Post on 23-Sep-2020
DJBP: A Novel DJ-1-Binding Protein, Negatively Regulates theAndrogen Receptor by Recruiting Histone DeacetylaseComplex, and DJ-1 Antagonizes This Inhibition byAbrogation of This Complex
Takeshi Niki,1,3 Kazuko Takahashi-Niki,1,3 Takahiro Taira,1,3 Sanae M.M. Iguchi-Ariga,2,3
and Hiroyoshi Ariga1,3
1Graduate School of Pharmaceutical Sciences and 2College of Medical Technology, Hokkaido University, Kita-ku,Sapporo, Japan; and 3CREST, Japan Science and Technology Corporation, Kawaguchi, Saitama, Japan
AbstractDJ-1 was identified by us as a novel oncogene that
transforms mouse NIH3T3 cells in cooperation with ras .
We later identified PIAS (protein inhibitor of activated
STAT)xA as a DJ-1-binding protein, and found that DJ-1
restored androgen receptor (AR) transcription activity
that was repressed by PIASxA. To further characterize
the function of DJ-1, we cloned cDNA encoding a novel
DJ-1-binding protein, DJBP, by a yeast two-hybrid
system. DJBP mRNA was found to be specifically
expressed in the testis. In addition to the binding of
DJBP to the COOH-terminal region of DJ-1, DJBP was
also found to bind in vitro and in vivo to the DNA-
binding domain of the AR in a testosterone-dependent
manner and to be colocalized with DJ-1 or AR in the
nucleus. Furthermore, a co-immunoprecipitation assay
showed that the formation of a ternary complex between
DJ-1, DJBP, and AR occurred in cells in which DJ-1
bound to the AR via DJBP. It was found that DJBP
repressed a testosterone-dependent AR transactivation
activity in monkey Cos1 cells by recruiting histone
deacetylase (HDAC) complex, including HDAC1 and
mSin3, and that DJ-1 partially restored its repressed
activity by abrogating DJBP-HDAC complex. These
results suggest that AR is positively regulated by DJ-1,
which antagonizes the function of negative regulators,
including DJBP.
IntroductionDJ-1 was identified as a protein that transforms mouse
NIH3T3 cells in cooperation with activated ras (1). The human
DJ-1 gene, which is comprised of seven exons, is mapped to
1p36.2–36.3, where many chromosome aberrations in cancer
have been reported (2). DJ-1 was found to be more strongly
expressed in the testis than other tissues and also in sperm,
suggesting that DJ-1 has at least two functions, one function
in somatic cells, especially in germ cells, and one function in
sperm.
With regard to the function of DJ-1 in somatic cells, we have
identified PIAS (protein inhibitor of activated STAT)xa/ARIP3
(androgen receptor-interacting protein 3) as a DJ-1-binding
protein (3). PIASxa/ARIP3 belongs to the PIAS family of
proteins, including PIAS1, PIAS3, PIASxa, PIASxh, and
PIASy/g, and was characterized at first as a testis-specific AR
coregulator (4, 5). We have shown that PIASxa inhibits the
transcription activity of the AR by binding to the DNA-binding
domain of AR and that DJ-1 antagonizes this inhibition by
sequestering PIASXa from the AR in CV-1, Cos1, and TM4
Sertoli cells, indicating that DJ-1 is a positive regulator of the
AR (3). Although PIAS family proteins have been shown to
play roles in SUMO-1 conjugation (6–11) and in the regulations
of STAT (12, 13), nuclear receptors (4, 5, 14–17), and K
channels (18), the contribution of DJ-1 to such functions is not
clear.
RS, another name for human DJ-1, has been reported by
another group as a protein of an RNA-binding protein
regulatory subunit (19). It has recently been reported that
DJ-1/RS was strongly expressed and secreted from cells to
serum in about half of breast cancer patients, some of whom
possessed an antigen against DJ-1/RS, indicating that DJ-1/RS
is a biomarker for breast cancer (20). It has also been reported
that pI of DJ-1 changed from 6.2 to 5.8 by treatment of cells
with H2O2, paraquat, or lipopolysaccharide that resulted in the
production of reductive oxygen species, suggesting that DJ-1
might be a sensor to oxidative stresses (21, 22).
With regard to the function of DJ-1 in sperm, a rat
homologue of human DJ-1 has been identified by other
laboratories as a key protein related to infertility of male rats
that had been exposed to sperm toxicants such as ornidazole
and epichlorohydrin (23–26). DJ-1/RS/CAP1 has been shown
to be expressed in the sperm head and to be translocated to
the cytoplasm of sperm after treatment of rat sperm with the
above toxicants (27, 28). Furthermore, we and others have
reported that an anti-SP22 or anti-DJ-1 antiserum inhibited
fertility in vitro , suggesting that DJ-1 plays a role in
Received 7/16/02; revised 11/1/02; accepted 12/19/02.The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.Grant support: Ministry of Education, Science, Sport, Culture and Technologyof Japan.The nucleotide sequence reported in this paper has been submitted to the
DDBJ/GenBank/EBI Data Bank with accession number AB073862.Requests for reprints: Hiroyoshi Ariga, Graduate School of PharmaceuticalSciences, Hokkaido University, Kita 12, Nishi 6, Kita-ku, Sapporo 060-0812,Japan. Phone: 81-11-706-3745; Fax: 81-11-706-4988.E-mail: hiro@pharm.hokudai.ac.jpCopyright D 2003 American Association for Cancer Research.
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fertilization (29, 30). The results suggest that DJ-1 affects both
the production of sperm by activation of the AR to express
male-specific genes and the fertilization function of mature
sperm.
