the AR gene family. A search of the protein sequence data base supports this, with one exception. GR from human and Xenopus, but not GR from fish, contains sequence 77DLSKAVSLSMGL88 at human GR residues 77–88, which is homologous to 12 of the 14 residues in the AR NH2-terminal conserved motif (Fig. 1B). The closer evolutionary distance between AR and the progesterone receptor compared with AR and GR (4) and the absence of the conserved motif in the progesterone receptor suggest that AR and GR acquired the sequence independently for a common function by convergent evolution.
Analysis of the predicted order and disorder composition of the AR NH2-terminal domain using the PONDR Protein Disorder Predictor3 indicates that the FXXLF motif and the AR NH2-terminal conserved motif form short structurally ordered regions as indicated by PONDR scores of <0.1. Each motif is predicted to form an amphipathic -helix, indicating the presence of short structured segments in an NH2-terminal region that is otherwise predicted to be largely unstructured.
Intrinsic Activity of the AR NH2-terminal Conserved Motif— We tested for the presence of transcriptional activity inherent to the AR NH2-terminal conserved motif by expressing Gal4 DNA-binding domain-AR fusion proteins in mammalian cells (Fig. 2A). The NH2-terminal conserved motif residues 234–247 lacked activity, but a low level of activity was detectable when the sequence was extended in Gal-AR-(229–254). A larger fragment, Gal-AR-(220–270), elicited 20% the activity of the AR NH2-terminal domain in Gal-AR-(1–503). When mutations at four residues (L237A/K239M/V241A/V243A) were introduced into the NH2-terminal conserved motif in Gal-AR-(220–270) (Gal-AR-(220–270m4)) (Fig. 2A), the transcriptional response was reduced to background levels. Expression levels of the Gal-AR fusion proteins were similar as determined by immunoblotting (Fig. 2C, left panel).
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Contribution of the core conserved motif to AR transcriptional activity was investigated further by testing several mutants of full-length AR. Deletion of region 220–270 (AR220–270) caused a 
50% decrease in AR transcriptional activity using the MMTV-Luc and PSA-Enh-Luc reporter vectors (Fig. 2B), confirming the importance of the region for AR activity. The decrease in transcriptional activity by AR220–270 and additional mutants was independent of AR expression levels (Fig. 2C, right panel). Deleting only the NH2-terminal conserved motif (AR234–247) caused less of a decrease in activity, and the decrease was similar to that seen with ARm4. Mutating the flanking Ser256 (Fig. 1B)to alanine to inhibit phosphorylation at this previously reported AR phosphorylation site (10) or to aspartic acid to mimic the negative charge of a phosphate group had no effect on the AR transcriptional response. The results suggest that the AR NH2-terminal conserved motif and closely flanking sequence contribute to AR transactivation.
AR NH2-terminal Conserved Motif Interaction with CHIP— The high sequence conservation of the AR NH2-terminal motif in AR gene evolution, its predicted structure, and evidence for a role in AR transcriptional activation led us to perform a yeast two-hybrid screen using a 51-amino acid residue fusion peptide as bait to probe a human testis cDNA library. Yeast strain HF7c was initially transformed with pGBT8-AR-(220–270), coding for the Gal4 DNA-binding domain and human AR residues 220–270. In agreement with the transcriptional response seen with Gal-AR-(220–270) in mammalian cells, the addition of 0.8 mM 3-amino-1,2,4-triazole was required to reduce intrinsic activity of pGBT8-AR-(220–270). Screening 106 clones of an amplified library revealed 138 positive clones that grew on synthetic medium lacking Leu, Trp, and His. Of these, 48 clones were positive in a -galactosidase filter assay performed in yeast. Sequence analysis revealed five identical clones coding for CHIP. The clones coded for CHIP residues 20–303, representing full-length CHIP lacking the first 19 NH2-terminal amino acids. That the interaction was specific was suggested by a parallel screen of the same library using a similarly sized AR fragment from a different region of the AR NH2-terminal domain. In this case, none of the positive clones that grew on synthetic medium lacking Leu, Trp, and His coded for CHIP.4
CHIP is a U-box-containing ubiquitin-protein isopeptide ligase (E3) (23–26) that interacts with heat shock proteins Hsp70 and Hsp90 through its NH2-terminal TPR domain (Fig. 3A) and has been reported to cause ubiquitylation of AR (13). To further investigate the role of the AR NH2-terminal conserved motif in the AR and CHIP interaction, we tested several AR mutants in a mammalian two-hybrid assay using a Gal-CHIP-(20–303) fusion protein. Deletion of AR residues 220–270 (AR220–270) strongly reduced the interaction with CHIP (Fig. 3B), supporting the role of the NH2-terminal conserved motif region in the AR interaction with CHIP. A small portion of this reduction was attributed to the slight decrease in background AR activity that resulted from the deletion of residues 220–270, in agreement with the results of Fig. 2 (A and B) showing that residues 220–270 harbor a transcriptional activation function. The background AR activity evident upon coexpression of the pGal0 empty vector results from a cryptic androgen response element in the 5xGal4-Luc3 reporter (19). Deleting the NH2-terminal conserved motif in AR234–247 or introducing the multiple mutations L237A/K239M/V241A/V243A into AR (ARm4) did not decrease background AR transcriptional activity and reduced (but did not eliminate) the AR and CHIP interaction.
