MOLECULAR AND CELLULAR BIOLOGY, July 2000, p. 4849–4858
0270-7306/00/$04.00⫹0
Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Vol. 20, No. 13
p53 Recruitment of CREB Binding Protein Mediated through
Phosphorylated CREB: a Novel Pathway of Tumor
Suppressor Regulation
HOLLI A. GIEBLER, ISABELLE LEMASSON,
AND
JENNIFER K. NYBORG*
Department of Biochemistry and Molecular Biology, Colorado State University,
Fort Collins, Colorado 80523-1870
Received 29 December 1999/Returned for modification 16 February 2000/Accepted 23 March 2000
CREB binding protein (CBP) is a 270-kDa nuclear protein required for activated transcription of a large
number of cellular genes. Although CBP was originally discovered through its interaction with phosphorylated
CREB (pCREB), it is utilized by a multitude of cellular transcription factors and viral oncoproteins. Both
CREB and the tumor suppressor p53 have been shown to directly interact with the KIX domain of CBP.
Although coactivator competition is an emerging theme in transcriptional regulation, we have made the
fortuitous observation that protein kinase A-phosphorylated CREB strongly enhances p53 association with
KIX. Phosphorylated CREB also facilitates interaction of a p53 mutant, defective for KIX binding, indicating
that CREB functions in a novel way to bridge p53 and the coactivator. This is accomplished through direct
interaction between the bZIP domain of CREB and the amino terminus of p53; a protein-protein interaction
that is also detected in vivo. Consistent with our biochemical observations, we show that stimulation of the
intracellular cyclic AMP (cAMP) pathway, which leads to CREB phosphorylation, strongly enhances both the
transcriptional activation and apoptotic properties of p53. We propose that phosphorylated CREB mediates
recruitment of CBP to p53-responsive promoters through direct interaction with p53. These observations
provide evidence for a novel pathway that integrates cAMP signaling and p53 transcriptional activity.
Although pCREB complexed with the KIX domain of CBP
has been well characterized, KIX has also been shown to interact with several other proteins, including the tumor suppressor p53 (36). p53 is a transcription factor that induces cell cycle
arrest or apoptosis in response to a variety of cellular stress
signals, thereby preventing the transmission of genetic mutations (reviewed in reference 22). Mutations within the coding
sequence of the p53 gene, leading to loss of p53 activity, have
been identified in 60% of the human malignancies examined
(17, 25). This statistic underscores the significance of p53 in
genome surveillance and suppression of malignant transformation. The transcriptional activity of p53, which is tightly linked
to its tumor suppressor function, appears to depend upon
efficient recruitment of CBP to p53 target promoters. Consistent with this observation, recent studies have shown that the
activation domain of p53 participates in CBP recruitment (16,
36). Together, these findings indicate that CBP plays a critical
role in supporting p53-dependent transcription function.
The recent study showing p53 binding to KIX led us to ask
whether both pCREB and p53 bind to KIX in a mutually
exclusive manner, possibly leading to coactivator competition
within the cell. From this line of investigation, we made the
surprising observation that pCREB strongly facilitates p53 association with KIX. We demonstrate that phosphorylated
CREB, but not unphosphorylated CREB, mediates p53 association with KIX, and that this is accomplished through
protein-protein interaction between the basic leucine zipper
(bZIP) domain of CREB and the amino terminus of p53. The
significance of this interaction is supported by studies showing
that p53 and CREB interact in vivo and that p53 function is
enhanced by forskolin, a stimulator of the PKA signal transduction pathway. These data support a model where phosphorylated CREB bridges the interaction between promoter-bound
p53 and CBP, furnishing an alternate mechanism of coactivator recruitment to p53-responsive genes. Finally, the evidence
CREB binding protein (CBP) is a large pleiotropic cellular
coactivator protein critical to the execution of virtually all
known cellular programs, including cell growth, differentiation,
the integration of both signal-dependent and -independent
cellular responses, and apoptosis. CBP and its sister protein
p300 are highly conserved in multicellular organisms from Caenorhabditis elegans to mammals and have been shown to have
profound effects on somatic differentiation during early embryogenesis (2, 20, 33, 38). CBP interacts with a multitude of
structurally unrelated cellular transcription factors, promoting
selective gene activation by providing communication between
promoter-bound transcription factors and components of the
basal transcription apparatus (34). Following promoter localization of CBP, the coactivator is also believed to directly
acetylate nucleosomes, leading to transcriptional activation
through localized chromatin remodeling. These acetylation
events are carried out through both intrinsic and associated
(P/CAF) histone acetyltransferase activities (7, 28).
