From the Cancer Research UK Laboratories, Department
of Molecular & Cellular Pathology, ¶ School of Life Sciences,
University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom
Received for publication, November 11, 2002, and in revised form, December 18, 2002
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ABSTRACT |
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Reconstitution of the stages in the assembly of
the p300·p53 transcription complex has identified a novel type
of DNA-dependent regulation of p300-catalyzed acetylation.
Phosphorylation at the CHK2 site (Ser20) in the
N-terminal activation domain of p53 stabilized p300 binding. The
phosphopeptide binding activity of p300 was mapped in vitro to two domains: the C-terminal IBiD domain and the N-terminal IHD
domain (IBiD homology domain). The
IHD or IBiD minidomains can bind to the p53 activation domain in
vivo as determined using the mammalian two-hybrid VP16-GAL4
luciferase reporter assay. The IHD and IBiD minidomains of p300 also
functioned as dominant negative inhibitors of p53-dependent
transcription in vivo. Upon examining the affects of p300
binding on substrate acetylation, we found that the p53 consensus site
DNA promotes a striking increase in p53 acetylation in
vitro. Co-transfection into cells of the p53
gene and plasmid DNA containing the consensus DNA binding site of p53
activated DNA-dependent acetylation of p53 in
vivo. The phosphopeptide binding activity of p300 is critical for
DNA-dependent acetylation, as p53 acetylation was inhibited
by phospho-Ser20 peptides. Consensus site
DNA-dependent acetylation of p53 stabilized the p300·p53
protein complex, whereas basal acetylation of p53 by p300 in the
presence of nonspecific DNA resulted in p300 dissociation. These data
identify at least three distinct stages in the assembly of a p300·p53
complex: 1) p300 docking to the activation domain of p53 via the IBiD
and/or IHD domains; 2) DNA-dependent acetylation of p53;
and 3) stabilization of the p300·p53AC complex
after acetylation. The ability of DNA to act as an allosteric ligand to
activate substrate acetylation identifies a conformational constraint
that can be placed on the p300-acetylation reaction that is likely to
be an amplification signal and influence protein-protein contacts at a promoter.
Regulation of eukaryotic gene expression is a complex process
regulating growth control in normal cells and dysregulation of gene
expression contributes to diseases including cancer. Characterizing the
protein-protein interactions that assemble transcription complexes will
provide a molecular basis for combinatorial specificity at the promoter
level and provide an understanding of how transcriptosomes affect
disease progression. One of the most studied regulators of eukaryotic
gene transcriptional activation is the stress-activated tumor
suppressor protein p53 (1). The vast array of genes that p53 induces
and represses in response to cell injury provides an excellent model
system with which to identify the complex and transient associations
with co-activators/repressors and the core promoter transcriptional
apparatus that drive promoter activation.
The tumor suppressor activity of p53 is linked to its activity as a
stress-activated transcription factor. p53 is subject to multiple
post-translational modifications such as phosphorylation, acetylation,
and ubiquitination that modulate the function of p53 as a transcription
factor. p53 protein is tetrameric (2) and the oligomeric nature of p53
provides the basis for complex intra/interdomain control mechanisms
that modulate its activity (3, 4). The most widely studied
post-translational modification of p53 is phosphorylation via protein
kinases that target specific domains on the assembled tetramer that can
activate its function by a two-step process (5). The first identified
involves phosphorylation of the C-terminal regulatory domain of p53 by
CK2/FACT at Ser392 and cyclin-dependent protein
kinases at Ser315, which play a role in activating its
latent sequence-specific DNA binding activity in vitro (6,
7) and in vivo (8, 9). The second step involves
phosphorylation of specific N-terminal residues of p53 that reside
within the activation domain by an ATM-dependent signal
transduction cascade at Ser15 (10), which stimulates p300
binding to p53 in vitro (11) and in vivo (12).
Additionally, an adjacent Ser20 phosphorylation event by a
CHK2-dependent pathway activates p53 (13) by stabilizing
the transcription coactivator p300·p53 complex formation (14). The
specific interaction of p300 in the p53-dependent transactivation pathway became apparent when it was demonstrated that
ectopically expressed p300 stimulated p53-dependent gene expression and that adenoviral E1A protein inhibited
p53-dependent transcription by virtue of binding to p300
(15-17). Although this initial p300-p53 interaction was mapped to the
N-terminal BOX-I domain of p53, an additional role for p300
in the control of p53 activity came from the observation that
acetylation of p53 by p300 in the C-terminal negative regulatory domain
adjacent to the CK2/FACT phosphorylation site activated the specific
DNA binding function of p53 to short oligonucleotides containing the
consensus binding site for p53 (18). Together, these data suggest the existence of a phosphorylation-acetylation cascade that targets p53 in
response to genotoxic stress (19).
The acetyltransferase activity of p300 and its homolog CBP on histone
and nonhistone substrates has been well documented and is important for
co-activator function (20, 21). The role for acetylation on the
N-terminal tails of histones is somewhat more widely appreciated, with
the acetylation of lysine residues potentially weakening the
nucleosome-DNA interaction, thereby allowing promoter access for the
core transcriptional machinery such as TFIID and RNA polymerase II.
Acetylation of transcription factors is now a widely observed
phenomenon (22-25) but the role and mechanism of acetylation of
transcription factors remains relatively elusive (26). However, more
recent studies have also suggested that acetylation may function to
stabilize coactivator complexes at a promoter (27, 28), presumably in
part through the Bromo-homology domain of p300/CBP that has the
potential to bind to acetylated residues (29).
The molecular mechanism underlying how p53 tetramers interact with p300
and how this modulates acetylation has not been defined biochemically.
Such analysis will likely give novel insights into how p300 protein
interacts with an oligomeric substrate as well as information into how
p300 links transcription to cell cycle control. We have started to
reconstitute the p300·p53 protein complex in vitro by
first examining how full-length p300 interacts with various
phosphorylated regions in the N-terminal BOX-I activation domain of p53 (14). This study has indicated that of three N-terminal phosphorylation sites in the BOX-I activation domain,
phosphorylation by CHK2 at Ser20 has the most stabilizing
affect on p300·p53 complex formation. In this report, we have used
biochemical assays to develop a consensus phosphopeptide-binding site
for p300 and to map the phosphopeptide-binding domain of p300 to two
distinct domains. One of the domains maps to a previously undefined
N-terminal region on p300 we name IBiD homology
domain (IHD), which has homology to the established
p53-binding domain in the C terminus of p300 previously called IBiD or
SRC-1 (30, 31). Further characterization of how p300 docking to the
activation domain of p53 influences acetylation has revealed that p300
docking is essential for sequence-specific DNA-dependent acetylation of p53 that in turn stabilizes the p300·p53AC
complex. These data reveal three distinct stages in the assembly of the
p300·p53 complex: (i) p300 docking, (ii) p300 catalyzed DNA-dependent acetylation, and (iii)
acetyl-CoA-dependent stabilization of the p300·p53
complex. These data further suggest that p53 protein conformation when
DNA bound at promoters may play a role in modulating the rate of its
acetylation and recruitment of chromatin remodeling factors.
