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INTRODUCTION |
The androgen receptor
(AR)1 is a
ligand-dependent transcription factor that belongs to a
family of steroid receptors along with the glucocorticoid (GR),
progesterone, and mineralocorticoid receptors. These steroid receptors
share similar domain structures and mechanism of action. Steroid
receptors including the AR have three functional domains: a
COOH-terminal ligand-binding domain (LBD), a central DNA-binding domain
(DBD), and an NH2-terminal domain (1). In the absence of
androgens, the AR is localized to the cytoplasm in an inactive complex
that includes heat shock proteins (HSP). Upon binding to its cognate
ligand, the AR undergoes a conformational change that results in a more
compact and stable form of the AR. The activated AR dissociates from
HSPs and translocates to the nucleus where it interacts with consensus
DNA sequences as a homodimer to influence transcription of downstream
genes (2). The estrogen receptor (ER) belongs to a different steroid receptor subfamily because it resides predominantly in the nucleus, even in its unliganded form, and does not require translocation across
the nuclear membrane following activation (3). There are two major
transactivation regions in the AR. 1) The activation function-1 (AF-1)
domain is found at the NH2 terminus. 2) AF-2 is located in
the LBD. AF-2 is a weak transactivator that is dependent on the
presence of androgens for its activation. AF-1, on the other hand, is
capable of ligand-independent transactivation, and fragments of the AR
that contain AF-1 show high levels of transcriptional activity when
ectopically expressed in cell lines that are devoid of endogenous AR
(4, 5).
Upon DNA binding, AR recruits components of the basal transcriptional
machinery and influences either the up-regulation or down-regulation of
gene expression. The exact mechanism of AR-specific gene expression is
not fully understood. Each steroid receptor regulates unique sets of
genes. However, in vitro assays have shown that these
receptors recognize similar DNA sequences known as steroid response
elements. These elements are comprised of a palindrome that contains
two half-sites based on the 5'-TGTTCT-3' motif that are separated by a
three nucleotide spacer (6). Detailed analysis has demonstrated that
both GR and AR bind with highest affinity to a steroid response element
that has an imperfect palindrome, 5'-GGTACAnnnTGTTCT-3' (7). The
quandary is that although activated steroid receptors bind to highly
homologous response elements on DNA, they still demonstrate an ability
to regulate the expression of unique gene sets.
There are several mechanisms by which receptors can specifically
regulate gene expression. One mechanism suggests that co-regulatory proteins interact with steroid receptors to direct their activity. The
search for AR-specific co-regulatory molecules has led to the
identification of several AR-interacting proteins. Early studies identified ARA70/ELE1 as a ligand-dependent
co-activator of AR (8). Subsequently, CREB-binding protein/p300, which
has histone acetyltransferase activity, was also shown to interact with
AR and enhance receptor activity in prostate cells (9). More recent AR-interacting proteins that have been identified include 
-catenin (10), caveolin (11), BAG-1L (12), SMAD3 (13), cyclin D1 (14),
and several others. These proteins have been shown to either positively
(caveolin and BAG-1L) or negatively (SMAD3 and cyclin D1) affect AR transactivation.
Identification of proteins that specifically interact with the AR has
been a challenge because the AR and other steroid receptors share a
high degree of sequence homology at their DBD and LBD. The DBD of human
AR shares as much as 80% homology with that of progesterone receptor
and over 70% with GR (15). The LBD of the steroid receptors are highly
homologous as well with up to 55% similarity at the amino acid level.
Therefore, many co-regulatory proteins that interact with the AR at the
LBD and DBD are promiscuous in their ability to interact with and
influence activity of other steroid receptors. A report by Alen
et al. (16) has demonstrated that ARA70 is not specific to
the AR and that this protein interacts with the ER and GR as well.
Likewise, the steroid receptor co-activator-1/NCoA1, which is
the founding member of the p160 family of transcriptional co-activators, interacts indiscriminately with the LBD of steroid receptors to enhance activity (17). Other members of the p160 family
such as TIF-2/GRIP-1 enhance AR, GR and ER activity alike (18).
Nevertheless, steroid receptor family members show the greatest degree
of sequence variability at the NH2-terminal domain (<15%). Little is known regarding the role of the
NH2-terminal domain in AR transactivation. Therefore, we
used an NH2-terminal fragment of the AR, which is devoid of
transcriptional activity (AR1-232), as bait in a yeast
two-hybrid assay, and RanBPM was identified as an AR-interacting
protein. Although RanBPM was initially described as a 55-kDa protein
(BPM55), a subsequent report has shown it to be a 90-kDa protein
(BPM90) (19, 20). Here we demonstrate that the larger form of RanBPM,
BPM90, is able to bind to multiple domains of the AR and that this
interaction occurs in vivo. Furthermore, overexpression of
RanBPM in prostate cancer cell lines shows that RanBPM can enhance AR
transactivation. This property of RanBPM does not appear to be
exclusive to the AR because BPM90 also enhances GR activity, although
neither ER-
nor ER- activity is affected. These experiments
clearly demonstrate that RanBPM is capable of interacting with and
modifying the activity of selective steroid receptors.
