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Journal of Virology, May 1999, p. 3574-3581, Vol. 73, No. 5
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Characterization of an E1A-CBP Interaction Defines
a Novel Transcriptional Adapter Motif (TRAM) in CBP/p300
Mark J.
O'Connor,1,*
Holger
Zimmermann,1
Søren
Nielsen,2
Hans-Ulrich
Bernard,1 and
Tony
Kouzarides2,3
Institute of Molecular and Cell Biology,
Singapore 117 609, Singapore,1 and
Wellcome/CRC Institute,2 and
Department of Pathology, University of Cambridge, Cambridge CB2
1QR,3 United Kingdom
Received 3 November 1998/Accepted 26 January 1999
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ABSTRACT |
The adenovirus E1A protein subverts cellular processes to induce
mitotic activity in quiescent cells. Important targets of E1A include
members of the transcriptional adapter family containing CBP/p300.
Competition for CBP/p300 binding by various cellular transcription
factors has been suggested as a means of integrating different
signalling pathways and may also represent a potential mechanism by
which E1A manipulates cell fate. Here we describe the characterization
of the interaction between E1A and the C/H3 region of CBP. We define a
novel conserved 12-residue transcriptional adapter motif (TRAM) within
CBP/p300 that represents the binding site for both E1A and numerous
cellular transcription factors. We also identify a sequence (FPESLIL)
within adenovirus E1A that is required to bind the CBP TRAM.
Furthermore, an E1A peptide containing the FPESLIL sequence is capable
of preventing the interaction between CBP and TRAM-binding
transcription factors, such as p53, E2F, and TFIIB, thus providing a
molecular model for E1A action. As an in vivo demonstration of this
model, we used a small region of CBP containing a functional TRAM that
can bind to the p53 protein. The CBP TRAM binds p53 sequences targeted
by the cellular regulator MDM2, and we demonstrate that an MDM2-p53
interaction can be disrupted by the CBP TRAM, leading to stabilization
of cellular p53 levels and the activation of p53-dependent
transcription. Transcriptional activation of p53 by the CBP TRAM is
abolished by wild-type E1A but not by a CBP-binding-deficient E1A mutant.
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INTRODUCTION |
The major role of the adenovirus E1A
protein is to induce cell entry into the S phase of the cell cycle,
thus providing an optimal environment for viral replication
(48). The biological activity of E1A is dependent on
interactions with a number of cellular proteins, most notably, the
pocket-containing proteins characterized by the Rb tumor suppressor
protein and members of the CBP/p300 family of transcriptional
coactivators (22, 34).
CBP and the closely related protein p300 are thought to play a
fundamental role in a variety of signal-modulated cellular events
(20, 31, 38). First described as a coactivator of the cyclic
AMP-responsive regulator CREB (8), CBP has since been shown
to interact with a large and diverse set of transcription factors
(21).
The activation of gene expression by CBP has been attributed to two
very different properties. First, CBP interacts with proteins that
contain acetyltransferase (AT) activity (55) and possesses intrinsic AT activity (3, 41) capable of modifying histones (3, 41) and nonhistone transcription factors (12,
19). This intrinsic AT activity of CBP was very recently shown to
be directly involved in stimulating gene transcription (32).
For histone acetylation, activation has been suggested to result from an increase in the accessibility of the target promoter to
transcription factors (27, 37). A second mechanism by which
CBP has been suggested to activate transcription is by bridging the gap
between DNA-bound transcription factors and components of the general transcription machinery. CBP is a large protein (2,441 amino acids), and its ability to interact simultaneously with a number of factors has
led to its description as a transcriptional adapter protein (20). An important insight into how CBP might activate
transcription as an adapter molecule was recently provided when CBP was
shown to activate CREB-dependent expression through an interaction with RNA helicase A, a component of an RNA polymerase II complex
(36). Both of the above mechanisms of activation depend on
the recruitment of CBP to a particular promoter by specific
protein-protein interactions.
