Steroid Receptor Coactivator-1 and Its Family Members Differentially Regulate Transactivation by the Tumor Suppressor Protein p53
Soo-Kyung Lee,
Han-Jong Kim,
Jung Woo Kim and
Jae Woon Lee
Center for Ligand and Transcription (J.W.L, H.-J.K) Hormone
Research Center (J.W.L) Department of Biology (S.-K.L) Chonnam
National University Kwangju 500757, Korea
Department of
Biochemistry (J.W.K) Paichai University Daejeon 302735,
Korea
 |
ABSTRACT
|
---|
The tumor suppressor protein p53 exerts its cell
cycle-regulatory effects through its ability to function as a
sequence-specific DNA-binding transcription factor. Herein, we show
that p53 physically interacts with specific subregions of steroid
receptor coactivator-1 (SRC-1) and its family members, p/CIP
(p300/CBP interacting protein), xSRC-3, and AIB1 (amplified in breast
cancer), originally isolated as transcription coactivators of
nuclear receptors, as demonstrated by the yeast and mammalian
two-hybrid tests as well as glutathione S-transferase
pull-down assays. Interestingly, cotransfection of HeLa cells with
SRC-1- or p/CIP expression vector potentiated the p53-mediated
transactivation, whereas AIB1 and xSRC-3 were repressive. All of these
SRC-1 members, however, similarly stimulated transactivation mediated
by nuclear receptors and AP-1, as previously described. These results
suggest that SRC-1 and its family members may differentially modulate
the p53 transactivation in vivo.
 |
INTRODUCTION
|
---|
Mutations within the p53 gene represent one of the most common
genetic aberrations in tumorigenesis (reviewed in Ref. 1). The
wild-type p53 negatively regulates cell growth and division, whereas
the mutant forms are unable to suppress or control cell cycle
progression. The tumor suppressor function of p53 is thought to result
from its ability to act as a cell cycle checkpoint protein, thus
halting the cell cycle in the G1 phase if and when DNA
damage occurs to a normal cell. Considerable evidence has accumulated
for regulation of transcription as one of the primary mechanisms of the
p53 action, in which p53 binds to a specific motif on gene promoters
and thus transactivates the genes required to suppress cellular
transformation (2, 3). Two repeats of a 10-bp motif, PuGPuCATGPyCPy, in
which the G and A at positions 2 and 5 are critical determinants in
p53-DNA binding, has been reported (2). Mutant p53 proteins are
reported to show a dominant-negative effect by forming oligomeric
complexes with the wild-type p53 before DNA binding, which results in a
change in conformation and subsequently a loss of affinity for DNA.
However, recent reports also suggest that, at least in some cases, the
mutant forms can even promote the growth of the parental tumor cell and
therefore exhibit an oncogenic gain of function of their own
(4, 5, 6).
Transcription coactivators bridge transcription factors and the
components of the basal transcriptional apparatus (reviewed in Ref. 7).
Functionally conserved proteins, CREB binding protein (CBP) and p300,
have been shown to be essential for the activation of transcription by
a large number of regulated transcription factors, including CREB,
nuclear factor-
B (NF
B), basic helix-loop-helix (bHLH) factors,
signal transducers and activators of transcription (STATs), AP-1,
ternary complex factor (TCF)/serum response factor (SRF),
nuclear receptors, and p53 (8). In particular, 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 promoters of target genes (reviewed in
Ref. 9). Transcriptional regulation by nuclear receptors depends
primarily upon a ligand-dependent activation function, AF2, with its
core located in the C terminus and predicted to undergo an
allosteric change upon ligand binding. Consistent with this, CBP and
p300 have been found to interact directly with nuclear receptors in a
ligand- and AF2-dependent manner (10, 11, 12, 13). In addition, a series of
factors that exhibit ligand- and AF2-dependent binding to nuclear
receptors have been identified both biochemically and by expression
cloning. Among these, a group of highly related proteins have been
shown to form a complex with CBP and p300 and enhance transcriptional
activation by several nuclear receptors; i.e. steroid
receptor coactivator-1 (SRC-1) (12, 14), xSRC-3 (15), AIB1 (amplified
in breast cancer) (16), TIF2 (transcription intermediary factor 2)
(17), RAC3 (18), ACTR (19), TRAM-1 (20), and p/CIP
(p300/CBP-interacting protein) (21). In particular, AIB1 was cloned as
a gene whose expression and copy number were significantly elevated in
human breast and ovarian cancers (16). We and others have recently
shown that SRC-1 and p/CIP also mediate transactivation by other
transcription factors including AP-1 (22), SRF (23), NF
B (24), CREB,
and STATs (21). Based on this rather broad spectrum of action, we
proposed that SRC-1 should be renamed transcription integrator, like
CBP and p300 (22). Interestingly, SRC-1 (25) and its homolog ACTR (19),
along with CBP and p300 (26, 27), were recently shown to contain
histone acetyltransferase activities themselves and associate with yet
another histone acetyltransferase protein p/CAF (28). In contrast, it
was shown that SMRT (29) and N-CoR (30), nuclear receptor
corepressors, form complexes with Sin3 and histone deacetylase proteins
(31, 32). From these results, it was suggested that cofactors also
exploit chromatin remodeling for transcriptional regulation, through
histone acetylation-deacetylation.
