Activating Protein-1, Nuclear Factor-
B, and Serum Response Factor as Novel Target Molecules of the Cancer-Amplified Transcription Coactivator ASC-2
Soo-Kyung Lee,
Soon-Young Na,
Sung-Yun Jung,
Ji-Eun Choi,
Byung Hak Jhun,
JaeHun Cheong,
Paul S. Meltzer,
Young Chul Lee and
Jae Woon Lee
Center for Ligand and Transcription (S.-K.L., S.-Y.N., S.-Y.J.,
J.H.C., Y.C.L., J.W.L.) Department of Biology (S.-K.L., S.-Y.N.)
and Hormone Research Center (J.H.C., Y.C.L., J.W.L.) Chonnam
National University Kwangju 500757, Korea College of
Pharmacy (J.-E.C., B.H.J.) Pusan National University Pusan
609735, Korea Cancer Genetics Branch (P.S.M.)
National Human Genome Research Institute National Institutes of
Health Bethesda, Maryland 20892-4470
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ABSTRACT
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ASC-2 was recently discovered as a
cancer-amplified transcription coactivator molecule of nuclear
receptors, which interacts with multifunctional transcription
integrators steroid receptor coactivator-1 (SRC-1) and CREB-binding
protein (CBP)/p300. Herein, we report the identification of three
mitogenic transcription factors as novel target molecules of ASC-2.
First, the C-terminal transactivation domain of serum response factor
(SRF) was identified among a series of ASC-2-interacting proteins from
the yeast two-hybrid screening. Second, ASC-2 specifically interacted
with the activating protein-1 (AP-1) components c-Jun and c-Fos as well
as the nuclear factor-
B (NF
B) components p50 and p65, as
demonstrated by the glutathione S-transferase pull-down
assays as well as the yeast two-hybrid tests. In cotransfection of
mammalian cells, ASC-2 potentiated transactivations by SRF, AP-1, and
NF
B in a dose-dependent manner, either alone or in conjunction with
SRC-1 and p300. In addition, ASC-2 efficiently relieved the previously
described transrepression between nuclear receptors and either AP-1 or
NF
B. Overall, these results suggest that the nuclear receptor
coactivator ASC-2 also mediates transactivations by SRF, AP-1, and
NF
B, which may contribute to the putative, ASC-2-mediated
tumorigenesis.
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INTRODUCTION
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Transcription coactivators either bridge transcription factors and
the components of the basal transcriptional apparatus and/or remodel
the chromatin structures (reviewed in Ref. 1). In particular, CREB
binding protein (CBP) and its functional homolog p300 were shown to be
essential for the activation of transcription by a large number of
regulated transcription factors (reviewed in Ref. 2). Similarly,
steroid receptor coactivator-1 (SRC-1) and its family members were
recently found to stimulate transactivation by many different
transcription factors, including CREB and signal transducer and
activator of transcription (STAT)s (3), activating protein-1 (AP-1)
(4), nuclear factor
B (NF
B) (5, 6), p53 (7), and serum response
factor (SRF) (8). SRC-1 (9) and its homolog ACTR (10), along
with CBP and p300 (11, 12), were recently shown to contain histone
acetyltransferase activities and associate with yet another histone
acetyltransferase protein p/CAF (13). In contrast, silencing mediator
of retinoid and thyroid hormone receptor (SMRT) (14) and nuclear
receptor corepressor (N-CoR) (15), nuclear receptor
corepressors, form complexes with Sin3 and histone deacetylase proteins
(16, 17). These results are consistent with the notion that acetylation
of histones destabilizes nucleosomes and relieves transcriptional
repression by allowing transcription factors to access to recognition
elements, whereas deacetylation of the histones stabilizes the
repressed state (1, 2).
We have recently isolated a novel transcription coactivator
molecule of nuclear receptors (designated ASC-2) (18). In particular,
microinjection of anti-ASC-2 antibody abrogated the ligand-dependent
transactivation of retinoic acid receptor, and this repression was
fully relieved by coinjection of ASC-2-expression vector, consistent
with an idea that ASC-2 is essential for the nuclear receptor function
in vivo (18). Interestingly, ASC-2 was found to be highly
amplified and overexpressed in colon, breast, and lung cancers (18),
although it was not clear whether the altered expression of ASC-2
directly contributed to the development of cancers. Based on the
interactions with multifunctional transcription integrators SRC-1 and
CBP/p300 (1, 2), ASC-2 was suspected to mediate transactivations by
transcription factors other than nuclear receptors. In this regard, it
was interesting to note that high levels of the ASC-2 expression in
various human breast cancer cell lines were not strictly correlated
with the estrogen receptor-
(ER
) positivity (18), an important
criterion in breast and ovarian cancers. Thus, if the altered
expression of ASC-2 plays any role in tumorigenesis, it is likely to
involve transcription factors other than ER
.