Evidence from studies of CBP-associated disease (11, 43) and
knockout mice (50, 56) suggests that cellular levels of CBP
may be rate limiting. Support for such an idea comes from observations
that different signal transduction pathways with a mutual dependence on
CBP antagonize one another, but not when intracellular CBP levels are
artificially raised. These observations were first noted for the
repression of AP-1 activity by nuclear receptors (23), and
subsequent examples have been described for Stat2 and NF
B
(18) and the AP-1 and JAK/STAT pathways (17). Together, these observations have led to the proposals that CBP acts as
an integrator of different signalling pathways and that sequestration
of CBP by particular transcription factors functions to select which
set of genes is to be expressed (6, 23).
This integration model for the regulation of signal transduction
pathways is also consistent with results obtained from studies with the
adenovirus E1A protein. An increasing body of evidence suggests that
one mechanism by which E1A functions to subvert cellular processes is
to displace cellular transcription factors from CBP (2, 10, 13,
28, 29, 45, 49). Consistent with this idea is the initial
observation that E1A binds a region of CBP (between amino acids 1621 and 1877 and often referred to as the C/H3 region) (9) that
is recognized by a number of important transcriptional regulators,
including p53 (1, 13, 29, 49), E2F (51), TFIIB
(25), MyoD (45, 47, 57), RNA helicase A
(36), and P/CAF (55).
We wished to establish a molecular basis for the mechanism by which E1A
controls the CBP-dependent integration of signal transduction pathways.
We therefore set about defining precisely the sequences responsible for
the E1A-CBP C/H3 interaction. From this analysis, we have now defined
an interaction motif within the CBP C/H3 region that is responsible for
binding E1A as well as a number of diverse transcription factors. This
transcriptional adapter motif (TRAM) is conserved in all members of the
CBP/p300 family of proteins. Moreover, we present data showing that the
sequence FPESLIL in E1A is required for E1A binding to the CBP TRAM and
for the ability of an E1A peptide to competitively inhibit the
interaction of cellular transcription factors with the CBP TRAM both in
vitro and in vivo.
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MATERIALS AND METHODS |
Plasmids and fusion proteins.
Glutathione
S-transferase (GST)-CBP constructs were created by cloning
PCR-amplified fragments or double-stranded oligonucleotides into
pGEX2TKP (a modified version of the Pharmacia vector pGEX2TK that
contains a new polylinker). GST-E1A constructs contained either
wild-type sequences from adenovirus type 12 (Ad12) E1A residues 14 to
72 or sequences containing alanine substitutions.
The GST-MDM2 (residues 1 to 125) construct was a gift from B. Li. GST
fusion proteins were expressed in Escherichia coli, extracted with lysis buffer (50 mM Tris-HCl [pH 8.0], 0.5 mM EDTA, 5 mM dithiothreitol, 15% glycerol, 1 mg of lysozyme per ml, 1 mM
phenylmethylsulfonyl fluoride), sonicated, centrifuged, and stored at
70°C.
Detection of protein-protein interactions with microaffinity
columns.
Bacterial lysate containing GST fusion proteins was
incubated with glutathione-Sepharose beads (Pharmacia) for 30 min at
4°C in 1× NENT buffer (100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, Tris-HCl [pH 8.0]). After being centrifuged and washed with 1 ml of
1× NENT, the beads were loaded into a yellow Gilson pipette tip
containing a glass bead (BDH; catalog no. 332134Y) to create a 25-µl
GST microaffinity column. In vitro transcription and translation (IVT)
of proteins incorporating 35S-methionine were performed
with TNT kits (Promega) in accordance with the manufacturer's
recommendations. Forty microliters of a 50-µl IVT reaction mixture
was diluted with 360 µl of IPD buffer (50 mM KCl, 40 mM HEPES [pH
7.5], 5 mM 2-
-mercaptoethanol, 0.1% Tween 20, 0.5% milk) before
being passed through the GST microaffinity column. After the column was
washed twice with 200 µl of wash buffer (IPD buffer containing 150 mM
KCl), proteins were eluted from the column by adding 25 µl of 2×
sodium dodecyl sulfate (SDS) loading dye, heating to 95°C for 5 min,
chasing with 25 µl of water, and centrifuging in a microcentrifuge.
