Two Distinct Nuclear Receptor-Interaction Domains and CREB-Binding Protein-Dependent Transactivation Function of Activating Signal Cointegrator-2
Soo-Kyung Lee1,
Sung-Yun Jung,
Youn-Sung Kim,
Soon-Young Na2,
Young Chul Lee and
Jae Woon Lee
Center for Ligand and Transcription (S.-K.L., S.-Y.J., Y.-S.K.,
S.Y.N. Y.C.L., J.W.L.) Department of Biology (S.-K.L.,,
S.-Y.N.) and Hormone Research Center (Y.C.L., J.W.L.) Chonnam
National University Kwangju 500757, Korea
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ABSTRACT
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ASC-2 is a recently isolated transcriptional
cointegrator molecule, which is amplified in human cancers and
stimulates transactivation by nuclear receptors, AP-1, nuclear factor
B (NF
B), serum response factor (SRF), and numerous other
transcription factors. ASC-2 contained two nuclear receptor-interaction
domains, both of which are dependent on the integrity of their core
LXXLL sequences. Surprisingly, the C-terminal LXXLL motif specifically
interacted with oxysterol receptor LXRß, whereas the N-terminal motif
bound a broad range of nuclear receptors. These interactions appeared
to be essential because a specific subregion of ASC-2 including the N-
or C-terminal LXXLL motif acted as a potent dominant negative mutant
with transactivation by appropriate nuclear receptors. In addition, the
autonomous transactivation domain (AD) of ASC-2 was found to consist of
three separable subregions; i.e. AD1, AD2, and AD3. In
particular, AD2 and AD3 were binding sites for CREB binding protein
(CBP), and CBP-neutralizing E1A repressed the autonomous
transactivation function of ASC-2. Furthermore, the receptor
transactivation was not enhanced by ASC-2 in the presence of E1A and
significantly impaired by overexpressed AD2. From these results, we
concluded that ASC-2 directly binds to nuclear receptors and
recruits CBP to mediate the nuclear receptor transactivation in
vivo.
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INTRODUCTION
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The nuclear receptor superfamily is a group of ligand-dependent
transcriptional regulatory proteins that function by binding to
specific DNA sequences named hormone response elements in the promoters
of target genes (1). The superfamily includes receptors for a variety
of small hydrophobic ligands such as steroids,
T3, and retinoids, as well as a large number of
related proteins that do not have known ligands, referred to as orphan
nuclear receptors. Functional analysis of nuclear receptors has shown
that there are two major activation domains. The N-terminal domain
(AF-1) contains a ligand-independent activation function, whereas the
ligand-binding domain (LBD) (AF-2) exhibits ligand-dependent
transactivation. The AF-2 core region, located at the extreme C
terminus of the receptor LBDs, is conserved among nuclear receptors and
undergoes a major conformational change upon ligand binding. Notably,
deletion or point mutations in this region impair transcriptional
activation without changing ligand and DNA binding affinities. This
region has been shown to play a critical role in mediating
transactivation by serving as a ligand-dependent interaction interface
with many different coactivators (2, 3).
Transcriptional coactivators either bridge transcription factors and
the components of the basal transcriptional apparatus and/or remodel
the chromatin structures (2, 3). 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 (4). 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 transducers and activators of transcription (STATs)
(5), AP-1 (6), nuclear factor
B (NF
B) (7, 8), p53 (9), and serum
response factor (SRF) (10). SRC-1 (11) and its homolog ACTR
(12), along with CBP and p300 (13, 14), were recently shown to contain
histone acetyltransferase (HAT) activities and associate with yet
another HAT protein P/CAF (15). In contrast, SMRT (16) and N-CoR (17),
nuclear receptor corepressors, form complexes with Sin3 and histone
deacetylase proteins (18, 19). 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 (2, 3).
We have recently described an AF-2 and ligand-dependent coactivator
molecule of nuclear receptors that we named activating signal
cointegrator-2 (ASC-2) (20, 21). Similar or identical molecules were
subsequently reported by other groups and variously named TRBP (for
thyroid hormone receptor-binding protein) (22), PRIP (for peroxisome
proliferator-activated receptor interacting protein) (23), and RAP250
(for nuclear receptor-activating protein 250) (24). Like SRC-1 and
CBP/p300, ASC-2 also enhanced transactivation by a large number of
other transcription factors, including CREB, SRF, NF
B, and AP-1 (21, 22). In particular, microinjection of anti-ASC-2 antibody abrogated the
ligand-dependent transactivation of retinoic acid receptor (RAR) and
the TPA-inducible AP-1 transactivation, suggesting that ASC-2 is
essential for the nuclear receptor and AP-1 functions in
vivo (20, 21). Northern blot and in situ hybridization
analyses revealed that ASC-2 is widely expressed with the highest
expression in reproductive organs and brain (24). Interestingly, ASC-2
was found to be highly amplified and overexpressed in human colon,
breast, and lung cancers (20). However, it still remains an important,
unresolved issue whether or not the altered expression of ASC-2
directly contributes to the development of cancers.
In this report, we analyzed various functional domains of ASC-2 in an
effort to understand its role in mediating transactivation of nuclear
receptors. Surprisingly, ASC-2 contained two LXXLL-type nuclear
receptor-interaction domains (5, 25), each of which was differentially
used by different member(s) of the nuclear receptor superfamily. ASC-2
also appeared to directly bind nuclear receptors and subsequently
recruit CBP to exert the receptor transactivation function in
vivo.
