From the George Whipple Lab for Cancer Research, Departments of Pathology, Urology, and Radiation Oncology and the Cancer Center, University of Rochester Medical Center, Rochester, New York 14642
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ABSTRACT |
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Androgen receptor (AR) is a member of the steroid
receptor superfamily that may require coactivators for proper or
maximal transactivation. Using a yeast two-hybrid screening followed by mammalian cell analyses, we identified a novel
ligand-dependent AR-associated protein, ARA54, which
consists of 474 amino acids with a molecular mass of 54 kDa. We
demonstrated that ARA54 might function as a preferential coactivator
for AR-mediated transactivation in human prostate cancer DU145 cells.
Interestingly, our data also showed that ARA54 could significantly
enhance the transcriptional activity of LNCaP mutant AR (ARt877a) but
not wild type AR or another mutant AR (ARe708k) in the presence of 10 nM 17 Androgen receptor (AR)1
is an androgen-dependent transcription factor that belongs
to the steroid receptor superfamily (1, 2). Although several studies
have revealed how steroid hormone-bound receptors can recognize and
interact with hormone-response elements (3-5), the mechanism of how
receptors activate their target genes is not fully understood.
Recently, it has been reported that several nuclear receptors can
interact directly with components of the basal transcription machinery
apparatus, such as TBP (6), TFIIB (7), and TFIIF (8). In addition,
specific sets of proteins were recruited by the steroid receptors as
coactivators that may function as bridge factors between the receptors
and general transcription factors in the preinitiation complex
(9-11).
Identifying and understanding the function of individual components of
these complexes are part of the key to answer how nuclear receptors
regulate their target genes. Indeed, several cofactors, such as ARA70
(12), SRC-1 (13), CBP/p300 (14), GRIP1/TIF2 (15, 16), Rb tumor
suppressor (17), and RAC3/ACTR (18, 19), have been identified as being
able to modulate the transactivation of the steroid receptors. More
significantly, recent progress in the study of coactivators further
linked the transcriptional activation of steroid receptors to chromatin
acetylation. Some of these coactivators, such as RAC3/ACTR (19),
CBP/p300 (20), and SRC-1 (21), have been found to either have intrinsic
histone acetyltransferase activity or have the capacity to recruit the p300/CBP-associated factor (pCAF) that has histone acetyltransferase activity. However, the physiological significance of these cofactors and their involvement in development, differentiation, and reproductive disease remains to be further studied.
Prostate cancer is the most common malignant neoplasm in aging males in
the United States. Surgical or chemical castration in combination with
antiandrogens, such as hydroxyflutamide (HF), has been widely used for
the treatment of this disease (22). However, most prostate cancers
undergoing androgen ablation treatment progress from an
androgen-dependent to an androgen-independent state (23).
Indeed, previous studies using transient transfection assays also
showed that antiandrogens might have agonist activity to stimulate
mutant AR-mediated transcription (24, 25). It has been hypothesized
that cellular factors and mutant ARs might contribute to the
alterations of the antiandrogen specificity and the development of
resistance to androgen ablation therapy in prostate cancer (26, 27).
Therefore, it will be an important issue to investigate whether AR
cofactors are involved in the progression of androgen resistance.
Here we report a new AR-associated protein, ARA54, which was isolated
from human prostate cDNA library by using a mutant AR, ARt877s
(codon 877 threonine to serine), to screen the yeast two-hybrid system.
We further provide evidence to demonstrate that ARA54 can function as a
coactivator for androgen-dependent transcription on both wild
type (wtAR) and mutant AR (mtAR). However, in the presence of 10 nM E2 or 1 µM HF, ARA54 can only
significantly enhance the transcriptional activity of LNCaP ARt877a but
not wtAR or mtAR ARe708k (codon 708 glutamic acid to lysine). These results imply that the specificity of antagonist versus
agonist of antiandrogens could be conferred via the specific
configuration between AR structure and different cofactors.