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EXPERIMENTAL PROCEDURES |
Yeast Two-hybrid Screening--
A 696-bp fragment coding
for the first 232 amino acids of the human AR (AR1-232)
was cloned into the BamHI site of the pGBT9 vector for
expression as a fusion protein with the GAL4 DBD
(Clontech). A cDNA library made from normal
human prostate tissue that was fused to the transactivation domain of
GAL4 in the pACT2 expression vector was used for screening
(Clontech). Expression plasmids were transformed
into the Y190 yeast strain, and transformants were selected on
SD minimal medium lacking tryptophan, leucine, and histidine.
Clones that grew on minimal medium agar plates were subjected to
-galactosidase assays by colony filter-lift according to the
manufacturer's instructions. Clones that tested positive for
-galactosidase were sequenced using Big Dye Terminator cycle
sequencing reactions (Applied Biosystems) and were then compared with
known sequences available in GenBankTM.
Plasmid Construction--
The full-length RanBPM expression
vector, pcDEB
-BPM90, and empty vector, pcDEB, were provided by
Dr. H. Nishitani (Kyushu University) (20). The longest RanBPM library
clone that was isolated in the yeast two-hybrid assay contained a
2152-bp fragment that coded for amino acids 148-729 of BPM90
and the 3'-untranslated region. This fragment of BPM90, herein referred
to as BPML, was cloned into the pRC/cytomegalovirus (CMV)
mammalian expression vector (Invitrogen) in which transcription is
driven by the CMV promoter. BPML was cloned in-frame with
an upstream ATG for translation initiation and a Kozak sequence
to enhance translation efficiency.
The NH2-terminal region of the human AR spanning the
NH2-terminal domain and DBD (hAR1-646) was
generated by PCR and cloned into the multiple cloning site of pRC/CMV
for expression in mammalian cells. The full-length rat AR cDNA was
expressed from the pRC/CMV mammalian expression plasmid,
pCMV/AR6 (21). The rat glucocorticoid receptor was
expressed from the pGR mammalian expression vector as described
elsewhere (22). The human ER- expression vector (pSVMT:wER) has also
been described previously (23). The pcDNA4/HisMax-hER1 vector (a
gift from Dr. L. Murphy, University of Manitoba) was used for the
expression of human ER- in mammalian cells.
Northern Blot Analysis--
Multiple tissue Northern blots
(Clontech) were probed with
[32P]dCTP-labeled RanBPM cDNA. Poly(A) RNA was
prepared from the LNCaP, PC3, MCF7, and HeLa cell lines using an
Oligotex mRNA kit (Qiagen). For the Northern blot, 5 µg of
poly(A) RNA were separated by electrophoresis on a 1% agarose gel with
30% formaldehyde and transferred to a Biodyne B nylon membrane (Pall
Corporation) by capillary action in 10 mM NaOH. The
membrane was hybridized with a [32P]dCTP-labeled
1100-bp cDNA fragment coding for RanBPM.
Glyceraldehyde-3-phosphate dehydrogenase was used to normalize the
loading of poly(A) RNA.
GST and His-tag Pull-down Assay--
Various
NH2-terminal and DBD fragments of the human AR
(AR1-232, AR1-559, and
AR559-646) and a COOH-terminal fragment of rat AR
(ARDBD/LBD) were cloned into the pGEX vector (Amersham
Biosciences) for expression as GST fusion proteins. GST fusion proteins
were expressed in the BL21 Escherichia coli strain and
purified as described previously (5). Radiolabeled RanBPM protein was
prepared from the pRC/CMV-BPML vector using the Quick
Coupled T7 TNT in vitro
transcription/translation kit (Promega Corporation) in the
presence of [35S]Met. Equimolar amounts of GST-AR fusion
protein coupled to glutathione-agarose beads were incubated with
radiolabeled RanBPM at 4 °C for 2 h in binding buffer (20 mM HEPES, pH 7.6, 150 mM KCl, 5 mM
MgCl2, 1 mM EDTA, 0.05% Nonidet P-40). Beads
were washed four times with binding buffer, and bound proteins
were eluted into protein sample buffer (2% SDS, 5%
-mercaptoethanol) for analysis by SDS-PAGE followed by autoradiography.
A fragment spanning the NH2-terminal domain and DNA-binding
domain of the AR (AR1-646) was cloned into the pTrcHisC vector (Invitrogen) for expression with an NH2-terminal His
tag, which consists of six histidine residues in tandem. His-tagged proteins were expressed in bacteria and purified using the
nickel-nitrilotriacetic acid-agarose column according to the
manufacturer's protocol (Qiagen). [35S]Met-RanBPM
fragments were incubated with His-AR1-646 at 4 °C for
4 h in binding buffer (see above). His-AR1-646 was
immunoprecipitated using an anti-His antibody (Qiagen) as described below.
Cell Culture and Transfection--
PC3, HeLa, and MCF7 cells
were maintained in Dulbecco's modified Eagle's medium (Sigma)
supplemented with 5% fetal bovine serum (FBS) (Invitrogen) at 37 °C
in 5% CO2. The LNCaP prostate carcinoma cell line was
cultured in RPMI 1640 medium containing 5% FBS. For transient
transfection, 3 × 105 cells were seeded in six-well
plates and were transfected the following day using Lipofectin reagent
(Invitrogen) as described previously (24). Transfection occurred for
16 h at 37 °C. Following transfection, cells were re-fed with
fresh medium containing 5% dextran-coated charcoal-stripped
FBS ± 1 nM R1881, 10 nM dexamethasone (Dex), 10 nM E2 or vehicle alone and incubated
at 37 °C for an additional 24 h. After induction with hormone,
cells were harvested and lysed in passive lysis buffer (Promega
Corporation) for luciferase assay and for Western blot analysis.