Peptide competition assays.
To study the influence of
specific peptides on protein-protein interactions, GST fusion proteins
were bound to glutathione-Sepharose beads as described previously. For
E1A peptide competition, the washed beads were incubated (rotated) in
200 µl of IPD buffer containing a peptide (final concentration, 10 µM to 100 mM) at 4°C for 1 h. Forty microliters of an in vitro
translation reaction mixture was diluted with 60 µl of IPD buffer and
added to the sample before further incubation for 30 min at 4°C. The
glutathione-Sepharose beads were centrifuged and washed as described
above before the bound proteins were eluted by heating at 95°C for 5 min in 50 µl of 1× SDS loading dye. For the CBP TRAM peptide
competition studies, the peptide was preincubated with diluted in vitro
translation reaction mixture for 15 min at 4°C before being incubated
with GST fusion proteins.
Transfections and chloramphenicol AT assays.
U-2 OS cells
were plated onto 10-cm-diameter culture dishes and transfected at 50 to
70% confluence with Lipofectin reagent (GIBCO-BRL) as described
previously (39). Chloramphenicol AT assays have been
described previously (39), and the data presented represent
between 3 and 10 independent transfection experiments.
p53 stability experiments.
U-2 OS cells were transfected
with 0.1 µg of CMV-p53, 2 µg of either CMV-MDM2 or empty
cytomegalovirus (CMV) expression vector, and 5 µg of either
CMV-GST-CBP (residues 1808 to 1852), CMV-GST-CBP (residues 1808 to
1852) Mut, or empty GST expression vector. At 24 h after
transfection, cells were shifted to medium containing 25 µg of
cycloheximide per ml and were harvested at various times.
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RESULTS |
E1A binds a 12-amino-acid motif in the CBP C/H3 region.
Many
transcription factors that interact with CBP bind the same
257-amino-acid, cysteine-rich region (C/H3 region) between residues
1621 and 1877 (Fig. 1A). The adenovirus
E1A protein has also been shown to bind this region, and it has been
proposed that E1A might repress the activity of these cellular factors by competing for binding to CBP. We therefore sought to establish the
binding site for E1A within CBP in the hope of providing some insight
into the molecular mechanism of E1A action.
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FIG. 1.
E1A binds a 12-amino-acid motif in CBP (amino acids 1811 to 1822). (A) Schematic representation of CBP and some of the
CBP-interacting proteins. (B) Use of GST-CBP fusion constructs to
define sequences capable of binding 12S E1A. Microaffinity column
experiments are described in Materials and Methods. Approximately 10%
of the E1A translation reaction mixture was run in the input lane, the
GST lane represents a control column, and lanes 1 to 9 represent the
eluate obtained after passage of E1A over columns containing the nine
GST-CBP fusion constructs. (C) Further deletion analysis of CBP amino
acids 1808 to 1826. Thick lines indicate constructs that bind E1A; thin
lines those that do not. A minimal construct of 12 amino acids
(construct 10; CBP amino acids 1811 to 1822) still retains E1A-binding
activity.
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Using glutathione-Sepharose microaffinity columns and a series of
GST-CBP fusion proteins, we initially identified a 19-amino-acid
region
of CBP (1808 to 1826) that was sufficient for the binding
of E1A (Fig.
1B, lane 8). Deletion into this region abolished
E1A binding (Fig.
1B,
lane 9). Further fine-deletion analysis
of CBP (amino acids 1808 to
1826) identified a 12-residue sequence
(1811 to 1822) that was
sufficient for E1A binding (Fig.
1C),
although this sequence bound with
a slightly lower affinity than
the larger, 19-residue sequence (1808 to
1826). A comparison of
analogous regions of CBP/p300 from a number of
species revealed
a high degree of conservation of this sequence. We
have therefore
termed the sequence between amino acids 1811 and 1822 of
CBP a
transcriptional adapter motif
(TRAM).
