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INTRODUCTION |
The tumor suppressor p53 plays an important role in cell cycle
progression and apoptosis by binding to p53 response elements and
functioning as a transcriptional activator (1, 2). Genes with p53
responsive elements include p21 (3), growth arrest and DNA damage
(GADD45) (4), Bax1 (5), cyclin G, (6) and murine double minute
(mdm2) genes (7, 8). Transactivation of the p21 gene by p53
has been correlated with cell cycle control by inducing G1
arrest or apoptosis (3, 9). In addition to its transactivation
function, when overexpressed, p53 can indirectly act as a
transcriptional repressor through its interaction with the TATA-binding
protein (10), or by squelching coactivators (11).
Transcriptional coactivators p300 and CREB-binding protein
(CBP/p300)1 interact with
wild-type as well as mutant forms of p53 (12-14). CBP/p300 function as
transcriptional coactivators by linking a number of cellular activators
to components of the basal transcription machinery (15, 16). CBP and
p300 are 63% identical overall at the amino acid level. Greater
similarity is observed in specific regions, including the N-terminal
C/H1 region encompassing the CREB binding site (93%), and the
C-terminal C/H3 region, which includes the E1A binding site (15, 16).
Accumulating evidence suggests that regions within these coactivators
may have similar functions. The C-terminal region of CBP/p300 binds to
the activation domain of p53, a region where several other basal
transcription factors interact with p53 (12-14, 17). These
interactions are necessary for p53 to function as a transcription
factor (12-14). The CBP associated factor (p/CAF), a CBP-binding
protein, is also a coactivator for p53-dependent
trans-activation (18). Coactivators like CBP and p/CAF have histone
acetyltransferase activity, suggesting a role for histone acetylation
in transcriptional regulation (19, 20). p300 acetylates the C-terminal
region of p53, resulting in increased DNA binding and transactivation
function of p53 (21).
Overexpression of p53 induces the expression of the mdm2
proto-oncogene, the product of which binds to the trans-activation region of p53 and inhibits its ability to stimulate transcription (22-24). Additionally, recent studies have shown that MDM2 is involved in degradation of p53 via the ubiquitin-proteasome pathway (25, 26).
This negative feedback loop is necessary to control p53 activity
because mdm2 knockout mice die during development, but mice
with mutations in both the mdm2 and p53 genes are normal (27). These observations demonstrate a key role for MDM2 in controlling
p53 activity, and illustrate the importance of understanding how p53 is
regulated by MDM2. Repression of p53-mediated transcription by MDM2 is
thought to involve two mechanisms. First, MDM2 may act by inhibiting
interactions between the transactivating region of p53 and components
of the basal transcriptional machinery (24). Second, MDM2 possesses an
additional intrinsic inhibitory function that directly represses basal
transcription in the absence of p53 (28). The relative importance of
these mechanisms in the repression of p53 activity by MDM2 is not well understood.
Here we focused on the interaction of p53 with a coactivator necessary
for its transactivation ability. We found that the transactivation
domain of p53 selectively interacts with the N- as well as the
C-terminal sections of CBP/p300. The functional significance of the
N-terminal region of CBP/p300 in p53 transactivation was suggested by
findings from transient transfections with dominant negative forms of
the coactivator. A mutant of CBP/p300 lacking just the N terminus was
unable to stimulate p53 transactivation. Similarly, overexpression of
the N-terminal region of CBP/p300 was able to abrogate
p53-dependent transactivation of a p21 promoter-reporter construct. Remarkably, MDM2 also interacts with the N-terminal region
of CBP/p300. In both p53 null Saos2 cells, and in UV-irradiated MCF7
cells, we observed that MDM2 can associate with the N-terminal region
of CBP/p300. The functional significance of these interactions was
demonstrated in vivo in mammalian cells. MDM2 inhibited the interaction of the transactivating region of p53 with either the N- or
C-terminal regions of CBP/p300 in a mammalian two-hybrid assay. These
observations suggest that MDM2 may be inhibiting p53
trans-activation by concealing its activation domain from the
coactivators, a new mechanism for the inhibition of
p53-dependent gene expression.
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EXPERIMENTAL PROCEDURES |
Plasmid Constructions--
For in vitro translation,
cDNAs for wild-type p53 and p53 mutants were cloned into the
BamHI site of the expression vector pSP64A+ (Promega Corp.)
GST-p53 was constructed by subcloning wild-type p53 into the
EcoRI site of pGEX-1
T (Amersham Pharmacia Biotech).