CBP was originally discovered, and thus named, through its
interaction with the kinase-inducible activation domain (KID)
of the cellular transcription factor CREB (4, 11, 23). Protein
kinase A (PKA) phosphorylation of a critical serine residue on
CREB (amino acid [aa] 133) is required for complex formation
between these two proteins (29). A small subdomain of CBP
(aa 586 to 679), called the KIX domain, participates in proteinprotein interaction with pCREB (31). KIX is composed of
three interacting ␣ helices that come together to form a hydrophobic core. A shallow groove on the surface of the KIX
structure provides the site for molecular interaction with
pCREB.
* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins,
CO 80523-1870. Phone: (970) 491-0420. Fax: (970) 491-0494. E-mail:
jnyborg@lamar.colostate.edu.
4849
4850
GIEBLER ET AL.
supports a unique form of transcription factor organization,
leading to multilayered regulation of gene expression, and
underscores the importance of CBP in mediating convergent
signaling pathways.
MATERIALS AND METHODS
Cloning, expression and purification of recombinant proteins. Glutathione
S-transferase (GST)-KIX (CBP aa 588 to 683), His6-p53, His6-p53 (L22Q-W23S)
(36), and KID (CREB aa 100 to 160) (27) were expressed and purified as
previously described. pGST-CREB-His6 expression plasmid was provided by
C.-Z. Giam and was expressed and purified as described (18). GST-p53 was
made by PCR amplification of the p53 gene, followed by insertion into pDEST
15 for expression in Escherichia coli and pDEST 10 for use in the Bac-To-Bac
baculovirus expression system (Life Technologies). Proteins were purified to
near homogeneity, dialyzed against TM 0.1 M KCl buffer (50 mM Tris-HCl [pH
7.9], 100 mM KCl, 12.5 mM MgCl2, 1 mM EDTA [pH 8.0], 20% glycerol, 0.025%
[vol/vol] Tween 20, 1 mM dithiothreitol), aliquoted, and stored at ⫺70°C. p53
proteins were dialyzed against 20 mM Tris-HCl [pH 8.0]–100 mM KCl–0.5 mM
EDTA–20% glycerol. PKA phosphorylation of CREB and KID was performed
as previously described (15).
GST pull-down assays. All GST pull-down experiments were performed using
12.5 ␮l of glutathione-agarose beads equilibrated in 0.5⫻ Superdex buffer (1⫻
Superdex buffer is 25 mM HEPES [pH 7.9], 12.5 mM MgCl2, 10 ␮M ZnSO4, 150
mM KCl, 20% [vol/vol] glycerol, 0.1% Nonidet P-40, and 1 mM EDTA). The
purified GST fusion protein was incubated with the beads for 1 to 2 h at 4°C and
then washed with 0.5⫻ Superdex buffer. The second protein(s) was then added
to the washed beads and incubated for 1 to 2 h (or overnight) at 4°C. The beads
were washed as before, and bound proteins were eluted with sodium dodecyl
sulfate (SDS) sample dyes. Bound proteins were separated by electrophoresis on
a 10% or 12% SDS gel or a 10% Tris-Tricine gel, transferred to nitrocellulose,
and probed with the appropriate antibody. The following antibodies were used in
this study: anti-p53 (DO-1 [epitope corresponding to aa 11 to 25]; Santa Cruz
Biotechnology), anti-p53 (Ab-1 [epitope corresponding to aa 371 to 380]; Calbiochem), anti-CREB (C-21 [epitope corresponding to the carboxy terminus of
human CREB-1]; Santa Cruz Biotechnology), anti-phosphoserine 133-CREB
(New England Biolabs), and anti-His (H-15; Santa Cruz Biotechnology).