Identification of amino acids within E1A required for the
interaction with the CBP TRAM.
Having identified the E1A-binding
region within the C/H3 domain of CBP, we were interested in identifying
the residues in E1A responsible for binding the CBP TRAM. Previous
dissection of E1A implicated residues 63 to 67 in binding the C/H3
domain of CBP. Figure 2 represents an
alanine substitution analysis of this E1A region and shows that the
sequence FPESLIL could be defined as essential for binding to CBP
(amino acids 1621 to 1877). Mutagenesis of any two residues within this
sequence (FE, PS, EL, SI, or LL) to alanine abolished binding to CBP.
However, while single-residue substitutions E67A, S68A, and L69A
resulted in a small reduction in CBP binding, no single substitution
was able to prevent the E1A-CBP complex, indicating that the
interaction between these proteins is dependent upon a combination of
residues. The mutations that altered residues outside the FPESLIL
sequence (F64P66 and E63F65) resulted in a very slight increase in
binding over that seen with the wild-type E1A sequence. However,
corresponding variations in the loading of the GST-E1A proteins suggest
that this difference in binding affinity is probably not significant.
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FIG. 2.
Identification of the amino acids in E1A involved in the
interaction with CBP. (Top) Schematic representation of E1A. Contained
within conserved region 1 (CR1) are residues 63 to 67, previously
implicated in the binding of CBP. GST-E1A proteins containing wild-type
or mutated (alanine-substituted) sequences were tested for their
ability to interact with 35S-labelled CBP (amino acids 1621 to 1877). Double substitution mutants within sequences F65 to L71
failed to bind CBP. (Bottom) Coomassie blue-stained SDS-polyacrylamide
gel revealing quantities of GST-E1A fusion proteins recovered from the
microaffinity columns. WT, wild type.
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The peptide competition studies depicted in Fig.
3 confirmed the importance of the FPESLIL
residues for the interaction between
E1A and the CBP TRAM. Peptides
containing wild-type or mutant
E1A sequences were analyzed for their
ability to prevent the binding
of full-length radiolabelled 12S E1A
protein to a GST-CBP fusion
protein. While E1A binding was detected in
the absence of a competitor
peptide, increasing amounts of the
wild-type E1A peptide abolished
the interaction. In contrast, a peptide
containing mutations in
E67L69 (peptide Mut 1) was abrogated in this
capacity. Additional
mutations (in F65L71) within the FPESLIL residues
(peptide Mut
2) resulted in a corresponding reduction in the ability of
this
peptide to inhibit the E1A-CBP TRAM interaction. Together, these
results demonstrate that the interaction of E1A with the CBP TRAM
involves this small E1A region from residues F65 to L71. It should
be
noted that previously published studies have also suggested
the
importance of L20 and R2 in the binding of p300 by E1A (
53,
54). In our E1A mutagenesis studies and E1A peptide competition
experiments, the L20 residue, but not the R2 residue, was present
and
contributed to the stability of the E1A-CBP TRAM interaction
(
40). Thus, while residues F65 to L71 play a critical role
in
the interaction with the CBP TRAM, L20 and other residues may
also
play an important role by stabilizing the interaction with
the CBP
TRAM.
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FIG. 3.
Peptide competition assays confirming the involvement of
sequences F65 to L71 in the interaction with CBP. Peptides of 30 amino
acids and containing the sequences shown were used in competition
studies (described in Materials and Methods). Only the wild-type (WT)
peptide prevented the E1A-CBP interaction.
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Peptides containing the FPESLIL sequence of E1A can prevent the
interaction of cellular factors with the CBP TRAM.
The CBP TRAM is
within a region of CBP (1621 to 1877) that is a "hot spot" for the
binding of transcription factors (Fig. 1A). We wished to know whether
the TRAM was also the target of these interactions. Figure
4A shows that three transcription
factors, namely, p53, E2F-1, and TFIIB, that bind this region of CBP
were able interact with the CBP TRAM (GST-CBP, 1808 to 1826). Figure 4A
also demonstrates that the interaction of these cellular factors with
the CBP TRAM could be prevented in competition experiments by an E1A
peptide containing the wild-type CBP-binding site but not by a mutant
E1A peptide that was unable to bind CBP (Mut 2).