Wild-type p53, pG13-luciferase and the p21 promoter-luciferase reporter
vectors were provided by Dr. Bert Vogelstein (The Johns Hopkins
University). The full-length human MDM2 expression vector was provided
by Dr. Arnold Levine (Princeton, University). A full-length RSV-based
CBP expression vector was provided by Dr. Richard Goodman (Oregon
Health Science University, Portland). Regions of CBP were assembled
into the GAL4 and VP16 vectors provided in the mammalian Matchmaker
two-hybrid assay kit (CLONTECH), as described
(29).
Reticulocyte Translation--
Wild-type and deletion mutants of
p53 were in vitro translated using
[35S]methionine in TNT SP6/T7-coupled rabbit reticulocyte
lysate according to the manufacturer instructions (Promega).
GST Fusion Protein Expression and Pulldown Experiments--
GST
fusion proteins were expressed, and extracts were prepared as
recommended by the manufacturer (Amersham Pharmacia Biotech). Bacterial
extracts containing GST, GST-p53, and GST-CBP were incubated with 20 µl of glutathione-Sepharose in 200 µl of buffer S
(phosphate-buffered saline plus 0.1% Nonidet P-40, containing 1 mM phenylmethylsulfonyl fluoride, 5 mM
dithiothreitol, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1.5 µg/ml
pepstatin) for 3 h at 4 °C. The fusion protein bound beads were
washed three times with 200 µl of 0.5× buffer B (20 mM
HEPES, pH 7.6, 500 mM NaCl, 0.5 mM EDTA, and
0.1% Nonidet P-40). For GST pulldown experiments, fusion proteins
bound to the beads were incubated with proteins from total cell
extracts as well as reticulocyte translated proteins for 3 h at
4 °C. The beads were washed using buffer B containing 150 mM NaCl and eluted with 25 µl of SDS sample buffer (75 mM Tris-HCl, pH 6.8, 0.5% glycerol, 1% SDS, 4%
-mercaptoethanol, 0.01% bromphenol blue), and boiled for 5 min
before separating on an 8% SDS-polyacrylamide gel. Eluted proteins
were subjected to Western blot analysis.
Immunoprecipitation/Western Blot--
Whole cell extracts were
prepared from transfected Saos2 cells by mechanical disruption in 500 µl of lysis buffer (100 mM Tris-HCl, pH 7.6, 150 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1.5 µg/ml pepstatin A, 0.2 mM
levamisole, 10 mM
-glycerophosphate, and 0.5 mM benzamidine) per 100-mm2 plate of confluent
cells. Particulate matter was removed by centrifugation at 10,000 × g for 20 min. Extracts were precleared with rabbit or
mouse secondary antibody and IgG/IgA-agarose beads. Supernatants were
incubated with 10 µg/ml MDM2 (Calbiochem or Santa Cruz Biotechnology) or CBP (Santa Cruz Biotechnology) antibodies, respectively. The antibodies were bound to 50 µl of IgG/IgA-agarose beads. These beads
were washed three times with 1 ml of lysis buffer and resuspended in
SDS sample buffer. The eluted proteins were boiled for 3 min and
separated by SDS-polyacrylamide gel electrophoresis (6-10%). Separated proteins were transferred to nitrocellulose membranes (Schleicher and Schuell), blocked with 5% nonfat dry milk in TBST buffer (20 mM Tris-HCl, pH 7.6, 150 mM NaCl,
0.5% Tween 20), and incubated with either anti-CBP or anti-MDM2
antibodies for 3 h at room temperature. Blots were washed three
times in TBST buffer, incubated with a donkey anti-rabbit or -mouse
secondary antibody conjugated to horseradish peroxidase (Amersham
Pharmacia Biotech), and washed three times in TBST. Bound proteins were
visualized using the ECL chemiluminescence reagent (Amersham Pharmacia
Biotech) followed by autoradiography for 30 s to 60 min.
Tissue Culture, Transfections, and Reporter
Assays--
Approximately 106 osteosarcoma (Saos2) cells
were seeded in 60-mm dishes in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum. Transfections were performed
by the calcium phosphate procedure (30), as described in the figure legends. All transfections contained 0.5 µg of the plasmid
CMV/
-gal as an internal control to monitor transfection efficiency.
Cells were harvested 24-36 h after transfection, and
-galactosidase activity was determined by a colorimetric assay. Mammalian matchmaker two-hybrid assays were performed according to manufacturer protocol (CLONTECH).