Materials and Plasmids--
5 Screening of Prostate cDNA Library by a Yeast Two-hybrid
System and RACE-PCR--
A pACT2-prostate cDNA library (a gift
from Dr. S. Elledge) that consists of GAL4 activation domain (amino
acids 768-881) fused with human prostate cDNA library was
transformed into Y190 yeast cells with pAS2-mtARt877s that contains the
C-terminal domain of ARt877s (amino acids 595-918) fused with GAL4
DNA-binding domain (DBD) (amino acids 1-147). Transformants were
selected for growth on nutrition selection plates with 20 mM 3-aminotriazole and 100 nM DHT without
adding histidine, leucine, and tryptophan. Colonies were also
filter-assayed for
The missing 5' coding region was isolated by 5' RACE-PCR according to
the manufacturer's protocol of the Marathon cDNA Amplification kit
(CLONTECH). The gene-specific antisense primer used
for 5' RACE-PCR was 5'-ttctgtagtttaattttctgaacctttggc-3'. The specific PCR reaction conditions were: 94 °C for 1 min; 5 cycles of 94 °C
for 5 s, 72 °C for 3 min; 5 cycles of 94 °C for 5 s, 70 °C for 3 min; and then 25 cycles of 94 °C for 5 s, 68 °C for 3 min. The PCR product was subcloned into pT7-Blue vector
(Novagen) and sequenced.
Northern Blot Analysis--
The blot (Fig. 2) containing
approximately 2 µg of poly(A)+ RNA from human spleen
(lane 1), thymus (lane 2), prostate (lane 3), testis (lane 4), uterus (lane 5), small
intestine (lane 6), colon (lane 7), and
peripheral blood leukocyte (lane 8) was purchased from
CLONTECH and hybridized with an ARA54-specific
cDNA probe. A Mammalian Two-hybrid Assay--
The mammalian two-hybrid system
used in our test system mainly followed the
CLONTECH protocol, with some modifications. To obtain better expression, the GAL4 DBD (amino acids 1-147) was fused
to pSG5, which was driven by the SV40 promoter, and named GAL0. The
hinge and ligand-binding domain of wtAR were then inserted into GAL0.
Similarly, the VP16 activation domain was fused to pCMX, which was
driven by the cytomegalovirus promoter, and named pCMX-VP16 (provided
by Dr. R. M. Evans). This pCMX-VP16 was then used to construct
full-length fusion of ARA54. The construction junction of each plasmid
was verified by sequencing.
Co-immunoprecipitation of AR and ARA54--
Lysates from
in vitro translated 35S-labeled wild type and
mutant ARs were incubated with or without 10 Cell Culture and Transfections--
Human prostate cancer DU145
cells and lung carcinoma H1299 cells were maintained in Dulbecco's
minimum essential medium containing penicillin (25 units/ml),
streptomycin (25 µg/ml), and 5% fetal calf serum. Transfections were
performed using the calcium phosphate precipitation method as described
previously (28). Briefly, 3.5 × 105 cells were plated
on 60-mm dishes for 24 h, and the medium was changed to
Dulbecco's minimum essential medium with 5% charcoal-stripped fetal
calf serum 1 h before transfection. A Cloning of the Androgen Receptor-associated Protein,
ARA54--
Estrogen, progesterone, and flutamide were able to activate
several AR mutants, including the LNCaP AR mutated in codon 877 (25).
The hypothesis that these mutant ARs may change the antiandrogen specificity and contribute to the progress of prostate cancer from an
androgen-dependent to an androgen-independent stage has been widely accepted. Therefore, we were interested in investigating whether cofactors are required for mtARs to exert this distinct function. A fusion protein (GAL4-ARt877s) containing the GAL4 DBD and
the C terminus of mtARt877s was used as a bait to screen the potential
positive clones from a human prostate cDNA library by the yeast
two-hybrid system. One of the positive cDNA clones, which can
interact with ARt877s, was further isolated and characterized.
Using the 5' RACE-PCR method, we were able to isolate a full-length
cDNA sequence (1701 base pairs) that encodes a novel protein with
an open reading frame of 474 amino acids (Fig.