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FIG. 4.
Multiple cellular factors bind to the CBP TRAM and can
be inhibited by an E1A FPESLIL-containing peptide. (A) p53, E2F, and
TFIIB all bind to CBP amino acids 1808 to 1826, containing the TRAM.
Binding can be inhibited by competition with the wild-type (WT) E1A
peptide but not the Mut 2 (Mut) E1A peptide. , no competitor. (B)
Alignment of adenovirus E1A, p53, and E2F sequences showing the
conservation (boldface) of FXE/DXXXL residues implicated in the
interaction with the CBP TRAM.
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Since E1A, p53, E2F, and TFIIB all recognize the same small motif
within CBP, we wondered whether or not these proteins might
have common
sequences that facilitate the recognition of the CBP
TRAM. In fact,
mutagenesis studies have already defined residues
in two of these
transcription factors (p53 and E2F) that are required
for the
interaction with CBP (
13,
14,
16,
30,
52). These
residues in
p53 and E2F show marked similarity to those identified
in this study
for the adenovirus type 12 E1A protein (and those
of adenovirus type 2 and type 5) (Fig.
4B). In each case, a conserved
sequence (FXE/DXXXL),
when mutated, resulted in an inability to
bind CBP. These findings
provide a possible model to explain how
E1A might regulate the activity
of certain cellular transcription
factors: that is, by utilizing the
FXE/DXXXL sequence and competing
for the same CBP-binding
site.
A small region of CBP (amino acids 1808 to 1852) containing a
functional TRAM is sufficient to bind to p53, stabilize cellular p53
levels, and activate p53-dependent transcription.
Recently, a
number of reports have demonstrated that the expression of full-length
CBP activates p53-dependent transcription in vivo and that this
activation is abolished by E1A (1, 13, 29, 49). Our results
shown in Fig. 4A demonstrated that p53 binds the CBP TRAM and that this
interaction is disrupted in vitro by an E1A peptide containing a
wild-type FPESLIL sequence but not a mutated version. We wished to
extend our in vitro studies and investigate this particular interaction
in more depth in vivo, since p53 is a potentially important target for
adenovirus E1A function.
Cellular p53 protein levels and p53 transcriptional activity are, under
normal circumstances, regulated by the MDM2 protein
(
26),
since the binding of MDM2 to p53 results in both the repression
of p53
transactivation capacity (
33,
42) and the degradation
of the
p53 protein (
15,
24). A previous study has shown that
a
double point mutation within the p53 FSDLWKLL sequence abolishes
the binding of p53 to the C/H3 region of CBP (
13).
Interestingly,
dissection of MDM2 has identified at the MDM2 N terminus
a p53-binding
site that recognizes p53 residues overlapping the p53
FSDLWKLL
sequence (
7,
44). In order to investigate whether
identical
contacts were made with p53 by MDM2 and the CBP TRAM, we
tested
whether a previously described p53 mutant (L14Q/F19L) that
abolishes
the MDM2 interaction had an effect on the binding of the CBP
TRAM.
As shown in Fig.
5A, the p53 mutant
L14Q/F19L (
30), while abolishing
the binding of the MDM2 N
terminus (residues 1 to 125), did not
affect the binding of the CBP
TRAM contained within residues 1715
to 1852. This result demonstrates
that the MDM2 N-terminal domain
and the CBP TRAM recognize overlapping
but distinct residues within
the p53 activation domain.
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FIG. 5.