In light of the fact that SRC-1 is capable of functional
interaction with CBP and p300, which in turn coactivate p53 (33, 34), we tested whether SRC-1 is also involved with the p53-mediated
transactivation. In this report, we showed that SRC-1 specifically
bound to p53 and coactivated the p53-mediated transactivation, either
alone or in synergy with p300. Surprisingly, we found that p/CIP acted
as a strong coactivator of p53 while AIB1 and xSRC-3 were repressive.
These results are in marked contrast to earlier reports (12, 14, 15, 16, 17, 18, 19, 20, 21) in
which these different SRC-1 family members similarly modulated
transactivation by nuclear receptors and suggest that each of these
different SRC-1 proteins may have a specific set of target
transcription factors in vivo.
 |
RESULTS
|
---|
Interactions of SRC-1 and p53
We have recently shown that the AP-1 components c-Jun and c-Fos
(22), SRF (23), and the NF
B component p50 (24) functionally interact
with specific subregions of SRC-1 (Fig. 1
). In addition, we have also found that
xSRC-3 interacts with p53 in the LexA-based yeast two-hybrid system
(35) (results not shown). To localize the p53 interaction domain of
SRC-1, we examined LexA proteins fused to a series of SRC-1 fragments
we recently described (22, 23, 24) (Fig. 1
). Consistent with an idea that
p53 interacts with SRC-A, SRC-D, and SRC-E, coexpression of a B42
fusion to the full-length p53 further stimulated the LexA/SRC-A,
LexA/SRC-D-, and LexA/SRC-E-mediated LacZ expression in the
yeast two-hybrid tests (Fig. 2A
). In
contrast, the LacZ expressions mediated by LexA alone or
LexA fusions to SRC-B or C were not further stimulated by coexpression
of B42/p53. As previously described (8), a LexA fusion to the
C-terminal subregion of CBP (i.e. LexA/CBP-E) efficiently
interacted with B42/p53. As previously reported (12, 14), LexA/SRC-C
also interacted with a B42 fusion to thyroid hormone receptor or
retinod X receptor in a ligand-dependent manner (results not shown),
indicating that the lack of interactions between B42/p53 and LexA/SRC-C
is not due to altered expression of the SRC-C fragment in this system.
Similar results were also obtained with a B42 fusion to p53
C
(i.e. the p53 residues 1290), except that the interaction
with SRC-A was not observed (Fig. 2A
). These interactions were further
probed in the mammalian two-hybrid tests (36). In HeLa cells, a Gal4
fusion to the full-length p53 (Gal4/p53) was a powerful transactivator
of a reporter construct controlled by upstream Gal4 binding, increasing
the activation up to 200-fold depending on the amount of Gal4/p53
cotransfected (results not shown). To easily assess the
fold-activation, however, we used only 50 ng of Gal4/p53, which
increased the activation approximately 10-fold (Fig. 2A
). Coexpression
of a VP16 fusion protein to the full-length SRC-1 further augmented
this Gal4/p53-stimulated transactivation in a dose-dependent manner,
supporting the idea that SRC-1 associates with p53 in vivo.
Similar results were also obtained with Gal4/p53
C, consistent with
the yeast results.

View larger version (14K):
[in this window]
[in a new window]
|
Figure 1. Schematic Representation of the SRC-1 Constructs
The full-length human SRC-1 (12 14 ) and a series of five SRC-1
fragments are as depicted. The nuclear receptor-interacting-,
CBP-p300-interacting-, bHLH/PAS-, serine-threonine-rich-, and
glutamine-rich domains, along with the recently identified histone
acetyltransferase domain (25 ), the NF B component p50-binding domain
(24 ), c-Jun/c-Fos-binding domain (22 ), and SRF-binding domain (23 ), are
as indicated. The amino acid numbers for each construct are shown.
|
|

View larger version (23K):
[in this window]
[in a new window]
|
Figure 2. Interactions of p53 with SRC-1 in
Vivo
A, The yeast two-hybrid tests were employed to map the
p53-SRC-1-interation domains. The indicated B42- and LexA plasmids were
transformed into a yeast strain containing an appropriate
LacZ reporter gene (35 ). Open, hatched,
and solid bars indicate coexpression of the constitutive
transactivation domain B42 (35 ) alone, a B42 fusion to the full-length
p53, and a B42 fusion to p53 C, respectively. The results are
expressed as induction (n-fold) over the value obtained with B42/- and
LexA/-, which was given an arbitrary value of 1. The data are
representative of three similar experiments. B, The p53-SRC-1
interactions were probed in the mammalian two-hybrid tests (36 ). HeLa
cells were transfected with LacZ expression vector and
VP16/SRC-1-expression vector along with a reporter gene Gal4-Luc
reporter (36 ), as indicated. Gal4/N represent parental Gal4 expression
vector and numbers indicate the amount of each
expression vector used (in nanograms). The results are expressed as
induction (n-fold) over the value obtained with VP16/SRC-1 alone, which
was given an arbitrary value of 1. The data are representative of three
similar experiments.