UV Irradiation and Preparation of Cell Extracts--
After
removing the media, 80% confluent control and transfected MCF7 cells
were exposed to UV light. The source of UV light was a Stratalinker
(Stratagene), and total exposure was 20 J/m2 at 254 nm UV.
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RESULTS |
The N-terminal Region of CBP/p300 Interacts with p53--
We
studied p53-CBP interaction and its effect on p53-dependent
transactivation as a model to determine the role of coactivators in
p53-dependent cell survival and apoptosis. To define the
interaction domains of CBP with p53 in more detail, CBP was divided
into six overlapping regions (1-6) which were linked to GST and
expressed as fusions in bacteria (Fig.
1A). An in vitro
translated labeled wild-type p53 was incubated with the GST-CBP fusion
proteins. Wild-type p53 showed an interaction with three different
regions of CBP (Fig. 1B), but not GST alone. We found that
the C-terminal region of CBP showed strong (amino acids 1452-1892) and
moderate (amino acids 1892-2441) interactions with p53, consistent
with previous studies (12-14). In addition to these regions of p53 and CBP interaction, the N-terminal region of CBP (amino acids 1-771) also
showed strong binding to p53 (Fig. 1B, lane 3).
This is consistent with the recent report of a p53 binding site in the
N terminus of p300 (15), and the striking homology of the proteins in
this region.

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Fig. 1.
p53 interacts with the N terminus of
CBP. A, schematic representation of functional
interaction domains of CBP. Deletion fragments of CBP were fused to GST
and GAL4 expression vectors and were used in GST pulldown as well as
one- and two-hybrid assays. Functional domains of CBP and some
activator binding sites are indicated. NHR, nuclear hormone
receptor binding region. C/H,
cysteine-histidine-rich region. B, wild-type p53 interacts
with CBP. Various regions of CBP were tested for interaction with
reticulocyte translated wild-type p53. Glutathione-Sepharose beads
containing GST, or GST CBP-(1-771), -(706-1069), -(1069-1452),
-(1452-1892), and -(1892-2441) were mixed with equal amounts of
[35S]methionine-labeled reticulocyte translated wild-type
p53. Input (lane 1) represents one-fourth of the lysates
used for GST pulldown assays. C, wild-type p53 interacts
with N terminus of CBP. Various subsections of the N terminus of CBP
were tested for interaction with reticulocyte translated wild-type p53.
Glutathione-Sepharose beads containing equal amounts of GST, GST
CBP-(1-100), -(100-446), -(1-446), -(373-771), or -(1-771) were
incubated with equal amounts of [35S]methionine-labeled
reticulocyte translated wild-type p53. All GSTs were tested on
Coomassie Blue stained SDS-polyacrylamide gels for equal loading (data
not shown).
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To further define the interaction between p53 and CBP, various
N-terminal deletion mutants of the coactivator (Fig. 1A)
were fused to GST and used in GST pulldown assays with full-length wild-type p53. As described in Fig. 1C, the extreme
N-terminal CBP (amino acids 1-100), which is involved in nuclear
hormone receptor binding, did not show an interaction with p53 (Fig.
1C, lane 3), but the 1-446 region of CBP, and a
smaller section within this region (CBP amino acids 100-446), showed
strong interactions with p53 (Fig. 1C, lanes 4 and 5). The N-terminal domain from amino acids 373-771,
which is important for c-JUN and c-MYB binding, interacted poorly with
p53 (Fig. 1C, lane 6). These results suggest that the
N-terminal region of CBP between amino acids 100-446 is required for
complex formation with p53.
Interaction between the N Terminus of CBP/p300 and the
Transactivation Domain of p53 in Vivo--
To understand the
importance of the interaction between the N-terminal region of CBP and
p53, we verified the binding by mammalian two-hybrid analysis.
Wild-type p53, either containing the transactivation domain (amino
acids 1-393), or lacking this region (amino acids 79-393), were fused
with the transcriptional activator VP16 (Fig. 2A). The N-terminal region of
CBP (amino acids 1-771) and another region (amino acids 500-1000)
were fused to the DNA-binding protein GAL4 (Fig. 2A) and
were tested for interaction with full-length and truncated p53 in
transfected Saos2 cells. In control studies, the ability of CBP-GAL4
and VP16-p53 constructs to activate GAL4-luciferase reporter were
examined first. The N-terminal CBP-GAL4 construct demonstrated some
endogenous transcriptional activating capacity (Fig. 2B,
lane 2), as reported previously by our group (29) and others
(31). However, the GAL4-CBP 1-771 construct showed strong
transactivation in the presence of full-length p53, but not with
truncated p53 (Fig. 2B, compare lanes 6 and
7). There was no interaction between another region of CBP
(amino acids 500-1000) and full-length p53 (Fig. 2B,
lane 8), demonstrating the specificity of N-terminal
interaction. The central section CBP (amino acids 1069-1892), as well
as the C-terminal region of CBP (amino acids 1892-2441), were also
tested for p53 interaction, and they confirmed the GST pulldowns
findings (Fig. 1B) and the previously reported results
(Ref.13, and data not shown). Collectively, these observations suggest
that the transactivation domain of p53 interacts with the N-terminal
section of CBP.