The CBP TRAM and the N-terminal domain of MDM2 recognize
distinct but overlapping residues on p53. (A) Differential effects of
the p53 mutant L14Q/F19L (p53 mut 14/19) on the MDM2 N-terminal domain
and the CBP TRAM. Binding of p53 to the N-terminal domain of MDM2 was
inhibited by the L14Q/F19L mutation, while binding to the CBP TRAM
remained unaffected. (B) A CBP TRAM peptide can inhibit the binding of
the N-terminal MDM2 domain to p53. CBP peptides of 27 amino acids (1806 to 1832) and containing either wild-type (WT) or mutant (Mut) (R1811,
K1812, and N1814) TRAM sequences were used in competition assays to
prevent the interaction of in vitro-translated p53 and GST-MDM2 (1 to
125). The wild-type peptide completely inhibited the p53-MDM2
interaction over the concentration range used (10 to 100 µM), while
the ability of the mutant TRAM peptide to inhibit the interaction was
severely impaired.
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Since distinct residues are involved in the interaction with p53, we
tested whether or not the binding of the CBP TRAM and
the binding of
MDM2 were mutually exclusive events. Figure
5B
shows the ability of a
peptide containing the CBP TRAM (residues
1806 to 1832) to successfully
inhibit the binding of the MDM2
N-terminal domain (residues 1 to 125)
to p53 in vitro. This competition
was dependent upon an intact TRAM,
since a peptide containing
mutations in this motif (R1811A, K1812A and
N1814A) that inhibit
the binding of E1A (
40) was severely
abrogated in this
capacity.
As the CBP TRAM inhibited the MDM2-p53 interaction in vitro, we next
tried to establish if the CBP TRAM was able to reverse
the functions of
full-length MDM2 in vivo. Recently, the binding
of MDM2 to p53 has been
shown to result in the degradation of
p53 (
15,
24). Figure
6 shows that indeed, as reported, the
introduction of MDM2 into U-2 OS cells resulted in a rapid degradation
of p53. However, the simultaneous expression of the CBP TRAM sequence
(residues 1808 to 1852) disrupted this effect. In contrast, a
mutant
version of the CBP TRAM that was compromised in MDM2 displacement
(Fig.
5B) was unable to reverse the MDM2-mediated degradation
of p53. These
results support the conclusion that the CBP TRAM
contained within
residues 1808 to 1852 is capable of displacing
MDM2 from p53 in vivo
and, as a consequence, stabilizes cellular
p53 protein.
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FIG. 6.
Introduction of the CBP TRAM, but not a mutant version,
into U-2 OS cells stabilizes cellular p53 levels. Transfection of U-2
OS cells and p53 stability experiments are described in Materials and
Methods. The wild-type [CBP (1808-52)] and mutant [CBP (1808-52)
Mut] proteins were expressed at similar levels (data not shown). Only
the CBP protein containing the wild-type TRAM was able to alleviate the
MDM2-mediated degradation of cellular p53.
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We next wished to determine whether or not the stabilization of
cellular p53 levels resulted in an increase in p53 transactivation
potential, as has previously been reported (
4). Figure
7 shows
that expression of the CBP TRAM
(residues 1808 to 1852) could
stimulate the activity of a
p53-responsive promoter (PG13CAT)
in a dosage-dependent manner, whereas
a TRAM mutant was compromised
in p53 stimulation. Thus, the expression
of a small domain containing
the CBP TRAM is sufficient to functionally
compete for the binding
of MDM2 to p53 and supports a model in which
MDM2 and the CBP
TRAM occupy overlapping and mutually exclusive sites
on p53 (Fig.
8).
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FIG. 7.
A CBP fragment (residues 1808 to 1852) containing a
functional TRAM can stimulate p53-dependent transcription. Transient
transfection of U-2 OS cells was carried out with 2 µg of either a
vector containing a p53-responsive promoter (PG13CAT) or a control
vector (MG15CAT). Indicated is the cotransfection of 1, 2, or 4 µg of
a CMV-GST-CBP (residues 1808 to 1852) vector containing either a
wild-type TRAM or a mutant (Mut) version. Introduction of the wild-type
TRAM resulted in a dose-dependent increase in p53-dependent
transcription. Cotransfection of wild-type (WT) 12S E1A but not a
CBP-binding-deficient mutant (del 63-67) of 12S E1A abolished the
activation of p53, while no activation was obtained with the mutant
TRAM construct. Error bars indicate standard error. CAT,
chloramphenicol AT.