Immunoprecipitation and Western Blotting--
LNCaP cells were
grown to 80% confluency in RPMI 1640 medium + 5% FBS. Cells were then
cultured in RPMI 1640 medium containing 5% dextran-coated
charcoal-stripped FBS for 16 h at 37 °C. The following day,
cells were induced in the presence or absence of 10 nM
R1881 for 4 h at 37 °C before scraping and lysis in radioimmune precipitation buffer (150 mM NaCl, 50 mM
Tris-Cl, pH 7.5, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1%
SDS). Whole cell extracts (1 mg of protein) were incubated with a
polyclonal rabbit anti-AR antibody or with normal rabbit IgG as a
negative control (Santa Cruz Biotechnology, Inc.). Immunocomplexes were
pulled down using protein A/G-agarose beads (Santa Cruz Biotechnology,
Inc.) and washed four times with radioimmune precipitation buffer.
After the final wash, proteins were solubilized in SDS sample buffer and analyzed by Western blot.
Western blots were carried out as described previously (21). Protein
samples were resolved on a polyacrylamide gel and transferred to a
polyvinylidene difluoride membrane (Millipore). Membranes were blocked
in TBS (20 mM Tris-Cl, pH 7.6, 137 mM NaCl)
with 5% skim milk. Blots were incubated with appropriate primary
antibody, diluted to 2 µg/ml in TBS + 5% milk for 4 h at room
temperature, washed three times in TBS + 0.5% Tween 20, and then
incubated for 45 min in horseradish peroxidase-conjugated secondary
antibody (1:10,000) (Santa Cruz Biotechnology, Inc.). Blots were
developed using the ECL chemiluminescence kit (Amersham Biosciences).
Transcription Assays--
AR and GR constructs were
co-transfected with the pARR3-tk-Luc reporter construct in which
the promoter has three androgen response regions (ARRs) in tandem (5).
In addition, the prostate-specific antigen (PSA) and probasin (PB)
luciferase reporter constructs were used to determine transcriptional
activity of the AR (4, 24). ER expression vectors were co-transfected
with the pERE-Luc reporter plasmid as described previously. The
pERE-Luc plasmid contains a single vitellogenin estrogen response
element upstream of the thymidine kinase (TK)
promoter.2 Transfected cells
were incubated in the presence or absence of hormone at 37 °C for
24 h prior to analysis. Transfection efficiency was normalized
using the Renilla luciferase expression vector, pRL-TK (Promega Corp.).
Firefly and Renilla luciferase activities were assayed with the Dual
Luciferase assay kit (Promega Corp.). 20 µl of cell lysate were
analyzed for luciferase activity using MicroLumiatPlus
luminometer (EG&G Berthold).
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RESULTS |
RanBPM Interacts with the NH2 Terminus of AR in
Yeast--
A fragment coding for the first 232 amino acids of the
AR (AR1-232) was used as bait in a yeast two-hybrid
assay to screen a human prostate library for interacting proteins. Over 3 × 105 transformants were screened on selective
media, and positive clones were identified by -galactosidase assay.
Sequence analysis of three independent positive clones revealed a gene
that was highly homologous to a known human protein, RanBPM
(GenBankTM accession number AB055311) (19). Both 55- and
90-kDa forms of RanBPM have been reported in the literature and are
referred to as BPM55 and BPM90, respectively (19, 20). Sequences
isolated from the three library clones isolated by the yeast two-hybrid assay had sequences upstream of the published BPM55 translation start
codon (Fig. 1). The longest sequence of
the three library clones (ARBP1) was 2346 bp in length
and coded for a protein from amino acid 148 of BPM90 to the termination
codon. This fragment is referred to as BPML.
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Fig. 1.
Amino acid sequence of BPM90
(GenBankTM accession number
BAB62525). AR1-232 was used as bait to screen a
normal prostate cDNA library for interacting proteins. Three
independent cDNA clones coded for RanBPM. The underlined
portion shows the protein encoded by the longest cDNA sequence that
was isolated (herein referred to as BPML). The
asterisk denotes the translation start site of BPM55
(GenBankTM accession number BAA23216). Shaded
sequence is the putative SPRY domain.
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RanBPM Interacts with AR in Vitro and in Vivo--
To confirm the
interaction between RanBPM and AR that was identified by yeast
two-hybrid screening, various AR constructs were expressed as GST
fusion proteins and coupled to glutathione-agarose beads for use in GST
pull-down assays. The 2346-bp fragment from ARBP1
(BPML) was cloned into pRC/CMV to allow for in
vitro transcription/translation from the T7 promoter.
[35S]Methionine-labeled BPML protein was
allowed to interact with the GST-AR fusion proteins and analyzed by
SDS-PAGE followed by autoradiography. As seen in Fig.
2A, RanBPM interacts with the 232-amino acid fragment that was originally used as bait in the yeast
two-hybrid screen (AR1-232) (Fig. 2A,
lanes 3 and 4). Equimolar amounts of AR-GST
fusion proteins were used to determine relative binding between RanBPM
and various domains of AR, and the results show that RanBPM interacts
most strongly with the DBD (AR559-646) (Fig.