The results support a role for the NH2-terminal conserved motif in the AR and CHIP interaction, but also suggest that additional direct or indirect interactions occur between AR and CHIP. One possible interaction site is the AR ligand-binding domain, which was shown to bind the Hsp70·Hsp90 heterocomplex (27, 28), and Hsp70·Hsp90 interacts with CHIP (23). A recent study demonstrating that CHIP functions as a dimer provides a basis for this type of interaction (29).
GST in Vitro Adsorption Assay—To confirm a direct interaction between the AR NH2-terminal conserved motif and CHIP, GST affinity matrix binding assays were performed. Wild-type GST-AR-(220–270) and GST-AR-(220–270m4) were expressed in E. coli, purified, and incubated with 35S-labeled in vitro translated full-length CHIP and several CHIP mutants. A strong interaction was evident between GST-AR-(220–270) and 35S-labeled CHIP that was not detected using the pGST0 empty vector control (Fig. 3C, lanes 1 and 5). The AR interaction with CHIP was eliminated by deleting the CHIP TPR domain (lane 6), by deleting the CHIP U-box domain (lane 7), or by introducing a K30A mutation into the CHIP TPR domain (lane 8). Lys30 is highly conserved in the TPR domain (30) and was shown to be required for the interaction between the CHIP TPR domain and Hsp70 or Hsp90.5
Two AR mutations identified in the transgenic adenocarcinoma of mouse prostate (TRAMP) model for prostate cancer, E236G and A234T (17), also reduced the AR and CHIP interaction by 43 and 16%, respectively (Fig. 3D). In contrast, no decrease in the AR and CHIP interaction was seen using GST-AR-(220–270m4), in which four mutations (L237A/K239M/V241A/V243A) were introduced into the NH2-terminal conserved motif (Fig. 3C).
The data indicate a direct interaction between AR and CHIP mediated by the short AR NH2-terminal fragment that contains the conserved motif. A reduced AR and CHIP interaction by AR mutations derived from the TRAMP model suggests that the AR NH2-terminal conserved motif functions to limit AR activity and that mutations in this region confer increased AR activity, which contributes to prostate cancer growth. However, unlike the transcriptional activity detected in this region that depended on four conserved residues of the NH2-terminal conserved motif, the AR and CHIP interaction was not diminished by the L237A/K239M/V241A/V243A mutations.
CHIP-induced Inhibition of AR Transcriptional Activity and AR Steady-state Levels—Coexpression of full-length CHIP decreased both AR and GR transcriptional activity (Fig. 4A). This is in contrast to the activity observed without any further DNA addition, with coexpression of the pCMV5 empty vector control, or with coexpression of the CHIP mutant lacking the TPR domain (CHIPTPR) (Fig. 4A, TP). Surprisingly, deletion of the conserved sequence in AR234–247 or AR220–270 did not eliminate the inhibitory effect of CHIP on AR transactivation (data not shown). Furthermore, the constitutive transcriptional activities of AR truncation mutant AR-(1–660), which lacks the ligand-binding domain, and AR-(1–660)234–247, which lacks, in addition, the NH2-terminal conserved motif, were both strongly inhibited by CHIP (Fig. 4A). That this inhibitory effect of CHIP was mediated by sequences outside of the NH2-terminal conserved motif was also supported by our observations that CHIP inhibited, to a similar extent, transactivation by the progesterone receptor, a receptor that lacks the conserved motif (data not shown). When the activity of a pCMV-Luc constitutive reporter was tested in the presence of CHIP expression, there was no significant decrease in transcriptional activity above that observed with the empty vector control or with pCMV5-CHIPTPR (data now shown), suggesting that the inhibitory effect of CHIP is not due to inhibition of general transcription mechanisms.