Cell culture, transient cotransfection assays, and expression plasmids. Jurkat
T cells and Molt 4 T cells were cultured in Iscove’s modified Dulbecco’s medium
supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and
penicillin-streptomycin. The H24 p53-3 cells were grown in Dulbecco’s modified
Eagle’s medium supplemented with 10% FBS, tetracycline (2 ␮g/ml), puromycin
(1 ␮g/ml), and geneticin (250 ␮g/ml). The MCF7 breast cancer cells were cultured in Eagle’s minimum essential medium supplemented with 2 mM L-glutamine, penicillin-streptomycin, Earle’s BBS adjusted to contain 1.5 g of sodium
bicarbonate per liter, a 0.1 mM concentration of nonessential amino acids, 1.0
mM sodium pyruvate, 10% FBS, and 0.01 mg of bovine insulin per ml. For
transient cotransfection assays (36), cells were grown to a density of 106 cells/ml
and transfected with Lipofectamine (Life Technologies, Inc.) and a constant
amount of DNA for 5 h. The cells were allowed to recover for 24 h before
harvest, in the absence or presence of 20 ␮M forskolin. Cells were lysed, and
luciferase activity was measured using the Dual-Luciferase reporter assay system
with a Turner Designs model TD 20-e luminometer. Luciferase activity was
normalized to pRL-TK vector (Promega), which encodes the Renilla luciferase
from HSV-TK promoter, as an internal control. Expression plasmids for p53
(pC53-SN3 [6]), RSV-PKA, or Gal4-p53 fusion proteins carrying p53 aa 1 to 393
(pGalp53) or aa 1 to 52 (pGalp53N) (12) have been previously described. The
luciferase reporter plasmids pG13-Luc (21), pGalTK-Luc (12), and viral CRELuc (15) have also been described. The Bax-Luc reporter plasmid was prepared
by cloning the Bax promoter (⫺340 to ⫹31) upstream of the luciferase gene.
Western blots. MCF7 cells were grown to 70% confluency, medium was removed, and fresh medium was added to the cells in the absence or presence of
20 ␮M forskolin, and the cells were incubated for the indicated amount of time.
Cells were lysed and resuspended in SDS sample dyes. Proteins were separated
on a 10% Tris-Tricine SDS-polyacrylamide gel electrophoresis and analyzed by
Western blot analysis.
Northern blots. MCF7 cells were grown to ⬃70% confluency, the medium was
removed and fresh medium was added to the cells in the absence or presence of
20 ␮M forskolin and incubated for 45 min. The cells were harvested in a solution
containing 4 M guanidinium thiocyanate, 0.5% sarcosyl, and 25 mM sodium
citrate, pH 7.0, and total RNA was isolated using acidic phenol extraction
followed by isopropanol precipitation and resuspension in FORMAzol (Molecular Research Center, Inc.). Purified total RNA (15 ␮g) was separated by
electrophoresis on a 1% agarose–formaldehyde-MOPS (morpholinepropane sulfonic acid) gel, transferred to Genescreen (DuPont-NEN) membrane, and crosslinked for 3 min with UV radiation at a wavelength of 320 nm. Membranes were
hybridized at 43°C with 32P-labeled cDNA probes complimentary to either the
p21 or GAPDH mRNAs. The membrane was washed several times, dried, and
analyzed by Phosphorimager analysis.
Coimmunoprecipitation (co-IP) assays. Molt 4 T-cell lysates were prepared in
RIPA buffer (50 mM Tris-HCl [pH 8.0], 1% Triton X-100, 100 mM NaCl, 1 mM
MOL. CELL. BIOL.
MgCl2, 2 mM benzamidine, 2 ␮g of leupeptin per ml, and 1 mM phenylmethylsulfonyl fluoride). Antibody-bound beads (20 ␮l) (Ab-1 [epitope corresponding
to aa 371 to 380] [Calbiochem] or DO-1 [epitope corresponding to aa 11 to 25]
[Santa Cruz Biotechnology]) were washed in RIPA buffer. Cell lysates (500 ␮g)
were added to each antibody column, incubated overnight, and washed several
times in RIPA buffer. The bound proteins were analyzed on an SDS–10%
polyacrylamide gel, and detected by Western blotting using anti-CREB-1 antibody (C-21 [epitope corresponding to aa 295 to 321]; Santa Cruz Biotechnology)
and anti-p53 (DO-1; Santa Cruz Biotechnology).
Apoptotic analysis. Molt 4 T-cells (5 ⫻ 105 cells) and H24 p53-3 cells (reference 10) were treated with the indicated concentrations of forskolin (or dimethyl
sulfoxide carrier) for 24 h. In the p53-inducible cell line H24 p53-3, tetracycline
was removed at the time of forskolin addition. Cells were harvested and treated
with YO-PRO-1 (from Vybrant apoptosis assay kit no. 4; Molecular Probes).
Cells were deposited on microslides coated with poly-L-lysine (Poly-Prep slides;
Sigma), and the fluorescent apoptotic cells were analyzed and photographed
using a fluorescence microscope.