1A). The in vitro
translated product expressed a polypeptide that matched the calculated
molecular mass of 53.8 kDa (data not shown), we thus named this
AR-associated protein ARA54. The middle portion of ARA54 (amino acids
220-265) contains a cysteine-rich region that may form a zinc finger
motif called the RING finger, defined as
CX2CX9-27CXHX2CX2CX6-17CX2C
(29) (Fig. 1B). Indeed, several human transcriptional
factors or proto-oncogene proteins, including BRCA1 (30), RING1 (31),
PML (32), and MEL-18 (33), have been shown to contain this domain. In
addition, ARA54 contains a second cysteine-rich motif that has a
B-box-like structure located 43 amino acids downstream from the RING
finger motif. However, ARA54 does not possess a predicted coiled coil
domain immediately C-terminal to the B-box domain (34). This coiled
coil domain is highly conserved among the members of the RING finger
B-box family; therefore, ARA54 may represent a new subgroup member of this family.
ARA54 mRNA Expression in Different Human Tissues--
Northern
blot analysis showed that ARA54 mRNA is expressed at the highest
level in testis but at a very low level in small intestine and blood
leukocyte (Fig. 2). Based on quantitation and normalization to Specific Interaction between ARA54 and ARs--
To test whether
ARA54 could interact with AR in an androgen-dependent
manner, we first applied yeast two-hybrid assay. The results showed
that DHT and testosterone, at concentrations of greater than 1 nM, promoted the specific interaction between ARA54 and
both wtAR and mtAR (data not shown). A mammalian two-hybrid system with
CAT reporter gene assay in prostate DU145 cells was further applied to
confirm this DHT dose-dependent interaction between wtAR
and ARA54 in vivo. As shown in Fig.
3A, transient transfection of
either ARA54 or wtAR alone showed negligible activity (lanes 2 and 5). However, the CAT activity was significantly induced only
when AR was co-expressed with ARA54 in the presence of 10 nM DHT (lane 3). Our previously identified AR
coactivator ARA70 (lane 6) and SV40 large T antigen
(lane 7) were used here as positive and negative controls,
respectively. Together, these data indicate that the specific
interaction between ARA54 and wtAR is an androgen-dependent process in both in vitro and in vivo assays.
To determine whether the interaction that occurred in yeast or
mammalian two-hybrid systems was due to a direct interaction between
ARA54 and ARs, co-immunoprecipitation assays were performed by an
in vitro transcription/translation system that expressed various deletion mutants of AR and full-length ARA54 fused to S·Tag
protein. The S·Tag-agarose beads used to precipitate the protein
complexes were then subjected to SDS-polyacrylamide gel electrophoresis. As shown in Fig. 3B, both the wtAR and
LNCaP mtAR could be co-immunoprecipitated by incubation with ARA54
(lanes 1 and 3), and the addition of 10 nM DHT
could further enhance this interaction (lane 1 versus lane
2). Moreover, the data showed that the C-terminal region of AR was
sufficient to interact with ARA54, whereas neither the N-terminal
domain nor DBD plus ARA54 Shows Preferential Coactivation with AR and PR--
Because
several identified cofactors could enhance the transcriptional activity
of most steroid hormone receptors (12-17), it is important to
investigate whether ARA54 could function as a general coactivator on
the transcriptional activity of other steroid receptors through their
cognate ligands and response elements. As shown in Fig.
4, among all the classic steroid
receptors we tested, ARA54 could further induce the transcriptional
activity of AR and PR up to 6- and 4-fold, respectively. Compared with another AR coactivator, ARA70 that can show higher specificity to AR,
ARA54 may represent a less specific coactivator for AR-mediated transactivation. Although ARA54 showed only marginal effects (less than
2-fold) on GR and ER in DU145 cells, we certainly cannot rule out the
possibility that ARA54 might be a more general coactivator to other
steroid receptors in other cell types under different conditions.
Co-expression of ARA54 with SRC-1 or ARA70 Additively Enhances AR
Transcriptional Activity--
It has been demonstrated that
co-expression of SRC-1 and CBP could stimulate ER and PR
transcriptional activity in a synergistic manner (35). In addition,
both ARA70 and SRC-1 could act as coactivators for AR transcriptional
activation (28). Therefore, we were interested to know if ARA54,
together with ARA70 or SRC-1, could synergistically enhance the
AR-mediated transcriptional activity. As shown in Fig.