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RESULTS
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Identification of Two Nuclear Receptor-Interaction Domains
We originally proposed the ASC-2 residues 586860 to be a minimum
receptor-interacting domain, based on its interaction with thyroid
hormone receptors (TRs) in the yeast two-hybrid assays (20). However,
this region was subsequently found to bind TR
very poorly in the
glutathione S-transferase (GST) pull-down assays (results
not shown), which prompted us to reexamine the nuclear
receptor-interaction domains of ASC-2. Two potentially
-helical
LXXLL motifs and a region distantly related to this motif
(i.e. IXXIM) as indicated in Fig. 1A
were suspected to be responsible for
the nuclear receptor interactions. This LXXLL motif, in which L and X
denote leucine and any amino acid, respectively, was recently shown to
be responsible for the AF-2 and ligand-dependent interactions of
nuclear receptors with coactivators (5, 25). However, the ASC22b
fragment (i.e. the ASC-2 residues 622849) containing the
wild-type IXXIM motif or its mutated version (m1 in Fig. 1B
) bound none
of the nuclear receptors tested in yeast, as shown in Table 1
. However, the ASC22c fragment
(i.e. the ASC-2 residues 8491,057), which includes the
first LXXLL motif, showed a broad range of target specificity in yeast,
binding retinoid X receptor (RXR), RAR, TR
, TRß, estrogen receptor
(ER
), ERß, peroxisome proliferator-activated receptor
(PPAR
), and PPAR
(Table 1
and results not shown). Notably, this
fragment did not interact with oxysterol receptor LXRß/RIP15 (26, 27). As suspected, the LXXLL motif (5, 25) was essential in these
interactions, as demonstrated by the complete loss of the receptor
interactions by mutations in this region (i.e. m2 in Fig. 1B
) (Table 1
). Consistent with these results, similar conclusions were
independently drawn from other laboratories (22, 23, 24). Finally, the
second LXXLL motif contained in the ASC-2 residues 1,1721,511
(i.e. ASC24C), which previously showed a very weak
ligand-dependent interaction with RXR in the GST pull-down assays (20),
did not interact with any of the receptors tested in yeast, as also
shown by other groups (22, 23, 24). Surprisingly, however, this region
strongly bound LXRß (26, 27), and mutation of the LXXLL motif to
LXXAA (i.e. m3 in Fig. 1B
) resulted in a complete loss of
the ASC-2-LXRß interactions (Table 1
). Similarly, ASC24
(i.e. the ASC-2 residues 1,1721,729), ASC24a
(i.e. the ASC-2 residues 1,4291,729) and ASC24LR
(i.e. the ASC-2 residues 1,4291,511) showed a relatively
strong basal interaction with LXRß in yeast, which was further
stimulated by ligand (Table 1
and results not shown). As expected,
ASC24b (i.e. the ASC-2 residues 15591729) that does not
contain the second LXXLL motif did not interact with LXRß (results
not shown). Further corroborating with these results, we have
independently isolated a partial cDNA clone encoding the LBD of LXRß
by using ASC24 as bait in a separate yeast two-hybrid screening of a
mouse liver cDNA library (results not shown).

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Figure 1. Schematic Representation of ASC-2
A, 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), and serine/threonine-rich
(S/T-rich), as well as three LXXLL-like motifs (5 25 ) that are known
to be important for the ligand-dependent interactions between
coactivators and nuclear receptors, are as indicated. The identified
interaction domains for nuclear receptors, along with the amino acid
numbers for each construct, are also shown. B, The sequences of three
LXXLL-like motifs are as indicated, along with their mutated sequences.
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These interaction results were further confirmed in the mammalian
two-hybrid tests as well as the GST pull-down assays. In the mammalian
two-hybrid tests, ASC22c and ASC24a showed a ligand-stimulated
interaction with LXRß and RAR, respectively (Fig. 2
, A and C). In the GST pull-down assays,
radiolabeled ASC22c but not ASC22c/m2, the LXXAA mutant
version, specifically bound GST-TRß in a
T3-dependent manner but neither GST alone nor
GST-LXRß (Fig. 2B
). In contrast, GST-LXRß specifically associated
with radiolabeled ASC24a, but not the LXXAA mutant form ASC24a/m3,
only in the presence of its ligand 22-OH-cholesterol (Fig. 2D
).
Interestingly, the basal interactions observed in yeast were not
visible in this in vitro assay. Neither GST alone nor GST
fusion to RAR or TRß was able to bind this region of ASC-2. Overall,
these results led us to conclude that ASC-2 contains at least two
LXXLL-dependent receptorinteraction domains, the N-terminal LXXLL
motif associating with a broad range of nuclear receptors and the
C-terminal motif specifically binding LXRß.

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Figure 2. Interactions of Nuclear Receptors with ASC-2
A and C, HeLa cells were transfected with LacZ
expression vector (100 ng) and VP16 fusion to the full-length LXRß or
RAR (100 ng), along with expression vectors for Gal4 alone,
Gal4/ASC22c and Gal4/ASC24a (100 ng), and a reporter gene
Gal4-TK-Luc (100 ng), as indicated. All-trans-RA and
22-OH-cholesterol (at a concentration of 100
nM) were used as ligands for RAR and
LXRß, respectively. Normalized luciferase expressions from triplicate
samples were calculated relative to the LacZ
expressions. The experiments were repeated at least three times and the
representative results were expressed as fold-activation
(n-fold) over the value obtained with Gal4 alone,
with the error bars as indicated. Similar results were
also obtained with CV-1 cells. B and D, The full-length ASC-2 and
various ASC-2 fragments were labeled with [35S]methionine
by in vitro translation and incubated with glutathione
beads containing GST alone, GST-TRß, GST-RAR, and GST-LXRß, as
indicated. ASC22c/m2 appears slightly larger than its wild-type
version because it is hemagglutinin-tagged and contains the
nuclear localization signal of SV40 T antigen. Beads were washed, and
specifically bound material was eluted with reduced glutathione and
resolved by SDS-polyacrylamide gel electrophoresis. Approximately 5%
of the total reaction mixture were loaded as input. and + indicate
the absence and presence of 100 nM of
all-trans-RA, T3, or 22-OH-cholesterol, respectively.