RESULTS
pCREB enhances p53 association with the KIX domain of
CBP. In a recent structural study characterizing the phosphorylated-kinase-inducible domain of CREB bound to the hydrophobic groove on the surface of KIX, the authors noted key
sequence and structural similarities between phosphorylated
KID (pKID) and the activation domain of p53 (31). This led to
the prediction that the hydrophobic groove on the surface of
KIX may also serve as the binding site for p53. To test this
hypothesis, we performed a GST pull-down competition experiment to determine whether titration of pCREB can displace p53 bound to KIX. Purified GST-KIX (aa 588 to 683)
was bound to glutathione-agarose beads and incubated with
recombinant, purified p53. Increasing amounts of PKA-phosphorylated CREB were added into the binding reaction mixtures, and the amount of p53 remaining bound to KIX was
determined by Western blot analysis (Fig. 1A). Surprisingly,
the addition of pCREB did not compete with p53 for binding
to KIX but produced a dramatic increase in the amount of p53
associated with the complex (compare lanes 4 to 7). As expected, neither p53 nor pCREB bound to GST beads alone
(Fig. 1A, lanes 2 and 3).
We were next interested in determining whether the increase in p53 association with KIX was specific to pCREB or
if unphosphorylated CREB could also promote the interaction. To test this hypothesis, we again compared the binding of
p53 to KIX following the addition of increasing amounts of
either unphosphorylated or phosphorylated CREB. As shown
in the GST pull-down assay in Fig. 1B (upper panel), we
observed enhancement of p53 binding to KIX only in the
presence of pCREB (compare lanes 2 to 5). In fact, increasing
amounts of unphosphorylated CREB appeared to reduce the
p53-KIX interaction in a dose-dependent manner (compare
lane 2 with lanes 6 to 8). Interestingly, we observed that the
enhancement of p53 association with KIX correlated precisely
with pCREB binding to KIX, suggesting that both pCREB and
p53 are simultaneously in complex with this region of CBP
(Fig. 1B, compare lanes 3 to 5 of the upper and lower panels).
Further, these data strongly support a role for a direct pCREBKIX molecular interaction in ternary complex formation. As
expected, we did not detect the binding of unphosphorylated
CREB to the KIX domain (Fig. 1B, lower panel, lanes 6 to 8).
To better characterize the ternary complex containing
pCREB, p53, and KIX, we performed the reciprocal experiment and tested whether p53 could also enhance pCREB binding to KIX. We performed a GST pull-down assay in which p53
was titrated into binding reaction mixtures containing GSTKIX and a constant amount of either phosphorylated or unphosphorylated CREB. As shown in Fig. 1C, p53 had only a
modest effect on pCREB binding to KIX and no detectable
VOL. 20, 2000
pCREB MEDIATES p53 TRANSCRIPTIONAL ACTIVITY
4851
FIG. 1. (A) CREB enhances p53 binding to the KIX domain of CBP. Purified p53 (5 pmol) was incubated with either GST alone (1 pmol) (lanes 2 and 3)
or GST-KIX (aa 588 to 683) (1 pmol) (lanes 4 to 7) in the absence (⫺) and
presence (⫹) of the indicated amount of purified, PKA-phosphorylated CREB.
Bound p53 protein was detected by Western blot analysis. Onput protein (6%)
is shown in lane 1 and protein standards (in kilodaltons) are indicated at left. (B)
pCREB, but not unphosphorylated CREB, enhances p53 binding to the KIX
domain of CBP. Purified p53 (5 pmol) was incubated with GST-KIX (1 pmol) in
the absence or presence of the indicated amounts of either purified pCREB
(lanes 3 to 5) or purified, mock-phosphorylated CREB (lanes 6 to 8). p53 (upper
panel) was detected by Western blot analysis, and bound protein is indicated.
The blot was reprobed using an anti-CREB antibody, and bound protein is
indicated in the lower panel. Onput protein (2%) is shown in lane 1 and protein
standards (in kilodaltons) are indicated at left. (C) p53 does not enhance the
binding of CREB to the KIX domain of CBP. Purified, phosphorylated or
mock-phosphorylated CREB (10 pmol) was incubated with GST-KIX (10 pmol)
in the absence or presence of the indicated amount of purified p53 (lanes 3 to 5
and 7 to 9). CREB (upper panel) was detected by Western blot analysis. The blot
was reprobed using an anti-p53 antibody, and bound protein is indicated in the
lower panel. Onput protein (2%) is shown in lane 1, and protein standards (in
kilodaltons) are indicated at left.
effect on CREB binding to KIX (lanes 2 to 9). These data
suggest that pCREB plays the primary role in facilitating formation of the ternary complex. This conclusion is further supported by the observation that significantly more KIX-associated p53 was detected in the presence of pCREB than in the
presence of unphosphorylated CREB (Fig. 1C, lower panel,
compare lanes 3 to 5 with lanes 7 to 9).