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Fig. 2.
The N-terminal domain of CBP functionally
interacts with the activation domain of p53. A,
structure of the CBP-GAL4 and VP16-p53 fusion constructs. The indicated
regions of CBP were cloned into a vector containing the GAL4 DNA
binding domain. Full-length (amino acids 1-393) and truncated (amino
acids 79-393) forms of wild-type p53 were fused with VP16 activation
domain (AD). B, the N-terminal CBP-(1-771)
region interacts with p53. Saos2 cells were cotransfected with 2 µg
of a GAL4-luciferase reporter gene, expression plasmids for
GAL4-CBP-(1-771) (25 ng), or GAL4-CBP-(500-1000) (25 ng), either
alone, or with an equivalent amount of full-length or truncated p53
(tp53). Total DNA was kept constant at 5 µg per 60-cm
tissue culture dish. Data are representative of three different
experiments.
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The N Terminus of CBP/p300 Is Required for p53
Transactivation--
To determine whether the N terminus of CBP was
functionally important in p53 transactivation, two approaches were
taken. In the first, overexpression experiments were performed with
intact and specifically altered (Fig.
3A) forms of CBP (32).
Overexpression of p53 activated transcription from a p21
promoter-reporter construct approximately 10-fold (Fig. 3B,
lanes 2 and 3). Co-transfection of expression
vectors for intact CBP and p53 further stimulated transcription from
this reporter construct about 4-fold (Fig. 3B, lane
5). This suggests that levels of CBP limit
p53-dependent gene expression, consistent with previous
reports (12-14). Interestingly, both the CBP deletion mutants (
468
and
C/H3) had reduced ability to stimulate p53-dependent
transcription (Fig. 3B, compare lanes 6 and
7, with lane 2). The N-terminal CBP mutant
(
468) had no activity, either activation or repression (Fig.
3B, compare lane 6 with lane 2), while
the
C/H3 region retained some activation capacity, relative to
full-length CBP (Fig. 3B, compare lane 7 with
lane 2).

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Fig. 3.
p53 transactivation involves the N terminus
of CBP. A, schematic of the wild type and mutant CBP
expression constructs. B, expression of an N-terminal CBP
deletion mutant blocks p53 transactivation. CV1 cells were transiently
transfected with 1 µg of a p21 luciferase reporter construct, 10 ng
of p53 (lanes 2 and 4-7), 20 ng of p53
(lane 3), and 1 µg of either intact CBP (lane
5), the N-terminal deletion (lane 6), or the C/H3
region deletion (lane 7). Forty-eight hours
post-transfection, the cells were harvested and luciferase activity was
assayed as described under "Experimental Procedures." Portions of
some of the cellular extracts were analyzed for p53 by Western blot
analysis (insets). Intact CBP, but not the deletions,
increased p53-dependent gene expression. C,
overexpression of the 100-446 region of CBP inhibits p53
transactivation. Saos2 cells were cotransfected with the
pG13-luciferase reporter containing p53 binding sites and increasing
amounts of a p53 expression vector (5, 10, and 20 ng, lanes
2-4). A reporter plasmid was cotransfected with a CBP expression
vector alone (2 and 5 µg, lanes 5 and 6,
respectively), or in the presence of 10 ng of wild-type p53 (2 or 5 µg, lanes 7 and 8, respectively). In
lanes 9 and 10, the p53 reporter was
cotransfected with wild-type p53 (10 ng) and CBP (5 µg) expression
vectors, and increasing amounts of a CMV-based expression vector
directing production of only a small region (100-446) of CBP (1 and 2 µg, respectively). Plasmid concentrations were balanced by using
PCR3-based expression vector. Portions of some of the cellular extracts
were analyzed for p53 by Western blot analysis (insets).
D, overexpression of the 100-446 region of CBP does not
alter expression of -galactosidase activity. Portions of the
cellular extracts described above were analyzed for -galactosidase
activity. The data presented above are representative of at least three
independent transfections.