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FIG. 8.
Model for the activation of p53-dependent transcription
by the CBP TRAM. (A) p53 under normal physiological conditions is
regulated by the MDM2 protein, which binds p53 and facilitates p53
degradation. (B) The expression of full-length CBP in vivo activates
p53, possibly through multiple pathways. These may include the
stabilization of the binding of p53 to its cognate DNA recognition
sites through the acetylation of p53, the bridging of DNA-bound p53 and
components of basal transcription factors, and the displacement of MDM2
from p53, preventing p53 degradation. (C) A small CBP fragment
containing a functional TRAM can lead to the stabilization of p53
protein and the activation of p53-dependent transcription. This diagram
suggests a role for the competitive inhibition of MDM2 binding to p53
during the activation of p53 by full-length CBP.
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E1A containing the wild-type FPESLIL sequence can counteract p53
transcriptional activation by the CBP TRAM.
Also demonstrated in
Fig. 7 is the ability of wild-type 12S E1A protein to abolish the CBP
TRAM-mediated activation of p53. This result is consistent with studies
showing that the activation of p53 by full-length CBP is also abolished
by E1A (13, 29, 49). Moreover, cotransfection of the del
63-67 mutant of E1A (54) demonstrated that abrogation of
this activation was dependent on a functional FPESLIL sequence.
Together, these results provide evidence that both the activation of
p53 transcriptional activity by CBP and the ability of E1A to inhibit
this activation are based on FXE/DXXXL-TRAM interactions.
Consequently, our in vivo evidence confirms the importance of the
FXE/DXXXL-TRAM interactions demonstrated in our in vitro binding assays.
| |
DISCUSSION |
The identification of a small TRAM within the C/H3 region of CBP
explains to a large extent why this region represents a hot spot for
transcription factor binding. In addition to interacting with E1A, p53,
E2F, and TFIIB, the CBP TRAM also binds to the cellular transcription
factors MyoD, YY1, c-fos, c-jun, and P/CAF (40). In principle, E1A could inhibit the binding of these
and other factors to the CBP TRAM, whether or not they interact with residues similar to the FXE/DXXXL sequence. At least for MyoD (47) and YY1 (40), sequences similar to those in
E1A, p53, and E2F have been shown to be necessary for the binding of
CBP. In MyoD, the sequence has been referred to as the FYD motif, which is present in the N-terminal region of a number of myogenic
transcription factors (36).
The modification and/or concentration of any given protein interacting
with the CBP TRAM is likely to be important because the increased
binding of one transcription factor may alter cellular responses by
competing for a limiting TRAM-containing regulator, as has been
suggested by recent studies (17, 18, 23). This strategy
would appear to have been adopted by adenoviruses, which express high
levels of the FXE/DXXXL-containing E1A protein; this protein
competitively inhibits the binding of CBP to cellular proteins,
resulting in both the inhibition of cellular differentiation and the
activation of cell proliferation. Our characterization of the E1A-CBP
interaction now provides a molecular basis for these observations.
One cellular regulator targeted by E1A in this way is p53. Previous
studies have demonstrated that p53-dependent transcription is activated
by CBP/p300 and that this activation is abrogated by E1A. Stimulation
of p53 activity by CBP/p300 has been attributed to the properties of
these transcriptional adapters, namely, the ability to provide AT
activity (12) and/or to bridge the gap between p53 and
components of the basal transcription machinery. In this study, we show
for the first time that binding per se is an important part of the
mechanism by which CBP/p300 activates p53-dependent transcription.
Consequently, E1A binding to the CBP TRAM and the concomitant
prevention of a p53-CBP interaction may be sufficient to explain the
abrogation of CBP-mediated p53 transcriptional activity.
The mechanism by which p53-dependent transcription is activated by a
relatively small region of CBP (residues 1808 to 1852) does not appear
to involve either the acetylation of p53 (since CBP sequences
responsible for AT activity are not present) or bridging to components
of the basal transcription machinery. Rather, activation results from
the competitive inhibition of MDM2 binding to p53 and the resulting
stabilization of cellular p53 protein levels (as demonstrated in Fig.