pCREB contacts KIX in the ternary complex. The solution
structure of the pCREB-KIX complex reveals intimate molecular contacts between the hydrophobic groove of KIX and the
pKID domain of CREB (31). Although we do not know where
p53 binds on the surface of KIX, it seems unlikely that the
hydrophobic groove could accommodate both pCREB and p53
simultaneously. The strong dependence on CREB phosphorylation in ternary complex formation suggests that pCREB
directly contacts the hydrophobic groove, as previously described (31). To determine whether, in the ternary complex,
p53 also directly contacts KIX, we utilized an activation domain mutant of p53 (L22Q-W23S) that we have previously
shown to be defective for KIX binding (36). We reasoned that
if p53 makes direct contacts with KIX in the ternary complex,
then the mutant p53 should also be defective for ternary complex formation. On the other hand, if pCREB alters or abolishes the p53-KIX interaction, then we may observe enhanced
binding of both wild-type and mutant forms of p53. To sort out
these questions, we compared the binding of the wild-type and
mutant forms of p53 to GST-KIX in the presence of pCREB.
We found that both forms of p53 bound well in the ternary
complex, with the binding of each molecule dependent upon
pCREB (Fig. 2A, lanes 3 and 5). These data indicate that aa 22
and 23 of the p53 activation domain are not involved in ternary
complex formation and support the idea that pCREB alters or
abolishes the p53-KIX interaction.
Two possible scenarios are consistent with the data presented in Fig. 2A. In the presence of pCREB, p53 forms
different contacts with KIX, or alternatively, p53 binding to
KIX is abolished. The second possibility would therefore require a direct CREB-p53 interaction as a means to incorporate
p53 into the ternary complex. To test this possibility, we utilized a highly truncated form of CREB that encompassed only
the Ser-133 phosphorylated-kinase-inducible domain (CREB
aa 115 to 148). Although this region of CREB lacks the bZIP
and the Q domains, it is competent for interaction with KIX
(29, 31). To determine whether pKID alone can enhance p53
association with KIX, we compared pKID in parallel with
full-length pCREB in a GST-KIX pull-down assay (Fig. 2B).
Interestingly, the pKID domain had no effect on p53 association with KIX (Fig. 2B, lane 3). Both pCREB and pKID were
fully competent for KIX binding, as assayed in a GST-KIX
pull-down reaction (data not shown). These data indicate that
a region of CREB, outside of the KIX-interacting kinaseinducible domain, is required for efficient formation of the
ternary complex. Furthermore, they are consistent with the
idea that CREB and p53 interact directly.
4852
GIEBLER ET AL.
MOL. CELL. BIOL.
FIG. 2. (A) A p53 mutant, defective for KIX binding, binds KIX in the presence of pCREB. Purified wild-type (wt) (10 pmol) (lanes 2 and 3) or mutant (mut)
(L22Q-W23S) (10 pmol) (lanes 4 and 5) p53 was incubated with GST-KIX (2 pmol) in the absence or presence of 30 pmol of purified, phosphorylated CREB. p53 and
CREB were detected simultaneously by Western blot analysis using an anti-His–anti-CREB antibody mixture. Onput wt p53 (2%) is shown in lane 1, and protein
standards (in kilodaltons) are indicated at left. At the exposure presented, p53 binding to KIX was not detected. (B) The PKA-phosphorylated kinase-inducible domain
of CREB does not support enhanced binding of p53 to KIX. Purified p53 (10 pmol) was incubated with GST-KIX (10 pmol) in the absence or presence of 30 pmol
of phosphorylated full-length CREB (lane 2) or pKID domain of CREB (lane 3). p53 was detected by Western blot analysis. Protein standards (in kilodaltons) are
indicated at left.