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In the second approach, to demonstrate that the N terminus of CBP/p300
was functionally relevant to p53 transactivation, we determined the
effect of overexpressing the N terminus of CBP on p53 transactivation.
A small region of CBP (amino acids 100-446) capable of binding p53,
but incapable of interacting with MDM2, was used in overexpression
studies (see Fig. 4A). Saos2
cells were transiently transfected with a reporter plasmid containing multiple p53 response elements (PG13) and increasing amounts of wild-type p53. The tumor suppressor activated expression of the p53-luciferase reporter (Fig. 3C, lanes 1-4). In
the presence of p53, CBP stimulated the activity of the reporter gene
severalfold (Fig. 3C, lanes 7 and 8).
When the CBP deletion mutant (amino acids 100-446) was co-transfected
with p53 and CBP, a dramatic inhibition of CBP co-activation was noted
(Fig. 3C, lanes 9 and 10). The
dominant negative form of CBP decreased p53 transactivation to below
levels seen with p53 alone (Fig. 3C, compare lane
3 with lanes 9 and 10). Western
blot analysis demonstrated that this effect was not because of
diminished levels of p53 (Fig. 3C, inset). Additionally, the 100-446 region did not inhibit expression of a
co-transfected
-galactosidase vector (Fig. 3D),
suggesting that the inhibition of CBP co-activation was not a
nonspecific effect. Thus, both approaches suggest that the N-terminal
section of CBP is necessary for p53 transactivation.

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Fig. 4.
MDM2 interacts with CBP. A,
interaction of MDM2 and p53 with the N terminus of CBP. Several regions
of the N terminus of CBP were tested for interaction with p53 and MDM2.
Confluent cultures of MCF7 cells were exposed to ultraviolet light (20 J/m2 at 254 nm), and total cell extracts were prepared.
Glutathione-Sepharose beads containing GST or GST-CBP (amino acids
1-100, 1-446, and 1-771) were mixed with MCF7 cell lysates. Western
blot analysis for p53 (top) and MDM2 (bottom) was
preformed as described under "Experimental Procedures."
B, interaction of MDM2 with N-terminal of CBP in p53 null
Saos2 cells. Saos2 cells were transfected with 10 µg of a CMV-MDM2
expression vector, total cell lysates were prepared, and GST pulldown
experiments were performed. N terminus of CBP (amino acids 1-771) with
GST and GST retinoic acid receptor were tested for MDM2 binding.
C, interaction of MDM2 and CBP in vivo in p53
null cells. Saos2 cells were transfected with 10 µg of CMV-MDM2
expression vector, and total cells extracts were prepared and
precleared using secondary antibody and IgG/IgA-agarose beads for
4 h at 4 °C with gentle agitation. Supernatants were
immunoprecipitated using an anti-MDM2 antibody or using nonimmune IgG
and IgG/IgA-agarose beads for 4 h at 4 °C with gentle
agitation. After extensive washing, samples were detected by Western
blot analysis using a CBP antibody. Anti-MDM2, but not control IgG,
retained CBP (compare lanes 2 and 3).
D, interaction of MDM2 and CBP in vivo in cells
expressing p53. ECV-304 cells were treated with ultraviolet light (20 J/m2 at 254 nm). Total cell extracts were prepared,
precleared, and subjected to immunoprecipitation with an anti-MDM2
antibody followed by Western blot analysis with antibodies to CBP,
MDM2, or p53, as described above. HC, IgG heavy chain.
E, interaction of CBP and MDM2 in vivo.
Supernatants of MDM2-transfected Saos2 cells were immunoprecipitated
using anti-CBP, anti-p300, or nonimmune IgG with IgG/IgA-agarose beads.
After extensive washing, retained proteins were detected by Western
blot analysis using an MDM2 antibody. Both anti-CBP and anti-p300
antibodies retained MDM2 (lanes 2 and 3).
Identical quantities of nonimmune IgG did not retain MDM2 (lane
4). Input (lane 1) represents one-tenth of the lysate
used in immunoprecipitation assay.
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Association of MDM2 and CBP--
The cellular proto-oncogene MDM2
forms an autoregulatory loop with p53 because the MDM2 gene
contains a promoter that is responsive to wild-type p53 (22). Synthesis
of MDM2 protein increases in a p53-dependent manner in
response to cellular stress such as DNA damaging agents like UV light.