2A, lane 2). In addition, RanBPM interacts with
the AR fragment that spans the DBD and LBD (ARDBD/LBD)
(Fig. 2, lane 5). However, presence of the LBD does not
enhance interaction, which suggests that the LBD does not have
additional domains for RanBPM binding.
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Fig. 2.
RanBPM interacts with the
NH2-terminal domain and DBD of the AR. A,
[35S]Methionine-labeled BPML was allowed to
interact with GST, GST-AR559-646,
GST-AR1-232, GST-AR 1-559, and
GST-ARDBD/LDB (lanes 1-5, respectively) coupled
to glutathione-agarose beads at 4 °C overnight. Proteins were eluted
from washed beads with protein sample buffer and resolved on an
SDS-PAGE gel before analysis by autoradiography. B, RanBPM
co-immunoprecipitates with AR. LNCaP cells were induced with
(lanes 2, 5, and 6) or without
(lanes 1, 3, and 4) 10 nM
R1881 for 4 h prior to lysis in radioimmune precipitation buffer.
Whole cell extracts were incubated with normal rabbit IgG (lanes
3 and 5) or an antibody to the COOH terminus of AR
(lanes 4 and 6). Input protein lysates from LNCaP
cells grown,  or + R1881, are shown in lanes 1 and
2, respectively. Proteins were pulled down using protein A/G
coupled to agarose beads and resolved on an 8% SDS-PAGE gel prior to
Western blot with an antibody that recognizes BPM90 (top
panel) or the NH2 terminus of AR (bottom
panel). C, the pRC/CMV-BPML vector was used
for in vitro transcription/translation in the presence of
[35S]methionine (top panel). Radiolabeled
truncated fragments of BPML were generated by restriction
digest of the expression vector with EcoRI
(BPM148-408; middle panel) or NdeI
(BPM148-251; bottom panel) followed by in
vitro labeling. The fragments of RanBPM were incubated with
His-AR1-646 for 4 h prior immunoprecipitation with an
anti-His antibody (lane 2) or normal mouse IgG as a negative
control (lane 3). 10% input of the labeled protein is seen
in lane 1.
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To assess the relevance of this interaction in vivo, we
performed a co-immunoprecipitation assay using LNCaP cell lysates. The
LNCaP prostate carcinoma cell line expresses high levels of functional
AR and displays androgen-dependent gene expression activity. Cells were treated for 4 h with 10 nM R1881
or with vehicle alone prior to preparation of protein lysates. Whole
cell lysates were immunoprecipitated with normal rabbit IgG as a
negative control (Fig. 2B, lanes 3 and
5) or with an antibody that recognizes the COOH terminus of
the AR (lanes 4 and 6) (Santa Cruz Biotechnology, Inc.). Protein complexes were pulled down with protein A/G coupled to
agarose beads. Proteins of the AR complex were resolved by SDS-PAGE
prior to Western blotting with an anti-BPM90 antibody (provided by Dr.
H. Nishitani). A 90-kDa protein that is seen in the input lanes (Fig.
2B, lanes 1 and 2) is BPM90. The
results in Fig. 2B show that BPM90 interacts with AR
specifically in the presence of hormone (lanes 3-6). We
confirmed that AR was pulled-down in this assay by blotting the same
membrane with an antibody that recognizes the NH2 terminus
of the AR (Affinity BioReagents) (Fig. 2B).
The NH2 Terminus of RanBPM Is Essential For interaction
with AR--
RanBPM encodes a putative SPRY domain at the
NH2 terminus (Fig. 1) (19). This domain has been implicated
in protein-protein interactions. To determine the importance of the
SPRY domain, His-tag pull-down assays were carried out to map the
interacting domains between RanBPM and the AR. Radiolabeled
COOH-terminal truncations of RanBPM were generated by restriction
digest of the pRC/CMV-BPML vector with EcoRI or
NdeI followed by in vitro transcription/translation in the presence of
[35S]methionine. The truncated RanBPM proteins
(BPM148-408 and BPM148-251) were incubated
with purified recombinant His-tagged AR1-646, which
includes the NH2-terminal domain and DBD. An antibody that
recognizes the His-tag (Qiagen) was used to immunoprecipitate AR and
its interacting proteins. After sufficient washing, samples were eluted
into protein sample buffer, resolved by SDS-PAGE, and analyzed by
autoradiography. Fig. 2C, Input lanes, show that
radiolabeled proteins have molecular masses of 64, 28, and 11 kDa (BPML, BPM147-408, and
BPM147-251, respectively). Both BPML and the
truncated peptide generated by EcoRI digest,
BPM147-408, interacted with the AR (Fig. 2C, top and middle panels). However,
BPM147-251 in which the SPRY domain is disrupted was no
longer able to interact with AR (Fig. 2C, bottom
panel). This finding agrees with yeast two-hybrid results in which
-galactosidase activity and, therefore, interaction with
AR1-232 are lost when an NH2-terminal
truncation of RanBPM, which does not have a complete SPRY domain, is
used as bait (data not shown).
RanBPM Is Ubiquitously Expressed--
To determine expression
levels of RanBPM, a [32P]cDNA probe was generated
from the ARBP1 yeast two-hybrid library clone and hybridized to a
multiple tissue Northern blot (Clontech). This Northern blot allows for analysis of RanBPM expression in several different tissue types. Fig.
3A shows that RanBPM is
expressed as a 2.9-kb transcript in multiple different tissue types.
High levels of RanBPM message are seen in the prostate and ovaries, and
highest levels of expression are observed in the testes (lanes 3, 5, and 4, respectively).