SRF, AP-1, and NF
B are known to control a surprisingly diverse set
of genes. However, it is interesting to note that these factors share
at least one common property, i.e. stimulation of cellular
proliferation processes. SRF, along with ternary complex factor (TCF),
binds to and activates the serum response element (SRE), present in the
upstream regulatory sequences of myogenic genes as well as a number of
immediate early genes, including c-fos, which in turn
activate genes critical for cell proliferation (reviewed in Ref. 19).
SRF belongs to the MADS box family of proteins and recognizes a CArG
box in the SRE, whereas TCF does not bind autonomously to the element,
but requires the assistance of SRF to efficiently contact the DNA. The
AP-1 complex, immediate early response genes, consists of a heterodimer
of a Fos family member and a Jun family member (reviewed in Ref. 20).
This complex binds the consensus DNA sequence (TGAGTCA) (termed AP1
sites) found in a variety of promoters. The Fos family contains four
proteins (c-Fos, Fos-B, Fra-1, and Fra-2), whereas the Jun family is
composed of three (c-Jun, Jun-B, and Jun-D). Fos and Jun are members of
the basic-leucine zipper (bZIP) family of sequence-specific dimeric
DNA-binding proteins. The C-terminal half of the bZIP domain is
amphipathic, containing a heptad repeat of leucines that is critical
for the dimerization of bZIP proteins. The N-terminal half of the long
bipartite-helix is the basic region that is critical for
sequence-specific DNA binding. Finally, NF
B is composed of homo- and
heterodimeric complexes of members of the Rel (NF
B) family
of polypeptides (reviewed in Ref. 21). In vertebrates, this family
comprises p50, p65 (RelA), c-rel, p52, and RelB. These proteins share a
300-amino acid region, known as the Rel homology domain, which binds to
DNA and mediates homo- and heterodimerization. This domain is also a
target of the I
B inhibitors, which include I
B
, I
Bß,
I
B
, Bcl-3, p105, and p100. In the majority of cells, NF
B
exists in an inactive form in the cytoplasm, bound to the inhibitory
I
B proteins. Treatment of cells with various inducers results in the
degradation of I
B proteins. The bound NF
B is released and
translocates to the nucleus, where it activates appropriate target
genes. Interestingly, several lines of evidence suggested that
constitutive activation of NF
B contribute to the malignant phenotype
of tumor cells. A naturally occurring splice variant of RelA was shown
to transform Rat-1 cells (22), whereas antisense oligonucleotides to
RelA were shown to inhibit proliferation and tumorigenicity of several
tumor cell lines, including the human breast cancer cell lines MCF7 and
T47D (23). In addition, activation of NF
B through the disruption of
I
B
regulation was shown to result in malignant transformation
(24).
Herein, we report the identification of SRF, AP-1, and NF
B as new
target molecules of ASC-2, in which ASC-2 directly interacts with these
factors. In cotransfections, ASC-2 potentiated their transactivations
in a dose-dependent manner and appeared to be involved with the
previously characterized transrepression between nuclear receptors and
either AP-1 (25) or NF
B (26, 27). These results indicate that ASC-2
may directly regulate the cellular proliferation or tumorigenesis
processes in vivo, by acting as a novel coactivator molecule
of these mitogenic transcription factors.