5, ARA54, SRC-1, and ARA70 all induced AR
transcriptional activity to 3-5-fold in DU145 cells (lane
3-5). Moreover, when ARA54 was co-expressed with SRC-1 or ARA70,
an additive, but not synergistic, induction of AR-mediated
transactivation was observed (lane 4 versus lane 9 and
lane 5 versus lane 10). These results indicate that these
cofactors may contribute individually to proper or maximal AR-mediated
transcriptional activity.
The C-terminal Domain of ARA54 Serves as a Dominant Negative
Inhibitor--
It has been suggested that the interaction domain of
coactivators could interfere with the receptor-mediated target gene
expression by squelching the endogenous coactivators present in limited
cellular concentration (13, 35). Because the C-terminal region (amino acids 361-474) of ARA54 isolated from prostate cDNA library has been shown to be sufficient to interact with AR in yeast two-hybrid assays, we were interested to test whether it could squelch the effect
of endogenous ARA54 on AR-mediated transcription in prostate PC-3
cells. As shown in Fig. 6, the C-terminal
region of ARA54 significantly inhibited AR-mediated transactivation
(lane 2 versus lane 6) and the co-expression of exogenous
full-length ARA54 could reverse this squelching effect in a
dose-dependent manner (lanes 7-9). These
results clearly indicate that the C-terminal domain of ARA54 can serve
as a dominant negative inhibitor and that ARA54 is required for proper
or maximal AR transactivation in human prostate PC-3 cells.
The Effects of ARA54 on the Transcriptional Activities of wtAR and
mtARs in the Presence of DHT, E2, and HF--
Prostate cancer patients
treated with maximal androgen ablation therapy showed undetectable
androgens but much higher concentration of antiandrogens (as high as
1-5 µM) in their serum (36). Thus, it is of interest to
us to investigate whether ARA54 can modulate the agonist-antagonist
activity of these antiandrogens on wtAR and mtARs. As shown in Fig.
7, wtAR responded well to DHT at 0.1-10 nM, and ARA54 enhanced these transactivations by another
3-5-fold (lanes 6-8). However, wtAR responded only
marginally to 1-100 nM E2 and 0.1-10 µM HF
in the presence of ARA54 (Fig. 7, lanes 14-17 and
23-26). These results are in contrast to the previous identified coactivator, ARA70, that could enhance the wtAR
transcriptional activity in the presence of 1 to 10 nM E2
and 1 µM HF (27, 28, 37). We further extended these
findings to two different AR mutants: ARt877a, which was found in many
prostate tumors including LNCaP cells (38), and ARe708k, which was
found both in a yeast genetic screening (39) and in one Reifenstein
syndrome patient with partial androgen insensitivity (28). Previous
reports showed that LNCaP ARt877s could be stimulated by E2,
progesterone, and flutamide (38). In comparison, ARt877a responded to
E2 from 1 to 100 nM and to HF from 0.1 to 10 µM. Moreover, ARA54 can further promote this E2- or
HF-mediated agonist activity on ARt877a (lanes 10-17).
These results suggest that LNCaP AR might require ARA54 for proper or
maximal DHT-, E2-, or HF-mediated transcriptional activity.
Unexpectedly, although DHT can still enhance the transcriptional activity of another AR mutant, ARe708k, E2- and HF-mediated agonist activity on ARe708k, even in the presence of ARA54 (lanes
5-9), is extremely low. Because the codon 708 is located on the
helix 3 of the ligand-binding domain and helix 3 has been suggested to
play an essential role for the formation of ligand-binding cavity (28),
our findings may therefore demonstrate that the residue 708 in helix 3 might be the key component to distinguish the binding between DHT and
E2/HF for AR. Together, these results suggest that the AR structure and
coactivators, such as ARA54, are both important factors in determining
the specificity of sex hormones and antiandrogens.
ARA54 described here represents a new AR coactivator with a novel
sequence, because neither the nucleotide nor amino acid sequence was
found in the EMBL and GenBankTM data bases. Moreover, ARA54
contains a conserved RING finger motif and a B-box-like structure.