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The influence of CHIP on AR steady-state levels was also tested. The low level of AR detected in the absence of androgen was further reduced by coexpression of CHIP (Fig. 4B, lanes 1 and 3). CHIP also decreased the stabilizing influence of androgen on AR levels (lanes 2 and 4). In contrast, deletion of the CHIP TPR region eliminated the reduction in AR levels induced by coexpression of CHIP. The results indicate that the TPR region of CHIP is required for the inhibitory effects of CHIP on AR steadystate levels and AR transcriptional activity.
Colocalization of CHIP and AR—The subcellular localization of CHIP and AR was determined in the absence and presence of DHT by immunocytochemistry. In the absence of DHT, transiently expressed AR (green) and FLAG-tagged CHIP (red) localized in the cytoplasm, with no evidence of nuclear staining (Fig. 5A). This is in agreement with a previous report that CHIP is a cytoplasmic protein (23). The partial colocalization of CHIP and AR in the cytoplasm supports their interaction in the absence of hormone. In the presence of DHT, AR was nuclear, whereas CHIP remained primarily in the cytoplasm (Fig. 5B). The data suggest that CHIP regulates AR degradation prior to androgen binding and that androgen-induced nuclear targeting sequesters AR from the inhibitory effects of CHIP.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, we have identified an extraordinarily conserved 14-amino acid NH2-terminal sequence at human AR amino acid residues 234–247 that is common to all published sequences of AR, including the lower vertebrate fish species. The conserved motif lacks intrinsic transcriptional activity per se, but contributes to the transcriptional response of a larger region between residues 220 and 270. The AR NH2-terminal conserved motif lies within a previously described -helical region (2) and is NH2-terminal to a recently reported hydrophobic motif identified among cellular and viral transcription factors that act as binding sites for transcription factor TFIIF (14), a general transcription factor reported to interact with AR (39, 40). To study the function of the AR NH2-terminal conserved motif, we performed a yeast two-hybrid screen and identified CHIP as the interacting partner for the conserved AR NH2-terminal conserved motif. CHIP is highly conserved across species and interacts with the NH2-terminal motif that is conserved throughout the entire AR gene family. Coexpression studies have shown that CHIP limits AR function by promoting AR degradation most likely through the previously described role of CHIP in ubiquitylation (13).
CHIP, named for its interaction with Hsp70, was originally identified from a screen for TPR domains. The TPR, first described in yeast proteins (41, 42), is a tandem 34-amino acid repeat composed of antiparallel -helices (30). The TPR domains of CHIP mediate its interaction with Hsp90 and Hsp70 to redirect incompletely folded proteins for degradation (31). TPR protein interaction surfaces on the chaperone cofactors Hip (Hsp70-interacting protein) (43) and Hop (Hsp90/Hsp70-organizing protein) (44) interact with the COOH-terminal EEVD motif of Hsp70 and Hsp90 during the chaperone heterocomplex formation that mediates protein folding (30, 45, 46). CHIP inhibits Hsp70 ATPase and protein refolding activities, although paradoxically, its overexpression increases chaperone activity (47). CHIP contains a U-box domain at its COOH terminus (25). Structurally similar to a RING finger domain, U-box-containing proteins are a third group of ubiquitin ligases with E3 activity that direct ubiquitylation of chaperone protein complex substrates, leading to increased degradation by the proteasome (24, 26, 48). CHIP was shown to promote ubiquitylation and degradation through the proteasome of GR (31), AR (13), and other proteins (26, 49), including ErbB2/Neu (50, 51). However, it was previously suggested that the predominant effect of CHIP on AR is inhibition of protein folding and reduced degradation (13). CHIP may act as a molecular link between the protein folding chaperone functions and proteasome-mediated protein degradation processes of the Hsp70·Hsp90 heterocomplex (52). CHIP also directly modulates the stress response by activating heat shock factor-1 (53) and influences protein trafficking (54).