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Fig. 3.
RanBPM is ubiquitously expressed.
A, A multiple tissue Northern blot
(Clontech) was analyzed with a
[32P]cDNA probe coding for RanBPM to determine the
size and expression profile of the RanBPM transcript. Tissue samples on
the membrane included spleen (lane 1), thymus (lane
2), prostate (lane 3), testes (lane 4),
ovaries (lane 5), small intestine (lane 6), colon
(lane 7), and peripheral blood lymphocytes (lane
8). B, poly(A) RNA was isolated from four
carcinoma cell lines for Northern blot analysis. 5 µg of mRNA
from each cell line was run on a 1% agarose gel with 30% formaldehyde
and transferred to a nylon membrane by capillary action. Samples
include PC3 and LNCaP prostate cancer cell lines (lanes 1 and 2, respectively), the MCF7 breast carcinoma cell line
(lane 3), and a cervical cancer cell line, HeLa (lane
4). The membrane was hybridized with a radiolabeled RanBPM
cDNA probe (top panel). Loading efficiency was
normalized with glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) (bottom panel). C,
RanBPM protein expression was assessed for the four cancer
cell lines described above. Total protein (50 µg) was resolved on a
10% SDS-PAGE gel and transferred to a polyvinylidene difluoride
membrane. Samples include PC3, LNCaP, MCF7, and HeLa cell lines
(lanes 1-4, respectively). An antibody specific for BPM90
(20) was used for Western blot analysis (top panel). Protein
loading efficiency was normalized by -actin (bottom
panel).
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RanBPM expression levels were determined for various cancer cell lines
by both Northern and Western blots (Fig. 3, B and
C). Cell lines that were used included the ovarian carcinoma
HeLa cell line from which BPM90 was first identified, two prostate cancer cell lines, PC3 and LNCaP, and the MCF7 breast cancer cell line.
Both Northern and Western blots show that RanBPM is highly expressed in
all of the cell lines. In Fig. 3C, the 90-kDa protein seen
in lanes 1-4 is BPM90. Although BPM90 is expressed in all of the cell lines tested, the highest levels were observed in HeLa cells (lane 4). PC3 and MCF7 cells expressed lower
levels of BPM90 as compared with the LNCaP cell line (compare
lanes 1 and 3 with lane 2). The
membrane was probed with an antibody for -actin (Sigma) to
demonstrate that loading efficiency was consistent in all lanes (Fig.
3C).
RanBPM Enhances the Activity of the Androgen Receptors--
Our
results have demonstrated that RanBPM interacts with the androgen
receptor at the NH2 terminus and at the DNA-binding domain
(Fig. 2A). To determine whether this interaction has a biological impact on AR activity, transcriptional assays were carried
out using the PC3 cell line. PC3 cells represent a relatively undifferentiated stage of prostate cancer. These cells express very
little to no androgen receptor and do not demonstrate
androgen-regulated growth. Cells were transiently transfected with an
AR expression vector (pCMV/AR6), the pARR3-tk-Luc reporter
plasmid, and increasing amounts of RanBPM (pcDEB-BPM90) and then
were induced in the presence or absence of 1 nM R1881 for
24 h prior to harvesting for luciferase assays (Fig.
4A). In the absence of
hormone, negligible AR-mediated transcriptional activity was observed
from the pARR3-tk-Luc reporter plasmid. An addition of hormone resulted
in an ~9-fold induction of AR activity. In the presence of hormone,
the overexpression of BPM90 resulted in an AR activity that was three
times greater than in the absence of RanBPM. AR activity in the absence
of hormone was unchanged by the addition of RanBPM, even with a 20-fold
excess of BPM90 (Fig. 4B). Western blot analysis of cell
lysates was carried out to ensure equivalent levels of AR expression in
all samples (data not shown). Additional transactivation assays were carried out using an NH2-terminal truncated form of RanBPM
(BPML) in which expression is under the control of a CMV
promoter (Fig. 4A). The presence of BPML
increases basal AR activity by 4-fold when androgens are present.
BPML did not affect the activity of AR in the absence of
ligand when the AR was transcriptionally inactive.
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Fig. 4.
RanBPM enhances AR activity. A,
PC3 cells were transfected with full-length AR (250 ng/well),
pARR3- tk-Luc (167 ng/well), and increasing amounts of pcDEB-BPM90
or pRC/CMV-BPML (0, 250, 500, or 2500 ng/well). DNA was
kept constant at 3 µg/well by the addition of empty vector.
Transfection was performed using Lipofectin reagent (Invitrogen) for
16 h at 37 °C. Cells were induced in the presence ( ) or
absence ( ) of 1 nM R1881 for 24 h before harvesting
for luciferase assay. B, RanBPM does not change AR activity
in the absence of ligand. PC3 cells were transfected with AR (125 ng/well) and pARR3-tk-Luc (167 ng/well) and BPM90 at increasing ratios
with AR (0:1, 2:1, 20:1) as described above. Transfected cells were
maintained in Dulbecco's modified Eagle's medium + 5% stripped FBS
without additional androgens () or with 1 nM R1881 ()
for 24 h prior to analysis. DNA was kept constant at 3 µg/well
by the addition of empty vector. C, RanBPM can enhance the
activity of a constitutively active fragment of the AR.