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RESULTS
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Identification of SRF as an ASC-2-Interactor
We have recently described the molecular cloning of ASC-2, a
cancer-amplified transcription coactivator molecule of nuclear
receptors (18). Based on the functional interactions with
multifunctional transcription integrators SRC-1 and CBP/p300 (1, 2),
ASC-2 was suspected to function with other transcription factors. In an
attempt to identify such factors, we have screened a mouse liver cDNA
library by using the yeast two-hybrid screening, in which the
previously described ASC-2 fragment (i.e. ASC24) (Fig. 1
) was used as bait. Consistent with the
notion that ASC-2 is a multifunctional transcription integrator
molecule, we have isolated a series of different transcription factors
and cofactors, identical or homologous to the previously characterized
factors (our unpublished results). Among these, two independent
isolates encoded the C-terminal region of SRF (i.e. the SRF
residues 203504, designated SRF
N), as shown in Fig. 2A
. This interaction was confirmed in the
in vitro glutathione S-transferase (GST)
pull-down assays. GST alone and GST-fusions to ASC24, ASC24.5, and
SRF were expressed, purified, and tested for interaction with in
vitro translated luciferase, SRF, and ASC-2. As shown in Fig. 2B
, the radiolabeled SRF readily interacted with GST-ASC24 (the ASC-2
residues 11721729) and GST-ASC24.5 (the ASC-2 residues 14292063),
but not with GST alone. Similarly, the radiolabeled ASC-2 interacted
with GST-SRF, but not with GST alone. In contrast, the radiolabeled
luciferase bound neither of the GST proteins, as expected (results not
shown). We have also examined whether the endogenous ASC-2 can bind to
SRF in vitro. As shown in Fig. 2C
, GST-SRF but not GST alone
retained the endogenous ASC-2 from HeLa nuclear extracts, as revealed
in Western analysis with the previously described specific monoclonal
antibody against ASC-2 (18). As a positive control, GST fusion to
thyroid hormone receptor (TR) was shown to retain the endogenous ASC-2
from HeLa nuclear extracts in a T3-dependent
manner. From these results, we concluded that ASC-2 specifically binds
to SRF.

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Figure 1. Schematic Representation of ASC-2
The full-length human ASC-2 and a series of 10 ASC-2 fragments are as
depicted. The glutamic acid/aspartic acid-rich (D/E), glutamine-rich
(Q-rich), glutamine/proline-rich (Q/P-rich), serine/threonine-rich
(S/T-rich), and basic amino acids (+/+) domains, as well
as two LxxLL motifs (3 46 ) that are known to be important for the
ligand-dependent interactions between coactivators and nuclear
receptors, are as indicated. The previously defined interaction domain
for nuclear receptors (RID) (18 ) and the interaction domains for c-Jun
and c-Fos (c-Jun/c-Fos-ID), p65 (p65-ID), SRF (SRF-ID), and p50
(p50-ID), along with the amino acid numbers for each construct, are
also shown.
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Figure 2. Interactions of SRF with ASC-2
A, The full-length SRF as well as SRF N (i.e. the SRF
residues 203504), which was isolated from the yeast two-hybrid
screening as an ASC-2-interactor, are as depicted. The
DNA-binding/dimerization (DBD/DD) and transactivation domains as well
as the amino acid numbers for each construct are as indicated. B, SRF
and ASC-2 were labeled with [35S]methionine by in
vitro translation and incubated with glutathione beads
containing GST alone, GST-ASC24, GST-AS24.5, or GST-SRF, as
indicated. Beads were washed, and specifically bound material was
eluted with reduced glutathione and resolved by SDS-PAGE. Approximately
20% of the total reaction mixture was loaded as input. C, HeLa nuclear
extracts were incubated with bacterially expressed GST fusions to TR
and SRF or GST alone, as indicated. - and + indicate the absence and
presence of 0.1 µM of T3, respectively.
Specifically bound proteins were released by glutathione, resolved by
SDS-PAGE, and probed with a monoclonal antibody against ASC-2 (18 ) in
Western analysis. Approximately 20% of the nuclear extracts used in
the binding reactions were loaded as input.
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Localization of the SRF-Interacting Interface of ASC-2
To localize the SRF-interaction interface of ASC-2, we
generated a series of ASC-2 fragments (Fig. 1
). In the yeast two-hybrid
tests, coexpression of a transactivation domain B42 (28) fusion to
the full-length SRF (i.e. B42-SRF) further stimulated
the LexA-ASC24 (the ASC-2 residues 11721729), LexA-ASC24.5 (the
ASC-2 residues 14292063), and LexA-ASC25 (the ASC-2 residues
15592063)-mediated LacZ expressions. However, coexpression
of B42-SRF did not affect the LexA-ASC21 (the ASC-2 residues 1557),
LexA-ASC22 (the ASC-2 residues 391-1057), and LexA-ASC23 (the ASC-2
residues 586-1310), and LexA-ASC23.5 (the ASC-2 residues
929-1511)-mediated LacZ expressions (Fig. 3A
). LexA fusions
to ASC21 and ASC22 showed autonomous transactivation function, as
previously noted (18). In addition, transactivation mediated by LexA
fusions to ASC24a (the ASC-2 residues 14291729) and ASC24b (the
ASC-2 residues 15591729) but not ASC24c (the ASC-2 residues
11721511) was enhanced by coexpression of B42 fusions to the
full-length SRF (Fig. 3A
). Similar
results were also obtained with a B42 fusion to the SRF residues
203504 (i.e. SRF
N). Consistent with these results, B42
fusions to ASC24.5, ASC24a, and ASC24b but not ASC24c
stimulated the LexA-SRF and LexA-SRF
N-mediated transactivation (Fig. 3B
). From these results, we concluded that the SRF transactivation
domain interacts with a region containing the ASC-2 residues 15591729
(as summarized in Fig. 1
).