The AR is a member of the nuclear receptor superfamily and
plays a role as a ligand-dependent transcription factor. After a
ligand binds to the AR, the AR is translocated into the nucleus
and binds to the androgen-responsive element, ARE, on the
androgen-activating gene that affects development, growth, and
regulation of male reproductive functions (31–34). The AR,
like other nuclear receptor family proteins, possesses domains
for transactivation, DNA-binding domain (DBD), and ligand-
binding domain (LBD) at the NH2-terminal, central, and
COOH-terminal regions, respectively. Transcriptional activity
of the AR is known to be regulated by various coregulators that
bind to the respective domain of AR. More than 30 proteins of
such coregulators have been reported (Ref. 35, see homepage at
http://ww2.mcgill.ca/androgendb).
To further characterize the function of DJ-1, we cloned
cDNA encoding a novel DJ-1-binding protein, DJBP, by a yeast
two-hybrid system using a human testis library in this study.
DJ-1, DJBP, and AR were found to form a ternary complex, in
which DJ-1 bound to AR via DJBP. DJBP repressed
testosterone-dependent AR transactivation activity and DJ-1
restored its repressed activity, suggesting that DJBP and DJ-1
have mutually antagonizing functions with respect to the AR.
ResultsCloning of cDNA Encoding DJBP From a Human TestiscDNA Library
To identify proteins that bind to DJ-1, a yeast two-hybrid
screening was carried out using a human testis cDNA library
with full-sized DJ-1 as a bait, and cDNA encoding a novel DJ-
1-binding protein, named DJBP, was cloned. DJBP cDNA
comprises 2119 bp and encodes 570 amino acids (Fig. 1). DJBP
contains three LXXLL-like motifs, LXXLL, YLXXL, and
ILXXL, located at amino acid numbers 74, 92, and 235,
respectively. The LXXLL motif is known to be the sequence
that is necessary for coactivators to interact with nuclear
receptors. To examine the expression of DJBP mRNA in human
tissues, Northern blotting was carried out using a premade
tissue blot with labeled DJBP cDNA as a probe (Fig. 2). Two
distinct bands, of 1.9 and 7 kb in length, the former of which
corresponds to the length of the DJBP cDNA obtained, were
detected only in the testis (Fig. 2, lane 12). DJ-1 was also
confirmed to be strongly and moderately expressed in the testis
and other tissues, respectively, as described previously (1).
Interaction of DJBP With DJ-1To assess the association of DJBP with DJ-1 in vivo , human
293T cells were transfected with expression vectors for Flag-
tagged DJ-1 (F-DJ-1) with or without HA-tagged DJBP (HA-
DJBP). Forty-eight hours after transfection, cell extracts were
prepared and subjected to an immunoprecipitation reaction with
an anti-Flag antibody. The precipitates were then blotted with
an anti-HA or -Flag antibody (Fig. 3A). The results showed that
HA-DJBP was coprecipitated with F-DJ-1 in cells cotransfected
with F-DJ-1 and HA-DJBP but not in cells transfected with
HA-DJBP alone (Fig. 3A, lanes 1 and 2), respectively),
indicating that DJBP binds to DJ-1 in 293T cells. To examine
the binding of DJP to DJ-1 in a physiological condition, the
proteins in the extracts prepared from bovine testes were first
immunoprecipitated with an anti-DJ-1 antibody or non-specific
IgG and the precipitates were immunoblotted against an anti-
DJBP antibody (Fig. 3B). The anti-DJ-1 antibody did
precipitate DJ-1 (Fig. 3B, lane 2 in lower panel). DJBP, on
the other hand, was detected in the immunoprecipitate with the
anti-DJ-1 antibody but not with IgG (Fig. 3B, lanes 1 and 2),
respectively). These results clearly indicate that DJBP binds to
DJ-1 in cells.
To examine the interaction of DJBP with DJ-1 in vitro ,
F-DJ-1 and DJBP were expressed in E. coli as glutathione
S-transferase (GST)-fusion proteins and purified after releasing
GST by digestion of GST-fusion proteins with PreScission
protease. Purified DJBP was mixed with or without F-DJ-1 and
subjected to immunoprecipitation reaction with the anti-Flag
antibody followed by the detection of proteins in the
precipitates with an anti-DJBP antibody (Fig. 3C). The results
showed that DJBP was coprecipitated with F-DJ-1 in the
mixture containing F-DJ-1 and DJBP but not in the mixture
containing DJBP alone (Fig. 3C, lanes 1 and 2), respectively),
indicating that DJBP directly binds to DJ-1.
To determine the DJ-1-binding region in DJBP, a yeast two-
hybrid assay was carried out using various deletion mutants of
DJBP fused to the GAL4-activating domain and DJ-1 fused to
the LexA DNA-binding domain as a bait (Fig. 3D). DJ-1 was
found to bind to COOH-terminal regions of DJBP spanning
amino acid numbers 193–570, 250–570, and 372-570 but not
to NH2-terminal regions, indicating that amino acids 372–570
of DJBP are bound by DJ-1. This was also confirmed by the
results of an in vitro pull-down assay using GST or GST-fusion
proteins of DJBP containing amino acids 1–570 (full length)
and 371–570, in which DJBP and its deletion mutant but not
GST were bound by DJ-1 (Fig. 3E).
Colocalization of DJBP and DJ-1To determine the localization of DJ-1 and DJBP, an
expression vector for HA-DJBP was transfected into monkey
Cos1 cells, and the cells were stained with anti-HA and anti-
DJ-1 antibodies to show the introduced HA-DJBP and
endogenous DJ-1, respectively, and visualized with FITC and
rhodamine-conjugated secondary antibodies under a confocal
laser microscope (Fig. 4). The results showed that endogenous
DJ-1 and HA-DJBP were localized mainly in the nucleus and
both proteins were colocalized in the nucleus (Fig. 4, A–C,
respectively).