MDM2 interacts with the activation domain of p53 and blocks the
transactivation ability of the tumor suppressor. Because MDM2 inhibits
p53 transactivation, we determined whether MDM2 interacts with CBP.
MCF7 cells were irradiated with UV to induce p53 expression, and total
cell extract was used for GST pulldown experiments with the N-terminal
deletion mutants of CBP. As shown in Fig. 4A, we observed
strong interaction of wild-type p53 with both the 1-446 and 1-771
portion of CBP. UV irradiation also induces MDM2 expression in these
cells 2-3 h after treatment (data not shown). MDM2 showed an
interaction with the N-terminal region of CBP-(1-771), but not with
GST alone, or either the 1-100 or the 1-446 regions of CBP (Fig.
4A). The poor interaction of MDM2 to CBP-(1-446) suggests
that the MDM2 site on CBP is distinct from that of p53. This raises the
possibility that the p53 and MDM2 binding sites on the N terminus of
CBP/p300 are not mutually exclusive.
It was important to know whether the association of MDM2 with CBP was
dependent on the presence of p53, or whether the interaction occurred
in the absence of the activator. To test this, p53 null Saos2 cells
were transfected with a MDM2 expression vector, and GST pulldown assays
were performed using the N-terminal region of CBP (amino acids 1-771).
In the absence of p53, MDM2 bound to the 1-771 region of CBP, but not
to GST alone (Fig. 4B). This suggests that the MDM2
interaction with CBP is independent of the tumor suppressor.
To demonstrate the physical association of MDM2 with CBP in a cellular
context, we overexpressed MDM2 in p53 null Saos2 cells and used
immunoprecipitation-Western blot analysis to detect interactions. Extracts from cells transiently transfected with a MDM2 expression vector were immunoprecipitated using an MDM2-specific antibody. The
coactivator was detected in immunoprecipitates with the MDM2 antibody,
but not with nonimmune IgG (Fig. 4C, lane 2).
This supports the finding that MDM2 can interact with CBP in the
absence of p53. The same approach was taken in an endothelial cell line
(ECV-304) that expresses p53. Whole cell extracts were prepared from
control cells, or cells exposed to ultraviolet light. Subsequent
immunoprecipitation-Western blot analysis revealed that in these
p53-containing cells, the tumor suppressor is contained in the MDM2
immunoprecipitation products (Fig. 4D).
To further demonstrate this association, the complementary study of
exchanging the antibodies was done. Cell lysates from MDM2-programmed
Saos2 cells were immunoprecipitated with either CBP or p300 antibodies,
and Western blot analysis was performed with an anti-MDM2-specific
serum. Antibodies specific for either CBP or p300, but not an
irrelevant antibody, contained MDM2 in the immunoprecipitated products
(Fig. 4E). These studies demonstrate that in a p53 null
background, MDM2 can associate with CBP in intact cells. These findings
are consistent with a recent report demonstrating that MDM2 can bind to
the N terminus of p300 (15).
MDM2 Blocks p53 Interactions with Both the N- and C-terminal
Regions of CBP/p300 in Vivo--
To study the effect of MDM2 on
p53-CBP interactions in vivo, we first verified that MDM2
inhibited p53-dependent transactivation. The
transactivation region of p53 (amino acids 1-100) was fused to the
Gal4 DNA binding domain and co-transfected with a GAL4-luciferase reporter (PF4). In this one-hybrid assay in Saos2 cells, the p53 GAL4
construct stimulated expression of the reporter (Fig.
5A, lane 2).
Expression of MDM2 inhibited transcriptional activation by GAL4-p53 in
a concentration-dependent manner (Fig. 5B,
lanes 3-5), although vector alone did not inhibit the GAL4
reporter activity (Fig. 5A, lanes 6-8). This
suggests that MDM2 blocked p53 transactivation, consistent with
previous studies (17, 22).

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Fig. 5.