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Figure 3. Localization of the SRF-Interacting Domain of ASC-2
The indicated B42- and LexA-plasmids were transformed into a yeast
strain containing an appropriate LacZ reporter gene, as
described (28 ). Open boxes indicate LexA (A) or B42 (B)
alone. Gray and closed boxes indicate the presence of
B42 (A) or LexA (B) fusions to SRF and SRF N, respectively, whereas
hatched box indicate the presence of LexA fusion to B42,
as a positive control. The results with a B42 fusion to SRF N were
not determined with regard to LexA fusions to ASC21, ASC22, and
ASC23. The data are representative of at least two similar
experiments, and the SDs were less than 5%.
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Stimulation of the SRF Transactivation by ASC-2
To assess the functional consequences of these interactions, ASC-2
was cotransfected into HeLa cells along with a reporter construct
SRE-c-fos-LUC. This reporter construct, previously
characterized to efficiently mediate the SRE-mediated transactivations
in various cell types (29), consists of a minimal promoter from the
c-fos gene and a single upstream consensus SRE. Serum shock
with 20% FCS resulted in approximately 10-fold increase in
transactivation of this reporter construct, relative to the level with
nonshocked cells (Fig. 4A
). Increasing
amounts of cotransfected ASC-2 enhanced the reporter gene expressions
in an ASC-2 dose-dependent manner, with cotransfection of 100 ng of
ASC-2 increasing the fold activation approximately 3-fold (Fig. 4A
). As
previously reported (8, 30), cotransfected p300 or SRC-1 also had
stimulatory effects on the reporter gene expressions. Consistent with
an idea that CBP/p300 and SRC-1 functionally cooperate with ASC-2,
coexpression of p300 or SRC-1 further increased the reporter gene
expressions above the levels observed with ASC-2 alone (Fig. 4A
). In
contrast, cotransfection of ASC-2 did not affect the LacZ
reporter expression of the transfection indicator construct
pRSV-ß-gal either in the presence or absence of serum shock (results
not shown). Similar results were also obtained with a reporter
construct driven by the previously described SRE-containing, natural
c-fos promoter (31) (Fig. 4B
). From these results, we
concluded that ASC-2 is a bona fide transcription
coactivator molecule of SRF.

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Figure 4. ASC-2 Stimulates the SRE-Mediated Transactivation
HeLa cells were transfected with LacZ expression vector
and ASC-2, p300, or SRC-1-expression vector along with a reporter gene
SRE-c-fos-LUC (29 ) (A) or the natural
c-fos promoter-driven reporter construct
c-fos-LUC (31 ) (B), as indicated. Cells were shocked
with 20% FCS before harvest, as described (8 ). Normalized luciferase
expressions from triplicate samples were calculated relative to the
LacZ expressions, and the results were expressed as
fold-activation (n-fold) over the value obtained with
unstimulated cells. The experiments were repeated at least three times,
and the SDs were as shown. Similar results were also
obtained with CV-1 and NIH3T3 cells.