Association of DJBP With the Androgen ReceptorTo determine whether DJBP associates with AR in vivo ,
293T cells cultured in the presence of 100 nM testosterone were
transfected with expression vectors for HA-DJBP with or
without F-AR, and 48 h after transfection, cell extracts were
immunoprecipitated with an anti-Flag antibody and the
precipitated protein was detected with an anti-HA or the anti-
Flag antibody (Fig. 5A). The results showed that HA-DJBP was
coprecipitated with F-AR in cells cotransfected with both F-AR
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FIGURE 1. Nucleotide and amino acidsequences of DJBP. The nucleotide sequence ofDJBP cDNA was determined by a dideoxy methodusing Long Reader 4200 (LI-Cor, Lincoln, Nebraska)and Gene Rapid (Amersham BioScience, Bucks,UK) autosequencers, and the deduced amino acidsequence is shown. LXXLL motifs within the aminoacid sequence are underlined .
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and HA-DJBP but not in cells transfected with HA-DJBP alone
(Fig. 5A, lanes 1 and 2), respectively), indicating that DJBP
binds to AR in 293T cells. To assess the direct interaction
between DJBP and AR, a GST pull-down assay was carried out
using purified GST-DJBP and GST. Purified proteins were
reacted in the presence of 100 nM testosterone with 35S-AR
synthesized in vitro in a reticulocyte lysate, and the bound
proteins were visualized by fluorography (Fig. 5B). The results
showed that labeled AR bound to DJBP but not to GST,
indicating that DJBP directly interacts with the AR (Fig. 5B,
lanes 1 and 2), respectively). Because it is known that some
coregulators for the AR bind to the AR in a ligand-dependent
manner, testosterone (a ligand of the AR)-dependent binding
activity of DJBP to AR was examined by a liquid h-galactosidase assay in yeast. Because DJBP binds to the
COOH-terminal half of the AR, as shown in Fig. 6B, this
truncated form of the AR, ARDN, was used in this assay
(Fig. 5C). The results showed that h-galactosidase activity
obtained between DJBP and ARDN increased with increase in
the dose of testosterone. To further confirm the testosterone-
DJBP
DJ-1
Actin
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FIGURE 2. Northern blot analysis ofDJBP in various human tissues. HumanNorthern RNA blot (12 major tissues,Origene, Rockville, MD) was hybridizedwith labeled cDNAs of DJBP (upperpanel ), DJ-1 (middle panel ), and h-actin(lower panel ) as probes. Positions of sizemarkers are shown on the right in theupper panel .
FIGURE 3. Interaction of DJBP with DJ-1. A. 293T cells were transfected with pcDNA3-F-DJ-1 and pcDNA3-HA-DJBP. At 48 h after transfection, cellextracts were prepared and subjected to an immunoprecipitation reaction using an anti-Flag antibody (M2, Roche Applied Science, Rotkreuz, Switzerland).The precipitates were blotted with an anti-HA or the anti-Flag antibody. B. The proteins in the extract from bovine testes were first immunoprecipitated with ananti-DJ-1 antibody or non-specific IgG. The proteins in the precipitates were separated in a 12.5% polyacrylamide gel and blotted with an anti-DJBP antibodyor the anti-DJ-1 antibody. Lane 1, the extract used for the binding reaction was applied to the same gel (Input). C. F-DJ-1 and DJBP were expressed inEscherichia coli as GST-fusion proteins and purified after releasing GST by digestion of GST-fusion proteins with PreScission protease (AmershamBioScience) as described in ‘‘Materials and Methods.’’ One microgram of each purified protein was mixed and subjected to an immunoprecipitation reactionusing an anti-Flag antibody. The precipitates were blotted with an anti-DJBP antibody. Lane 3, the DJBP used for the reactions (Input) was run in parallel.D. Yeast L40 cells were transformed with expression vectors for LexA, LexA-DJ-1, and various deletion mutants of DJBP fused to the GAL4-activationdomain. The h-galactosidase activity of each colony was measured. E. DJBP(1– 570), DJBP(372 – 570), and DJ-1 were expressed in E. coli as GST-fusionproteins and purified. GST-free DJ-1 was then prepared after releasing GST from GST-DJ-1 by digestion with PreScission protease. DJ-1 and GST-DJBPswere mixed together and subjected to a pull-down assay as described in ‘‘Materials and Methods.’’ The proteins bound to GST-DJBPs were blotted with ananti-DJ-1 antibody. Lane 4, the DJ-1 used for the reactions (Input) was run in parallel.
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dependent binding activity of DJBP to the AR in vivo , 293T
cells cultured in the various amounts of testosterone were
transfected with expression vectors for Flag-DJBP with or
without the AR, and 48 h after transfection, cell extracts were
immunoprecipitated with the anti-Flag antibody and the
precipitated protein was detected with the anti-Flag antibody
or an AR antibody (Fig. 5D). The results showed that AR was
coprecipitated in a testosterone-dependent manner with F-DJBP
in cells cotransfected with both AR and F-DJBP but not in cells
transfected with the AR alone (Fig. 5D, lanes 1, 2, 4, 5, 7, 8, 10,
and 11), indicating that DJBP binds to the ligand-binding form
of the AR in 293T cells.
To determine the AR-binding region of DJBP, 293T cells
were cotransfected with the AR and various Flag-tagged
deletion mutants of DJBP, and the AR that co-immunopreci-
pitated with DJBP in cells were detected with an anti-AR
antibody after the immunoprecipitation of cell extracts with an
anti-Flag antibody (Fig. 6A). In addition to the full-sized DJBP,
DJBP deletion mutants of amino acids 372–570 and 476–570
also immunoprecipitated the AR, whereas DJBP deletion
mutants of amino acids 1–371 and 372–475 did not (Fig. 6A,
lanes 1, 3, 5, 2, and 4), respectively), indicating that the AR
binds to amino acids 476–570 of DJBP which do not contain
LXXLL motifs. To determine the DJBP-binding region of the
AR, on the other hand, Cos1 cells were cotransfected with the
HA-tagged DJBP and various Flag-tagged deletion mutants of
the AR, and DJBP that co-immunoprecipitated with ARs in
cells was detected with an anti-HA antibody after the
immunoprecipitation of cell extracts with the anti-Flag antibody
(Fig. 6B). Deletion constructs of the AR are schematically
shown in Fig. 6C. The results showed that while two mutants of
the AR, DC and LBD, were not bound by DJBP, the deletion
mutants of the AR, DN and DBD, were, like the wild-type AR,
bound by DJBP (Fig. 6B, lanes 2, 5, 3, 4, and 1), respectively).