Characterization of the CREB-p53 interaction in vitro and
in vivo. Based on the data presented above, we began to formulate a model where the p53-pCREB-KIX ternary complex is
formed through direct pCREB interaction with the hydrophobic groove of KIX, with p53 incorporated into the coactivator
complex via protein-protein interaction with CREB. To directly test for a p53-CREB interaction, we examined the binding of p53 to GST-CREB, in the absence of KIX. As shown in
Fig. 3A, both wild-type and mutant p53 (L22Q-W23S) interacted equally well with GST-CREB (lanes 5 and 6). To confirm
FIG. 3. (A) Wild-type (wt) and mutant (mut) (L22Q-W23S) p53 directly interact with CREB in the absence of KIX. Purified wt or mutant p53 protein (10 pmol
each) was incubated with either GST alone (10 pmol) (lanes 3 and 4), or GST-CREB-His6 (10 pmol) (lanes 5 and 6). Bound p53 proteins were detected by Western
blot analysis. Onput protein (10%) is shown in lanes 1 and 2, and protein standards (in kilodaltons) are shown at left. (B) PKA-phosphorylated or mock-phosphorylated
CREB (25 pmol) was incubated with either GST alone (100 pmol) (lanes 1 and 3) or GST-p53 (100 pmol) (lanes 2 and 4). Bound CREB was detected by Western
blot analysis. Output proteins (10%) (lanes 5 and 6) and protein standards (in kilodaltons) are indicated. (C) CREB (5 pmol) was incubated with either GST alone
(10 pmol) (lane 2) or GST-p53 produced and purified from baculovirus-infected Sf9 cells (10 pmol) (lane 3). Bound CREB was detected by Western blot analysis. Onput
protein (10%) (lane 1) and protein standards (in kilodaltons) are indicated.
VOL. 20, 2000
the p53-CREB interaction, we performed the reciprocal GST
pull-down assay. As shown in Fig. 3B, both phosphorylated and
unphosphorylated forms of full-length CREB bound equally
well to GST-p53 in the absence of KIX (lanes 2 and 4). Taken
together, these results indicate that p53 interacts directly with
CREB, in a binding reaction that is independent of KIX. Together, these data provide direct evidence for a novel interaction between CREB and p53 and support the hypothesis that
pCREB enhancement of p53 binding to KIX is mediated
through a direct p53-CREB protein-protein interaction. Finally, we were interested in testing whether posttranslational
modifications of p53 might affect interaction with CREB. Figure 3C shows that GST-p53 derived from baculovirus-infected
Sf9 cells interacted with CREB in a manner indistinguishable
from that observed with p53 derived from E. coli (compare Fig.
3C, lane 3, with Fig. 3B, lane 2). These data suggest that Sf9
cells do not introduce modifications in p53 that affect interaction with CREB. We have also shown that GST-CREB interacts with non-GST-tagged forms of p53, derived from both E.
coli (Fig. 3A) and baculovirus-infected Sf9 cells (data not shown).
To determine which region of CREB interacts with p53, we
tested a series of CREB deletion mutants in the GST pulldown assay using GST-p53. Most of the CREB deletions were
centered in the region of KID and extended into both the Q1
and Q2 domains (Fig. 4A). Surprisingly, all the CREB deletion
mutants tested were competent for binding to GST-p53 (Fig.
4B, lanes 1 to 6). Since all of the mutants possessed the bZIP
domain (aa 254 to 327) (and a portion of Q1 and Q2), we directly
tested whether bZIP alone was competent for p53 binding. Figure
4C shows that both full-length CREB and bZIP bound GST-p53
with comparable relative affinities (lanes 2, 3, 5, and 6).
Although these studies establish an interaction between
CREB and p53 in vitro, we were interested in determining
whether this interaction could also be detected in vivo. To
investigate this possibility, we performed a co-IP assay in which
a carboxy terminus-reactive p53 antibody (epitope corresponding to aa 371 to 380) was immobilized on a resin. A lysate
prepared from the wild-type p53-expressing T-cell line, Molt4,
was then passed over the column, the column was washed, and
the bound proteins were analyzed by Western blot analysis
using an anti-CREB antibody. As shown in Fig. 4D (upper
panel), we detected several anti-CREB immunoreactive
polypeptides, ranging in molecular mass from about 35 to 45
kDa. The band with the highest molecular mass comigrated
with recombinant CREB, indicating that it likely represents
endogenous CREB in the Molt4 lysate (Fig. 4D, compare lanes
1 and 3). The other polypeptides are likely cross-reactive ATF/
CREB family members (CREM and ATF-1). In a parallel
reaction, we also performed the co-IP assay using an anti-p53
antibody reactive against the amino terminus of p53 immobilized on the resin (epitope corresponding to aa 11 to 25) and
probed for bound polypeptides using the anti-CREB antibody.
Interestingly, we did not detect the binding of any ATF/CREB
proteins when the amino-terminal p53 antibody was used in
the co-IP, suggesting that this antibody masks critical p53
amino acids that may be required for interaction with CREB
(Fig. 4D, upper panel, lane 2). p53 bound equally well to both
the amino-terminal and carboxy-terminal antibodies (Fig. 4D,
lower panel, lanes 2 and 3). These data support the in vitro
data and provide evidence for the binding of multiple ATF/
CREB proteins to p53 in vivo. Furthermore, the results suggest
that the amino terminus of p53 participates in the proteinprotein interaction with CREB.