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The N-Terminal LXXLL Motif Binds Similar TR Helices Recognized by
SRC-1 and SMRT
Surprisingly, similar LXXLL-type motifs have recently been shown
to mediate the receptor interactions of not only coactivators but also
corepressors, in which corepressors associated with specific residues
in the same TR pocket required for coactivator binding (28, 29). By
using the identical set of TRß-LBD mutants described in these works
(28, 29), we searched for any mutation of TR that might distinguish
ASC-2 from other receptor cofactors such as SRC-1 and SMRT, which would
be of great value in elucidating the ASC-2 function. As shown in Table 2
, the TR-ASC22c interactions were
abolished by specific mutations in the TRß helices 3, 5, and 6, which
were known to form the binding pocket for SRC-1 and SMRT (28, 29).
Notably, these mutant TRs were shown to be nonfunctional in mediating
the basal repression (28, 29) as well as the
T3-dependent transactivation (results not shown).
Interestingly, mut2 in the TRß helix 1 did not interact with SMRT in
yeast, although it was previously shown to bind N-CoR in the GST
pull-down assays (29). Overall, these results indicate that the
ASC-2-receptor interactions are very similar to those of other
coactivators and corepressors. However, a more extensive mutagenesis
study is warranted to find TR mutants that specifically lack its
ability to bind ASC-2 but not other cofactors.
Direct Bindings to Nuclear Receptors Required for the ASC-2
Function
As we have previously shown (20), ASC-2 is an essential
coactivator for the RAR transactivation. To assess the functional
importance of the N-terminal LXXLL motif in the RAR transactivation, we
cotransfected various ASC-2 fragments containing either the wild-type
LXXLL or mutated LXXAA sequences. Interestingly, ASC22c but not
ASC22c/m2 showed a dominant negative phenotype with the RAR or TR
transactivation (Fig. 3
and results
not shown). Similarly, expression of ASC-2 residues 7861,132
was recently shown to specifically repress the 9-cis-RA and
BRL 49653directed transactivation by RXR and PPAR
,
respectively (23). Consistent with the above interaction results,
ASC-2 is also a bona fide transcription coactivator molecule
of LXRß (26, 27). As reported previously (26), coexpression of
increasing amounts of LXRß showed a constitutive activation of the
LXRE-Luc reporter gene expression in a LXRß-dose dependent manner
(results not shown). This basal level of the reporter gene expression
was further enhanced by coexpressed ASC-2 (Fig. 4A
). 22-OH-cholesterol or 24,
25-epoxy-cholesterol, the recently described ligands for LXRß (26),
stimulated the reporter gene expression approximately 2- to 4-fold,
which was further enhanced by coexpressed ASC-2 (Fig. 4A
and results
not shown). As expected, the LXRE-Lucdependent transactivation was
not observed either in the absence or presence of 22-OH-cholesterol
when LXRß was not cotransfected (Fig. 4B
). Interestingly, a
full-length ASC-2 in which LXXAA sequences were incorporated into the
second LXXLL motif (i.e. ASC-2/m3) was completely inert,
supporting the importance of the second LXXLL motif in the LXRß
transactivation (Fig. 4A
). In contrast, ASC-2/m3 was as functional as
the wild-type ASC-2 with the RAR transactivation. Furthermore,
overexpression of ASC24LR but not ASC24LR/m3 directed a potent
dominant negative phenotype with the LXRß transactivation (Fig. 4B
).
From these results, direct bindings to receptors through the LXXLL
motifs appeared to be essential for the ASC-2 action with nuclear
receptors.

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Figure 3. The N-Terminal LXXLL Subregion as a Dominant
Negative Mutant for the Receptor Transactivation
HeLa cells were transfected with LacZ expression vector
(100 ng) and ASC22c or ASC22c/m2-expression vector along with a
reporter gene ßRARE-Luc (100 ng), as indicated. For optimal RA
response, 10 ng of RAR expression vector were also cotransfected.
Normalized luciferase expressions from triplicate samples were
calculated relative to the LacZ expressions. The
experiments were repeated at least three times, and the representative
results were expressed as fold-activation (n-fold) over the value
obtained with no ligand and ASC-2, with the error bars
as indicated. Open and closed boxes
indicate the absence and presence of 100 nM of
all-trans-RA, respectively. Similar results were also
obtained with TRE-Luc and CV-1 cells.
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Figure 4. The C-Terminal LXXLL Motif Required for the LXRß
Transactivation
HeLa cells were transfected with LacZ expression vector
(100 ng) and ASC-2, ASC-2/m3, ASC24LR, ASC24LR/m3, or
LXRß-expression vector along with a reporter gene ßRARE-Luc (100
ng) or LXRE-Luc (100 ng), as indicated. For optimal RA-response with
the ßRARE-Luc construct, 10 ng of RAR-expression vector were also
cotransfected. Normalized luciferase expressions from triplicate
samples were calculated relative to the LacZ
expressions. The experiments were repeated at least three times, and
the representative results were expressed as fold-activation (n-fold)
over the value obtained with no ligand and ASC-2, with the error
bars as indicated. Open and closed
boxes indicate the absence and presence of 100 nM
of all-trans-RA or 10 µM of
22-OH-cholestrerol, respectively. Similar results were also obtained
with CV-1 cells.