Plasmids and Constructs--
p21-Luc, Bax-Luc,
pGL3-Basic, pCMV- Immunochemical Assays--
The peptide or p53 tetramer binding
activity of p300 was examined by enzyme-linked immunosorbent assay
(ELISA),1 as described
previously (3, 14). Essentially, 96-well plates (Dynex Microlite 2)
were first coated with p53, antibody-captured p300, or streptavidin and
the indicated biotinylated peptide for 16 h, as described
previously (14). Nonreactive sites were blocked in 3% bovine serum
albumin in PBS/Tween 20 (0.02% v/v) to reduce the nonspecific
binding. This was followed by titrating increasing amounts of p53,
p300, MDM2, peptides, without or with RNA (poly(G); data now shown))
(as in Ref. 34) in 3% bovine serum albumin in PBS/Tween 20 (0.02%
v/v) for 1 h, followed by an extensive wash, and incubation with
the indicated IgG. All reactions were carried out at 4 °C and
detected by the appropriate secondary antibody linked to horseradish
peroxidase from DAKO. The signal detection by enhanced
chemiluminescence was developed using Fluoroscan Ascent FL. The
BOX-I transactivation domain of p53 or its
Ser20-phosphorylated derivative contains amino acids
14-27, as described previously (14). All synthetic peptides were
obtained from Chiron Mimitopes.
Cell Culture, Transfections, ELISAs, and Western Blots--
A375
and Saos-2 cells were maintained in Dulbecco's modified Eagle's
medium (Invitrogen), whereas HCT116 cells were in McCoys 5A medium,
both supplemented with 10% fetal bovine serum and incubated at
37 °C with an atmosphere of 10% CO2. Transient
transfections and ELISAs were carried out as previously described (14).
Full-length p300 and His-p300-infected Sf9 cells were harvested
72 h postinfection and purified as described previously (14).
Sf9-expressed wtp53 tetramers were purified by heparin-Sepharose
chromatography as described previously (3). Transfected lysates were
run on a 12% SDS-PAGE, transferred to nitrocellulose membrane, and
protein loading was confirmed by Ponceau S (Sigma) staining. The p300 antibody used was N-15 (Stressgen). Primary antibodies anti-p53 (DO-1
or ICA-9 (3)) were used and the appropriate secondary antibody
conjugated to horseradish peroxidase. The signal detected by enhanced
chemiluminescence was developed using autoradiography film (Amersham
Biosciences).
ELISA--
Microtiter wells were coated with 100 ng of
streptavidin and incubated overnight at room temperature. To prevent
nonspecific binding, 180 µl of 50 µl/well of 3% bovine serum
albumin + PBST (3BPBST) was added and incubated for 1 h at
4 °C. Wells were washed 3 times with 180 µl of PBST and
biotinylated peptides, resuspended in 3BPBST with 50 mM
NaF, 5 mM In Vitro Acetylation Reactions--
Purified p53 (50-400 ng)
from Sf9 cells was incubated with 300-400 ng of purified
His-p300 in 30-100 µl of AT buffer (50 mM Tris·HCl (pH
8), 10% glycerol, 0.1 mM EDTA, 1 mM
dithiothreitol, 5 µM trichostatin A, and 2 µM acetyl-CoA) for 4-10 min at 30 °C where the
enzymatic reaction was linear. The specific oligonucleotides (pG DNA)
and nonspecific oligonucleotides (NS-DNA) were described previously
(35). Reactions were incubated on ice for 10 min and started with the
addition of p300. Acetylation of p53 was detected by the
antibody-capture ELISA technique using anti-p53 (ICA9) or by direct
Western blot using anti-acetyl p53 (AcK373/382) and normalizing with
anti-p53 (19.1). Histone acetylation was carried out in a similar
manner but with the use of 1 µg of purified histone H4 (Upstate
Biotechnology) as the substrate for p300 and using anti-histone (Roche
Molecular Biochemicals) or anti-acetyllysine (Upstate Biotechnology) to
detect reaction products. Quantification of acetylation was carried out
using bioluminescence (Genegnome (Syngene Bioimaging System)).
In Vivo DNA-dependent p53 Acetylation
Assay--
HCT116 (p53 Developing a Consensus p300-binding Motif by Mapping Its Contact
Site in the Ser20-phosphorylated BOX-I Activation Domain of
p53--
The overlapping docking site for MDM2 and p300 on p53 (Fig.
1E) creates a negative and
positive effect, respectively, on its ability to function as a tumor
suppressor protein. The notable differences in the affinity of p300 and
MDM2 toward p53-derived Thr18 and Ser20
phosphopeptides suggested an intrinsic difference in their binding contacts in the BOX-I activation domain (14). To define
essential amino acid contacts, a series of alanine-substituted
Ser20-phosphorylated peptides were synthesized and the
protein-ligand binding assay was employed using full-length p300 and
full-length MDM2 proteins (Fig. 1, A and B,
respectively).
Ser20 phosphorylation of p53 is required for maximal
full-length p300 binding to this domain, whereas MDM2 is not inhibited (14) thus allowing the same phosphodomain of p53 to be used for
consensus site mapping of MDM2 and p300. If Gln16,
Trp23, Lys24, Leu25, or
Leu26 are individually mutated to alanine, then this
abrogates the ability of the p300 protein to bind to the
Ser20 phosphodomain (Fig. 1A). Certain alanine
substitutions in the Ser20 phosphodomain including
Ser15, Glu17, Thr18,
Phe19, and Asp21 stabilized the binding of p300
to the phospho-motif (Fig. 1A). In contrast to p300, a
Thr18, Phe19, Leu22,
Trp23, or Leu26 substitution to alanine on the
BOX-I-derived Ser20-phosphorylated domain
inhibits MDM2-binding (Fig. 1B), correlating with the
essential residues required for MDM2 protein binding to p53 identified
by the crystal structure of the MDM2-p53 peptide complex (37). This
evidence that MDM2 and p300 have distinct binding contacts explains, in
part, their differential binding to phospho-Thr18 or
phospho-Ser20 p53 (14).
Interestingly, a search for proteins in the data base that have
significant homology to the consensus BOX-I p300-binding
motif on p53, QXXXSPO3XXWKLL (Fig.
1C), identified the HMG box architectural factor, UBF1
(upstream binding factor 1; Fig. 1C). UBF necessitates major chromatin remodeling and the SXXWKLL region of UBF is within
a domain that is already known to interact with the p300 homologue, CBP
(CREB-binding protein) (38). To determine whether p300 can bind to this
region of UBF1 we employed the protein-ligand binding assay containing
two putative SPO3XXWKLL consensus peptides from
UBF1 (amino acids 231-246 and 321-336) with or without a phosphate
moiety at the predicted serine residue. Notably, when a phosphate group
was attached to Ser329 in UBF1-(321-336), which contains
strict homology to the SPO3XXWKLL motif, p300
bound with an affinity slightly less than the p53
BOX-I-Ser20 phosphopeptide (Fig. 1D),
suggesting that this may indeed be a consensus contact region for p300.
In contrast, UBF1-(231-246) did not bind p300 with or without a
phosphate substitution at Ser239 (Fig. 1D) nor
did p300 bind the BOX-I domain of p53 without a phosphate
(Fig. 1D). These differences may be because of the absence of a tryptophan residue at position 242 (or the presence of a destabilizing lysine) (Fig. 1C), which is required for p300
binding to the p53 BOX-I-Ser20 phosphopeptide
alanine scan (Fig. 1A).