Proteins in the RING finger family are ubiquitously expressed in
species ranging from human to virus, participate in diverse cellular
processes, and may be involved in some aspect of transcriptional
regulation and protein-protein interaction (40). In addition, it has
been reported that mutant PML proteins without the RING finger motif
could become the potential dominant negative inhibitors of the wild
type PML (41). In agreement with these findings, we have demonstrated
that the C-terminal region of ARA54, without the RING finger motif, can
serve as a dominant negative inhibitor to attenuate the AR-mediated
transactivation. Although the significance of the RING finger domain in
ARA54 remains unclear, it is possible that ARA54 might use this domain
to interact with other key factors in the activated nuclear
receptor-mediated signaling complex and function as a bridge factor
between AR and general transcription machinery. Thus, it will be of
great interest to further characterize the functions of the RING finger
region of ARA54.
The evidence that the C-terminal domain of ARA54 can function as a
dominant negative mutant to inhibit the AR transcriptional activity
strongly suggests that ARA54 might play some important roles in the
androgen action. Moreover, the incomplete blocking of AR-mediated
transcriptional activation by the dominant mutant of ARA54 (Fig. 6)
implies that other cofactors like ARA70 and SRC-1 might still function
as independent coactivators to induce AR-mediated transcription in the
absence of endogenous ARA54. This hypothesis was further supported by
the fact that ARA54, ARA70, and SRC-1 could additively induce the AR
transactivation. Therefore, each of these cofactors may function via
its own individual pathway to promote the AR transcriptional initiation
complex for optimal transcription. It will be of particular interest to
determine whether AR can simultaneously recruit both ARA54 and SRC-1 or other components of the general transcription factors in the
preinitiation complex.
So far, the most popular and effective treatment for advanced prostate
cancer is androgen ablation therapy using a combination of surgical or
chemical castration and antiandrogens such as HF. Unfortunately, most
of the prostate tumors in patients treated with this therapy may
relapse within 18 months (13). The mechanism by which prostate cancer
cells become resistant to hormone therapy remains unknown. One of the
explanations of how prostate cancer progresses from an
androgen-dependent to an androgen-independent stage is that
mutations in AR may change the specificity and sensitivity of AR to
antiandrogens, such as HF (13, 16). It is particularly interesting to
investigate whether cofactors may also play important roles in the
progression of prostate cancer to an androgen-independent stage. Here
we report that in the presence of 10 nM E2 or 1 µM HF, ARA54 can further enhance the transcriptional
activity of LNCaP ARt877a but not wtAR or ARe708k. This suggests that
in addition to particular amino acids within the structure of AR, other
cofactors such as ARA54 might also contribute to the partial agonist
activity of HF and E2 to the mtARs. The differential E2- or HF-mediated AR transcriptional activity between wtAR and mtAR in the presence of
ARA54 may provide a nice model for the development of antiandrogens, which will be able to specifically block the mtARs that occur only in
prostate tumors.
In summary, our findings suggest that a novel AR-associated protein,
ARA54, can function as a relatively specific coactivator for AR in
human prostate cancer DU145 cells. Its ability to further enhance E2-
or HF-mediated transactivation of ARt887a, but not wtAR, may not only
help us to further understand the complexity of the molecular mechanism
of androgen action in prostate but also to develop more specific
antiandrogens in the battle against prostate cancer.
-estradiol or 1 µM
hydroxyflutamide. These results imply that both ARA54 and the positions
of the AR mutation (877 versus 708) might contribute to the
specificity of AR-mediated transactivation. Our findings further
demonstrated that the C-terminal domain of ARA54 can serve as a
dominant negative inhibitor and exogenous full-length ARA54 can reverse
this squelching effect on AR transcriptional activity. Co-expression of
ARA54 with other AR coactivators, such as ARA70 or SRC-1, showed
additive stimulation of AR-mediated transactivation, which indicates
that these cofactors may function individually as AR coactivators to
induce AR target gene expression. Through our findings, we have
identified and characterized a novel AR coactivator, ARA54, which may
play an important role in the AR signaling pathway in human prostate.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Dihydrotestosterone (DHT),
dexamethasone, progesterone, and E2 were obtained from Sigma, and
HF was from Schering. pSG5 wtAR was used in our previous report (12).
pCMV-mtARt877a (mutant AR derived from prostate cancer tumors, codon
877 mutation threonine to alanine) and pSG5mtARe708k (mutant
AR derived from a partial androgen insensitive syndrome patient,
codon 708 mutation glutamic acid to lysine) were constructed in our
laboratory (28).