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Dissection of the Autonomous Transactivation Domains of ASC-2
We have previously mapped at least two distinct autonomous
transactivation domains of ASC-2 in yeast; i.e. a very
strong transactivation domain contained within ASC-2 residues 1557
and a rather modest transactivation domain within ASC-2 residues
391-1057 (20). Surprisingly, these ASC-2 fragments showed reversed
transactivation potency in mammalian cells, suggesting that their exact
mode of actions is distinct between yeast and mammalian cells. In
mammalian cells, the transactivation function within the ASC-2 residues
1557 was localized to the ASC-2 residues 1218 (as summarized in
Fig. 5A
). Notably, this transactivation
function was modest in contrast to its strong activity in yeast (20),
and we named this region autonomous transactivation domain 1 (AD1). As
shown in Fig. 5B
, ASC2-II (i.e. ASC-2 residues 391930)
contained a potent transactivation function in mammalian cells, which
was further localized to ASC22b (i.e. ASC-2 residues
622849) (designated AD2) (Fig. 5B
). In addition, ASC22c
(i.e. ASC-2 residues 849-1057) also showed a significant
activity. Thus, the modest autonomous transactivation function in yeast
(20), included within ASC-2 residues 3911,057, was mapped to two
separable subregions in mammalian cells; i.e. AD2 and AD3
(Fig. 5A
). Notably, the activity of AD3 was relatively poor, whereas
AD2 was much stronger than AD1 and AD3 (Fig. 5B
). Consistent with the
yeast results (20), the C-terminal region of ASC-2 (i.e.
ASC2-III and ASC24.5) did not show any evidence of autonomous
transactivation function. From these results, it was clear that ASC-2
contains three separable transactivation domains. Each of these
multiple transactivation domains may represent just part of a
consecutive, single transactivation domain, cooperating with each other
to achieve the maximum transactivation potential of ASC-2.
Alternatively, it is possible that each of these domains may represent
an independent transactivation function, which differentially responds
to various activating signals under certain conditions.

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Figure 5. Localization of the Autonomous Transactivation
Domains of ASC-2
A, The autonomous transactivation domains of ASC-2 (AD1, AD2, and AD3)
are schematically indicated, along with a series of nine ASC-2
fragments. B, HeLa cells were transfected with LacZ
expression vector (100 ng), expression vectors for various Gal4 fusions
(100 ng), and a reporter gene Gal4-TK-Luc (100 ng), as indicated.
Normalized luciferase expressions from triplicate samples were
calculated relative to the LacZ expressions. The
experiments were repeated at least three times, and the representative
results were expressed as fold-activation (n-fold) over the value
obtained with Gal4 alone, with the error bars as
indicated. Similar results were also obtained with CV-1 cells.
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AD2 and AD3 as CBP-Interaction Interfaces
We have previously mapped the SRC-1 and CBP-binding sites to the
N-terminal half of ASC-2 (20). In this report, we found that ASC22b
encompassing the strongest transactivation domain AD2 was able to
interact with the previously described CBP-E fragment (i.e.
CBP residues 1,8672,441) (Fig. 6A
). The
CBP-AD2 interactions were further mapped to the CBP-E/C region
(i.e. CBP residues 2,1712,441) and the ASC22b/C
(i.e. ASC-2 residues 737849) (Fig. 6
, A and B). In
addition, the ASC22b/AD2 region bound weakly to SRC1-D
(i.e. SRC-1 residues 7591,141) and very strongly to SRC-E
(i.e. SRC-1 residues 1,1011,441) (Fig. 6A
). Interestingly,
ASC22c encompassing the AD3 also bound CBP-E/C but not any of the
SRC-1 fragments (Fig. 6C
), whereas ASC2-I encompassing the AD1 did not
bind any CBP fragment in yeast (results not shown). The LXXLL motifs
similar to those involved with the receptor-coactivator interactions
(5, 25) were also found within the SRC-1-CBP binding domains (2, 3, 4).
Thus, we tested whether these motifs included within the ASC22b and
ASC22c are involved with the observed ASC-2-CBP interactions.
However, neither motif was involved with the CBP and SRC-1 bindings in
yeast, as demonstrated by the lack of any effect by mutations in these
motifs (Table 3
). The specific ASC-2
residues involved with the CBP and SRC-1 bindings are currently being
extensively mapped. Consistent with these results, the full-length
ASC-2 interacted with GST fusions to either CBP4 (i.e. CBP
residues 1,4591,891) or CBP5 (i.e. CBP residues
1,8912,441) in the GST pull-down assays, whereas ASC22bc and
ASC22 interacted only with GST-CBP5 (Fig. 6D
and results not shown).
Overall, these studies identified AD2 and AD3 as CBP-interaction
interfaces and AD2 as an SRC-1-binding domain.

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Figure 6. Localization of the CBP-Binding Domain of ASC-2
A, B, and C, The indicated B42- and LexA-plasmids were
transformed into a yeast strain containing an appropriate
LacZ reporter gene, as described (40 ).
Closed and hatched boxes indicate the
presence of LexA fusions to ASC22b (A) and ASC22c (C) or ASC22b/N
(i.e. the ASC-2 residues 622736) and ASC22b/C
(i.e. the ASC-2 residues 737849) (B), respectively.