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Interaction of ASC-2 with AP-1 and NF
B
The fact that ASC-2 is overexpressed in human cancers and mediates
transactivation by SRF, the well-characterized mitogenic transcription
factor (19), prompted us to examine whether ASC-2 is also involved with
other pro-proliferative transcription factors. Thus, GST alone and GST
fusions to the AP-1 components c-Jun and c-Fos as well as the NF
B
components p50 and p65 were expressed, purified, and tested for
interaction with in vitro translated ASC-2. As shown in Fig. 5A
, the radiolabeled ASC-2 interacted
with all of these GST fusion proteins except GST alone. In contrast,
the radiolabeled SRC-1 bound to GST fusions to c-Jun, c-Fos, and p50
but not p65, as we have previously reported (4, 5). These interactions
were further confirmed in yeast. Coexpression of B42 fusions to c-Jun
and c-Fos stimulated transactivation by LexA-ASC22 but not LexA
fusions to ASC23, ASC23.5, and ASC24.5 (Fig. 5B
and results not
shown). These results strongly suggest that interactions with c-Jun and
c-Fos are likely to involve the ASC-2 residues 391585, although this
prediction has yet to be independently confirmed. Coexpression of
B42-p50 stimulated transactivation by LexA fusions to ASC22, ASC23,
and ASC23.5 but not ASC24.5, suggesting that the p50 interactions
involve either the ASC-2 residues 929-1057 or multiple regions
throughout the ASC-2 residues 391-1429. Finally, coexpression of
B42-p65
N (i.e. the p65 residues 353550), which
encompasses the p65 transactivation domain, stimulated transactivation
by LexA-ASC22 but not LexA-ASC23.5 and LexA-ASC24.5, suggesting
that the p65 interactions involve the ASC-2 residues 391929. In
contrast, B42-p65
C (i.e. the p65 residues 1323) did not
show any interactions (Fig. 5B
). Overall, these results indicate that
specific subregions of ASC-2 are differentially recognized by the
AP-1/NF
B components c-Jun, c-Fos, p50, and p65, as summarized in
Fig. 1
.

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Figure 5. Interactions of ASC-2 with AP-1 and NF B
A, ASC-2 and SRC-1 were labeled with [35S]methionine by
in vitro translation and incubated with glutathione
beads containing GST alone or GST fusions to c-Jun, c-Fos, p50, and
p65, as indicated. Beads were washed, and specifically bound material
was eluted with reduced glutathione and resolved by SDS-PAGE.
Approximately 20% of the total reaction mixture were loaded as input.
B, The indicated B42- and LexA-plasmids were transformed into a yeast
strain containing an appropriate LacZ reporter gene, as
described (28 ). Open boxes indicate LexA alone, whereas
hatched, stippled, closed and gray boxes
indicate the presence of LexA fusions to ASC22, ASC23, ASC23.5,
and ASC24.5, respectively. The data are representative of at least
two similar experiments and the SDs were less than 5%.
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ASC-2 Stimulates Transactivations by AP-1 and NF
B
To assess the functional consequences of these interactions, ASC-2
was cotransfected into CV1 cells along with a reporter construct
(AP-1)4-TK-LUC (Fig. 6A
) and
(
B)4-interleukin-2 (IL-2)-LUC (Fig. 6C
), which
consist of a minimal promoter from the thymidine kinase gene and four
upstream consensus AP-1 sites (4) and a minimal promoter from the
interleukin-2 gene and four upstream
B sites from the IL-6 gene (5),
respectively. These reporter constructs were previously characterized
to efficiently mediate the AP-1 and NF
B-dependent transactivations,
respectively, in various cell types. Increasing amounts of
cotransfected ASC-2 enhanced the reporter gene expressions in an ASC-2
dose-dependent manner. As previously noted (2, 4, 5), cotransfected
SRC-1 or p300 also had stimulatory effects on these reporter gene
expressions. Consistent with an idea that CBP/p300 and SRC-1
functionally cooperate with ASC-2, coexpression of p300 or SRC-1
further increased the reporter gene expressions above the levels
observed with ASC-2 alone (Fig. 6
, A and C). ASC-2 also coactivated the
12-O-tetradecanoylphorbol-13 acetate (TPA)- or tumor
necrosis factor-
(TNF
)-induced level of transactivations, whereas
cotransfection of ASC-2 did not affect the LacZ reporter
expression of the transfection indicator construct pRSV-ß-gal either
in the presence or absence of TPA or TNF
(results not shown). To
investigate the function of ASC-2 in vivo, the
microinjection technique (32) was further used, in which the
LacZ reporter gene was placed under the control of an SV40
minimal promoter containing TPA-responsive AP-1 sites. Remarkably,
microinjection of anti-ASC-2 IgG significantly prevented TPA from
activating this TPA-dependent transcription unit (Fig. 6B
) but had no
effect on a promoter under the control of the cytomegalovirus promoter
(results not shown). The percentage of cells that expressed the
LacZ reporter (i.e. blue cells) was not
significant among cells microinjected with control IgG in the absence
of TPA but increased to approximately 45% in the presence of TPA.