-galactosidase activity. DNAs from positive
clones were recovered from yeast, amplified in Escherichia coli, and confirmed by sequencing.
-actin probe was used as a control for equivalent
mRNA loading.
8
M DHT in the modified RIPA buffer (50 mM
Tris-HCl, pH 7.4; 150 mM NaCl; 5 mM EDTA; 0.1%
Nonidet P-40; 1 mM phenylmethylsulfonyl fluoride;
aprotinin, leupeptin, pepstatin; 0.25% sodium deoxycholate; 0.25%
gelatin). The cell-free translated full-length ARA54 fused to S·Tag
protein was incubated with S·Tag protein-agarose beads (Novagen) at
4 °C. The conjugated beads were then washed four times with RIPA
buffer, boiled in SDS sample buffer, analyzed by 8% SDS-polyacrylamide
gel electrophoresis, and visualized by STORM 840 (Molecular Dynamics).
-galactosidase expression plasmid, pCMV-
-gal, was used as an internal control for transfection efficiency. The total amount of DNA was adjusted to 10.5 µg with pSG5
or pVP16 in all transfection assays. After 24 h, the medium was
changed again, and the cells were treated with various steroids. Cells
were harvested after 24 h for chloramphenicol acetyltransferase (CAT) assay as described previously (12). The CAT activity was visualized and quantitated by STORM 840 (Molecular Dynamics). At least
three independent experiments were carried out in each case.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Nucleotide and amino acid sequence of human
ARA54. A, the nucleotide sequence and the deduced amino
acid sequence are shown and numbered on the left. Residues
constituting the C3HC4 type of the putative RING finger and B-box-like
structure are underlined. Sequence data have been deposited
into GenBankTM (accession number AF060544). B,
alignment of proteins that contain the RING finger motif. Residues
conserved in all members are indicated in bold and represent
the putative zinc metal ion ligands. X represents any amino
acid, and the number of such residues is also indicated.
-actin mRNA using testis as 100%, the
expression levels of ARA54 mRNA in the following tissues are:
thymus, 17.8%; spleen, 16.6%; colon, 16%; prostate, 14.5%; uterus,
13.2%; small intestine, 5.1%; and blood leukocyte, 3.6%. The ARA54
mRNA also were strongly detected in two other prostate cell lines,
PC-3 and LNCaP (data not shown). These observations suggest that ARA54 expression may exhibit a certain degree of tissue or cell-type specificity. The major band that appeared at 3 kilobases was present in
spleen, thymus, prostate, and uterus. In addition, there was a second
band at 2 kilobases that only appeared in testis. Whether the
2-kilobase mRNA is due to the RNA alternative splice or simply the
degraded form of longer mRNA remains to be determined.
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Fig. 2.
Northern blot analysis of ARA54 mRNA
levels in different human tissues. The blot containing
approximately 2 µg of poly(A)+ RNA from human spleen
(lane 1), thymus (lane 2), prostate (lane
3), testis (lane 4), uterus (lane 5), small
intestine (lane 6), colon (lane 7), and
peripheral blood leukocyte (lane 8) was hybridized with an
ARA54-specific cDNA probe. A -actin probe was used as a control
for equivalent mRNA loading.
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Fig. 3.
Specific interaction of ARA54 with AR in
DHT-dependent manner. A, mammalian
two-hybrid assay. DU145 cells were co-transfected with 3 µg of GAL0
AR encoding the hormone-binding domain of wtAR fused to the GAL4 DBD
and 4.5 µg of VP16-ARA54 encoding full-length ARA54 fused to the
activation domain of VP16. Interaction was estimated by determining the
level of CAT activity from 3 µg of the reporter plasmid pG5CAT in the
presence of 10 8 M DHT. VP16-ARA70 and
VP16-SV40 large T antigens were used as a positive and negative control
(lanes 6 and 7), respectively. B, the
wtAR and different AR mutants used in the co-immunopreciptation assay
are shown schematically. The DBD and ligand-binding domain
(LBD) are indicated. C, in vitro
interactions between AR and ARA54. A series of 35S-labeled
mtARs incubated with ARA54 in the presence or absence of 10 nM DHT are shown in lanes 1-6. The S
protein-agarose beads were applied to precipitate the protein-antibody
complex. 10% of each input protein is shown on the left
panel. Molecular size markers are in kDa. IP,
immunoprecipitation.