AR1-646 encodes the full NH2-terminal domain
and DBD of the AR and exhibits ligand-independent transactivation. PC3
cells were transfected with pRC/CMV-AR1-646 (250 ng/well),
pARR3-tk-Luc, and BPM90 (1, 250, or 2500 ng/well) as described
above. Cells were induced for 24 h at 37 °C with 1 nM R1881 () or with vehicle alone (). For all
experiments, Western blot analysis was carried out on lysates to ensure
that AR levels were constant (data not shown). Transfection efficiency
was normalized with a Renilla luciferase expression vector
(pRL-TK). Values are the average of triplicates. Each graph is
representative of three independent experiments.
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Because RanBPM was identified by its interaction with the
NH2 terminus of the AR, similar transactivation experiments
were carried out with a form of the AR that is deleted for the LBD, pRC/CMV-AR1-646. This region of the AR has the AF-1 site and is capable of ligand-independent transcriptional activity. PC3
cells were transiently transfected with the truncated AR construct and
treated with or without 1 nM R1881. AR1-646 is
capable of high levels of transcriptional activity both in the presence and absence of ligand (Fig. 4C). Increasing amounts of BPM90
resulted in increased AR1-646 activity up to 2.5-times
greater than basal levels. This increase in AR activity was independent
of added hormone.
The ARR3 promoter of the reporter plasmid is a synthetic highly active
promoter that has three ARRs in tandem. The PB and PSA promoters,
however, may be more physiologically relevant. In vivo, the
expression of the rat probasin gene is restricted to the prostate and
is regulated by the AR (21, 25). PSA gene expression is also regulated
by AR activity and is used as a molecular marker for prostate cancer
progression (26). Luciferase reporter constructs under the control of
the PB and PSA promoters were also used to determine the effect of
BPM90 overexpression on AR activity (Fig.
5, A and B). PC3
cells transfected with pCMV/AR6 and PB-Luc demonstrated a
slight increase in ligand-dependent AR activity in the
presence of BPM90 (Fig. 5A). On the other hand, the
overexpression of BPM90 resulted in a 2-fold increase in
ligand-dependent AR transactivation from the PSA-Luc
reporter construct (Fig. 5B). AR expression levels remained
constant under the different treatments as determined by Western blot
(data not shown).
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Fig. 5.
BPM90 enhances AR activity on multiple
AR-regulated promoters. PC3 cells were transfected as described
previously with full-length AR, the pPB-Luc (A) or pPSA-Luc
(B) reporter plasmids (167 ng/well) with the addition (+) or
absence () of pcDEB-BPM90 (2500 ng/well). Relative luciferase
units were measured following a 24-h induction with 1 nM
R1881 () or with vehicle alone (). Transfection efficiency was
normalized with pRL-TK. Values are the average of triplicates. Each
graph is representative of three independent experiments.
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RanBPM Specifically Regulates AR- and GR-mediated
Transactivation--
To determine whether RanBPM could enhance
transactivation of AR alone or of other steroid receptors as well,
transcription assays were carried out with GR, ER-, and ER-. PC3
cells were transfected with expression plasmids for GR, ER-, or
ER- (250 ng/well) and with increasing amounts of BPM90 prior
to induction with 10 nM Dex or 10 nM
E2, respectively (Fig.
6A). In PC3 cells, GR shows an
11-fold induction in the presence of 10 nM Dex (Fig. 6A, lane 1). This activity increases with the
overexpression of BPM90 as seen in Fig. 6A, lane
2. High levels of BPM90 (2.5 µg/well) gave a 31-fold induction
of GR activity in the presence of Dex (Fig. 6A, lane
2). Similar to the AR, GR activity was unchanged by high levels of
BPM90 in the absence of ligand (data not shown).
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Fig. 6.
RanBPM enhances AR- and GR- but not
ER-mediated transactivation. A, PC3 cells were transfected
with GR (lanes 1 and 2), ER- (lanes
3 and 4), or ER- (lanes 5 and
6) (250 ng/well), appropriate reporter plasmids
(pARR3-tk-Luc for GR and pERE-Luc for ER- and ER-) (167 ng/well),
and increasing amounts of pcDEB-BPM90 (0, 250, or 2500 ng/well).
Cells were induced in the presence or absence of 10 nM Dex
or 10 nM E2 for cells transfected with GR or
ER, respectively, for 24 h prior to analysis for luciferase
activity. Empty pcDEB was used to keep DNA levels constant at 3 µg/well. Transfection efficiency was normalized with pRL-TK.
B, LNCaP cells were transfected with
pCMV/AR6 (lanes 1 and 2) or pGR
(lanes 3 and 4) (250 ng/well) and pARR3-tk-Luc as
described previously. Receptor activity was determined in the absence
() or presence (+) of pcDEB-BPM90 (2500 ng/well). Transfected
cells were induced in the presence of 1 nM R881 for AR, 10 nM Dex for GR, or vehicle alone for 24 h. Empty vector
was used to ensure that DNA was kept constant at 3 µg/well.
C, MCF7 cells were transfected with the ER- or
ER- expression vector and pERE-Luc as described above and in the
absence () or presence (+) of pcDEB-BPM90 (2500 ng/well). Cells
were treated with 10 nM E2 or vehicle alone for
24 h prior to analysis for luciferase activity. DNA content was
maintained at 3 µg/well with empty pcDEB plasmid. For all
experiments, protein content was used to normalize luciferase results
(relative luciferase units/µg protein). Results are expressed
at fold induction in the presence of ligand and are an average of three
replicates. Results are representative of three independent
experiments.