However, only approximately 10% of cells turned blue even in the
presence of TPA, when microinjected with anti-ASC-2 IgG (Fig. 6B
). This
ASC-2-mediated repression of the TPA response was fully relieved by
coinjected ASC-2-expression vector but not by pcDNA3 (Fig. 6B
). Taken
together with the transient transfection data (Fig. 6A
), we concluded
that ASC-2 is a molecule pivotal for the function of AP-1 in
vivo. These results, along with the results with SRF (Fig. 4
),
NF
B (Fig. 6B
), and nuclear receptors (18), strongly suggest that
ASC-2 is a multifunctional transcription integrator molecule, like
CBP/p300 and SRC-1.

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Figure 6. ASC-2 Stimulates the AP-1 and NF B
Transactivations
A and C, CV-1 cells were transfected with LacZ
expression vector and ASC-2, SRC-1 or p300-expression vector along with
a reporter gene (AP-1)4-TK-LUC (A) or
( B)4-IL-2-LUC (C), as indicated. Transfections were done
essentially as described (4 5 ). Normalized luciferase expressions from
triplicate samples were calculated relative to the LacZ
expressions, and the results were expressed as fold-activation
(n-fold) over the value obtained with unstimulated
cells. Similar results were also obtained with TPA and TNF as well
as other cell lines including HeLa and NIH3T3. B, Rat-1 cells were
microinjected with control IgG or anti-ASC-2 IgG, along with a reporter
construct (i.e. AP-1-SV40-ß-GAL), pcDNA3, and
pcDNA3-ASC-2, as indicated. The number of cells that express
LacZ is counted relative to the total number of
microinjected cells, and expressed as % blue cells, as indicated.
Experiments were done at least three times, with >200 cells injected;
error bars are ± 2 x SEM. Open and
closed boxes indicate the absence and presence of 0.1
µM of TPA, respectively.
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ASC-2 Is Involved with the Transrepression between Nuclear
Receptors and AP-1/NF
B
Mutual transcriptional inhibitions have been described between
various liganded nuclear receptors and either AP-1 or NF
B, which was
suggested to be due to an interaction among these factors that results
in mutual loss of DNA-binding activity (25, 26, 27). More recently,
competition for common transcription coactivators such as CBP/p300 and
SRC-1 was also proposed to be responsible for this mutual antagonism
(4, 33). ASC-2 shows similar effects. Coexpression of ASC-2 enhanced
the T3-dependent transactivation by TR in a
dose-dependent manner (Fig. 7A
). As
previously noted (4, 5, 27), however, the
T3-dependent transactivation by TR was repressed
by coexpression of c-Fos or p65 (Fig. 7B
), whereas liganded TR
efficiently repressed transactivation of both AP-1 and
NF
B-responsive reporter constructs (Fig. 7
, C and D). These
inhibitory effects were largely relieved upon addition of increasing
amounts of ASC-2. These results strongly indicate that ASC-2 is
directly involved with the transrepressions between nuclear receptors
and either AP-1 or NF
B.
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DISCUSSION
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ASC-2 was previously shown to have characteristics that are
typical of a bona fide transcription coactivator molecule of
nuclear receptors (18), i.e. ASC-2 showed a strong ligand-
and AF2-dependency in interactions with nuclear receptors, a potent
autonomous transactivation function, and specific interactions with the
basal transcription machinery. Most strikingly, microinjection of the
ASC-2 antibody completely abrogated the ligand-dependent reporter gene
activities in Rat-1 fibroblast cells, which was rescued by coexpressed
ASC-2 (18). In this report, our original hypothesis (18) that ASC-2
could function as a multifunctional transcription integrator molecule,
like CBP/p300 and SRC-1, has been further strengthened. First, we have
isolated a series of transcription factors and cofactors, including SRF
(Fig. 2
), as ASC-2-interacting proteins from the yeast two-hybrid
screening (our unpublished results). Second, ASC-2 was shown to
stimulate transactivations by SRF (Fig. 4
), AP-1, and NF
B (Fig. 6
),
likely through specific interactions with these factors (as summarized
in Fig. 1
). Third, ASC-2 appeared to be involved with the previously
defined transrepression between nuclear receptors and either AP-1 or
NF
B (Fig. 7
). This ASC-2-mediated reversal of the transrepression
between TR and AP-1/NF
B (Fig. 7
) suggests that a limiting amount of
ASC-2 is competitively recruited by these transcription factors.