These results indicate that DJBP binds to the DNA-binding
domain of the AR and that the LXXLL motif is not necessary
for DJBP to interact with the AR. To examine the colocalization
of DJBP with the AR in cells, Cos1 cells were cotransfected
with F-DJ-1 and HA-DJBP in the presence of 100 nM
testosterone, and transfected cells were stained with anti-AR
and anti-HA antibodies at 48 h after transfection. HA-DJBP
was found to be colocalized with the AR in the nucleus (data
not shown).
Repression of AR Transactivation Activity by DJBP andRestoration of Its Repression by DJ-1
Because we showed in this study that DJBP binds to both
DJ-1 and AR and in a previous study that DJ-1 does not directly
bind to the AR (3), we then examined the relationships among
DJBP, DJ-1, and AR. 293T cells cultured in the presence of
100 nM testosterone were transfected with various combinations
of F-AR, HA-DJBP, and DJ-1-HA, the cell extracts were
immunoprecipitated with an anti-Flag antibody, and the
precipitants were blotted with anti-HA and Flag antibodies
(Fig. 7A, lanes 1–8). HA-DJBP was co-immunoprecipitated
with F-AR as expected (Fig. 7A, lanes 2 and 3). DJ-1-HA, on
the other hand, was co-immunoprecipitated with F-AR in cells
transfected with both HA-DJBP and F-AR but not in cells
transfected with F-AR alone (Fig. 7A, lanes 3 and 4),
respectively). Expression levels of the respective proteins of
F-AR, HA-DJBP, and DJ-1-HAwere comparable in transfected
cells (Fig. 7A, see Input). Furthermore, the amounts of DJ-1
and HA-DJBP co-immunoprecipitated with F-AR were found
to be stimulated in the presence of 100 nM testosterone (Fig. 7A,
lanes 9 and 10). Thus, these results clearly indicate that DJ-1,
DJBP, and AR form a ternary complex, in which DJ-1
associates with the AR via DJBP.
To then assess the relationship between the biological
activities of DJ-1, DJBP, and AR, monkey Cos1 cells were
transfected with pARE2-TATA-Luc as a reporter and with or
without pcDNA3-F-AR together with various amounts of
pcDNA3-HA-DJBP or pEGFP-N1 in the presence or absence
of 100 nM testosterone. The plasmid, pCMV-h-gal, was also
cotransfected into cells to normalize the transfection efficiency.
Forty-eight hours after transfection, cell lysates were prepared
and their luciferase activities were measured (Fig. 7B). A
testosterone-dependent transcription activity of the AR was first
confirmed in the transfected cells without pcDNA3-HA-DJBP
(data not shown). It was found that neither DJBP nor EGFP
responded to pARE2-TATA-Luc reporter in the absence of the
AR. The results then showed that while introduction of EGFP
that did not bind to the AR, pEGFP-N1, did not affect the AR
activity, DJBP repressed the testosterone-dependent transcrip-
tion activity of the AR in a dose-dependent manner and about
70% of the AR activity was repressed after 1 Ag of pcDNA3-
HA-DJBP was transfected into cells (Fig. 7B), suggesting that
DJBP acts as a negative regulator of the AR. DJ-1-HA or EGFP
was then cotransfected into Cos1 cells together with 1 Ag of
pcDNA3-HA-DJBP and pARE2-TATA-Luc, and the luciferase
activities were determined (Fig. 7C). The results showed that
while introduction of vector, pEGFP-N1, did not affect the AR
activity, DJ-1 partially restored AR transcription activity
repressed by DJBP in a dose-dependent manner. Because
DJ-1 directly binds to DJBP but not the AR, these results
suggest that DJ-1 changes the AR from a transcriptional
negative form to an active form as we reported in the case of
DJ-1 and PIASxa (3).
Association of DJBP With Histone Deacetylase ComplexBecause transcriptional repression pathways of genes are
often connected with histone deacetylase (HDAC) or corepres-
sor complex, it is possible that the transcriptional repression of
the AR by DJBP uses the same or similar pathway connected
with HDAC. To explore this possibility, an expression vector
for FLAG-tagged DJBP was transfected into human 293T cells
cultured in the presence of 100 nM testosterone. Forty-eight
hours after transfection, the cell extract was prepared and the
proteins in the extract were first immunoprecipitated with the
anti-FLAG antibody or non-specific IgG. The precipitates were
immunoblotted against an anti-AR antibody (Fig. 8A). It was
first confirmed that the anti-FLAG antibody did not precipitate
proteins in the extracts from cells into which FLAG-DJBP had
not been transfected, and that the anti-FLAG antibody
precipitated FLAG-DJBP (Fig. 8A, lanes 3 and 1), respec-
tively). The endogenous AR, on the other hand, was detected in
the immunoprecipitate with the anti-FLAG antibody but not
with IgG (Fig. 8A, lanes 1 and 2), respectively), indicating
again that DJBP was associated with the AR in 293T cells. The
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same blot as that shown in Fig. 8A was reprobed with anti-
HDAC1 and-mSin3A antibodies (Fig. 8A). Endogenous
HDAC1 and mSin3A were detected in the immunoprecipitates
with the anti-FLAG antibody but not with IgG, indicating that
DJBP and the AR are complexed with a corepressor complex,
including HDAC1 and mSin3A. It was found that neither
HDAC1 nor mSin3A directly bound to DJBP by the pull-down
assay, in which GST-DJBP was reacted with 35S-HDAC1 or35S-mSin3A synthesized in vitro in a reticulocyte lysate (data
not shown), suggesting that the DJBP-bound AR or the DJBP-
AR complex recruits the corepressor complex.