Identification of Two p53-activation Motif Binding Domains on
p300--
Various techniques have been utilized to originally define
the p53 activation domain-binding sites in p300 to within the C/H1, KIX, and C/H3 minidomains of p300 (Fig.
2). However, the most recent mapping of
the p53-binding domain of p300 was localized to the C-terminal
interferon-binding domain (IBiD,
Fig. 2A), which is known to bind to peptides of apparently
unrelated primary amino acid homology including phosphorylated peptides
derived from interferon regulatory factor-3 (30). This motif on
IRF-3 has homology to the phosphorylated p53-activation domain (data
not shown) and in a recent study, a p53-activation domain-binding
region in p300 was mapped to the IBiD domain and independently called
SRC-1 (Fig. 2A) (31). An in vitro assay was
employed to localize the p53 peptide-binding domains of full-length
p300 to either previously known or unmapped regions of p300 protein by
assaying fragments of p300 for Ser20
phospho-BOX-I activation domain binding. Various N- and
C-terminal fragments of p300 that overlap, which are distinct from, or
that contain the CH/1, KIX, CRD1, Bromo, C/H2, C/H3, and IBiD
minidomains were tagged and tested for p53 binding activity. These
include (Fig. 2B): GAL4, GAL4-p300, GAL4-p300-(1-504),
GAL4-p300-(1-703), GAL4-p300-(192-504), GAL4-p300-(192-600),
GAL4-p300-(192-703), GAL4-p300-(192-1004), GAL4-p300-(504-1238),
GAL4-p300-(852-1071), GAL4-p300-(636-2414), GAL4-p300-(1064-2414),
or GAL4-p300-(1757-2414).
Cell lysates obtained from HCT116 (p53
To determine whether the N-terminal IHD domain of p300 exhibited
similar p53-binding characteristics to the C-terminal IBiD domain of
p300, we used the VP16-GAL4 two-hybrid system that was used originally
to fine map the C-terminal IBiD domain on p300 that binds to the
activation domain of p53 when fused to GAL4 (30, 31). The
co-transfection of pACT-VP16-IBiD or pACT-VP16-IHD along with
p53-TAD:GAL4 both supported GAL4-dependent transcription (Fig. 3A, right 5 panels; 1063 RLUs for IBiD and
877 RLUs for IHD), relative to the negative control using pACT-VP16
only (180 RLUs). The pACT-VP16-C/H3
As an independent test of the ability of IHD and IBiD as important
effectors of p300-coactivated p53-dependent transcription, the ability of pACT-IBiD and pACT-IHD were tested as dominant negative
inhibitors of p53-dependent transcription from the
p21-Luc reporter (Fig. 3B). The maximal
p53-dependent transcription upon co-transfection with p300
(Fig. 3B, 2483 RLUs) was significantly attenuated using
either pACT-IBiD (1002 RLUs) or pACT-IHD (1146 RLUs), relative to
pACT-C/H3 Consensus Site DNA-dependent Acetylation of
p53--
To establish a function for the IBiD and IHD domains of p300,
the contribution of the phosphopeptide-binding activity of p300 to p53
acetylation was examined. Specifically, does binding of p300 to the
BOX-I domain of p53 affect acetylation in the C-terminal NRD1 domain of p53 (see p53 domain structure in Fig. 1E).
Before setting up an in vitro p53 acetylation assay, the
purified p300 protein was first characterized enzymatically using a
well known substrate, histone H4. The kinetics of the acetylation
reaction between p300 and histone H4 were determined (Fig.
4, A and B). The
data indicate that p300 is behaving like a classical histone acetyltransferase by displaying monophasic kinetics on a histone substrate (39, 40) and this preparation of p300 was tested for the
ability to acetylate native p53 tetramers expressed in Sf9 cells
(41). Using an antibody against acetylated Lys373/382 of
human p53 that has been previously described as being specific for
acetyl-p53 (27) and normalizing to levels of p53 protein with an
anti-p53 antibody, acetyl-CoA-dependent acetylation of p53
was observed (Fig. 4C, lane 3 versus lane
2).
In a further characterization of p53 acetylation, we found that
including consensus site DNA in the acetylation buffer resulted in an
unexpected stimulation in p53 acetylation (see below). Using the basal
p300-acetylation reaction where acetyl-CoA promotes weak p53
acetylation (Fig. 4C, lane 3 versus lane
2), experiments demonstrated a stimulation of p53 acetylation by
p300 upon titration of an excess of consensus site oligonucleotide DNA
(pG DNA; Fig. 5A,
lanes 5-8 versus lane 4). Nonspecific
oligonucleotides could not stimulate p53 acetylation under the same
condition (NS-DNA; Fig. 5B, lanes 5-8
versus lanes 3 and 9). A titration of consensus site DNA demonstrated that at a ratio of DNA to p53 of ~1:1, the acetylation of p53 approached saturation (Fig. 5C,
lanes 4 and 5 versus lane 1). If
consensus site DNA were stimulating general p300 activity by an
allosteric mechanism via a sequence-specific DNA-binding domain on
p300, then basal histone acetylation (Fig. 4, A and
B) would be expected to be stimulated by DNA. However, histone acetylation by p300 was neither inhibited nor stimulated by the
addition of consensus site DNA (Fig. 5D, lanes
1-6). These data suggest that the acetylation of p53 was unique
to a change in the conformation of p53 when bound to consensus site
DNA.
The kinetics of p300 acetylation on histones has been published
recently (40) and displays a ping-pong mechanism, via an ordered
reaction involving first the formation of a stable p300-acetyl intermediate followed by binding of the histone substrate and transfer
of the acetyl moiety to a lysine residue. As a final control to define
the integrity of the acetyltransferase reaction, we determined whether
the acetylation of DNA-bound p53 by p300 displays similar kinetics to
that of histone substrates. The linearity of the p53-acetylation
reaction over time (Fig. 6A)
and as a function of DNA concentration (Fig. 6B) was
optimized using bioluminescent quantification of reaction products.
By changing the concentration of acetyl-CoA at a fixed p300
concentration and consensus site DNA concentration, varying the p53
levels resulted in linear changes in the levels of acetylation. After
quantification using bioluminescence, a double reciprocal plot of the
initial velocities was drawn using Prism kinetics software (Fig.
6C). p300 acts in a similar manner to p53·DNA complexes as
a substrate compared with histones suggesting that the p53-p300
acetylation reaction similarly displays a ping-pong mechanism as
opposed to a sequential (ternary complex) mechanism. Kinetics could not
be performed on p53 acetylation without DNA as the stoichiometry
of acetylation was too low to acquire statistically significant
data.2
Although acetylation can stimulate latent p53 binding to small
consensus site DNA oligonucleotides (18), acetylation does not change
the affinity of p53 for its consensus site in large fragments of DNA
(28). As such, the role of acetylation in the control of p53 function
has remained elusive (26). However, it is very difficult to acquire
pure isoforms of recombinant latent phosphorylated p53, latent
unphosphorylated p53, or kinase-activated p53 (41), especially in
insect cell expression systems where dozens of p53 isoforms exist (42).