MDM2 inhibits p53-CBP/p300 interaction
in vivo. A, MDM2 inhibits GAL4-p53
transactivation. 2 µg of a GAL4-luciferase reporter (pF4)
were cotransfected into Saos2 cells with 20 ng of a GAL4-p53 expression
vector containing the transactivating region of p53 fused to a GAL4 DNA
binding domain either together (lane 2) or with increasing
amounts (250 ng, 500 ng, or 1 µg) of an MDM2 expression vector
(lanes 3-5), or with equivalent amounts of an empty pCR3
expression vector (lanes 6-8). MDM2 inhibits p53
transactivation by the GAL4-p53 fusion protein. B, MDM2
negatively regulates GAL4-CBP-(1-771) mediated transactivation. A
GAL4-CBP-(1-771) expression vector (2.0 µg) was cotransfected into
Saos2 cells with the GAL4-luciferase reporter (pF4), either
alone (lanes 2 and 3), with increasing amounts
(500 ng or 1 µg) of an MDM2 expression vector (lanes 4 and
5, respectively), or with equivalent amounts of an empty
pCR3 expression vector (lanes 6 and 7). MDM2
inhibits the endogenous transcriptional activity of the N terminus of
CBP. C, MDM2 blocks the interaction between p53 and the N
terminus of CBP in a mammalian two-hybrid system. In Saos2 cells, 2 µg of the GAL4-luciferase reporter (pF4) was cotransfected
with 25 ng of the GAL4-CBP-(1-771) expression vector and 25 ng of the
VP16-p53 expression vector (200 ng), either alone (lane 4),
or with increasing amounts (100, 200, or 400 ng) of either MDM2
(lanes 5-7) or 500 ng of a control vector (lane
8). Total DNA concentration in all transfected plates was adjusted
to 5 µg of total DNA with empty pcDNA expression plasmid. MDM2
inhibits the interaction of p53 with the N terminus of CBP.
D, MDM2 blocks the interaction between p53 and the
C-terminal region of CBP in a mammalian two-hybrid system. In Saos2
cells, 2 µg of the GAL4-luciferase reporter (pF4) was
cotransfected with 20 ng of a GAL4-p53 expression vector, either alone
(lane 2), or with increasing amounts of a
VP16-CBP-(1452-1892) expression vector (200 or 500 ng), in the absence
(lanes 5 and 6), or presence of 400 ng of MDM2
(lanes 7 and 8). Total DNA concentration in all
transfected plates was adjusted to 5 µg of total DNA with empty
pcDNA expression plasmid. MDM2 inhibits the interaction of p53 with
the C terminus of CBP. In panels A-D, data are
representative of three different experiments. E, MDM2 bound
to p53 blocks binding of the N-terminal 100-446 region of CBP.
Glutathione-Sepharose beads containing GST alone, or GST-p53 were
incubated with reticulocyte translated
[35S]methionine-labeled CBP-(100-446), and GST pulldown
assays were performed. Input (lane 1) represents the amount
of CBP-(100-446) used in the assay. In lane 4,
MDM2-transfected Saos2 total cell extract was incubated with GST-p53
beads; after washing, the beads were further incubated with
35S-labeled CBP-(100-446). Prebinding of MDM2 to p53
blocks the interaction with CBP.
|
|
The findings outlined above demonstrated that MDM2 interacted with the
N-terminal region of CBP/p300. As noted previously, this section of the
coactivator has endogenous transcriptional activity. To determine
whether MDM2 binding blocks this transcriptional activity, we again
utilized a one-hybrid approach. As shown in Fig. 5B, the N
terminus of CBP activates transcription of a GAL4 reporter construct in
Saos2 cells. Expression of MDM2, but not vector, results in repression
of reporter gene activity. Because p53 is not present in these cells,
the decrease in reporter gene activity may be a direct effect of MDM2
on CBP 1-771 transactivation. This suggests that binding of MDM2 to
this region of CBP blocks the endogenous transcriptional activating
capacity of the coactivator.
To examine the effect of MDM2 on interactions between p53 and either
the N- or C-terminal regions of CBP, we utilized the mammalian
two-hybrid system. An MDM2 expression vector was cotransfected with
GAL4-CBP-(1-771) and VP16-p53-(1-100) expression constructs into
Saos2 cells. A dose-dependent down-regulation of GAL4
reporter construct activity was noted (Fig. 5C, lanes
5-7). Overexpression of an irrelevant vector did not diminish the
reporter gene activity (Fig. 5C, lane 8). In
control studies, Western blot analysis revealed that levels of the
VP16-p53 activator did not diminish with increasing amounts of MDM2
(data not shown). Similarly, levels of
-galactosidase did not
diminish with increasing amounts of MDM2 (data not shown).
A similar approach was used to examine the effect of MDM2 on
interactions between p53 and the C-terminal region of CBP. In a
mammalian two-hybrid assay in Saos2 cells, the MDM2 expression vector
was cotransfected with VP16-CBP-(1452-1892) and GAL4-p53 expression
constructs. MDM2 decreased the expression of the
GAL4-dependent reporter construct (Fig. 5D,
lanes 10-12). In control studies, levels of
-galactosidase did not diminish with increasing amounts of MDM2
(data not shown), indicating that the inhibition by MDM2 was not a
nonspecific effect.