However, it is also possible that ASC-2 may titrate out a putative,
secondary factor, which links TR to AP-1/NF
B and is required for the
transrepression. Alternatively, the exogenously supplied ASC-2 could be
blocking the protein-protein interactions of TR with AP-1/NF
B
(i.e. AP-1 and NF
B may directly compete with nuclear
receptors to recruit ASC-2). Consistent with this idea, we noticed that
the c-Jun/c-Fos, p65, and p50-interaction domains of ASC-2 are
clustered around the previously defined receptor interaction domain (as
summarized in Fig. 1
). Finally, its interesting to note that the
ASC-2 interaction domains of SRF (Fig. 2
) and p65 (Fig. 5B
) overlap
with their previously defined autonomous transactivation domains
(i.e. the N-terminal region of SRF and the C-terminal region
of p65) (20, 21).
Considering the fact that ASC-2 is highly amplified in human cancers
(18), its particularly interesting to note that these newly
identified target proteins of ASC-2 are mitogenic transcription
factors. Recently, AIB1, an SRC-1 family member, was identified as a
gene amplified and overexpressed in breast and ovarian cancers (34).
SRC-1 and its family members were also shown to serve as a novel
transcription coactivator molecule of AP-1 (4), NF
B (5, 6), and SRF
(8). Interestingly, ASC-2 maps to 20q11, substantially centromeric to
AIB1 (which maps to 20q12) (35), demonstrating that two distinct
coactivator molecules of nuclear receptors can be coamplified in cancer
cells. Such a co-selection process may favor genes that impinge on the
same cellular processes and result in significant effects on
transcriptional regulation within tumor cells. In particular,
overexpression of these multifunctional integrator molecules may
provide a selective advantage for tumor growth. Thus, it remains to be
determined whether these mitogenic factors (i.e SRF, NF
B,
and AP-1) are indeed targeted by increased levels of these coactivator
proteins in vivo to sustain tumor growth. However, it is
interesting to note that the inhibitory effects of the AP-1 and NF
B
transactivations by T3 or retinoids can be
significantly attenuated when ASC-2 is overexpressed (Fig. 7
and
results not shown). These results raise an interesting possibility that
overexpressed ASC-2 may contribute to the induction of retinoid
resistance in certain human cancers. In breast cancer cells, both
overexpression of c-Jun and decreased expression of retinoid receptors
were previously shown to result in such retinoid resistance (36, 37).
Our recent unpublished data indicate that ASC-2 forms a distinct
steady-state coactivator complex in vivo and
functionally communicates with other coactivator complexes such as
CBP/p300/SRC-1 (38) and ASC-1 (39). The ASC-2 complex was different
from the ASC-1, CBP/p300, and SRC-1-complexes (38, 39) when eluted from
a sizing column. Interestingly, this endogenous ASC-2 complex was
readily retained by GST fusions to SRF, TR, and p50 (Fig. 2C
and
results not shown). In particular, the TR interactions were strictly
T3-dependent, as expected (18). Similar to the
previously shown results with nuclear receptors (18), microinjection of
ASC-2 antibody effectively disrupted the AP-1 transactivation by TPA
(Fig. 6B
), suggesting that this ASC-2 complex is essential for at least
two classes of transcription factors in vivo,
i.e. AP-1 and nuclear receptors. Currently, one of our major
focuses is to elucidate the molecular mechanisms by which the ASC-2
complex functions and its activity is regulated. First, we are
biochemically dissecting the components of the ASC-2 complex. Second,
we have recently found that expression of ASC-2 mRNA is up-regulated in
certain cells by various cytokines and growth factors, including IL-1
and epidermal growth factor (EGF), activating signals for NF
B and
AP-1/SRF, respectively (our unpublished results). Similarly,
transcripts of the SRC-1 family member RAC3 (40) and the nuclear
receptor corepressor molecule SMRT (14) were recently shown to be
directly up-regulated by retinoids (41, 42). Expression of PGC-1, a
thermogenic tissue-specific coactivator of nuclear receptors, was also
shown to be temporally regulated (43). To understand this up-regulation
of ASC-2, we have recently isolated its full-promoter from a human
genomic library, which contains a series of interesting regulatory
sites, including AP-1 sites (results not shown). Finally, ASC-2
contains a numerous number of putative phosphorylation sites (18) and,
thus, the possible regulation of its inherent activity through various
signal transduction pathways is being examined. Recently, this notion
has been confirmed with CBP, in which a calcium flux was shown to
function as an activating signal for its stimulatory transcriptional
activity (44).