This heterogeneity in p53 modifications possibly explains, in part, the
apparent controversy on the role of acetylation in modulating the
specific activity of p53 using in vitro DNA binding assays.
It therefore is important to determine whether consensus site DNA
stimulation of p53 acetylation also could be promoted in large plasmid
of the DNA rather than small oligonucleotides.
Using a supercoiled plasmid DNA containing multiple copies of the p53
consensus site (pG13) (43), a DNA-dependent stimulation of
p53 acetylation by p300 was observed, compared with parallel reactions
containing pMG13 nonconsensus site control DNA (Fig. 7A, lane 4 versus lane 3). These data indicate that
DNA-dependent acetylation was not confined to small
oligonucleotides and indicate that although acetylation can stimulate
unphosphorylated and latent p53-DNA binding (18), our use of in
vivo kinase-activated, purified p53 protein isoforms (as in Ref.
41) demonstrates that acetylation is a post-DNA binding event.
It was next important to determine whether sequence-specific
DNA-dependent acetylation could occur in vivo.
Two p53 constructs (p53 and p53-6KR (the nonacetylatable p53 (56)))
were co-transfected with consensus site plasmid DNA (or plasmid control
containing the mutated consensus site) and the expressed p53 protein
was assayed for both binding to endogenous p300 protein and in
vivo acetylation (Fig. 7, B-E). The transfection of
the p53 gene with either pMG13 DNA or pG13 DNA, followed by
immunoprecipitation with a p300 antibody resulted in the same level of
p53 protein being co-precipitated with p300 as detected by blotting
with a p53 antibody (Fig. 7C, lanes 1 and
2). However, only consensus site pG13 DNA promoted a
substantial acetylation of p53 in the p300 immune complex as detected
by blotting with a p53-acetylation site antibody (Fig. 7B,
lane 2 versus lane 1). This is in contrast to the
control transfection of the p53 gene with the mutant
consensus site supercoiled plasmid pMG13, which did not produce a
stable complex between p300 and acetylated p53 (Fig. 7B,
lane 1). Thus, because significant amounts of nonacetylated
p53 protein bound to p300 upon co-transfection with the p53
gene and pMG13 plasmid DNA (Fig. 7, B and C,
lanes 1), these data indicate that acetylation of p53,
rather than p53 binding to p300, is promoted by consensus site plasmid
DNA. As a further control for total p53 acetylation, co-transfection of
the p53 gene with pG13 plasmid (versus the pMG13
plasmid) stimulated total acetylation of p53 after precipitation of
protein complexes with an anti-p53 antibody and immunoblotting with an
acetylation-specific p53 antibody (Fig. 7D, lane
2 versus lane 1). This is similar to the in
vitro stimulation of p53 acetylation by p300 with the inclusion of
pG13 DNA (Fig. 7A, lane 4 versus lane
3). Together, these data indicate that consensus site plasmid DNA-dependent acetylation can be observed in
vitro or vivo.
Acetyl-CoA Stabilizes the p300·p53AC
Complex--
With the DNA-dependent p53 acetylation assay
biochemically characterized, we could finally access the role of the
phospho-Ser20 peptide-binding domains of p300 to both
DNA-dependent p53 acetylation and DNA-independent histone
acetylation (Fig. 8). In the absence of
peptide, acetyl-CoA was required for DNA-dependent
acetylation of p53 by p300 (Fig. 8A, from left,
lane 2 versus lane 1). A titration of
BOX-I peptide displayed no effect on the p53 acetylation
reaction (Fig. 8A, from left, lanes
3-6 versus lane 2). However, a titration of the
BOX-I phospho-Ser20 peptide inhibited p53
acetylation with an IC50 of ~150 µM in this
assay (Fig. 8A, from left, lanes 7-10
versus lane 2). These data indicate that the
phosphopeptide-binding domain(s) of p300 interacts with the N-terminal
domain of p53 to facilitate acetylation in the C-terminal domain of
p53. As a control, histone acetylation was not affected by the
BOX-I phospho-Ser20 peptide (Fig. 8B)
indicating that the acetylation reaction is docking- and
DNA-independent with respect to histone acetylation. Using the p53
acetylation assay in an ELISA format where monoclonal-captured p53
could be probed with an anti-acetylation polyclonal antibody, the
phospho-Ser20 peptide similarly prevented
DNA-dependent acetylation of full-length p53 (Fig.
8C).
In summary, our reconstitution of the p300·p53 complex has indicated
that acetylation is DNA-dependent and requires p300 docking to the N terminus of p53 presumably via the IBiD and/or IHD domains of
p300. This data provides an alternate model for acetylation compared
with other studies showing that acetylation can either stimulate the
latent DNA binding activity of p53 (18), have no effect on p53 DNA
binding (28), or block MDM2-dependent ubiquitination of p53
(32). What then is the role of the acetylation of DNA-bound p53? One
previous study has suggested a clue, as acetylation of MyoD by CBP (in
the absence of DNA) can promote a high salt-resistant stable complex
between the two proteins presumably through the bromo-homology domain
(29). The stability of the p300·p53 complex was therefore quantitated
without or with acetyl-CoA using the ELISA solid phase assay to
determine whether p300 dissociates from p53 after acetylation in the
absence or presence of consensus site DNA. In contrast to the
immunoprecipitation of p300·p53 complexes that is a very sensitive,
but rapid assay not allowing significant time for dissociation of weak
protein-protein complexes (Fig. 7), the ELISA measures very
stable complex formation because 20-30-fold longer wash times permit
weakly bound proteins to dissociate. Using purified p300 and p53
proteins, we show that small oligonucleotides containing the p53
consensus site (Fig. 9A) or
supercoiled plasmid DNA containing the p53 consensus site (Fig.
9B), can promote a striking stabilization of the p300·p53
complex after the addition of acetyl-CoA (Fig. 9, A and
B, lane 12), relative to nonspecific DNA (Fig. 9,
A and B, lane 11 versus
12). As controls for this quantitation,
acetyl-CoA-dependent p53 acetylation was stimulated by
consensus site DNA in oligonucleotide or plasmid form (Fig. 9,
A and B, lane 9 versus 8 and lane 2 versus 3). Furthermore, p300 binding
to p53 (Fig. 9, lane 5) was de-stabilized by acetyl-CoA in
reactions with nonspecific DNA (Fig. 9, A and B,
lane 11 versus 5). By contrast, acetyl-CoA
stabilized the p300·p53 complex when p53 is pre-bound to consensus
site DNA (Fig. 9, A and B, lane 12 versus 11). The docking-dependent acetylation of
DNA-bound p53 that results in subsequent stabilization of the
p300·p53 complex is consistent with the data suggesting that one role
of acetylation is to allow p300 to nucleate transcriptional
coactivators like TRAPP or BRG-1 and to promote chromatin
remodeling.
The acetylation of histone and nonhistone chromosomal proteins is
critical for the fine-control of gene expression and the characterization of how p300 recognizes and acetylates these classes of
proteins will help to further define how gene expression is controlled.