6). Similar observations were made in vivo when the MDM2-p53
interaction was blocked by an alternative mechanism: that is, through
the expression of a small molecule (thioredoxin) that contains the
MDM2-binding domain of p53 in its active-site loop (4). From
these observations, we could predict that any protein or protein
fragment that inhibits the binding of full-length MDM2 to p53 should be
able to activate p53-dependent transcription. We tested this hypothesis
by expressing the N-terminal p53-binding domain of MDM2 that lacks the
sequences required to target p53 for degradation (24). As
predicted, the expression of the MDM2 N-terminal region from residues 1 to 125 in U-2 OS cells resulted in an almost identical level of
activation of p53-dependent transcription as the expression of the CBP
TRAM fragment (residues 1808 to 1852) (40).
Interestingly, E1A is not the only viral oncoprotein to bind the CBP
TRAM. We have recently shown that human papillomavirus E6 proteins also
target CBP/p300 and can interact directly with CBP amino acids 1808 to
1826 (58). This interaction results in the down-regulation
of p53 transcriptional activity to a level comparable to E1A-mediated
repression and is limited to E6 proteins from human papillomaviruses
associated with cervical cancer (58). Moreover, given that
the simian virus 40 large T antigen also binds the C/H3 region of CBP
(9a) and down-regulates p53-dependent transcription
(32a), it is likely that all three of these small DNA tumor
virus oncoproteins target the CBP TRAM, strongly suggesting an
important role for this motif in cell cycle regulation.
Other inhibitors of the cell cycle that bind the CBP/p300 TRAM and have
been shown to be targeted by adenovirus E1A include MyoD and P/CAF. The
interaction of MyoD with CBP/p300 has been shown to be essential for
cell cycle arrest and muscle-specific gene expression (45).
E1A, by binding to CBP/p300, inhibits these processes (5,
35). Our results suggest this activity may involve an E1A
FPESLIL-TRAM interaction. Similarly, our unpublished finding that P/CAF
binds CBP amino acids 1808 to 1826 suggests another TRAM interaction
targeted by E1A. Consistent with this idea is the previously described
ability of E1A to displace P/CAF from the C/H3 region of CBP
(55). Like MyoD and p53, P/CAF possesses cell cycle
inhibition and cellular differentiation properties (46, 55).
Thus, by targeting the CBP/p300 TRAM, E1A may affect multiple cellular
factors whose role it is to inhibit cell cycle progression.
Future studies that make use of TRAM mutants in the context of
full-length CBP/p300 should prove useful in the analysis of CBP/p300-mediated integration of different signalling pathways. Such an
approach may be facilitated by the interesting finding that not all CBP
TRAM-interacting transcription factors are affected to the same extent
by the same point mutations within the CBP TRAM sequence
(40). Thus, by use of variants of the TRAM sequence, it
might be possible to selectively block the binding of certain transcription factors to CBP/p300.
In summary, we initiated this study in order to gain greater insight
into the molecular mechanism of E1A action and how this protein
functions to subvert cellular pathways. Through detailed mapping of the
E1A-CBP C/H3 interaction, we have identified a TRAM that is conserved
in all CBP/p300 proteins. This motif, targeted by E1A, is used by
various cellular factors and may prove to play an important role in the
integration of multiple signal transduction pathways.
| |
ACKNOWLEDGMENTS |
We thank R. Goodman and Oh Hue Kian for materials, Anita
Kathiresan and Alistair Cook for technical assistance, and Benjamin Li
and Ed Manser for helpful discussions and critical reading of the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Molecular and Cell Biology, 30 Medical Dr., Singapore 117 609, Singapore. Phone: (65) 874-3363. Fax: (65) 779-1117. E-mail:
mcbomark{at}imcb.nus.edu.sg.
| |
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Journal of Virology, May 1999, p. 3574-3581, Vol. 73, No. 5
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