In conclusion, we identified three mitogenic transcription factors
(i.e. SRF, AP-1, and NF
B) as novel targets for ASC-2,
which may contribute to the putative, ASC-2-mediated tumorigenesis. We
have also shown that ASC-2 is a multifunctional transcription
integrator molecule, like SRC-1 (1, 3, 4, 5, 6, 7, 8) and CBP/p300 (2). Thus,
further characterization of ASC-2 may provide important insights into
the tumorigenesis processes as well as the molecular mechanisms by
which multiple transcription factors are coordinately regulated within
the cell.
 |
MATERIALS AND METHODS
|
---|
Plasmids
To express LexA- and B42-fusions, PCR fragments encoding
ASC21, ASC22, ASC23, ASC23.5, ASC24, ASC24.5, ASC25,
ASC24a, ASC24b, ASC24c, and p65
N (i.e. the p65
residues 353550) and p65
C (i.e. the p65 residues
1323) were cloned into EcoRI and
XhoI/SalI restriction sites of pEG202PL and
pJG45 (28), respectively. For GST fusion vectors, PCR fragments
encoding ASC24 and ASC24.5 were cloned into EcoRI and
XhoI restriction site of pGEX4T (Pharmacia Biotech, Piscataway, NJ). The AP-1-SV40-ß-GAL reporter
construct was a gift from Dr. Dave Rose (University of California, San
Diego, CA). GST fusion vectors encoding SRF, TR, c-Jun, c-Fos, p50, and
p65 as well as the mammalian expression vectors for SRF, ASC-2, p300,
SRC-1, c-Fos, p65, and TR, along with the transfection indicator
construct pRSV-ß-gal and reporter constructs
SRE-c-fos-LUC, c-fos-LUC, T3RE-TK-LUC,
(AP-1)4-TK-LUC, and
(
B)4-IL-2-LUC, were as previously described
(4, 5, 7, 8, 18, 27, 29, 31, 39).
The Yeast Two-Hybrid Screening and Yeast ß-Galactosidase
Assay
The LexA-ASC24 (Fig. 1
) was used as a bait to screen a mouse
liver cDNA library in pJG45 (28) for ASC-2-interacting proteins, and
the screening was executed essentially as previously described (45).
The yeast ß-galactosidase assay was done as described (28). For each
experiment, at least three independently derived colonies expressing
chimeric proteins were tested.
GST Pull-Down Assays
The GST-fusions or GST alone was expressed in Escherichia
coli, bound to glutathione-Sepharose-4B beads (Pharmacia Biotech), and incubated with labeled proteins expressed by
in vitro translation by using the TNT-coupled
transcription-translation system, with conditions as described by the
manufacturer (Promega Corp., Madison, WI). Specifically
bound proteins were eluted from beads with 40 mM
reduced glutathione in 50 mM Tris (pH 8.0) and
analyzed by SDS-PAGE and autoradiography as described (28).
Cell Culture and Transfections
HeLa, NIH3T3, and CV-1 cells were grown in 24-well plates with
medium supplemented with 10% FCS for 24 h and transfected with
100 ng of LacZ expression vector pRSV-ß-gal and 100 ng of
an indicated reporter gene, along with indicated amounts of various
mammalian expression vectors. Total amounts of expression vectors were
kept constant by adding pcDNA3. Transfections and luciferase assays
were done as described (4, 5, 8), and the results were normalized to
the LacZ expression. Similar results were obtained in more
than two similar experiments.
Single-Cell Microinjection Assay
Rat-1 fibroblast cells, made quiescent by incubating in
serum-free medium for 24 h, were microinjected with either
preimmune IgG or the affinity-purified anti-ASC-2 IgG along with
AP-1-SV40-ß-GAL reporter construct (25 µg/ml). About 1 h after
injection, cells were stimulated, where indicated, with 0.1
µM TPA. After 4 h incubation, cells were fixed and
stained to detect injected IgG by using fluorescein
isothiocyanate (FITC)-conjugated antibodies and examined for
ß-galactosidase expression as previously described (32).
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Dave Rose for the LacZ reporter
construct.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Jae Woon Lee, Ph.D., Center for Ligand and Transcription, Chonnam National University, Kwangju 500757, Korea.
This work was supported by a grant from the National Creative Research
Initiatives Program from the Korean Ministry of Science and Technology,
Republic of Korea. B.H.J was supported by a grant from the Korean
Ministry of Public Health (HMP-98-B-30022).
Received for publication November 16, 1999.
Revision received January 24, 2000.
Accepted for publication February 24, 2000.
 |
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