CBP-A, B, C, D, E, E/N, and E/C indicate the CBP residues 1446,
452721, 7221,166, 1,1611,752, 1,8672,441, 1,8672,170, and
2,1712,441, respectively. SRC-A, B, C, D, and E indicate the SRC-1
residues 1361, 361568, 568779, 7591,141, and 1,1011,441,
respectively. The data are representative of at least two similar
experiments, and the error bars are as indicated. D, The
full-length ASC-2 and ASC22bc were labeled with
[35S]methionine by in vitro translation
and incubated with glutathione beads containing GST alone, GST-CBP4,
and GST-CBP5, as indicated. Beads were washed, and specifically bound
material was eluted with reduced glutathione and resolved by SDS-PAGE.
Approximately 5% of the total reaction mixture were loaded as input.
Similarly, the full-length CBP labeled with
[35S]methionine by in vitro translation
specifically bound GST-ASC22bc (results not shown).
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The Autonomous Transactivation Function of ASC-2 Requires CBP
Interestingly, transactivation by Gal4 fusion to AD2 in the
context of ASC22b or ASC22bc was significantly stimulated by
coexpressed CBP but neither SRC-1 nor P/CAF (Fig. 7A
). Consistent with the lack of CBP
interactions, however, transactivation by Gal4 fusion to ASC2-Ia
(i.e. AD1) was not enhanced by coexpressed CBP (results not
shown). Unexpectedly, however, transactivation by Gal4 fusion to
ASC22c (i.e. AD3) was not stimulated by coexpressed CBP,
despite the fact that this fragment also binds to CBP (Fig. 6C
).
Interestingly, a mutant CBP defective for its HAT activity
(i.e. F1541A) (30) stimulated the AD2-dependent
transactivation (Fig. 7A
). In addition, the AD2-dependent
transactivation, either alone or in combination with AD1 or AD3, was
specifically inhibited by CBP-neutralizing E1A but not E1A(
236),
an E1A N-terminal deletion mutant that lacks the CBP bindings (30)
(Fig. 7B
and results not shown). Similarly, transactivation by Gal4
fusion to ASC-2 residues 1929 that includes AD1, AD2, and part of AD3
was also inhibited by E1A (results not shown). In contrast, E1A or CBP
alone had no effect on the significant basal activity directed by Gal4
alone (results not shown). Overall, these results clearly suggest that
recruitment of CBP is essential for the autonomous transactivation
function of ASC-2.

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Figure 7. CBP Required for the Autonomous Transactivation
Function of ASC-2
HeLa cells were transfected with LacZ expression vector
(100 ng) and a reporter gene Gal4-TK-Luc (100 ng), along with
expression vectors for various Gal4 fusion proteins (100 ng) and
increasing amounts of expression vectors (100 and 200 ng) for CBP, the
HAT-negative mutant mCBP (F1541A) (30 ), SRC-1, P/CAF, E1A, and
E1A( 236) (30 ), as indicated. Open, shaded, hatched,
and closed boxes indicate the presence of Gal4 alone,
Gal4/ASC22b, Gal4/ASC22c, and Gal4/ASC22bc, respectively.
Normalized luciferase expressions from triplicate samples were
calculated relative to the LacZ expressions. The
experiments were repeated at least three times and the representative
results were expressed as fold-activation (n-fold) over the value
obtained with Gal4 alone, with the error bars as
indicated. Similar results were also obtained with CV-1 cells.
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The CBP Recruitment Is Essential for the ASC-2 Function with
Nuclear Receptors
The results presented in Figs. 6
and 7
, along with the result in
Figs. 3
and 4
in which direct recruitment of ASC-2 to receptors appears
to be important, indicated that ASC-2 may act as an adaptor molecule
between CBP and nuclear receptors, and these interactions could be
essential for the coactivation function of ASC-2 with nuclear
receptors. Consistent with this idea, ASC22bc but not ASC22bc/m2
led LexA/TRß-LBD to interact, in a strictly
T3-dependent manner, with B42 fusion to CBP-E/C
in yeast (Fig. 8
). It should be noted
that the receptor-interaction domain was previously mapped to the
N-terminal region of CBP (4), and thus LexA/TRß-LBD was incapable of
directly interacting with B42/CBP-E/C in this yeast system. However,
despite our extensive effort, we were not able to show formation of a
ternary complex between ASC-2, receptor, and CBP in the GST pull-down
assays (results not shown), suggesting that the ternary complex
observed in yeast (Fig. 8
) could have been mediated by other yeast
protein(s). In addition, overexpressed ASC22b, which contains the
potent transactivation function AD2 serving as a CBP-interaction
interface, significantly repressed the 9-cis-RA-dependent
RAR transactivation (Fig. 9A
), indicating
that the CBP-ASC-2 interactions are pivotal for the nuclear receptor
transactivation. As we have previously shown for the RAR
transactivation with p300 (20), CBP also synergized with ASC-2 to
mediate the T3-dependent transactivation (Fig. 9B
). It is noted that only 25 ng of CBP, which alone exerted negligible
effects on the T3 transactivation, was used in
this specific experiment, mainly because the endogenous level of CBP
and ASC-2 within the cells interferes with readily observing such
synergistic effects in standard cotransfections. Furthermore, the
ASC-2-dependent enhancement of the RAR or TR transactivation was not
observed in the presence of CBP-neutralizing E1A, whereas E1A(
236)
was inert (Fig. 9C
and results not shown). From these results, along
with the results in which CBP was shown to be required for the
autonomous transactivation function of ASC-2 (Fig. 7
), we concluded
that ASC-2 should recruit CBP, either directly or indirectly, to
mediate the nuclear receptor transactivation in vivo.