Histone acetylation reduces the extent of nucleosome assembly
presumably by minimizing electrostatic DNA-protein interactions thus assisting in stabilizing the RNA polymerase preinitiation and
transcription complex. By contrast, acetylation of sequence-specific transcription factors including E2F, p53, and MyoD have been shown to
enhance their affinity for specific sequences in promoter regions (44).
The molecular mechanism of p300 binding to and acetylation of
sequence-specific transcription factors has not been defined. The relatively large size of p300 suggests that an
intrinsically complex intradomain and/or interdomain communication
exists between itself and the variety of target proteins at a promoter.
The interferon-responsive enhanceosome is one such example of
multiprotein nucleation at a promoter (45). Given that molecular
reactions in vivo likely involve large protein complexes, or
protein machines (46), it is likely that p300 will be regulated and
function in a complex fashion. Furthermore, because acetylation may
prove to be a covalent modification as important as phosphorylation
mechanistic insight into acetylation function and regulation is of
fundamental importance. In this report, we have assembled in
vitro the p300·p53 complex, defined co-factors that modulate the
acetylation of p53, and reconstituted a docking-dependent
and DNA-dependent acetylation reaction required to clamp
the p300·p53 complex.
The oligomeric nature of p53 provides a unique model with which to
define conformational elements that modulate the binding and
acetylation of a target protein by the transcriptional co-activator p300. The N-terminal BOX-I domain of p53 was originally
shown to contain a p300-binding site, as mutation of this region
produces a transcriptionally inert protein. The first evidence for a
multidomain component for the interaction between p53 and p300 came
from data showing that phosphorylation of p53 at Ser15 by
DNA-PK stimulates acetylation in the C terminus (11). Subsequent studies have shown that phosphorylation of p53 in the BOX-I
domain at the CHK2 Thr18 and Ser20
phosphorylation sites can stabilize the p300·p53 protein complex (14). As different class of p53-activating kinases target the Ser15, Thr18, or Ser20 residues
(ATM, CK1, and CHK2, respectively), these phosphorylation events
provide a method for kinase signaling networks to regulate gene
expression by altering the stability of the p300·p53 complex (14).
Developing a consensus phosphate-binding motif for p300 that is
distinct from the contact sites involved in the MDM2·p53 complex
(Fig. 1) explains why kinase phosphorylation can differentially affect
p300·p53 and MDM2·p53 complex stability and led to the identification of phosphorylated-UBF as a p300-binding sites (Fig. 1,
C and D).
Our mapping of one of the p53-binding domains of p300 to IBiD is
consistent with recent studies (30), whereas our identification of a
second domain with homology to IBiD (IHD) was previously undefined. The
original studies on mapping the p53-p300 interactions have focused on
the use of glutathione S-transferase fused N- and C-terminal
fragments of p53 in pull-down and immunoprecipitation experiments. For
example, the C-terminal region of p300 (amino acids 1990-2414)
containing the Gln-rich domain and IBiD/SRC-1 binds p53 (amino
acids 1-72) in vitro; consistent with our findings that
p300-GAL4-C2 binds p53 (amino acids 1945-2414). Other groups have
shown interactions between p53 and the KIX domain and C/H1 or C/H3
domain exclusively (47), which are not observed in vitro using our p300-phospho-ligand binding assays. However, weak binding of
the C/H3 The observation that DNA promotes p53 acetylation brings to mind a
recent discussion of allosteric regulation of transcription regulators
through specific DNA binding (48). In the latter report, it was
proposed that DNA response elements contain information that is
interpreted by bound transcription factors. In particular, DNA could
act as an "allosteric ligand whose binding alters the regulator's
affinity for other ligands, such as co-activators or co-repressors."
Presumably, this allosteric control of p53 acetylation is brought about
by a conformational change in p53 after DNA binding. The conformational
changes in p53 that occur upon specific DNA binding remain to be
determined. Additionally, after acetylation of p53, p300 binding is
further stabilized by consensus site DNA (Fig. 9) identifying a role
for acetylation as a stabilizer of protein-protein interactions. The
docking dependence in p300 acetylation of a substrate further addresses
the poorly understood nature of modularity in a native transcription
regulator. Transactivation domains are defined, in part, as minimal
motifs that can drive transcription when fused to a heterologous
DNA-binding protein, like GAL4, and such domain swap experiments are
usually used as arguments for a modular or scaffold nature of a
transcription factor. However, such domain swap experiments
underestimate the complexity of a transcription factor DNA-binding
domain and transactivation domain and do not rule out intramolecular or
allosteric associations on the native protein. For example, the USF2
transactivation domain activates transcription when fused to its native
DNA-binding domain, but when the USF2 transactivation domain is fused
to GAL4 it is inactive or even repressive (49). ATF-2 and OCT-4
transactivation domains are thought to function through an interaction
with their DNA-binding domains, as defined using the OCT-4
transactivation domain and the chimera of the DNA-binding domain (50,
51). The N terminus of p53 has an activation motif that drives
transcription when fused to the GAL4 DNA-binding domain in the yeast
transactivation system (52). However, when the p53 transactivation
domain is fused to a p53 DNA-binding domain mutant, this blocks
transcription (58), further indicating that the p53 activation domain
is allosterically regulated by its DNA-binding domain. Although such
widespread evidence that the allosteric control of transactivation by
DNA-binding domains is lacking, there is growing data showing that
transcription factors change their conformation when DNA-bound, based
on proteolytic fingerprints, CD, and NMR studies. The observation that
DNA binding by p53 affects its acetylation by p300 indicates that the
DNA-binding domain is pivotal in linking the activation domain to
acetylation and coactivator recruitment. A recent study has confirmed
using NMR that the conformation of p53 changes upon DNA binding;
presumably this conformational change in the core domain and surface of
p53 also creates a conformationally stable binding site for p300 or releases the acetylation motif into the acetyltransferase active site.
In summary, the use of biochemical techniques to
reconstitute the p300·p53 complex using active forms of full-length
p300 has identified specific stages in the assembly reaction that
include a docking-dependent and consensus site
DNA-dependent acetylation of p53. The docking dependence in
the DNA-dependent acetylation of p53 identifies a
conformational constraint imposed on the p300-acetylation reaction at a
promoter. Identification of the mechanism of conformational regulation
of p53 acetylation may reveal novel factors that modulate acetylation
of the DNA-bound transcription factor.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-gal, pCMV-p53, and pCMV
-p300 have been
previously described (14). pCMV-P53-6KR (32) was a gift of Dr. Ron
Hay, St. Andrews University, Scotland, UK. Vectors used for the p300
minidomain two-hybrid studies (Fig. 3A), pACT,
pG5luc, and pBIND, were obtained from Promega and used according to the manufacturers instructions and as described previously (41). pACT-IBiD encoded amino acids 2050-2094 of human p300, pact-IHD
encoded amino acids 406-566 of human p53, and pACT-C/H3
encoded a
region of the standard C/H3 domain from amino acids 1709 to 1913. GAL4,
GAL4-p300, GAL4-p300-(1-504), GAL4-p300-(1-703), GAL4-p300-(192-504), GAL4-p300-(192-600), GAL4-p300-(192-703), GAL4-p300-(192-1004), GAL4-p300-(504-1238), GAL4-p300-(852-1071), GAL4-p300-(636-2414), GAL4-p300-(1064-2414), and
GAL4-p300-(1757-2414) have been previously described (33). GAL4-N1,
GAL4-N2, GAL4-N3, GAL4-C1, GAL4-C2, and GAL4-C3 were a gift from Y. Shi
(Harvard Medical School, Boston, MA).