To confirm the existence of the HDAC complex in the
transrepression pathway of the AR to DJBP, TSA, an inhibitor
of HDAC1, was added to the cells transfected with the AR and
various amounts of DJBP in the presence of 100 nM
testosterone, and the luciferase activity was measured
(Fig. 8B). The results showed that TSA abrogated the DJBP-
repressed transcription activity of the AR and further increased
the transcription activity of the AR to the level comparable to
that obtained after introduction of the AR alone, suggesting that
repression of the AR transcription activity by DJBP is carried
out by the HDAC complex.
Abrogation of the DJBP-HDAC Complex by DJ-1To determine the mechanism underlying the abrogation of
the DJBP-repressed AR transcription activity by DJ-1, FLAG-
tagged DJBP and various amounts of HA-tagged DJ-1 or
EGFP were transfected into human 293T cells cultured in the
presence of 100 nM testosterone. Forty-eight hours after
transfection, the cell extract was prepared and the proteins in
the extract were first immunoprecipitated with an anti-FLAG
antibody. The precipitates were immunoblotted against anti-
HA, anti-HDAC1, anti-GFP, and anti-FLAG antibodies as
described in Fig. 8A (Fig. 9). It was first confirmed that the
constant amounts of endogenous HDAC1 were expressed and
that DJ-1-HA and EGFP were expressed in a dose-dependent
manner in transfected cells (Input, Fig. 9, lanes 9–16). It was
also confirmed that FLAG-DJBP was precipitated with the
anti-FLAG antibody (Fig. 9, lanes 1–3, 5, and 6). While EGFP
was not detected in the precipitates, DJ-1-HAwas also detected
with the anti-HA antibody in the immunoprecipitates from cells
that had been transfected with FLAG-DJBP (Fig. 9, lanes 2
and 3), indicating that DJBP was associated with DJ-1 in
ectopic expressed 293T cells. It was found that endogenous
HDAC1 was, however, first detected in the immunoprecipitates
from cells that had been transfected with FLAG-DJBP alone
and then the amounts of immunoprecipitated HDAC1
decreased in a dose-dependent manner (Fig. 9, lanes 1–3),
indicating that DJ-1 abrogated the DJBP-HDAC complex,
thereby leading to the restoration of the AR transcription
activity repressed by DJBP.
DiscussionIn this study, we identified a novel DJ-1-binding protein,
named DJBP, by a yeast two-hybrid system using a human
testis cDNA library. DJBP contains three LXXLL-like motifs
that are known to be necessary for coactivators to interact with
nuclear receptors. Expressions of DJBP mRNAs of 2 and 7 kb
in length were found to be limited to the testis of human tissues,
FIGURE 4. Colocalization of DJBP with DJ-1 in Cos1 cells. Cos1 cells were transfected with pcDNA3-HA-DJBP, stained with anti-HA (A) and anti-DJ-1(B) antibodies at 48 h after transfection, and visualized under a confocal laser microscope. C. Figs. A and B are merged.
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and the cDNA cloned in this study appears to correspond to the
2-kb mRNA. Although the cDNA corresponding to the 7-kb
mRNA is not available at present, it might be of an alternative
splicing form of DJBP mRNAs.
From the results of various binding experiments in vitro and
in vivo , it was found that DJBP directly binds to both DJ-1 and
AR in a testosterone-dependent manner and that DJ-1
associates with AR via DJBP in a ternary complex in cells.
Because AR binds to the COOH-terminal region spanning
amino acids 476–570 of DJBP, in which three LXXLL-like
motifs are not present, it is thought that these motifs in DJBP
are not necessary for binding to the AR, like several AR-
binding proteins, including BRCA1 (36), Cyclin E (37), RAF
(38), TFIIH (39), CAK (39), RB (40), Ubc9 (41), c-Jun (42),
and Cyclin D1 (43). DJBP, on the other hand, binds to the
DNA-binding domain of the AR (AR-DBD). Because the
FIGURE 5. Interaction of DJBP with AR. A. 293T cells cultured in the presence of 100 nM testosterone were transfected with pcDNA3-F-AR and pcDNA3-HA-DJBP. Forty-eight hours after transfection, the proteins in cell extracts were precipitated with an anti-Flag antibody and blotted with anti-HA or -Flagantibodies. B. GST-DJBP and GST were expressed in E. coli and purified as described in ‘‘Materials and Methods.’’ GST-DJBP or GST was mixed with35S-labeled AR synthesized in vitro in reticulocyte lysate and subjected to a pull-down assay. Labeled proteins bound to GST-DJBP or GST were visualizedby fluorography. Lane 4, the AR used for the reactions (Input) was run in parallel. C. Yeast L40 cells were transformed with various combinations ofLexA-DJBP, GAD-ARDN, LexA, and GAD. The h-galactosidase activity of each colony was measured in the presence of various concentrationsof testosterone. D. 293T cells cultured in the various amounts of testosterone were transfected with pcDNA3-AR and pcDNA3-F-DJBP. Forty-eight hoursafter transfection, the proteins in cell extracts were precipitated with an anti-Flag antibody and blotted with anti-AR or -Flag antibodies.
FIGURE 6. Determination ofregions of binding between DJBPand AR. A. 293T cells cultured inthe presence of 100 nM testosteronewere transfected with various dele-tion mutants of Flag-tagged DJBPand AR. Forty-eight hours aftertransfection, the proteins in the cellextract were precipitated with ananti-Flag antibody and blotted withan anti-AR antibody. Lane 7, thecell extract used for the reactions(Input ) was run in parallel. B. Cos1cells cultured in the presence of 100nM testosterone were transfectedwith various deletion mutants ofFlag-tagged AR and HA-taggedDJBP. Forty-eight hours after trans-fection, the proteins in the cellextract were precipitated with ananti-Flag antibody and blotted withan anti-HA antibody. Lane 6, thecell extract used for the reactions(Input ) was run in parallel. C. TheAR and its deletion mutants usedfor the reactions are schematicallyshown, and the DJBP-binding activ-ities are shown on the right .