A direct interaction between CHIP and the AR NH2-terminal conserved motif was confirmed in mammalian two-hybrid and GST affinity matrix binding assays, supporting the initial identification of CHIP in a yeast two-hybrid screen as a bona fide AR-interacting protein. A BLAST search of the protein data base showed that besides AR, GR from Xenopus and higher species is the only other protein that contains a homologous sequence. However, in the yeast two-hybrid interaction assay, CHIP did not interact with a GR fragment containing the partial sequence (data not shown), even though CHIP decreased GR transcriptional activity to an extent similar to the decrease in AR activity. It is conceivable that CHIP also associates with AR and GR indirectly via the ligand-binding domain through an association with Hsp70 and Hsp90. The requirement of the TPR region of CHIP for inhibition of AR and GR transcriptional activity suggests a mechanism involving CHIP interaction with the heat shock protein complex. The AR·CHIP·Hsp complex may be further stabilized by the NH2-terminal conserved motif. On the other hand, CHIP also decreased the transcriptional activity of an AR deletion mutant lacking the ligand-binding domain, and deletion of the conserved sequence did not eliminate the inhibitory effect. The lack of a requirement for the AR NH2-terminal conserved motif and the ligand-binding domain for the inhibitory effect of CHIP on AR transactivation suggests that other factors are involved.
Our immunostaining results show that, in the absence of androgen, AR and CHIP partially colocalized in the cytoplasm. The addition of DHT translocated AR to the nucleus, but CHIP remained predominantly cytoplasmic. The data suggest the model shown in Fig. 6, whereby CHIP, in association with the heat shock protein complex, targets AR for degradation in the cytoplasm prior to hormone binding, thereby contributing to the rapid turnover of AR in the absence of ligand (55). Ligand-induced targeting of AR to the nucleus would favor its stabilization not only by sequestering it in the nuclear compartment away from CHIP, but also by the androgen-induced interdomain AR NH2- and COOH-terminal interaction that increases AR stabilization (56, 57). The subcellular segregation of CHIP and androgen-bound AR provides an additional mechanism for AR stabilization subsequent to hormone binding.
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Several spontaneous somatic AR mutations were reported in the TRAMP model of prostate cancer during tumor progression, two of which (E236G and A234T) are within the AR NH2-terminal conserved motif. TRAMP tumors were derived in transgenic mice in response to overexpression of the SV40 large and small tumor antigens under the control of the minimal -426 to +28 regulatory sequence of the prostate-specific rat probasin promoter (60). The interaction between AR and CHIP was reduced as a result of these AR mutations. This is in support of the AR NH2-terminal conserved motif functioning to limit AR activity. Interference with the AR and CHIP interaction would be expected to increase AR activity, which could lead to increased prostate cancer growth. In agreement with this, recent studies have shown that the E236G mutation, which we found inhibited the AR and CHIP interaction to the greatest extent, is associated with an increased incidence of tumor formation when the AR mutant is overexpressed.6 Furthermore, it was reported that the E236G mutant increases AR transactivation in the presence of AR coactivators (17). The results support the inhibitory role of the AR NH2-terminal conserved motif mediated by CHIP. With the recent report of increased AR levels as a common feature of hormone refractory recurrent prostate cancer (61), down-regulation of or mutations in CHIP may contribute to increased AR steady-state levels during prostate cancer progression.
However, increased degradation of AR cannot necessarily be construed as a decrease in AR functional activity. In many instances, ligand-activated transcription factors are rapidly degraded. Among the steroid receptors, AR (55) and the vitamin D receptor (62) are exceptions. AR is distinguished by its rapid degradation in the absence of ligand (55) and androgen-induced stabilization (56). Part of the mechanism for androgen-induced AR stabilization involves the androgen-dependent NH2- and COOH-terminal interaction (57, 63) mediated by the FXXLF and WXXLF motifs mentioned above (18, 21). It is plausible that through the interaction of CHIP and the AR NH2-terminal conserved motif, CHIP helps to maintain low levels of AR in normal cells. We have shown that CHIP is mainly in the cytoplasm, and CHIP has been reported to be highly expressed in non-proliferating cells (23). In contrast to the low level of AR in non-proliferating cells, AR levels in prostate cancer are elevated (61), and AR has increased stability, which results in a greater sensitivity to low levels of circulating androgen (64) or to androgens present in prostate cancer tissue (65). It is possible that nuclear localization of AR that is detected in prostate cancer cells in the absence of androgen (66) contributes to increased AR levels by sequestering AR in the nucleus away from the degrading influence of CHIP in the cytoplasm. Alterations in CHIP levels, mutations in CHIP, or, as shown here, mutations in AR could decrease AR degradation, resulting in increased AR-dependent proliferation of recurrent prostate cancer cells.