Activation of the PKA pathway stimulates p53 function. Our
biochemical characterization of the ternary complex led us to
address the question of whether the binding of p53 to pCREB
pCREB MEDIATES p53 TRANSCRIPTIONAL ACTIVITY
4853
and the formation of a complex with CBP might facilitate the
biological effects of p53 in the cell. To first address this question, we were interested in testing whether forskolin, a stimulator of adenylate cyclase and the cyclic AMP (cAMP) pathway, might induce apoptosis. We treated p53-positive Molt4 T
cells with increasing concentrations of forskolin, and monitored the cells undergoing apoptosis. Figure 5A shows that
forskolin treatment produced a significant increase in the number of apoptotic cells in a concentration-dependent fashion.
Treatment of p53-negative Jurkat T cells had no effect on cell
death (data not shown). However, since these experiments did
not address whether the observed apoptotic effects were directly mediated through endogenous p53, we also tested
whether forskolin induced apoptosis in the p53-inducible (tetregulated) cell line, H24 p53-3 (10). Figure 5B shows that,
upon induction of p53, forskolin treatment produced a significant increase in the number of cells undergoing apoptosis.
Although the effect was not as dramatic as that observed in the
Molt4 T cells, Western blot analysis indicated that p53 expression was significantly lower in the H24 p53-3 cell line than in
Molt4 cells (Fig. 5C).
To more directly address the role of forskolin on p53 transcription function, we performed transient-transfection assays.
Jurkat T cells were transiently transfected with a reporter plasmid carrying 13 copies of a p53-responsive promoter driving
expression of the luciferase gene (pG13-Luc). As expected,
transfection of a p53 expression plasmid dramatically stimulated luciferase expression from the p53-responsive reporter
plasmid (Fig. 6A, lane 4). Treatment of the cells with 20 ␮M
forskolin further increased p53-dependent transcriptional activity, resulting in an additional twofold stimulation. The effect
of forskolin was only observed in the presence of transfected
p53, suggesting that the effect was dependent upon transcriptionally competent p53 in the cell (Fig. 6A, compare lanes 2
and 5). The effect of forskolin was likely mediated through
CREB (or a related factor in the cell), as there is no evidence
for direct PKA phosphorylation of p53 (1). Additionally, since
pG13 carries only reiterated p53 response elements upstream
of a minimal promoter, it is likely that the observed stimulation
by forskolin was mediated through these elements. Figure 6B
shows that forskolin treatment had little or no effect on p53
levels produced in the transfection assay.
Since forskolin is an activator of adenylate cyclase, we were
interested in determining more specifically whether the p53dependent transcriptional stimulation was mediated directly
through PKA. To address this question, we simultaneously
treated the transfected cells with both forskolin and the drug
H-89, a specific pharmacological inhibitor of PKA. H-89 appeared to abolish the forskolin stimulation of pG13-Luc, reducing transcription to the level observed with transfected p53
alone (Fig. 6A, lane 6). Figure 6C shows that forskolin and
H-89 were functioning correctly in the assay, as both drugs
appropriately activated and inhibited transcription initiated
through a minimal cAMP responsive promoter (lanes 1–3).
We were also interested in testing a natural p53-responsive
promoter in the transient-transfection assay, as pG13 is an
artificial promoter construct and may not behave in a physiologically appropriate fashion. To address this question, we
tested the effect of forskolin on the p53-responsive Bax promoter, linked to luciferase. Figure 6D shows that expression
from the Bax promoter was strongly stimulated by forskolin, in
a p53-dependent manner (lanes 1 to 4).
In Fig. 4D, we present a co-IP experiment that provides
evidence suggesting that the amino terminus of p53 is involved
in interaction with CREB. Based on this observation, we were
interested in testing whether this region of p53 is sufficient for
4854
GIEBLER ET AL.
MOL. CELL. BIOL.
FIG. 4. (A) Schematic of the CREB deletion mutants. (B) CREB deletion
mutants are competent for p53 binding. The indicated CREB deletion mutants
(10 pmol) were incubated with GST-p53 (25 pmol) (lanes 1 to 6). Bound CREB
proteins were detected by Western blot analysis. Protein standards (in kilodaltons) are indicated. (C) The bZIP domain is sufficient for interaction with p53.