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FIGURE 7. Effect of transcription activity of the AR by DJBP and DJ-1. A. 293T cells cultured in the presence of 100 nM testosterone were transfectedwith various combinations of pcDNA3-F-AR, pcDNA3-HA-DJBP, and pEF-DJ-1-HA (lanes 1 –8). Lanes 9 and 10, plasmids used in lane 3 were transfectedinto 293T cells in the absence or presence of 100 nM testosterone. Forty-eight hours after transfection, the proteins in the cell extract were precipitated with ananti-Flag antibody and blotted with anti-Flag and anti-HA antibodies. In the lower two panels (Input), the cell extract used for the reactions was run in paralleland blotted with the anti-HA antibody. B. Cos1 cells cultured in the presence of 100 nM testosterone were transfected with 0.12 Ag of pCMV-h-gal, 0.03 Ag ofpcDNA3-F-AR, and 1.2 Ag of pARE2-TATA-Luc together with various amounts of pcDNA3-HA-DJBP or pEGFP-N1 (HA-DJBP + AR or EGFP + AR,respectively). Cos1 cells were also transfected with the same DNAs as those above except for omitting pcDNA3-F-AR (HA-DJBP or EGFP, respectively). Forty-eight hours after transfection, cell lysates were prepared and their luciferase activities were measured. C. Cos1 cells cultured in the presence of 100 nM
testosterone were transfected with 0.12 Ag of pCMV-h-gal, 0.03 Ag of pcDNA3-F-AR, 1.2 Ag of pARE2-TATA-Luc, and 1 Ag of pcDNA3-HA-DJBP together withvarious amounts of pEF-DJ-1-HA or pEGFP-N1. Forty-eight hours after transfection, cell lysates were prepared and their luciferase activities were measured.
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amino acid sequences of DBD among nuclear receptor family
proteins were well conserved, DJBP might also bind to other
nuclear receptors. Of the proteins that bind to AR-DBD,
SNURF (44), Ubc9 (41), and ARIP3/PIASXa (4) have been
reported to associate with multiple nuclear receptors, including
the AR.
DJBP was found to repress the transcription activity of AR
to 30% of that without DJBP. The mechanism underlying this
repression of AR activity by DJBP was assessed by the
findings that DJBP recruited the corepressor complex,
including HDAC1 and mSin3A. These results suggest that
an active form of the AR changes into an inactive form by
translocation to an inactive structure within the chromatin.
Repression activity of DJBP toward the AR was sensitive to
TSA, a specific inhibitor of HDAC, indicating that HDAC is
involved in the transrepression pathway of the AR. The
introduction of DJ-1 relieved the repressed the AR tran-
scription activity by abrogation of DJBP-HDAC complex. It
was reported that TSA augments dihydrotestosterone induc-
tion of AR levels (45). Although the results in this report
suggested that the androgen-induced chromatin remodeling
and transcription occur in the AR-responsive genes, the
results in our study here is a first report to identify the AR-
HDAC complex bridged by DJBP.
FIGURE 8. Binding of DJBP to the ARin the HDAC complex in vivo and effect oftrichostatin A (TSA ) on the repressionactivity of DJBP toward the AR. A. 293Tcells cultured in the presence of 100 nM
testosterone were transfected with anexpression vector for FLAG-DJBP bythe calcium phosphate precipitation tech-nique, and the cell extract was prepared48 h after transfection. Proteins in theextract were first precipitated with an anti-FLAG mouse monoclonal antibody ornon-specific IgG, and the precipitateswere immunoblotted with an anti-ARrabbit polyclonal antibody (N-20, SantaCruz Biotechnology, Santa Cruz, CA).Proteins in the extract from non-trans-fected cells were similarly treated withantibodies. The same blot as thatdescribed above was reprobed with ananti-HDAC1 antibody (H-51, Santa CruzBiotechnology) and an anti-mSin3A anti-body (AK-11, Santa Cruz Biotechnology).Lanes 4 and 5, the proteins used for thereactions (Input ) were run in parallel. B.Cos1 cells cultured in the presence of 100nM testosterone were transfected with0.12 Ag of pCMV-h-gal, 0.03 Ag ofpcDNA3-F-AR, and 1.2 Ag of pARE2-TATA-Luc together with various amountsof pcDNA3-HA-DJBP. Two-hundrednanomolars of TSA was added to theculture 40 h after transfection. After 48 h,cell lysates were prepared, and theluciferase activities in the lysates weremeasured.
Molecular Cancer Research 257
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Functions of the AR are regulated by various complex
formations. PIASxa, a member of the PIAS family of
proteins, was characterized at first as a testis-specific AR
coregulator, ARIP3 (4, 5). We have shown that PIASxa
inhibits the transcription activity of the AR by binding to the
DNA-binding domain of the AR and that DJ-1 antagonizes
this inhibition by sequestering PIASXa from the AR in CV-1,
Cos1, and TM4 Sertoli cells, indicating that DJ-1 is a positive
regulator of the AR (3). DJBP was found to bind to the AR,
leading to formation of a ternary complex between DJ-1,
DJBP, and the AR. Different to the mode of action of
PIASxa on the AR, DJBP was found to inhibit AR
transcription activity by recruiting histone-deacetylase
complexes, including HDAC1 and mSin3, and DJ-1 was
found to restore the repressed activity of the AR. These
findings suggest, as schematically shown in Fig. 10, that DJ-1
restores AR activity that has been repressed by different
mechanisms.
DJBP and DJ-1 were found to be expressed specifically and
strongly, respectively, in the testis. Furthermore, it was found
that DJ-1 was also expressed in sperm and played roles in
fertilization reactions. These results suggest that DJBP also
plays a role in spermatogenesis or fertilization along with DJ-1.