MDM2 Blocks Recruitment of the N Terminus of CBP/p300 to
p53--
The previous studies suggest that MDM2 blocks the interaction
between p53 and both the N- and C-terminal regions of CBP. This could
be because of MDM2 binding to p53 and inhibiting the interaction between the activator and CBP. Alternatively, MDM2 may be binding to
CBP and blocking its interaction with p53. Because MDM2 does not
repress the expression of other CBP-dependent inducible
genes in overexpression studies (33), and CBP must be recruited to p53
to perform its role as a coactivator, we investigated whether MDM2
blocked CBP recruitment to p53. GST-p53 beads were first saturated with
unlabeled MDM2 transfected Saos2 cell extracts and then were incubated
with a labeled region of the N terminus of CBP (amino acids 100-446).
As expected, GST protein alone did not show any binding of the CBP, but
GST-p53 alone showed significant binding to this portion of the
coactivator (Fig. 5E, lane 3). Binding of
CBP-(100-446) to GST-p53 was blocked by MDM2 (Fig. 5E,
lane 4). This suggests that recruitment of the N-terminal region of CBP to p53 can be inhibited by MDM2.
 |
DISCUSSION |
In this study we found the transactivation domain of p53, and its
inhibitor MDM2, functionally interacts with the N-terminal region of
CBP/p300. MDM2 inhibited the interaction between the transactivating
region of p53 and both the N- and C-terminal regions of CBP/p300
in vivo in a mammalian two-hybrid assay, and blocked p53
recruitment of CBP/p300 in vitro in an interaction study. These findings provide new insights into how MDM2 negatively regulates p53-dependent gene expression.
Repression of p53-mediated transcription by MDM2 is thought to involve
three mechanisms. First, MDM2 could act by inhibiting interactions
between the transactivating region of p53 and components of the basal
transcriptional machinery (24). This is consistent with studies
reporting that the transactivation domain of p53 interacts with various
nuclear proteins from basal transcription machinery, including
TATA-binding protein, TAFII 31, and TAFII 70 (10, 34, 35). The
interaction of the basal factors with p53 involves residues
Leu22 and Trp23. This region of p53 is also
involved in MDM2 interaction (36), which shows the complexity and
importance of this region in transcriptional activity of wild-type p53.
A second mechanism by which MDM2 represses p53 transcription involves
an intrinsic inhibitory function that directly represses basal
transcription in the absence of p53 (28). When MDM2 is recruited to
p53, it may provide a potent negative effect on the function of the
basal transcriptional machinery. The third mechanism by which MDM2 can
regulate p53 function is by stimulating the degradation of the
activator. Recent studies demonstrated that MDM2 acts as a ubiquitin
ligase E3 for rapid degradation of p53 via ubiquitin-proteasome pathway
(25, 41). In this model, MDM2 binds to p53 and inhibits gene expression by stimulating proteosome-mediated degradation of the transcription factor. Association of MDM2 with CBP/p300 may also have an important modulatory role in p53 stability. Each of these mechanisms contributes to the overall inhibitory effect of MDM2, although the relative importance of these p53 control systems is uncertain.
The new findings outlined here indicate that MDM2 has an additional
mechanism of action. When p53 stimulates the transcription of target
genes, it is probably positioned in a collection of other transcription
factors that generates a precise network of interactions that are
unique to a given p53 target gene. This assembly of activators presents
a distinct surface that is required for CBP/p300 recruitment, as well
as a surface that is displayed to the basal transcriptional machinery
(16, 37). This leads to cooperative recruitment of RNA polymerase II
and chromatin remodeling factors to DNA, and to synergistic activation
of transcription (38, 39). Any change in the ability of the
transcription factors to interact with CBP has a dramatic effect on the
activation of gene expression (40). The data reported here are
consistent with this type of model, in that MDM2 inhibited the
interaction between both the transactivating region of p53 and the N-
and C-terminal regions of CBP/p300 and blocked p53 recruitment of the
coactivator. The new observations are also consistent with previous
mechanisms of MDM2 repression (28). p53 containing enhancer assemblies
could also generate another distinct surface that interacts with the
general transcription factors and their associated proteins. By binding
to p53, MDM2 also decreases interactions with the basal transcriptional
machinery. Thus, binding of MDM2 would block two parallel pathways by
which p53-containing enhancers engage the transcriptional apparatus,
resulting in stringent control of transcription from p53-responsive promoters.