|
|
To further characterize these interactions in vitro,
glutathione-S-transferase (GST) alone and GST fusion to p53
were expressed, purified, and tested for interaction with in
vitro translated luciferase and various SRC-1 proteins. The
radiolabeled SRC-1 interacted with GST/p53 and GST/p53
C, but not
with GST alone (Fig. 3A
). In contrast,
the radiolabeled luciferase did not bind to any of the GST proteins, as
expected. In agreement with the yeast two-hybrid results (Fig. 2A
),
only SRC-A, SRC-D, and SRC-E, among various SRC-1 fragments,
specifically interacted with GST/p53, but not with GST alone, whereas
only SRC-D and SRC-E interacted with p53
C (Fig. 3A
). CBP-E also
interacted with GST/p53 and GST/p53
C, but not with GST alone, and
GST/TR interacted with SRC-1 and SRC-C only in the presence of
T3. The full-length AIB1 (16), p/CIP (20), and xSRC-3 (15),
as well as the C-terminal fragments from these proteins, were also
found to specifically interact with GST/p53 (Fig. 3B
and results not
shown). Overall, these results suggest that SRC-1 and its distinct
family members directly associate with p53, and the interaction
interfaces include the p53 residues 1290 and, at least, the
C-terminal subregions of SRC-1 containing the previously shown
CBP-binding and histone acetyltrans-ferase domains (12, 14, 15, 16, 17, 18, 19, 20, 21).

View larger version (18K):
[in this window]
[in a new window]
|
Figure 3. Interactions of p53 with SRC-1 in
Vitro
A, Luciferase, SRC-1, a series of five SRC-1 fragments, and CBP-E
were labeled with [35S]methionine by in
vitro translation and incubated with glutathione beads
containing GST alone, GST/p53, GST/p53 C, or GST/TR, as indicated.
Beads were washed, and specifically bound material was eluted with
reduced glutathione and resolved by SDS-PAGE. -/+ Indicates the
absence and the presence of 0.1 µM T3,
respectively. Approximately 20% of the labeled proteins used in the
binding reactions were loaded as inputs. B, The C-terminal domains of
distinct SRC-1 family members were labeled with
[35S]methionine by in vitro translation
and incubated with glutathione beads containing GST alone or GST/p53,
as indicated. AIB1 N, p/CIP N, and xSRC-3 N contained the AIB1
residues 11021420, the p/CIP residues 11091402, and the xSRC-3
residues 10931391, respectively. Approximately 20% of the labeled
proteins used in the binding reactions were loaded as inputs.
|
|
Cotransfection of SRC-1 Stimulates p53-Mediated Transactivation
To assess the functional consequences of these interactions, SRC-1
was cotransfected into HeLa cells along with a reporter construct
p53RE-Luc that consists of a minimal SV40 early promoter and 17
upstream consensus p53 sites. Increasing amounts of cotransfected p53
efficiently enhanced the reporter gene expressions up to 200-fold, as
expected (results not shown). SRC-1 enhanced the p53-dependent
transactivation in an SRC-1 dose-dependent manner, with cotransfection
of 200 ng of SRC-1 increasing the fold activation approximately 9-fold
relative to the level with 10 ng of p53 alone (Fig. 4A
). Consistent with the reports that CBP
and p300 are transcription coactivators of p53 (33, 34), cotransfected
p300 also had stimulatory effects on the reporter gene expressions,
with cotransfection of 50 ng of p300 increasing the fold activation
approximately 3.5-fold relative to the level with 10 ng of p53 alone.
Coexpression of both p300 and SRC-1 further increased the reporter gene
expressions above the levels observed with SRC-1 or p300 alone,
suggesting that SRC-1 and p300 cooperate to coactivate the p53-mediated
transactivation (Fig. 4A
). Similar results were also obtained with
reporter constructs that contain promoters of MDM2 (37, 38) and
p21waf (39), previously characterized target genes of p53.
In particular, synergistic action of SRC-1 and p300 was apparent with
the p21waf promoter (Fig. 4B
). In contrast, cotransfection
of SRC-1 did not affect the LacZ reporter expression of the
transfection indicator construct pRSV-ß-gal (results not shown).

View larger version (41K):
[in this window]
[in a new window]
|
Figure 4. Effects of SRC-1 and p300 Cotransfection on the
p53-Mediated Transactivations
HeLa cells were transfected with p53-, LacZ-, SRC-1-, or
p300 expression vector along with a reporter gene p53RE-Luc (A),
p21waf-Luc (B), or MDM2-Luc (C), as indicated. The results
are expressed as induction (n-fold) over the value obtained with a
reporter construct alone, which was given an arbitrary value of 1. The
data are representative of three similar experiments.
|
|
Distinct SRC-1 Family Members Differentially Regulate the p53
Transactivation
Recently, three distinct CBP/p300-containing coactivator
complexes were described that differentially mediate transactivation by
retinoic acid receptor, CREB, and STATs (40). In this study,
coactivator complex mediating transactivation by retinoic acid receptor
was demonstrated to require p/CIP and SRC-1, whereas two distinct
complexes containing p/CIP regulated CREB and STATs. These results led
us to test whether distinct SRC-1 family members show such differences
in their potential regulation of the p53-dependent transactivation.