To clarify this possibility, we are investigating the expression
patterns, including location and timing, of DJBP in the testis
and sperm.
FIGURE 9. Abrogation of recruitmentof HDAC complex to DJBP by DJ-1.293T cells cultured in the presence of100 nM testosterone were transfectedwith 5 Ag of pcDNA3-F-DJBP, variousamounts of pEF-DJ-1-HA and pEGFP-N1 by the calcium phosphate precipita-tion technique, and the cell extract wasprepared 48 h after transfection. Proteinsin the extract were first precipitated withan anti-FLAG mouse monoclonal anti-body, and the precipitates were immuno-blotted with an anti-HA rabbit polyclonalantibody, an anti-HDAC1 antibody, or theanti-FLAG antibody as described in Fig. 8.The 0.25-, 1-, and 1-Ag pEF-DJ-1-HAor pEGFP-N1 was transfected into cellsin lanes 2, 3, and 4, respectively. Lanes9 – 16, the cell extracts used for thereactions (Input) were run in parallel.
AR
DJBP
DJ-1
HDAC
mSin3
corepressor complex
PIASxα
histone deacetylation
ARE target genes
FIGURE 10. Schematic drawings ofmechanisms by which DJ-1 antagonizesnegative modulators of the AR by DJBPand PIASxa.
DJBP, a Negative Regulator of the Androgen Receptor258
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Materials and MethodsCells
Human 293T and monkey Cos1 cells were cultured in
DMEM supplemented with 10% calf serum.
PlasmidsNucleotide sequences of the oligonucleotide used for PCR
primers were as follows:
DJBP-ATG(Bam), 5 V-GGATCCATGGGTCATTTT-
ACAA-3V;DJBP-ATG(Eco), 5V-GGGAATTCATGGGTCATTTT-
ACAA-3V;DJBP-193(Eco), 5V-GGGAATTCTATATAAATTGG-
AAAT-3V;DJBP-250(Eco), 5V-GGGAATTCATCACCCAGGAG-
TTTG-3V;DJBP-372(Eco), 5V-GGGAATTCGGCACTCCACCC-
TTGC-3V;DJBP-372(Bam), 5V-GGGGATCCGGCACTCCACCC-
TTGC-3V;AR-DN(Eco), 5 V-GGGAATTCGGACCTTATGGG-
GACA-3V;DJBP-372(Bam), 5V-GGGGATCCGGCACTCCACCC-
TTGC-3V;DJBP-476(Bam), 5V-GGGGATCCCAGAATGCACAC-
AAGA-3V;DJBP-475(Xho), 5V-GGGAGCTCGATCTTCATCCT-
GTGC-3V;AR-DBD(Eco), 5V-GGGAATTCTACGGAGCTCTC-
ACTT-3V;AR-DBD(Xho), 5V-GGCTCGAGTACAGTCATCTT-
CTGG-3V; andAR-LBD(Eco), 5V-GGGAATTCCAGAAGATGACT-
GTAT3V.
Combinations of plasmids constructed, primers, templates,
and restriction enzyme sites and plasmids inserted are shown in
Table 1. pGEX-F-DJ-1: The EcoRI-XhoI fragment of pGlex-
DJ-1 (3) was inserted into the EcoRI-XhoI sites of pGEX-6P-1-
F (46). pGAD-DJBP(1–371), pGAD-DJBP(193–371), and
pGAD-DJBP(250–371): the EcoRI-SmaI fragments of pGAD-
DJBP(1 – 570), pGAD-DJBP(93 – 570), and pGAD-
DJBP(250–570) were inserted into the EcoRI-SmaI sites of
pBluescript SK(-), and the EcoRI-XhoI fragments of these
plasmids were then inserted into the EcoRI-XhoI sites of
pGAD-GHX, respectively. pGLex-DJBP: The EcoRI-XhoI
fragment of pGAD-DJBP(1–570) was inserted into the
EcoRI-XhoI sites of pGLex (47). pcDNA3-F-DJBP(1–371):
The EcoRI-Xho I fragment of pGAD-DJBP(1–371) was
inserted into the EcoRI-XhoI sites of pcDNA3-F. pcDNA3-F-
AR-DC: The EcoRI-HindIII fragment of pcDNA3-F-AR was
subcloned into the EcoRI-SmaI sites of pBluescript SK(-), and
the EcoRI-XhoI fragments of this plasmid were then inserted
into the EcoRI-XhoI sites of pcDNA3-F. pcDNA3-F-AR-DN:
The EcoRI-XhoI fragment of pGAD-AR-DN was inserted into
the EcoRI-XhoI sites of pcDNA3-F.
AntibodyA fusion protein of GST and DJBP was expressed in E. coli
MN524 and purified as described previously (3). Rabbits were
immunized by the purified GST-DJBP. The anti-DJBP antibody
from the rabbit serum was prepared by the affinity chromatog-
raphy containing GST-DJBP, absorbed by GST, and used as the
polyclonal anti-DJBP antibody.
Cloning of DJBP cDNA From a Human Testis cDNALibrary by a Yeast Two-Hybrid System
Yeast L40 cells were co-transformed with expression vectors
for DJ-1 fused to the LexA DNA-binding domain, pGLex-DJ-1,
and human testis cDNAs from the MATCHMAKER cDNA
library (Clontech Laboratories, Inc., Palo Alto, CA), which
express human testis cDNAs fused to the GAL4-activating
domain. Approximately 1.5 � 106 colonies were screened for
expressions of HIS3 and LacZ.
Northern BlottingA human Northern RNA blot (12 major tissues, Origene)
was hybridized with 32P-labeled DJBP, DJ-1, and h-actincDNAs as probes under a highly stringent condition, and then
hybridized bands were detected by autoradiography.