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Figure 8. The Ternary Interactions of ASC-2, Receptor, and
CBP in Yeast
Expression vectors for B42/CBP-E/C, ASC22bc, ASC22bc/m2, and
LexA/TRß-LBD were transformed into a yeast strain containing an
appropriate LacZ reporter gene, as described previously
(40 ). Open and closed boxes indicate the
absence and presence of 100 nM of T3,
respectively. The data are representative of at least two similar
experiments and the error bars are as indicated.
|
|

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Figure 9. CBP Is Essential for the Nuclear Receptor
Transactivation
HeLa cells were transfected with expression vectors for
LacZ, ASC-2, ASC22b, CBP, E1A, and E1A( 236) along
with a reporter gene ßRARE-Luc (A and C) or TRE-Luc (B). For optimal
RA- or T3-response, 10 ng of RAR- or TR-expression vector
were also cotransfected. and + indicate the absence and presence of
100 nM of all-trans-RA (A and C) or
T3 (B), respectively. Normalized luciferase expressions
from triplicate samples were calculated relative to the
LacZ expressions. The experiments were repeated at least
three times, and the representative results were expressed as
fold-activation (n-fold) over the value obtained with unstimulated
cells, with the error bars as indicated. Similar results
were also obtained with CV-1 cells.
|
|
 |
DISCUSSION
|
---|
ASC-2 is a novel transcription cointegrator molecule that mediates
transactivation by a variety of nuclear receptors, AP-1, CREB, NF
B,
SRF, and numerous other transcription factors (Refs. 20, 21, 22, 23, 24 and our
unpublished results). The LXXLL-type motifs have recently been shown to
mediate the receptor interactions of coactivators and corepressors (5, 25), although residues immediately adjacent to the LXXLL motif
modulated the affinity of the interaction (31). Accordingly, a few
different classes of the LXXLL-containing peptides that showed a
variety of distinct receptor-binding selectivity have been isolated by
a combinatorial phage display approach (32). Notably, ASC-2
contains two LXXLL motifs (5, 25). In this report, we have shown that
both of these motifs constitute the functional receptor interaction
domains of ASC-2. The N-terminal LXXLL motif turned out to be
responsible for binding many different nuclear receptors (Table 1
and
Refs. 22, 23, 24). In contrast, the C-terminal LXXLL motif was capable of
binding only LXRß. These interactions were absolutely dependent on
the integrity of the LXXLL motifs (Table 1
and Fig. 2
). Interestingly,
the C-terminal LXXLL motif resembled none of the LXXLL sequences of
known receptor cofactors or the subclasses of LXXLL peptides isolated
from a combinatorial phage display (32). Thus, we believe that this
C-terminal LXXLL motif of ASC-2 may represent a distinct subtype that
specifically recognizes LXRß. In contrast, the N-terminal motif
resembled the class III peptides (32), which contain the residues
proline and leucine preceding the LXXLL motif (i.e. PLLXXLL,
as shown in Fig. 1B
). This class of peptides was shown to prefer RXR
and the RXR partner receptors (32), consistent with our results.
The current model for the nuclear receptor-mediated transactivation
involves two-step mechanisms (2, 3). According to this model, SRC-1
appears to be directly recruited to the liganded receptors first, which
serves as a platform to recruit CBP. Consistent with this idea, the
receptor-interacting LXXLL motif located at the N terminus of CBP could
be deleted without significantly affecting transactivation by RAR-RXR
heterodimers, whereas the SRC-1 LXXLL motifs were absolutely essential
(33, 34). These factors and associated proteins such as P/CAF (15), by
using their HAT activities (11, 12, 13, 14, 15), are known to remodel the
nucleosomal structures so that the TRAP/DRIP complex (35, 36)
can replace SRC-1/CBP and bind the liganded receptors. Subsequent
recruitment of RNA polymerase II complex to TRAP/DRIP (35, 36)
completes the second step in the nuclear receptor transactivation. The
results from this and other reports (20, 23) are consistent with an
idea that ASC-2 may play a similar, essential role as SRC-1;
i.e. direct bindings to nuclear receptors and recruitment of
CBP to the receptor-ASC-2 complex, as summarized in Fig. 10
. First, we
have previously shown that ASC-2 is essential for the AP-1 and RAR
transactivation in vivo by using antibody-blocking
experiments (20, 21). In addition, ASC22c (but not its mutated
version in which the LXXLL motif was converted to LXXAA) and a fragment
slightly larger than this (i.e. the ASC-2 residues
7861,132) exhibited a potent dominant negative phenotype with the
receptor transactivation (Fig. 3
and Ref. 23). Similarly, ASC24LR
containing the LXRß-interacting LXXLL motif acted as a dominant
negative repressor of the LXRß transactivation in an
LXXLLdependent manner (Fig. 4B
). In addition, a full-length ASC-2
with the second LXXLL motif mutated to LXXAA was inert with the LXRß
transactivation (Fig. 4A
). Thus, direct bindings between receptors and
ASC-2 appear to be required for the ASC-2 function with the nuclear
receptor transactivation. Second, the autonomous transactivation
function of ASC-2 was dependent on CBP (Fig. 7
), in which AD2 and AD3
appear to play a pivotal role as CBP interaction interfaces (Fig. 6
).