-phosphoglycerate, and incubated for
1 h at 4 °C. Nonspecific binding sites were blocked by
adding 180 µl of 5% milk + PBS
(5MPBST)/NaF/
-phosphoglycerate for 30 min at 4 °C before
adding specific protein, resuspended in 50 µl/well of Sf9LB or
PBST for p300 and MDM2, respectively. The proteins were incubated for
1 h at 4 °C and wells were rigorously washed 3 times with 180 µl of PBST before incubating for 1 h with specific antibodies in
5MPBST/NaF/
-phosphoglycerate. Wells were given another 3 washes with PBST to remove unbound antibody and specific binding was
detected with secondary antibody (anti-mouse or anti-rabbit) conjugated
to horseradish peroxidase. The binding was detected by ECL and
quantified using a luminometer (Fluoroskan Ascent FL). The protocol for
the streptavidin capture ELISA was followed, except increasing amounts
of peptide was incubated with the protein for 30 min at 4 °C before
incubating with the substrate peptide.
/
) cells were transfected with
pcDNA3.1-p53 (or p53-6KR acetylation mutants as indicated (56))
and either pG13-CAT DNA (p53 consensus-sites) or pMG13-CAT DNA (mutated
p53 consensus-sites; vectors described as indicated (36)) for 48 h
before harvesting. After washing twice with 5 ml of ice-cold PBS,
lysates were incubated with acetylation immunoprecipitation lysis
buffer (50 mM Hepes, pH 7.8, 200 mM NaCl, 1%
(v/v) Triton X-100, 10 mM EDTA, 5 mM
dithiothreitol, 1× PIM, and 5 µM trichostatin A)
for 15 min on ice before scraping and passing through a 21-gauge
syringe needle 10 times and then transferring the supernatant to fresh
microcentrifuge tubes. The resultant lysates were then
immunoprecipitated using a p53-specific antibody (DO-1 or ICA-9) or
p300 antibody (N-15) and then probed for acetyl-p53. The specific
acetylation was normalized to the quantity of total p53 captured
assessed by probing with anti-p53 (DO-1 or CM1/5).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Developing a consensus phosphopeptide binding
motif for p300 protein. The binding of: A,
full-length p300 protein, and B, full-length MDM2 protein,
to biotinylated-Ser20 phosphopeptides (amino acids 14-27
of human p53), as described previously (14), substituted with alanine
at the indicated positions were analyzed by ELISA. Increasing amounts
of biotinylated peptide (0, 0.01, 0.1, or 1 ng) were titrated onto
streptavidin-coated surfaces as indicated (14). The amount of p300 or
MDM2 protein bound is quantitated as luciferase activity (RLU) using a
peroxidase-linked secondary antibody coupled to anti-p300 or anti-MDM2
antibodies. C, the alanine scan of p53 derived the
putative consensus site for p300 in blue
(underlined) as
QXXXSPO3XXWKLL. An e-motif
search of a protein data base with these amino acids identified the
transcription factor UBF as containing BOX-I homology
domains and are shaded in green. D, the binding
of p300 to UBF or the p53-BOX-I peptide with or without a
phosphate substitution at the highlighted serine residue was analyzed
by ELISA as indicated in A. The amount of biotinylated
peptide titrated onto streptavidin-coated surfaces is as indicated. The
amount of p300 protein bound is quantitated as luciferase activity
(RLU) using a peroxidase-linked secondary antibody coupled to an
anti-p300 antibody. UBF-1 has the strongest homology to the p300
consensus site in the C-terminal hydrophobic region
(SPO3XXWKLL). E, domain structure of
human p53. The conserved domains I through V, the tetramerization
motif, the polyproline domain, and C-terminal negative regulatory
domains (NRD1 and NRD2) are as indicated. The position of the p300- and
MDM2-binding sites in BOX-I, the p300 acetylation sites in
NRD1, and the ATM and CHK2 phosphorylation sites in BOX-I
are as indicated.
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Fig. 2.
Identification of two phosphopeptide-binding
domains on p300 using an in vitro p300-miniprotein
binding assay. A, a summary of the well characterized
peptide-binding domains of p300: C/H1, KIX, C/H2, Bromo, C/H3, IBiD,
and (see Fig. 3) the N-terminal phosphopeptide-binding domain with
homology to IBiD (IHD). B, mapping of the
phosphopeptide-binding domain to two large regions of p300. HCT116
p53 /
cells were transfected with 5 µg of GAL4,
GAL4-p300, or the indicated GAL4-p300 miniproteins and the p300 from
lysates was captured on ELISA wells with an anti-GAL4 antibody to
purify the indicated miniproteins. The indicated biotinylated peptide
(BOX-I, Ser20-phospho-BOX-I, or no
peptide) was added and the amount of p300 protein bound is quantitated
as luciferase activity (RLU) using a peroxidase-linked secondary
antibody coupled to an anti-p300 antibody. C, small
fragments of p300 used to fine map the phosphopeptide-binding domain to
two regions flanking the C/H1 and C/H3 domains of p300. HCT116
p53
/
cells were transfected with 5 µg of GAL4-p300
miniproteins (C1-C3 and N1-N3) and p300 miniproteins lysates were
captured on ELISA wells with an anti-GAL4 antibody to purify the
indicated polypeptides. The indicated biotinylated peptide
(BOX-I, Ser20-phospho-BOX-I, or no
peptide) was added and the amount of p300 protein bound is quantitated
as luciferase activity (RLU) using a peroxidase-linked secondary
antibody coupled to an anti-p300 antibody.
/
) cells
transfected with the indicated GAL4-p300 fusion constructs were
incubated with an anti-GAL4 antibody in ELISA wells to capture and
purify the p300 miniproteins. The corresponding biotinylated peptide
derived from p53 (BOX-I,
Ser20-phospho-BOX-I, or no peptide) was added,
and p300-peptide complex stability was verified and quantitated using
streptavidin-horseradish peroxidase coupled to luminography (Fig.