2 domain of AR (lane 5 versus lanes 4 and 6) could interact. Together, these data suggest that the
interaction domain within AR could be located between amino acids 652 and 919.
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Fig. 4.
Effect of ARA54 and ARA70 on the
transcriptional activities of AR, GR, PR, and ER. DU145 cells were
transiently co-transfected with 3 µg of reporter plasmids (MMTV-CAT
for AR, GR, and PR; ERE-CAT for ER), 1 µg of each receptor
constructed in pSG5, and 4.5 µg of pSG5-ARA54-FL (or 3 µg of
pSG5-ARA70) in the presence of 10 8 M of each
cognate ligand. Each CAT activity is presented relative to the
transactivation observed in the absence of ARA54, and an error
bar represents the mean ± S.D. of four independent
experiments. Dex, dexamethasone; P,
progesterone.
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Fig. 5.
Simultaneous co-expression of ARA54 and SRC-1
or ARA70 enhances AR transcriptional activity additively. DU145
cells were co-transfected with 1 µg of pSG5-AR, 3 µg of MMTV-CAT,
and 3 µg or 6 µg of pSG5-ARA54, pSG5-SRC-1, pSG5-ARA70 alone or
together with 3 µg of pSG5-SRC-1 or pSG5-ARA70 in the absence or
presence of 10 8 M DHT (lanes
1-8). Each CAT activity is presented relative to the
transactivation observed in the absence of DHT, and an error
bar represents the mean ± S.D. of four independent
experiments. Cells in which ARA54, SRC-1, or ARA70 expression vectors
were not introduced were transfected with pSG5 parent vector.
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Fig. 6.
C-terminal domain of ARA54 functions as a
dominant negative inhibitor, and full-length ARA54 reverses the
squelching effect. Increasing amounts of C-terminal domain (amino
acids 361-474) of ARA54 expression vector (pSG5-ARA54C 39) and 3 µg
of MMTV-CAT reporter plasmid with 1.5 µg of AR expression vector were
co-transfected into H1299 cells in the absence or presence of
10 8 M DHT (lanes 1-6). A fixed
amount of pSG5-ARA54C 39 (4 µg) was co-transfected with increasing
amounts of pSG5-ARA54-FL expression vectors (2.5, 3.5, and 5.5 µg) as
described in the text (lanes 7-9). Each CAT activity is
presented relative to the transactivation observed in the absence of
DHT, and an error bar represents the mean ± S.D. of
four independent experiments.
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Fig. 7.
Effect of ARA54 on the transcriptional
activities of wild type and mutant ARs. A, 1.5 µg of
wtAR was co-transfected with 4.5 µg of ARA54 in the absence or
presence of DHT, E2, or HF at indicated concentrations. B
and C, the LNCaP mtARt877a (B) and mtARe708k
(C) were used to replace the wtAR to perform the same
experiment as in A. All experiments were performed in DU145
cells, and 3 µg of MMTV-CAT was used as a reporter plasmid. Each CAT
activity is presented relative to the transactivation observed in the
absence of ligand, and an error bar represents the mean ± S.D. of four independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Drs. Balk, O'Malley, and Evans for plasmids, Juu-Chin Lu for RACE-PCR cloning, and Karen Wolf for manuscript preparation.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants CA55639, CA68568, and CA75732.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF060544.
These authors contributed equally to this paper.
§ To whom correspondence should be addressed. E-mail: chang{at}pathology.rochester.edu.
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ABBREVIATIONS |
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The abbreviations used are:
AR, androgen
receptor;
wtAR, wild type AR;
mtAR, mutant AR;
ER, estrogen receptor;
PR, progesterone receptor;
GR, glucocorticoid receptor;
ARA, AR-associated protein;
DHT, 5-dihydrotestosterone;
E2, 17
-estradiol;
HF, hydroxyflutamide;
DBD, DNA-binding domain;
CAT, chloramphenicol acetyltransferase;
RACE, rapid amplification of
cDNA ends;
PCR, polymerase chain reaction.
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REFERENCES |
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