It is also notable that the mutant CBP without apparent HAT activities
(i.e. F1541A) (30) was functionally intact in promoting the
autonomous transactivation function of AD2, suggesting that the
autonomous transactivation function of ASC-2 does not require the
nucleosomal remodeling activities of CBP. Third, ASC22b, which
contains the CBP interaction domain, acted as a potent dominant
negative mutant with the RAR transactivation (Fig. 9A
), suggesting that
the ASC-2-CBP interactions are critical components of the
ASC-2-mediated receptor transactivation. Consistent with this idea,
ASC-2-dependent enhancement of the RAR transactivation was not observed
in the presence of CBP-neutralizing E1A, but not E1A(
236) (30), a
mutated E1A incapable of associating with CBP (Fig. 9B
). Although the
exact molecular mechanism by which E1A blocks the CBP-dependent ASC-2
function is not clearly understood, one interesting possibility is that
E1A may impair the CBP-ASC-2 interactions, as was shown for the
CBP-SRC-1 interactions (37). However, our preliminary GST pull-down
experiments indicated that E1A does not interfere with the CBP-ASC-2
interactions. More importantly, it still remains unresolved whether the
interactions between ASC-2 and CBP are direct. In particular, the
ternary interactions of ASC-2, receptor, and CBP observed in yeast
(Fig. 8
) were not detected in the GST pull- down assays (our
unpublished results). These results suggest the requirement of other
protein(s) in these ternary interactions, although we could not easily
rule out the possibility of technical problems associated with
the lack of the ternary interactions in the GST pull-down assays.
Interestingly, the transcriptionally inert LexA/TRß-LBD became an
excellent T3-dependent transactivator upon
coexpression of ASC22bc in yeast (see the third column in
Fig. 8
), in which ASC22bc may have helped TRß to recruit yeast
proteins that are essential for the receptor function in yeast, such as
Swi/Snf proteins (38). These Swi/Snf proteins, conserved between yeast
and mammals, could also be responsible for the putative, indirect
ASC-2-CBP interactions in yeast (Fig. 8
) and thus essential components
in the ASC-2 function in mammalian cells.

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Figure 10. The Model for the ASC-2 Action
ASC-2 acts as a bridging molecule between nuclear receptors and CBP.
Supporting this model, ASC22c (i.e. a subregion of
ASC-2 containing the binding site for many different receptors) or
ASC22b (i.e. a subregion of ASC-2 containing the
binding site for CBP) acted as potent dominant negative mutants of the
RAR transactivation. Similarly, ASC24LR (i.e. a
subregion of ASC-2 containing the binding site for LXRß) also acted
as a dominant negative mutant of the LXRß transactivation. However,
neither the exact relationship of ASC-2 to different coactivators such
as SRC-1 and TRAP/DRIP (35 36 ) nor whether or not the CBP-ASC-2
interactions are direct are clearly understood. HRE indicates hormone
response elements, and CBP, ASC-2, TRAP, and SRC-1 are schematically
shown as a single polypeptide, although each of them is known to exist
as a steady state complex of multiple polypeptides in
vivo (Refs. 35, 36, 44, and our unpublished results).
|
|
Considering the fact that ASC-2 is expressed in relatively low amounts
in most cells but can be up-regulated in certain cells by various
cytokines and growth factors (our unpublished results), ASC-2 may serve
as an inducible factor that represents an alternative functional
homolog of SRC-1. Similarly, the ASC-2 overexpressing human cancer
cells (20) could be potential sites where ASC-2 may play a functionally
dominant role over SRC-1. As indicated in the model (Fig. 10
), however, it is not clear how ASC-2
functionally corroborates with other known coactivators of nuclear
receptors such as SRC-1 and TRAP/DRIP (35, 36). In the previously shown
antibody-blocking experiments (20), neutralization of the endogenous
ASC-2 in rat-1 fibroblast cells significantly abolished the RAR
transactivation, and coexpressed CBP but not SRC-1 was able to partly
overcome these
ASC-2 antibody-blocking effects. These results
indicate that either the RAR transactivation simply requires ASC-2 but
not SRC-1 within this cell type (i.e. rat-1 fibroblast
cells) or SRC-1 requires the presence of ASC-2 to function. The latter
possibility is consistent with an idea that ASC-2 may function as a
general platform to recruit other related coactivators such as SRC-1
and TRAP/DRIP. In fact, ASC-2 can interact with SRC-1 (Fig. 6A
and Ref.
20) although the functional role of these SRC-1-ASC-2 interactions is
not currently clear, and a component of the TRAP/DRIP complex and
CBP/p300 were recently found to associate with the C-terminal region of
ASC-2 (22). Interestingly, the latter results are in contrast to our
yeast two-hybrid-based findings in which CBP appeared to exclusively
bind to AD2 and AD3 located at the N-terminal region (Fig. 6
). This
discrepancy is not clearly understood, although it is possible that the
ASC-2 fragments used in our experiments may have impaired a cryptic
CBP-binding site. Indeed, in our own GST pull-down assays, the
full-length ASC-2 interacted with multiple regions of CBP, as opposed
to the yeast two-hybrid-based assays (Ref. 20 and Fig. 6D
).
Furthermore, ASC24a (i.e. the ASC-2 residues 1,4291,729)
but not ASC24a/m3 stimulated the LXRß transactivation (results not
shown), for which the putative TRAP/CBP interaction(s) at the C
terminus of ASC-2 could be responsible. Thus, more detailed analyses to
localize the exact CBP interaction interfaces of ASC-2 are currently
under way. Alternatively, the antibody used in the original
antibody-blocking experiments may have crossreacted with other
essential component(s) that are required for the SRC-1 and ASC-2
functions. We are currently reexamining this issue by using more
defined monoclonal antibody against ASC-2, other cell-types, and
various LacZ reporters responsive to different
receptors.