Table 1. Plasmid Construction
Plasmid Template 5V-Primer 3V-Primer Restriction Enzyme Site Plasmid Inserted
pcDNA3-HA-DJBP pME18S-DJBP DJBP-ATG(Bam) SP6 BamHI, Xho I pcDNA3-HApGEX-DJBP pME18S-DJBP DJBP-ATG(Bam) SP6 BamHI, Xho I pGEX-6P-1pGAD-DJBP(1–570) pME18S-DJBP DJBP-ATG(Eco) SP6 EcoRI, Xho I pGAD-GHXpGAD-DJBP(193– 570) pME18S-DJBP DJBP-193(Eco) SP6 EcoRI, Xho I pGAD-GHXpGAD-DJBP(250– 570) pME18S-DJBP DJBP-250(Eco) SP6 EcoRI, Xho I pGAD-GHXpGAD-DJBP(372– 570) pME18S-DJBP DJBP-372(Eco) SP6 EcoRI, Xho I pGAD-GHXpGEX-DJBP(372–570) pcDNA3-HA-DJBP DJBP-372(Bam) SP6 EcoRI, Xho I pGEX-6P-1pGAD-AR-DN pCDNA3-F-AR AR-DN(Eco) SP6 EcoRI, Xho I pGAD-GHXpcDNA3-F-DJBP(1 – 570) pcDNA3-HA-DJBP DJBP-ATG(Bam) SP6 BamHI, Xho I pcDNA3-FpcDNA3-F-DJBP(372 –570) pcDNA3-HA-DJBP DJBP-372(Bam) SP6 BamHI, Xho I pcDNA3-FpcDNA3-F-DJBP(476 –570) pcDNA3-HA-DJBP DJBP-476(Bam) SP6 BamHI, Xho I pcDNA3-FpcDNA3-F-AR-DBD pCDNA3-F-AR AR-DBD(Eco) AR-DBD(Xho) EcoRI, Xho I pcDNA3-FpcDNA3-F-AR-LBD pCDNA3-F-AR AR-LBD(Eco) SP6 EcoRI, Xho I pcDNA3-F
Note: Combinations of plasmids constructed, primers, templates, and restriction enzyme sites and plasmids inserted are shown.
Molecular Cancer Research 259
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Interaction of DJBP With DJ-1 and AR in Vivo293T cells were transfected with pcDNA3-F-DJ-1 and
pcDNA3-HA-DJBP in the presence or absence of 100 nM
testosterone by the calcium phosphate method (48). Forty-eight
hours after transfection, the cell extract was prepared and
subjected to an immunoprecipitation reaction using an anti-
FLAG antibody conjugated with agarose beads (M2, Sigma
Chemical Co., St. Louis, MO). The precipitates were separated
on 12.5% SDS-PAGE and blotted with an anti-HA antibody
(12CA5, Roche). To examine formation of a ternary complex
between DJ-1, DJBP, and AR, 5 Ag of pcDNA3-F-AR, 2.5 Agof pcDNA3-HA-DJBP, and 1 Ag of pEF-DJ-1-HA were
transfected into 293T cells in the presence of 10 mM
testosterone, and then the same immunoprecipitation assay as
that described above was carried out.
Interaction of DJBP with DJ-1 and AR in VitroF-DJ-1 and DJBP were expressed in E. coli as GST-fusion
proteins and purified after releasing GST by digestion of GST-
fusion proteins with PreScission protease (Amersham
BioScience) as described previously (49). Purified F-DJ-1
and DJBP were mixed and subjected to an immunoprecipita-
tion reaction using an anti-Flag antibody followed by an anti-
DJBP antibody as described above. The anti-DJBP antibody
used in this reaction was proteins of the IgG faction prepared
from rabbits immunized with purified DJBP as an immunogen.
To examine the binding of DJBP with the AR, purified GST-
DJBP or GST was incubated with 35S-AR synthesized in vitro
using a reticulocyte lysate of a TnT-transcription-translation
coupled system (Promega, Madison, WI) and subjected to a
pull-down assay using an glutathione-Sepharose as described
previously (49).
ImmunofluorescenceCos1 cells were transfected with 1 Ag of pcDNA3-HA-
DJBP or pcDNA3-F-AR by the calcium phosphate method
(48). Forty-eight hours after transfection, the cells were fixed
with 4% paraformaldehyde, stained with anti-DJ-1 (3), anti-AR
(N-20, Santa Cruz Biotechnology), and HA antibodies, and
visualized under a confocal laser microscope.
b-Galactosidase Liquid AssayL40 cells were transformed with expression vectors for DJ-1
fused to the LexA DNA-binding domain, pGLex-DJ-1, and for
various deletion mutants of DJBP fused to the GAL4-activation
domain, pGAD-GHX-DJBPs. Transformed cells were cultured
in YPAD medium in the presence of testosterone for 4 h, and
h-galactosidase assay using cell extracts was carried out.
Luciferase AssayCos1 cells in 6-cm dishes were transfected with 1.3 Ag of
pARE2-TATA-Luc, a reporter plasmid, and 0.13 Ag of pCMV-
h-gal, an expression vector for h-galactosidase, by the calcium
phosphate method (48). Forty-eight hours after transfection, the
luciferase activity in cells was measured after normalization of
the transfection efficiency with h-galactosidase activities as
described previously (49).
AcknowledgmentsWe thank Yoko Misawa and Kiyomi Takaya for their technical assistance.
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Molecular Cancer Research 261
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2003;1:247-261. Mol Cancer Res Takeshi Niki, Kazuko Takahashi-Niki, Takahiro Taira, et al. accession number AB073862.submitted to the DDBJ/GenBank/EBI Data Bank with
The nucleotide sequence reported in this paper has been 2 2Science, Sport, Culture and Technology of Japan.
Ministry of Education, 1 1Abrogation of This ComplexComplex, and DJ-1 Antagonizes This Inhibition bythe Androgen Receptor by Recruiting Histone Deacetylase DJBP: A Novel DJ-1-Binding Protein, Negatively Regulates
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