2B, GAL4 versus GAL4-full-length p300, 50-65
RLUs background versus 120 RLUs, respectively). The minimal
p300 fragments that bound to the phospho-BOX-I motif were from 1757 to 2414 in the C terminus (147 RLUs) and from 192 to 504 in
the N terminus (93 RLUs) (Fig. 2B). These p300 domains contain the C/H1, part of the C/H3 (C/H3
) domain, and the IBiD domain, which were previously reported to bind to p53 (20). Further
localization of the phosphopeptide-binding region of p300 was carried
out using portions of p300 from 1757 to 2414 in the C terminus and from
192 to 504 in the N terminus (Fig. 2C): GAL4-N1 (aa 2-337),
GAL4-N2 (aa 302-667), GAL4-N3 (aa 406-566), GAL4-C1 (aa 1737-2414),
GAL4-C2 (aa 1945-2414), and GAL4-C3 (aa 1709-1913). The minimal p300
fragment from the N terminus that bound to the phospho-BOX-I
motif encoded a 17-kDa miniprotein (Fig. 2C; GAL4-N3, aa
406-566, 118 RLUs), which is flanked by the C/H1 domain or KIX domain
(Fig. 2A) and defines a previously unreported region on p300
that binds to p53. The minimal p300 fragment from the C terminus that
bound to the phospho-BOX-I motif mapped out the C-terminal
C/H3 domain and encoded a 51-kDa miniprotein (Fig. 2C;
GAL4-C2, 94 RLUs) that displayed specificity for the BOX-I Ser20-phospho motif of p53, relative to the negative
control GAL4 alone (50-60 RLUs) or the positive control
GAL4-full-length p300 (122 RLUs). The C-terminal p53-binding domain in
our studies maps out the C/H3 domain, which is consistent with recent
studies showing that the p53-binding domain in the C terminus of p300
resides within a domain called IBiD or SRC-1 (30, 31). An alignment of
the minimal C-terminal IBiD domain with the N-terminal GAL4-N3 fragment
(amino acids 406-566) demonstrated a significant degree of identity
(Fig. 3C). We have named the
N-terminal domain of p300 that binds the phosphorylated activation
domain of p53 as IHD (Figs. 2A and 3C).
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Fig. 3.
The IBiD and IHD domains interact with p53
in vivo. A, binding avidity of IBiD
and IHD to the transactivation domain of p53 in vivo. The
IBiD domain (amino acids 2050-2094), IHD domain (406-566), and part
of the C/H3 domain (1709-1913; i.e. GAL4-C3 as in Fig.
2C) were subcloned into the pACT vector to produce a
VP16-transactivation domain fusion protein. In the left 5 panels, the indicated constructs were co-transfected (1 µg) into
HCT116 p53 /
cells with a
-galactosidase control
vector (pCMV-
-gal) and lysed 24 h later
to test for the ability to affect transcription from a GAL4-luciferase
reporter construct (pG5-luc). In the right 5 panels, the indicated pACT-VP16 fusion constructs (1 µg) were
co-transfected with a construct encoding GAL4 fused to the N-terminal
transactivation domain of p53 (amino acids 10-40) and a
-galactosidase control vector (pCMV-
-gal).
The activity is summarized as relative light units normalized to
-galactosidase activity. B, the IBiD and IHD domains
attenuate p53-dependent transcription in cells. HCT116
p53
/
cells were co-transfected with the indicated
constructs (1 µg; pCMV-p53, pCMV-p53 + pCMV-p300, alone, or with the
indicated pACT fusion constructs), pCMV-
-gal (1 µg), and the
p53-responsive p21-luc reporter vector. The activity is
summarized as relative light units normalized to
-galactosidase
activity. C, the identity between the C-terminal IBiD and
N-terminal IHD domains is summarized.
domain gave rise to p53-binding
activity above the background (324 RLUs), consistent with some studies showing that the C/H3 domain can bind to p53. Control transfection of
the same pACT constructs without the p53-TAD-GAL4 vector did not
support transcription (Fig. 3A, left 5 panels).
(1918 RLUs) or pACT only (2538 RLUs). Together, these data
identify IBiD as the major p53-binding domain of p300 and the novel
N-terminal domain IHD as an equally effective p53-binding domain. We
cannot rule out the previously identified p53-binding domains including
the C/H3, the KIX , or the C/H1 domains as effective p53-binding
domains of p300, as the folding and assembly of the relatively large
p300 protein with its various repressor and activator motifs
complicates a clear understanding of how p300 really binds to
p53. For example, there is some weak, but significant in
vivo p53-binding activity associated with the region of the
C/H3
domain used in this study (Fig. 3, A and
B). However, IBID and IHD are the predominate p53-binding
domains with similar specific activities as defined in: (i) in
vitro p53-binding assays (Fig. 2B); (ii) in
vivo p53-binding assays (Fig. 3A); (iii) dominant
negative inhibitors in p53-dependent transcription assays (Fig. 3B).
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Fig. 4.
p300 acetylation of p53 and histone H4.
A, monophasic kinetics of acetylation using purified p300,
p53, and the histone H4 substrate. 100 ng of purified p300 was
incubated over the indicated times (0 to 30 min) with 1 µg of histone
H4 and acetyl-CoA. The relative acetylation over time was determined by
immunoblotting the reaction products onto nitrocellulose and detecting
total histone protein (H4) or acetylated histone
(Ac-H4) using either histone antibody or an anti-acetylation
antibody. B, quantitation of the acetylation using
bioluminescence with a Genegnome-Syngene Bioimaging System.
C, acetylation of p53 by p300. 100 ng of p300 and 400 ng of
p53 were incubated for 10 min and relative acetylation was determined
by Western blot using an anti-p53 acetylation antibody: p53 alone
(lane 1), p53 + p300 (lane 2), and p53 + p300 + acetyl-CoA (lane 3).
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Fig. 5.
Consensus site oligonucleotide DNA stimulates
acetylation of p53. A, p53 acetylation by p300 is
stimulated by the addition of p53 consensus site DNA. Fully
reconstituted acetylation reactions were carried out with p300 and p53
for 10 min in the presence of acetyl-CoA without DNA (lane
4) and increasing amounts of consensus site oligonucleotide DNA
(pG-DNA, from 100 to 1000 ng, lanes 5-8).
Relative acetylation was quantitated by Western blot using an anti-p53
acetylation antibody and normalized to total p53 protein using the
monoclonal antibody DO-1. B, p53 acetylation by p300 is not
stimulated by the addition of nonspecific DNA. Acetylation reactions
were carried out with p300 and p53 for 10 min in the presence of
acetyl-CoA and increasing amounts of nonspecific DNA
(NS-DNA; lanes 5-8 versus lanes
1-4). A control including consensus site DNA (pG-DNA)
is in lane 9. Relative acetylation was quantitated by
Western blot using an anti-p53 acetylation antibody and normalized to
total p53 protein using the monoclonal antibody DO-1. C, p53
acetylation by p300 is stimulated by equimolar amounts of p53 protein
to consensus site DNA. Acetylation reactions were carried out with p300
and p53 for 10 min in the presence of acetyl-CoA without DNA
(lane 1) and increasing amounts of consensus site
oligonucleotide DNA (pG-DNA, from 1 to 200 ng, lanes
2-12). From 10 to 15 ng of 26-mer oligonucleotide DNA gave rise
to maximal acetylation of p53 that is at the 1:1 molar ratio of
DNA·p53 protein tetramer in the reaction. Relative acetylation was
quantitated by Western blot using an anti-p53 acetylation antibody and
normalized to total p53 protein using the monoclonal antibody DO-1.
D, DNA does not stimulate histone acetylation by p300.
Acetylation reactions were carried out as described in C but
with the inclusion of histone H4 in place of p53 and without DNA
(lane 1) or with the addition of p53 consensus site DNA
(lanes 2-6). The top panel measures acetylated
histone and the bottom panel measures total histone protein
levels.
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Fig. 6.
Kinetic studies on the consensus site
oligonucleotide DNA-dependent acetylation of p53.