In conclusion, we have shown that ASC-2 interacted with oxysterol
receptor LXRß in a manner specifically dependent on the C-terminal
LXXLL motif, whereas the N-terminal LXXLL motif of ASC-2 bound a broad
range of nuclear receptors. We have also demonstrated that the
CBP-ASC-2 interactions are essential for the autonomous transactivation
and the receptor coactivation functions of ASC-2, in which ASC-2
appears to serve as a bridging molecule between CBP and nuclear
receptors. Finally, we have noted that ASC-2 contains a numerous number
of putative phosphorylation sites (20) and, thus, the possible
regulation of its inherent activity through various signal transduction
pathways could play an important role. In this regard, it was
interesting to note that DNA-dependent protein kinase complex (39) was
recently copurified with GST-ASC-2 from HeLa nuclear extracts (22).
 |
MATERIALS AND METHODS
|
---|
Plasmids
PCR fragments encoding ASC2-I, ASC2-Ia, ASC2-Ib, ASC2-II,
ASC2-IIa, ASC22b/N, ASC22b/C, ASC22b/m1, ASC22bc, ASC22c/m2,
ASC2-III, ASC24a, ASC24b, ASC24c, ASC24c/m3, ASC24LR,
ASC24LR/m3, ASC24.5, CBP-E/N, CBP-E/C, and LXRß were inserted
into EcoRI and XhoI/SalI restriction
sites of the LexA-fusion vector pEG202PL (40), the B42-fusion vector
pJG45 (40), and the mammalian expression/in vitro
translation vector pcDNA3, respectively. PCR fragments, obtained by
using Gal4 fusion vectors to a series of TRß mutants (kind gift of
Drs. M. G. Rosenfeld and Chris Glass at University of California,
San Diego) as templates, were cloned into EcoRI and
SalI restriction sites of pEG202PL to express appropriate
LexA fusion proteins in yeast. PCR fragments encoding ASC24a and
ASC22c were cloned into BamHI and EcoRI
restriction sites of pCMX1 to express Gal4-ASC24a and Gal4-ASC22c,
respectively. Vectors encoding GST, LexA, and B42-fusions to ASC22,
ASC22a, ASC22b, ASC22c, ASC24, CBP-A, CBP-B, CBP-C, CBP-D,
CBP-E, CBP-4, CBP-5, SRC-A, SRC-B, SRC-C, SRC-D, SRC-E, RXR, RAR,
TRß, and ERß, along with ßRARE-Luc, TRE-Luc, LXRE-Luc,
Gal4-TK-Luc, the transfection indicator construct pRSV-ß-gal, and
expression vectors for ASC-2, E1A, E1A(
236), CBP, and mCBP
(i.e. F1541A), were as previously described (6, 7, 9, 10, 20, 21, 26, 30). To express GST fusion to LXRß, PCR fragment encoding
the full-length LXRß was cloned into EcoRI and
XhoI restriction sites of pGEX4T (Pharmacia Biotech, Piscataway, NJ). The site-directed mutagenesis kit
QuickChange (Stratagene, San Diego, CA) was used to create
a mutated, full-length ASC-2, in which the second LXXLL motif was
converted to LXXAA. ASC22bc and its m2 mutant were subcloned into the
HindIII and XhoI restriction sites of the yeast
expression vector p425-Gal1 (41).
The Yeast Two-Hybrid Screening and Yeast ß-Galactosidase
Assay
The LexA-ASC24 was used as a bait to screen a mouse liver cDNA
library in pJG45 (40) for ASC-2-interacting proteins, and the
screening was executed essentially as previously described (42). The
yeast ß-galactosidase assay was done as described (40). 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-Sepahrose-4B beads (Pharmacia Biotech) in binding buffer (25 mM HEPES,
pH 7.8, 0.2 mM EDTA, 20% glycerol, 100
mM KCl, and 0.1% NP40), 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 (43).
Cell Culture and Transfections
HeLa 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 previously (40), and the results were normalized
to the LacZ expression. Similar results were obtained in
more than two similar experiments.
Note Added In Proof.
While this manuscript was being revised, another paper describing
hASC-2 (hNRC) as well as the rat homolog rNRC was published in which
the CBP-ASC-2 associations in vivo were also suggested
(Mahajan MA, Samulels HH 2000 A new family of nuclear receptor
coregulators that integrate nuclear receptor signaling through
CREB-binding protein. Mol Cell Biol 20:50485063).
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. M. G. Rosenfeld, Chris Glass, Joonho Choe,
and David Mangelsdorf for various plasmids.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Jae Woon Lee, Ph.D., Center for Ligand and Transcription, Chonnam National University, Kwangju 500757, Korea. E-mail: jlee{at}chonnam.chonnam.ac.kr
This work was supported by a grant from the National Creative Research
Initiatives Program of the Korean Ministry of Science and Technology,
Republic of Korea.
1 Present address: Howard Hughes Medical Institute, Department of
Medicine, University of California, San Diego, La Jolla, California
92093-0651. 
2 Present address: Department of Molecular Biology, Massachusetts
General Hospital, Boston, Massachusetts 02114. 
Received for publication June 16, 2000.
Revision received October 23, 2000.
Accepted for publication October 25, 2000.
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