Surprisingly, p/CIP, originally described as a relatively weaker
coactivator of nuclear receptors (21), was a very potent coactivator of
p53, with cotransfection of 200 ng of p/CIP increasing the fold
activation approximately 150-fold (Fig. 5A
). Interestingly, xSRC-3 (23) and AIB1
(16) were not able to stimulate the p53-dependent transactivation,
despite the fact that these proteins readily interacted with p53 (Fig. 3B
). These differential effects may stem from different expression
levels of these SRC-1 proteins. In contrast to this notion, however,
all of these SRC-1 family members were capable of stimulating the
AP-1-dependent transactivation to similar extents, as we recently
described (22) (Fig. 5B
). These results are consistent with an idea
that coactivator complexes containing AIB1 may function with AP-1 and
nuclear receptors but not with p53, whereas complexes containing SRC-1
and p/CIP work for AP-1, nuclear receptors, and p53. Alternatively, p53
may preferentially interact with SRC-1 and p/CIP but not with AIB1
within the context of macromolecular coactivator complex in
vivo, although p53 is capable of interacting with all of these
proteins on a one-to-one basis in vitro (Fig. 3B
).
Consistent with this hypothesis, p53-dependent inhibition of the AP-1
transactivation (34) was relieved with cotransfected SRC-1 and p/CIP,
but not with AIB1 (Fig. 5C
).

View larger version (42K):
[in this window]
[in a new window]
|
Figure 5. Differential Modulation of the p53 Transactivation
by Distinct SRC-1 Family Members
HeLa cells were transfected with LacZ-, p53-, c-Fos-,
SRC-1-, p/CIP-, xSRC-3-, or AIB1 expression vector along with a
reporter gene p53RE-Luc (A) or TRE-Luc (B and C), as indicated. The
results are expressed as induction (n-fold) over the value obtained
with a reporter construct alone, which was given an arbitrary value of
1. The data are representative of three similar experiments.
|
|
AIB1 Inhibits the p53-Dependent Transactivation
AIB1 was originally isolated based on its amplification and
overexpression in human breast and ovarian cancers (16). Since
mutations within the p53 gene and perturbed transactivation potential
of the p53 protein represent one of the most common genetic aberrations
in tumorigenesis, we further examined the effects of overexpressed AIB1
on the p53 transactivation potential. As shown in Fig. 6A
, increasing amount of cotransfected
AIB1 inhibited the p/CIP-stimulated p53 transactivation in a
dose-dependent manner. With the reporter construct
p21waf-Luc or MDM2-Luc, p/CIP showed potent stimulation of
the p53-dependent transactivation, whereas AIB1 showed dose-dependent
inhibitory actions (Fig. 6
, B and C). Similarly, AIB1 and xSRC-3 also
inhibited the Gal4/p53-directed activation of Gal4-Luc reporter gene
expressions in a dose-dependent manner, whereas SRC-1 and p/CIP showed
stimulatory actions (Fig. 6D
). Consistent with the interaction results
(Figs. 2
and 3
), SRC-1 also coactivated transactivation by
Gal4/p53
C. We have also examined whether these results reflect
differential p53 expression levels directed by each SRC-1 family
member. As shown in Fig. 7
, cotransfection of each SRC-1 family member didnt significantly change
the level of p53 expression. Similar results were also obtained with
Gal4/p53 expressions (results not shown). From these results, we
concluded that overexpressed AIB1 may seriously perturb the p53
transactivation potential in vivo.

View larger version (38K):
[in this window]
[in a new window]
|
Figure 6. AIB1, as an Inhibitor of the p53 Transactivation
HeLa cells were transfected with LacZ-, p53-, c-Fos-,
SRC-1-, p/CIP-, xSRC-3-, Gal4/p53-, Gal4/p53 C-, or AIB1 expression
vector along with a reporter gene p53RE-Luc (A), p21waf-Luc
(B), MDM2-Luc (C), or Gal4-Luc (D), as indicated. The results are
expressed as induction (n-fold) over the value obtained with a reporter
construct alone, which was given an arbitrary value of 1. The data are
representative of three similar experiments.
|
|

View larger version (11K):
[in this window]
[in a new window]
|
Figure 7. Western Analysis of p53 Expression
HeLa cells were transfected with p53 alone or together with SRC-1-,
p/CIP-, or AIB1 expression vector, and the resulting nuclear extracts
were subjected to Western analyses for the p53 expression levels. The
data are representative of two similar experiments. Similarly, the
expression level of Gal4/p53 was not also affected by coexpression of
each SRC-1 family member (results not shown).
|
|
 |
DISCUSSION
|
---|
SRC-1 (12, 14) and its family member p/CIP (21), originally
isolated as transcription coactivators of nuclear receptors, were
recently shown to mediate transactivation by AP-1 (22), SRF (23),
NF
B (24), CREB, and STATs (21). In this report, we added the tumor
suppressor p53, as a new member to the list of transcription factors
that are regulated by SRC-1 (as summarized in Fig. 1
). These results
are not surprising, considering the recent reports (12, 14, 15, 16, 17, 18, 19, 20, 21) in
which SRC-1 and its family members were shown to functionally form a
complex with CBP or its human homolog p300, which in turn binds and
coactivates a wide spectrum of different transcription factors. These
include nuclear receptors, CREB, NF
B, bHLH factors, STATs, TCF/SRF,
AP-1, and p53 (reviewed in Ref. 8). Based on this broad spectrum of
action, we have recently proposed to regroup SRC-1 into a class of
proteins, such as CBP/p300, called integrator (22).