A and B, acetylation reactions were carried out
as described in the legend to Fig. 5 to define parameters (time and DNA
concentration) for linearity. By varying the concentrations of
acetyl-CoA (500 to 2000 µM) at different concentrations
of p53 (0.3, 0.6, and 1.2 µM), as indicated, acetylation
was quantitated using bioluminescence with a Genegnome-Syngene
Bioimaging System and a resultant double-reciprocal plot was drawn
(C).
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Fig. 7.
Consensus site
DNA-dependent acetylation of p53 using supercoiled plasmid
DNA in vitro and in vivo.
A, in vitro p53 acetylation. Human recombinant
p53 (lanes 2-4) was purified from insect cells and
incubated with p300 and supercoiled plasmid containing the
p53-consensus site (pG13, lanes 4) or nonspecific
DNA (pMG13, lanes 3). Relative acetylation was
quantitated by Western blot using an anti-p53 acetylation antibody and
normalized to total p53 protein using the monoclonal antibody DO-1.
B-E, in vivo DNA-dependent
acetylation of p53 by p300. pCMV-P53 (lanes 1 and
2) or pCMV-p53-6KR (lanes 3 and 4;
mutated acetylation sites) were co-transfected into
p53 /
cells in the presence of consensus site plasmid
DNA (pG13-CAT, lanes 2 and 4) or
plasmid DNA without the consensus site (pMG13-CAT,
lanes 1 and 3). The amount of total p53 in each
transfection was quantified by direct immunoblotting with the
monoclonal antibody DO-1. B and C, IP p300
panel: endogenous p300 protein was immunoprecipitated with an
anti-p300 antibody and levels of total p53 protein (C) or
acetylated p53 bound to p300 (B) were quantitated by
immunoblotting, as indicated, with either the acetylation-specific
antibody or a p53 protein antibody to normalize to the total p53
protein. D and E, IP p53 panel: the
total p53 protein was immunoprecipitated with a mixture of ICA-9 and
DO-1 monoclonal antibodies and the total levels of acetylated p53
(D) or p53 protein (E) were quantitated by
immunoblotting, as indicated, with either the acetylation-specific
antibody or a p53 protein antibody to normalize to the total p53
protein.
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Fig. 8.
N-terminal phosphopeptides inhibit
acetylation of DNA-bound p53. A,
p300-dependent acetylation of p53 is inhibited by the
Ser20 phosphopeptide derived from p53. Acetylation
reactions were carried out as described before except with the addition
of varying concentrations of peptides: BOX-I or
Ser20 phospho-BOX-I. The top panel
measures acetylated p53 and the bottom panel measures total
p53 protein levels. B, histone acetylation is not inhibited
by the Ser20 phosphopeptide derived from p53. Acetylation
reactions were carried out as in A but with 1 µg of
histone H4 as the substrate for p300. The insensitivity of histone
acetylation to the p300 Ser20 phosphopeptide derived from
p53 indicates that p53 acetylation inhibition by this peptide is not
necessarily allosteric. C, p53 acetylation is inhibited
using the ELISA p53 capture assay. Reaction products from A
were incubated in ELISA wells with monoclonal antibody ICA-9 to capture
p53 protein (30). The amount of acetylated p53 captured was quantitated
by incubating the wells with polyclonal antibody to acetyl-p53 or to
total p53 protein and depicted in relative light units (ac-p53/total
p53).
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Fig. 9.
Acetyl-CoA stabilizes the p300·p53 protein
complex only in the presence of consensus-site DNA. A,
acetyl-CoA-dependent p300·p53 protein complex
stabilization by consensus site oligonucleotides. Insect cell-purified
human recombinant p53 was captured in a microtiter well with the ICA-9
anti-p53 monoclonal antibody (3) in the presence of consensus site
pG-DNA or NS-DNA oligonucleotides. The captured p53 isoforms were
incubated with buffer containing insect cell-purified p300 protein in
the absence (lanes 1-6) or presence of 1 mM
acetyl-CoA (lanes 7-12). The level of p53 acetylation:
( /+)-acetyl-CoA, (
/+)-NS-DNA, or (
/+)-pG-DNA was quantitated
using the anti-acetylation antibody (lanes 1-3 and
7-9). The amount of p300 protein stably bound to p53
protein: (
/+)-acetyl-CoA, (
/+)-NS-DNA, or (
/+)-pG-DNA was
determined using an antibody to p300 and quantitated as p300
bound/total p53 using enhanced chemiluminescence expressed as relative
light units (RLU). B,
acetyl-CoA-dependent p300·p53 protein complex
stabilization by consensus site supercoiled plasmid DNA. Insect
cell-purified human recombinant P53 was captured in a microtiter well
with the ICA-9 anti-p53 monoclonal antibody (3) in the presence of
pG13-CAT plasmid DNA or pMG13-CAT plasmid DNA. The captured p53
isoforms were incubated with buffer containing insect cell-purified
p300 protein in the absence (lanes 1-6) or presence of 1 mM acetyl-CoA (lanes 7-12). The level of p53
acetylation: (
/+)-acetyl-CoA, (
/+)-NS-DNA, or (
/+)-pG-DNA was
quantitated using the anti-acetylation antibody (lanes 1-3
and 7-9). The amount of p300 protein stably bound to p53
protein, (
/+)-acetyl-CoA, (
/+)-NS-DNA, or (
/+)-pG-DNA was
determined using an antibody to p300 and quantitated as p300
bound/total p53 using enhanced chemiluminescence expressed as relative
light units. The notable effects include the dissociation of p300 from
p53 after acetylation in reactions with nonconsensus site DNA
(lanes 11 versus 5), whereas a
stabilization of the p300·p53 complex occurs after acetylation in
reactions with consensus site DNA (lanes 12 versus 11).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
domain of p300 to the N-terminal activation domain of p53
was observed in cells (Fig. 3A), but not in vitro
(Fig. 2). Additionally, a more recent study has shown that the minimal IBiD domain (or SRC-1) is another peptide-binding domain within p300
that can also interact with p53 (31). Such differences may be explained
by the fact that previous p300-p53 interactions were mapped by protein
binding assays, or nonphosphorylated p53 protein domains, whereas our
assays utilize a combination of phosphopeptide binding assays and
full-length p300 protein to measure p53·p300 complex stability. The
use of truncations of p300 protein may also complicate studies, as we
have previously shown that the C-terminal acetyltransferase domain of
p300 displays significant binding activity toward the
Ser15-BOX-I activation domain but little
activity toward Ser20-BOX-I domain fragments
(14).
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ACKNOWLEDGEMENT |
---|
We thank Dr. Deborah French (University of Strathclyde, UK) for critical reading of the manuscript and helpful discussions.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a BBSRC PhD studentship. Current address: Dept. of Molecular Oncology, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080.
Supported by a Royal Society University Fellowship.
** Supported by a Program Grant from the Cancer Research UK and a Career Establishment Grant from the UK Medical Research Council. To whom correspondence should be addressed. Tel.: 44-0-1382-496430; Fax: 44-0-1382-633952; E-mail: t.r.hupp@dundee.ac.uk.
Published, JBC Papers in Press, December 23, 2002, DOI 10.1074/jbc.M211460200
2 D. Dornan and T. R. Hupp, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; UBF1, upstream binding factor 1; CBP, CREB-binding protein; IHD, IBiD homology domain; RLU, relative light units; IBiD, interferon-binding domain.
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