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In addition, estrogen receptor was ectopically expressed in PC3 cells
by transient transfection of either the pSMVT:wtER or pcDNA4-hER1 expression vector (250 ng/well) (Fig. 6A,
lanes 3-6). ER- activity in PC3 cells showed a greater
than 1.7-fold induction in the presence of ligand (10 nM
E2) (Fig. 6A, lane 3). This activity was relatively unchanged by the expression of RanBPM. Even when BPM90
was present at a 10:1 ratio with the receptor (2500 ng of BPM90/well),
ER--mediated transactivation remained the same (lane 4).
Similarly, ER- showed a 2-fold induction in the presence of 10 nM E2 regardless of the presence of BPM90 (Fig.
6A, lanes 5 and 6).
To assess the affect of RanBPM in alternate cell environments, prostate
cancer LNCaP and breast carcinoma MCF7 cell lines were used (Fig. 6,
B and C). Cells were transiently transfected with
the expression vector for BPM90 or with empty vector pcDEB and
steroid receptor expression vectors (250 ng/well) to determine whether
RanBPM overexpression could modify steroid receptor activity. In LNCaP
cells, AR has very high levels of activity when induced with 1 nM R1881 for 24 h (~64-fold induction). AR activity
was 2.8-times greater when high levels of BPM90 (2500 ng/well) were expressed in LNCaP cells as determined by luciferase assay (Fig. 6B). A similar enhancement of transcriptional activity,
2-fold increase in the presence of 10 nM Dex, was seen in
LNCaPs that were co-transfected with GR and BPM90 (Fig. 6B).
On the other hand, the difference in either ER- or ER- activity
in MCF7 cells in the presence or absence of BPM90 was not statistically
significant (Fig. 6C). These results demonstrate that RanBPM
is able to modify AR and GR activity in at least two different prostate
cancer cell lines but did not influence ER activity in either prostate
or breast cancer cell lines.
| |
DISCUSSION |
The mechanism that dictates AR-specific transactivation is still
unclear. Upon ligand binding, the AR translocates to the nucleus, binds
to DNA, and recruits members of the basal transcription machinery.
Unique protein-DNA interactions in the promoter/enhancer region of
AR-regulated genes are one mechanism by which AR-specific transcriptional regulation may be attained. Recent studies by Reid
et al. (27) show that the AR interacts with two different classes of androgen receptor response elements in the rat probasin promoter. The expression of the probasin gene in rats is highly restricted to the prostate and is regulated by AR. In addition to known
interactions between the AR and conventional class I androgen receptor
response elements, these studies have demonstrated novel interactions
that occur with a class II androgen receptor response elements, which
may add specificity to AR transactivation responses. Other studies have
demonstrated that unique receptor-DNA interactions can occur for
different steroid receptors as well (28, 29).
Co-regulatory molecules that interact with AR and the transcription
apparatus to enhance or repress gene expression may also impart
a degree of receptor-specific activity. Although several AR-interacting
co-regulatory proteins have been identified, the majority of these
proteins interacts with AR at the LBD and DBD, which are regions of
high homology between steroid receptor family members. AR and
progesterone receptor share up to 80% homology at the DBD, whereas AR
and GR share ~55% homology at the LBD (15). As a result, several
co-regulatory proteins such as steroid receptor co-activator-1, ARA70,
CREB-binding protein/p300), and the SWI/SNF chromatin-remodeling
proteins are not unique to the AR and have been shown to interact with
other steroid receptors (reviewed in Ref. 30).
In this study, RanBPM was identified as an AR-binding protein. An
NH2-terminal fragment of the AR (AR1-232) was
used as bait in the yeast two-hybrid assay, and subsequent GST
pull-down assays have confirmed the interaction (Figs. 1 and
2A). Traditionally, the NH2-terminal domain of
the AR is incompatible with yeast two-hybrid assays because of the
ligand-independent transcriptional activity of the AF-1 domain (5).
However, the AR fragment that was used as bait is truncated for AF-1
and is devoid of transactivation properties. Furthermore,
AR1-232 is of interest because there are no homologous
regions found in other steroid receptors.
RanBPM was originally identified by its interaction with Ran, a small
Ras-like GTPase (19). Although RanBPM was initially identified as a
55-kDa protein (BPM55), a longer form has been reported more recently
(20). The novel RanBPM protein, BPM90, is a 90-kDa protein that
demonstrates nuclear and perinuclear localization in HeLa and KB cells
lines. The function of BPM90 is still unclear, but it has recently been
linked to the Ras/ERK signaling pathway because of its interaction with
the MET receptor protein tyrosine kinase (31).
The three RanBPM library clones that were isolated in the yeast-two
hybrid assay contain amino acid sequences that are upstream of the
BPM55 start codon but are within the coding region of BPM90, which
suggests that BPM90 interacts with AR in vivo. This finding was confirmed in LNCaP cells by co-immunoprecipitation of BPM90 with
endogenously expressed AR (Fig. 2B). A SPRY domain straddles the start codon of BPM55 such that part of the domain is upstream of
the start codon. Hence, the complete SPRY domain is not present in
BPM55 but is fully intact in BPM90. The SPRY domain, originally identified in the ryanodine receptor, is involved in protein-protein interactions (32). It is probable that the SPRY domain is the region of
BPM90 that interacts with the NH2 terminus of the AR. In
fact, an NH2-terminal truncation of the SPRY domain results in the loss of the RanBPM/AR interaction in yeast (data not shown). The
interaction between RanBPM and AR is also lost when the SPRY domain is
truncated at the COOH-terminal end (Fig. 2C).