Recently, p300 and CBP, despite their similarities, were shown to have
distinct functions during retinoid-induced differentiation of embryonic
carcinoma F9 cells (40). In addition, different classes of mammalian
transcription factors were shown to functionally require distinct
components of the CBP/p300 coactivator complex, based on their platform
or assembly properties; retinoic acid receptor was demonstrated to
require p/CIP and SRC-1, whereas CREB and STATs required only p/CIP
(41). In addition, these transcription factors were shown to require
different histone acetyltransferase activities within the CBP/p300
complex to activate transcription (41). Overall, these results suggest
that related but distinct CBP/p300-containing coactivator complexes
exist in the cell, which exhibit a different specificity for each
transcription factor. The results shown in Figs. 5
and 6
are consistent
with this view, for which a few different coactivator complexes, each
containing a distinct SRC-1 family member, can be proposed to exist.
Coactivator complexes containing SRC-1 and/or p/CIP may mediate the
nuclear receptor-, AP-1-, and p53-dependent transactivation, whereas a
complex containing AIB1 may function with nuclear receptors and AP-1
but not with p53. Alternatively, p53 may preferentially interact with
SRC-1 and p/CIP, but not AIB1, within the context of macromolecular
coactivator complex in vivo, although these proteins are
capable of interacting equally well with p53 in a one-to-one basis
in vitro (Fig. 3B
). Consistent with this hypothesis,
p53-dependent inhibition of the AP-1 transactivation (34) was relieved
with cotransfected SRC-1 and p/CIP, but not with AIB1 (Fig. 5C
).
However, it is interesting to note that p/CIP showed a much stronger
coactivation with the AP1 transactivation when p53 was coexpressed
(compare the results in panels B and C of Fig. 5
). Thus, the
p53-mediated repression of the AP-1 transactivation may also involve
formation of a novel transcription inhibitory complex between AP-1 and
p53, which is subjected to derepression and further coactivation by
p/CIP, but not by AIB1. The SRC-1 family members were recently proposed
to group into three subclasses based on their sequence homology;
i.e. SRC-1/NCoA-1 (17, 21, 42), SRC-2/TIF2/GRIP1/NCoA-2 (17, 42), and SRC-3/p/CIP/ACTR/AIB1/xSRC-3/Rac3 (15, 16, 18, 19, 21). In
particular, ACTR (19), Rac3 (18), p/CIP (21), and AIB1 (16), which form
an SRC-3 subfamily along with xSRC-3 (15), have been considered to
derive from the same gene. However, ACTR and xSRC-3 are known to have
multiple splicing isotypes (15, 19) that have not been extensively
characterized, such as xSRC-3a shown in Fig. 8
. Similarly, AIB1 and p/CIP may also
represent different splicing isotypes from the same gene, although AIB1
is a human gene and p/CIP is a mouse gene. Therefore, we believe that
this unexpected, differential regulation of the p53 transactivation by
each SRC-3 family member may represent isotype-specific effects,
although we can not exclude the possibility of species-specific
differences.

View larger version (24K):
[in this window]
[in a new window]
|
Figure 8. Multiple Isotypes of SRC-3 Subfamily Members
The nuclear receptor-interacting-, CBP-p300-interacting-, bHLH/PAS-,
and glutamine-rich domains are as indicated. The relative positions of
the glutamine-rich domains as well as specific insertions and deletions
are as indicated. The nucleotide sequences of xSRC-3a (only a partial
clone containing amino acids 252-1221 has been isolated) are identical
to those of xSRC-3 (15 ) except in-frame insertion of unrelated 51 amino
acids.
|
|
Overexpression of a component within an integrator complex could
potentially perturb signal integration by the complex and affect
multiple signal transduction pathways. Recently, AIB1 was identified as
a gene amplified and overexpressed in human breast and ovarian cancers
(16). Since AIB1 is capable of potentially regulating multiple
transcription factors (21, 22, 23, 24), such as CBP and p300 (8), it will be
important to identify specific target transcription factors of
overexpressed AIB1 that may lead to tumorigenesis. In particular, we
have shown that the overexpressed AIB1 severely impaired p53
transactivation (Fig. 6
). This may involve destabilizing the dynamic
equilibrium established among distinct coactivator complexes in
vivo, in which overexpressed AIB1 shifts the equilibrium to
formation of the putative p53-inhibitory complexes containing AIB1.
Alternatively, overexpressed AIB1 may lead to formation of a totally
different macromolecular complex, which is also inhibitory to the p53
transactivation. Given the importance of p53 in tumorigenesis in
general (1), p53 could be an excellent target factor of overexpressed
AIB1 in tumorigenesis processes in vivo, even though the
involvement of other factors cannot be excluded.