Sequence homology comparisons in the AR gene family show that Ser256 in human and rat AR replaces the negatively charged residue aspartic or glutamic acid in AR from lower species. Phosphorylation at Ser256 was reported for human AR (10) and effectively reintroduces the constitutive negative charge present in lower species. The proximity of Ser256 to the NH2-terminal conserved motif suggests that a negative charge at this position modulates protein interactions at the NH2-terminal conserved motif and that phosphorylation at this site is a regulatory switch. Although the interaction between CHIP and AR was not significantly influenced by the S256A and S256D mutations in mammalian two-hybrid assays, the transition from the constitutive negative charge of lower species to the conditional charge regulated by phosphorylation may influence other interacting partners at this site. Higher vertebrates gain the ability to regulate the negative charge by phosphorylation. This is also suggested by the human AR phosphorylation site Ser650 (8, 10), which is Asp in lower species. Similarly, the human estrogen receptor- phosphorylation site Ser118 (67, 68) is Asp in the lamprey estrogen receptor (4). Increased regulation introduced by phosphorylation likely helps to modulate protein interactions to increase the overall level of homeostatic control.
NH2-terminal domains of steroid receptors are largely unstructured, and amino acid sequence is poorly conserved; yet computational programs that predict protein structure can identify short 20-amino acid residue segments with ordered secondary structure, some of which are conserved across species. The PONDR Protein Disorder Predictor indicates that the FXXLF and WXXLF motifs are structured, and similarly, an ordered amphipathic -helical structure is predicted for the AR NH2-terminal conserved motif described in this work. We designed a yeast two-hybrid screening approach based on sequence conservation and structure predictions that short ordered regions, especially amphipathic -helices, are surfaces for protein-protein interactions. In support of this, the highly conserved AR FXXLF motif and flanking sequence mediate the androgen-dependent interaction with AF2 in the ligand-binding domain (18, 21) and the interaction of coregulatory proteins (19, 38). The predicted amphipathic -helices of the p160 coactivator LXXLL motifs were also confirmed by structural studies (69, 70). Thus, although transcriptional activation domains of regulatory proteins are largely unstructured and not conserved, short segments within these regions are predicted to form amphipathic -helices whose structure is likely stabilized by binding coregulatory proteins (2, 71). We previously proposed a reversal of the acidic transcriptional activation domain-coactivator interaction model, implicating charge interactions that precede nonpolar contacts of hydrophobic residues in protein-protein interactions mediated by amphipathic -helices (21). In the present study, we used comparative sequence alignment and structure predictions to identify a 14-amino acid AR NH2-terminal conserved motif conserved in all AR sequences throughout the lower vertebrates. We have demonstrated the usefulness of yeast two-hybrid library screening using short 50-amino acid fusion peptides containing conserved sequence and predicted structure in the identification of interacting partners. The results demonstrate a novel strategy to identify interacting partners and to probe the functional significance of short conserved sequences present in steroid receptor NH2-terminal domains.
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FOOTNOTES |
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¶ Present address: Dept. of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030.
To whom correspondence should be addressed: Labs. for Reproductive Biology, CB 7500, Medical Biomolecular Research Bldg., Rm. 3340, 103 Mason Farm Rd., University of North Carolina, Chapel Hill, NC 27599. Tel.: 919-966-5168; Fax: 919-966-2203; E-mail: emw{at}med.unc.edu.
1 The abbreviations used are: GR, glucocorticoid receptor; AR, androgen receptor; AF, activation function; CHIP, COOH terminus of the Hsp70-interacting protein; GST, glutathione S-transferase; TPR, tetratricopeptide repeat; MMTV, mouse mammary tumor virus; Luc, luciferase; DHT, dihydrotestosterone; TRITC, tetramethylrhodamine isothiocyanate; E3, ubiquitin-protein isopeptide ligase; TRAMP, transgenic adenocarcinoma of mouse prostate; PSA-Enh-Luc, prostate-specific antigen enhancer luciferase.
2 Available at ww2.mcgill.ca/androgendb/data.htm.
3 Available at www.pondr.com/PONDR/pondr.cgi.
4 S. Bai, B. He, and E. M. Wilson, unpublished data.
5 C. Patterson, unpublished data.
6 N. M. Greenberg, personal communication.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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