RanBPM may also have a role in Ran-dependent nuclear
transport. Ran/ARA24 is a small nuclear Ras-like GTPase that is
ubiquitously expressed (33). Quantitative analysis shows that
~107 molecules of Ran/ARA24 are expressed in an
individual cell (34). The Ran/ARA24 nuclear import/export pathway is
well defined and uses a RanGTP/GDP gradient along with carrier proteins
to shuttle proteins across the nuclear membrane (reviewed in Refs. 35
and 36). Interestingly, Ran/ARA24 was also found to interact with the
NH2 terminus of the AR, suggesting that AR, RanBPM, and
Ran/ARA24 work together as part of a multi-protein complex (34).
The LNCaP co-immunoprecipitation data presented in this paper show that
AR interacts with BPM90 in vivo only in the presence of
ligand when the receptor has been activated (Fig. 2B).
Therefore, it is probable that RanBPM and Ran/ARA24 enhance AR activity
either by promoting nuclear import of the activated receptor or by
discouraging AR export from the nucleus. These mechanisms are currently
under investigation.
The AR1-232 fragment that was used as bait in the yeast
two-hybrid assay contains unique elements such as the long polymorphic polyglutamine (poly-Gln) and polyglycine (poly-Gly) repeats (37). The
poly-Gln tract is of particular interest because an inverse relation
has been demonstrated between the length of the poly-Gln tract and AR
activity (38), which suggests that poly-Gln tracts have an important
role in directing AR activity.
Our GST- and His-tagged pull-down assays have demonstrated that RanBPM
interacts directly with AR1-232 and with larger AR
fragments that span the full NH2-terminal domain
(AR1-559) (Fig. 2, A and C). In
addition, RanBPM was found to interact with the DBD of the AR, whereas
the LBD did not contribute to the interaction (Fig. 2C). The
overexpression of BPM90 in the PC3 cell line, which does not express
endogenous AR, along with full-length AR resulted in a 3-fold increase
in androgen-induced AR activity from a synthetic promoter,
pARR3-tk-Luc, above basal AR activity (Fig. 4A). The functional domain of BPM90 is not located at the NH2
terminus because BPML, which is truncated for the first 147 amino acids of BPM90, also enhances AR activity (Fig.
4A).
Transcription assays using the full-length AR indicate that
BPM90-mediated enhancement of AR activity is
ligand-dependent because negligible activity was seen in
the absence of hormone, even at very high levels of BPM90 (Fig.
4B). These results are consistent with the nuclear
localization of BPM90 and suggest that AR must first be activated
before RanBPM can function. The ability of BPM90 to augment AR-mediated
transactivation is not only seen with the artificial ARR3 promoter,
which has three ARR motifs in tandem, but also with the naturally
occurring PSA promoter and, to a lesser degree, the rat probasin
promoter (Fig. 5).
Furthermore, it seems probable that the RanBPM is most likely to
influence the transcriptional activity of AF-1, which is found in the
NH2-terminal domain, and not the COOH-terminal AF-2 function. Additional transcriptional assays in PC3 cells using the
constitutively active AR, AR1-646, confirm the role of
BPM90 in AF-1 transactivation because enhanced AR activity was observed
irrespective of hormone (Fig. 4C). The
ligand-dependent interaction between AR and BPM90 is
confirmed because the co-immunoprecipitation from LNCaP cells only
occurs with R1881 treatment (Fig. 2B). These data are
consistent with the role of RanBPM in AR transport across the nuclear
membrane because full-length AR requires activation by its cognate
ligand before translocation, whereas AR1-646 is targeted
to the nucleus even in the absence of hormone.
Although RanBPM interacts with the unique NH2-terminal
domain of the AR, it also interacts with the DBD (Fig. 2A).
This finding suggests that RanBPM can interact with other steroid
receptors as well. Transcription assays carried out in PC3 cells show
that overexpression of BPM90 enhances the transactivation function of
GR, whereas the activity of ER- and ER- remains unchanged (Fig.
6A). These results demonstrate that RanBPM selectively
amplifies steroid receptor activity. These results are supported by
observations with receptor transactivation assays using LNCaP and MCF7
cell lines (Fig. 6, B and C). Again, the role of
RanBPM in receptor transport is supported by these data since
GR, but neither ER nor ER requires translocation to the
nucleus upon activation. Because ER- and ER- are already
localized in the nucleus (39, 40), the overexpression of RanBPM is not
likely to have an effect on ER activity.
The data presented here show that BPM90 is capable of enhancing AR
transactivation in a ligand-dependent manner. Nevertheless, the constitutively active form of AR is also influenced by the overexpression of BPM90. Furthermore, BPM90 influences the activity of
GR but has no effect on either ER- or ER- activity, which suggests that this protein selectively directs steroid receptor activity, possibly at the level of nuclear transport. Nevertheless, the
elucidation of RanBPM function will provide further insight into the
mechanism of AR-mediated gene expression.
§,
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed. Tel.:
604-875-4849; Fax: 604-875-5654; E-mail:
prennie@interchange.ubc.ca.
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