SRC-1 (25) and its homolog ACTR (19), along with CBP and p300 (26, 27),
were recently shown to contain histone acetyltransferase activities
themselves and associate with yet another histone acetyltransferase
protein p/CAF (28). In contrast, it was shown that SMRT (29) and N-CoR
(30), nuclear receptor corepressors, form complexes with Sin3 and
histone deacetylase proteins (31, 32). Thus, it was suggested that
chromatin remodeling by cofactors contributes to transcription
factor-mediated transcriptional regulation, through histone
acetylation-deacetylation. In addition, p300 was recently shown to be
involved with an acetylation-mediated change in the function of a
non-histone-regulatory protein; i.e. p300 acetylated p53
both in vivo and in vitro, which stimulated its
sequence-specific DNA-binding activity, possibly as a result of an
acetylation-induced conformational change (43). SRC-1 synergized with
p300 to stimulate the p53 transactivation (Fig. 4
). This synergy is
believed to reflect a cooperative recruitment of two different
coactivator molecules (i.e. SRC-1 and CBP-p300) by p53.
These two distinct histone acetyltransferases could either modify
selective sites on the histone tails or act in a concerted fashion to
control different aspects of transcriptional activation. However, SRC-1
may not affect the acetylation-induced stimulation of the p53 DNA
binding, like p300 (43), because SRC-1 efficiently coactivated
transactivation by chimeric p53 proteins fused to Gal4, a heterologous,
constitutive DNA- binding domain (i.e. Gal4/p53 and
Gal4/p53
C, Fig. 6D
). In particular, p53
C lacks the C-terminal
acetylation sites by CBP-p300 (43).
In summary, we have shown that SRC-1 and its family members interact
with p53 and differentially modulate p53-mediated transactivation. In
particular, our results suggest an interesting hypothesis for a novel
tumorigenesis mechanism, in which the overexpressed AIB1 perturbs
transactivation potential by p53.
 |
MATERIALS AND METHODS
|
---|
Plasmids
LexA-, B42-, T7-, or GST vectors to express fragments of SRC-1
(AE as depicted in Fig. 1
) were as previously described (22, 23, 24).
PCR-amplified fragments of CBP-E (i.e. the CBP residues
18672441), the full-length p53, and p53
C were subcloned into
EcoRIXhoI restriction sites of the B42 fusion
vector pJG45 (35), the GST fusion vector pGEX4T (Pharmacia Biotech, Piscataway, NJ), the Gal4 fusion vector pCMXGal4/N
(36), or the CMV/T7 vector pcDNA3 (Invitrogen, San Diego,
CA). The expression vectors for p53, c-Fos, AIB1 (human gene in pcDNA3,
kind gift of Paul Meltzer at NIH), p/CIP (mouse gene in pcMX, kind gift
of Chris Glass at University of California at San Diego), xSRC-3, p300,
SRC-1 (human SRC-1a in pCR3.1, kind gift from Ming Tsai at Baylor
College of Medicine, Houston, TX) and VP16/SRC-1, the transfection
indicator construct pRSV-ß-gal, the AP-1-responsive reporter
construct TRE-Luc, the Gal4-responsive reporter construct Gal4-Luc, and
the T3-responsive reporter construct TREpal-Luc were as
previously described (22, 23, 24). PCR fragment encoding AIB1
N (the AIB1
residues 11021420), p/CIP
N (the p/CIP residues 11091402),
and xSRC-3
N (the xSRC-3 residues 10931391) were subcloned into
EcoRIXhoI sites of the CMV/T7 vector pcDNA3
(Invitrogen). Reporter constructs p53RE-Luc, MDM2-Luc, and
p21waf-Luc were kind gifts of Moshe Oren (Weisman Institute
of Science, Rehovot, Israel) and Leonard Freedman (Cornell
University, New York, NY).
Yeast Two-Hybrid Tests
For the yeast two-hybrid tests, plasmids encoding LexA fusions
and B42 fusions were cotransformed into Saccharomyces
cerevisiae EGY48 strain containing the LacZ reporter
plasmid, SH/1834 (35). Plate and liquid assays of ß-gal expression
were carried out as described (35). Similar results were obtained in
more than two similar experiments.
GST Pull-Down Assays
The GST fusions or GST alone was expressed in Escherichia
coli, bound to glutathione-Sepahrose-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 previously (35).
Cell Culture and Transfections
HeLa 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 each
reporter gene, along with increasing amounts of expression vectors for
SRC-1, VP16/SRC-1, p53, c-Fos, Gal4/p53, Gal4/p53
C, AIB1, xSRC-3,
p/CIP, or p300 as indicated. After 12 h, cells were washed and
re-fed with DMEM containing 10% FCS. Cells were harvested 36 h
later, luciferase activity was assayed as described (35), and the
results were normalized to the LacZ expression. Similar
results were obtained in more than three similar experiments.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Moshe Oren for p53RE-Luc and MDM2-Luc plasmids and
Dr. Leonard Freedman for p21waf-Luc plasmid. We also thank
Chris Glass, Paul Meltzer, and Ming Tsai for plasmids.
This work was exclusively supported by the National Creative Research
Initiatives sponsored by the Korean Ministry of Science and
Technology.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Jae Woon Lee, Ph.D., Center for Ligand and Transcription, Chonnam National University, Kwangju 500757, Korea.
Received for publication April 7, 1999.
Revision received June 25, 1999.
Accepted for publication July 15, 1999.
 |
REFERENCES
|
---|
-
Levine AJ 1997 p53, The cellular gatekeeper for growth
and division. Cell 88:323331[Medline]
-
El-Deiry WS, Kern SE, Pietenpol JA, Kinzler KW, Vogelstein B 1992 Definition of a consensus binding site for p53. Nat Genet 1:4549[Medline]
-
Funk WD, Pak DT, Karas RH, Wright WE, Shay JW 1992 A
transcriptionally active DNA-binding site for human p53 protein
complexes. Mol Cell Biol 12:28662871[Abstract]
-
Hsiao M, Low J, Dorn E, Ku D, Pattengale P, Yeargin J, Haas M 1994 Gain-of-function mutations of the p53 gene induce
lymphohematopoietic metastatic potential and tissue invasiveness.
Am J Pathol 145:702714[Abstract]
-
Iwamoto KS, Mizuno T, Ito T, Tsuyama N, Kyoizumi S, Seyama T 1996 Gain-of-function p53 mutations enhance alteration of the T-cell
receptor following X-irradiation, independently of the cell cycle and
cell survival. Cancer Res 56:38623865[Abstract]
-
Gualberto A, Aldape K, Kozakiewicz K, Tlsty TD 1998 An
oncogenic form of p53 confers a dominant, gain-of-function phenotype
that disrupts spindle checkpoint control. Proc Natl Acad Sci USA 95:51665171[Abstract/Free Full Text]
-
Horwitz KB, Jackson TA, Bain DL, Richer JK, Takimoto GS, Tung
L 1996 Nuclear receptor coactivators and corepressors. Mol Endocrinol 10:11671177[Abstract]
-
Goldman PS, Tran VK, Goodman RH 1997 The multifunctional role
of the co-activator CBP in transcriptional regulation. Recent Prog Horm
Res 52:103119[Medline]
-
Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G,
Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans RM 1995 The
nuclear receptor superfamily: the second decade. Cell 83:835839[Medline]
-
Chakravarti D, LaMorte VJ, Nelson MC, Nakajima T, Juguilon H,
Montminy M, Evans RM 1996 Role of CBP/p300 in nuclear receptor
signaling. Nature 383:99103[CrossRef][Medline]
-
Hanstein B, Eckner R, DiRenzo J, Halachmi S, Liu H, Searcy B,
Brown M 1996 p300 is a component of an estrogen receptor coactivator
complex. Proc Natl Acad Sci USA 93:1154011545[Abstract/Free Full Text]
-
Kamei Y, Xu L, Heizel T, Torchia J, Kurokawa R, Gloss B, Lin
SC, Heyman RA, Rose DW, Glass CK, Rosenfeld MG 1996 A CBP integrator
complex mediates transcriptional activation and AP-1 inhibition by
nuclear receptors. Cell 85:403414[Medline]
-
Yao TP, Ku G, Zhou N, Scully R, Livingston DM 1996 The
nuclear hormone receptor coactivator SRC-1 is a specific target of
p300. Proc Natl Acad Sci USA 93:1062610631[Abstract/Free Full Text]
-
Onate SA, Tsai SY, Tsai MJ, OMalley BM 1995 Sequence and
characterization of a coactivator for the steroid hormone receptor
superfamily. Science 270:13541357[Abstract]
-
Kim HJ, Lee SK, Na S-Y, Choi HS, Lee JW 1998 Molecular
cloning of xSRC-3, a novel transcription coactivator from
Xenopus, that is related to AIB1, p/CIP, and TIF2. Mol
Endocrinol 12:10381047[Abstract/Free Full Text]
-
Anzick SL, Kononen J, Walker RL, Azorsa DO, Tanner MM, Guan
XY, Sauter G, Kallioniemi OP, Trent JM, Meltzer PS 1997 AIB1, a steroid
receptor coactivator amplified in breast and ovarian cancer. Science 277:965968[Abstract/Free Full Text]
-
Voegel JJ, Heine MJS, Zechel C, Chambon P, Gronemeyer H 1996 TIF2, a 160 kDa transcriptional mediator for the ligand-dependent
activation function AF-2 of nuclear receptors. EMBO J 15:36673675[Abstract]
-
Li H, Gomes PJ, Chen JD 1997 RAC3, a steroid/nuclear
receptor-associated coactivator that is related to SRC-1 and TIF2. Proc
Natl Acad Sci USA 94:84798484[Abstract/Free Full Text]
-
Chen H, Lin RJ, Schiltz RL, Chakravarti D, Nash A, Nagy L,
Privalsky ML, Nakatani Y, Evans RM 1997 Nuclear receptor coactivator
ACTR is a novel histone acetyltransferase and forms a multimeric
activation complex with P/CAF and CBP/p300. Cell 90:569580[Medline]
-
Takeshita A, Gardona GR, Koibuchi N, Suen CS, Chin WW 1997 TRAM-1, A novel 160-kDa thyroid hormone receptor activator molecule,
exhibits distinct properties from steroid receptor coactivator-1.
J Biol Chem 272:2762927634[Abstract/Free Full Text]
-
Torchia J, Rose DW, Inostroza J, Kamei Y, Westin S, Glass CK,
Rosenfeld MG 1997 The transcriptional coactivator p/CIP binds CBP and
mediates nuclear receptor function. Nature 387:677684[CrossRef][Medline]
-
Lee S-K, Kim H-J, Na S-Y, Kim T-S, Choi H-S, Im S-Y, Lee JW 1998 Steroid receptor coactivator-1 coactivates activating
protein-1-mediated transactivations through interaction with the c-Jun
and c-Fos subunits. J Biol Chem 273:1665116654[Abstract/Free Full Text]
-
Kim H-J, Kim JH, Lee JW 1998 Steroid receptor coactivator-1
interacts with serum response factor and coactivates serum response
element-mediated transactivations. J Biol Chem 273:2856428567[Abstract/Free Full Text]
-
Na S-Y, Lee S-K, Han S-J, Choi H-S, Im S-Y, Lee JW 1998 Steroid receptor coactivator-1 interacts with the p50 subunit and
coactivates nuclear factor
B-mediated transactivations. J Biol
Chem 273:1083110834[Abstract/Free Full Text]
-
Spencer TE, Jenster G, Burcin MM, Allis CD, Zhou J, Mizzen CA,
McKenna NJ, Onate SA, Tsai SY, Tsai MJ, OMalley BW 1997 Steroid
receptor coactivator-1 is a histone acetyltransferase. Nature 389:194198[CrossRef][Medline]
-
Bannister AJ, Kouzarides T 1996 The CBP coactivator is a
histone acetyltransferase. Nature 384:641643[CrossRef][Medline]
-
Ogryzko VV, Schiltz RL, Russanova V, Howard BH, Nakatani Y 1996 The transcription coactivators p300 and CBP are histone
acetyltransferase. Cell 87:953959[Medline]
-
Yang XJ, Ogryzko VV, Nishikawa J, Howard BH, Nakatani Y 1996 A p300/CBP-associated factor that competes with the adenoviral
oncoprotein E1A. Nature 382:319324[CrossRef][Medline]
-
Horlein AJ, Naar AM, Heinzel T, Torchia J, Gloss B, Kurokawa
R, Ryan A, Kamei Y, Soderstrom M, Glass CK, Rosenfeld MG 1995 Ligand-independent repression by the thyroid hormone receptor mediated
by a nuclear receptor corepressor. Nature 377:397404[CrossRef][Medline]
-
Chen JD, Evans RM 1995 A transcriptional corepressor that
interacts with nuclear hormone receptors. Nature 377:454457[CrossRef][Medline]
-
Heinzel T, Lavinsky RM, Mullen TM, Soderstrom M, Laherty CD,
Torchia J, Yang WM, Brard G, Ngo SD, Davie JR, Seto E, Eisenman RN,
Rose DW, Glass CK, Rosenfeld MG 1997 A complex containing N-CoR, mSin3
and histone deacetylase mediates transcriptional repression. Nature 387:4348[CrossRef][Medline]
-
Nagy L, Kao HY, Chakravarti D, Lin RJ, Hassig CA, Ayer DE,
Schreiber SL, Evans RM 1997 Nuclear receptor repression mediated by a
complex containing SMRT, mSin3A, and histone deacetylase. Cell 89:373380[Medline]
-
Lill NL, Grossman SR, Ginsberg D, DeCaprio J, Livingston DM 1997 Binding and modulation of p53 by p300/CBP coactivators. Nature 387:823827[CrossRef][Medline]
-
Avantaggiati ML, Ogryzko V, Gardner K, Giordano A, Levine AS,
Kelly K 1997 Recruitment of p300/CBP in p53-dependent signal pathways.
Cell 89:117584[Medline]
-
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith
JA, Struhl K (eds) 1995 Current Protocols in Molecular Biology. Greene
Associates, New York
-
Forman BM, Umesono K, Chen J, Evans RM 1995 Unique response
pathways are established by allosteric interactions among nuclear
hormone receptors. Cell 81:541550[Medline]
-
Barak Y, Juven T, Haffner R, Oren M 1993 mdm2 expression is
induced by wild type p53 activity. EMBO J 12:461468[Abstract]
-
Wu X, Bayle JH, Olsen D, Levine AJ 1993 The p53-mdm-2
autoregulatory feedback loop. Genes Dev 7:11261132[Abstract]
-
El-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parson R, Trent
JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B 1993 WAF1, a potential
mediator of p53 tumor suppression. Cell 75:817825[Medline]
-
Kawasaki H, Eckner R, Yao T-P, Taira K, Chiu R, Livingston DM,
Yokoyama KK 1998 Distinct roles of the co-activators p300 and CBP in
retinoic-acid-induced F9-cell differentiation. Nature 393:284289[CrossRef][Medline]
-
Korzus E, Torchia J, Rose DW, Xu L, Kurokawa R, McInerney EM,
Mullen TM, Glass CK, Rosenfeld MG 1998 Transcription factor-specific
requirements for coactivators and their acetyltransferase functions.
Science 279:703707[Abstract/Free Full Text]
-
Hong H, Koli K, Garbedian MJ, Stallcup MR 1997 GRIP1, a
transcriptional coactivator for the AF-2 transactivation domain of
steroid, thyroid, retinoid, and vitamin D receptors. Mol Cell Biol 17:27352744[Abstract]
-
Gu W, Roeder RG 1997 Activation of p53 sequence-specific DNA
binding by acetylation of the p53 C-terminal domain. Cell 90:595606[Medline]