The indicated amounts of either full-length or bZIP (aa 254 to 327) CREB were
incubated with GST alone (100 pmol) (lanes 1 and 4) or GST-p53 (100 pmol)
(lanes 2, 3, 5, and 6). Bound CREB was detected by Western blot analysis. Onput
proteins (5 pmol of CREB, 10 pmol of bZIP) (lanes 7 and 8), and molecular mass
standards (in kilodaltons) are indicated. (D) Anti-p53 immunoprecipitates ATF/
CREB proteins from a T-cell lysate. The upper panel shows a CREB Western
blot of the proteins bound to p53 immobilized using either the Ab-1 or DO-1
anti-p53 antibodies (lanes 2 and 3). Purified, recombinant CREB was electrophoresed as a control (lane 1). The lower panel shows a Western blot of p53
bound to the immobilized anti-p53 antibodies (lanes 2 and 3).
PKA-dependent transcription in transient-transfection assays.
We utilized a truncation mutant of p53, which carried only the
amino-terminal 52 aa of the activation domain of p53, fused to
the DNA binding domain of Gal4 (pGal4-p53N) (12). Figure
6E shows that cotransfection of Gal 4-wtp53 and Gal4-p53N
similarly enhanced transcription from the Gal4-Luc reporter
VOL. 20, 2000
pCREB MEDIATES p53 TRANSCRIPTIONAL ACTIVITY
4855
FIG. 5. Induction of apoptosis by forskolin. (A) Molt 4 cells were treated
with the indicated concentrations of forskolin for 24 h. (B) H24 p53-3 cells (10)
were removed from tetracycline-containing medium and immediately treated
with forskolin for 24 h. Apoptotic cells (upper panels) were detected under
fluorescent microscopy using YO-PRO-1. Lower panels correspond to brightfield microscopy of the cells presented in the upper panel. (C) Western blot
analysis of H24 p53-3 extracts derived from cells grown in the presence (uninduced) (⫹) and absence (induced) (⫺) of tetracycline. Extracts from Molt4 cells
were run in an adjacent lane for comparison. p53 protein was detected by
Western blot analysis (DO-1).
plasmid (compare lanes 3 and 5). This observation is not
unexpected, as the amino terminus of p53 encompasses the
primary activation domain of the protein. The addition of
forskolin to the transfection reaction strongly enhanced Gal4p53N-dependent transcription, with the stimulation exceeding
that observed with the full-length protein (Fig. 6E, compare
lanes 3 and 4 with lanes 5 and 6). Cotransfection of the catalytic subunit of PKA, which is constitutively active for CREB
phosphorylation, also produced strong pGAL4-p53N stimulation comparable to that observed with forskolin (Fig. 6E, lanes
6 and 7). These data provide strong supportive evidence for an
interaction between pCREB and the amino terminus of p53,
resulting in PKA-dependent transcriptional stimulation. The
data also indicate that p53 tetramerization is not required for
interaction with pCREB.
To further address the biological relevance of the cAMP
pathway on p53-dependent transcription function, we tested
the effect of forskolin on an endogenous p53-responsive target
gene. We selected the cell cycle inhibitor gene p21 for our
studies and examined p21 mRNA levels in the p53 wild-type
breast cancer cell line MCF7. We selected this cell line as the
p21 message was undetectable in the Molt4 T-cell lines used
above. Northern blot analysis revealed that treatment of MCF7
cells with forskolin strongly stimulated p21 mRNA synthesis in
vivo (fourfold) (Fig. 7A). Forskolin-induction of p21 mRNA
correlated with an increase in ATF/CREB phosphorylation in
vivo, as demonstrated by Western blot analysis using an antibody directed against the serine 133 phosphorylated form of
CREB (Fig. 7B, upper panel). We also detected a concomitant
increase in p21 protein levels, with little or no change in p53
protein levels (Fig. 7B, middle and lower panels). Interestingly,
forskolin did not increase p21 protein levels in two human
T-cell lymphotrophic virus type 1 infected T-cell lines, where
the presence of the Tax protein inactivates the endogenous
wild-type p53 (data not shown) (9, 14, 30, 36).
DISCUSSION
Deciphering the mechanism(s) of p53 function in the cell has
been one of the most intensively studied areas of modern
biology. This focus derives from the critically important role
p53 plays in genome surveillance and suppression of oncogenic
transformation. Understanding the role of p53 as a transcription factor and the identification of p53-responsive target
genes have greatly advanced our understanding of p53 function; however, many of the molecular events that contribute to
p53 transcriptional activity remain elusive. The recent identification of a direct p53-CBP interaction provides insight into
4856
GIEBLER ET AL.
MOL. CELL. BIOL.
FIG. 6. (A) Forskolin activates p53-dependent transcription in vivo. The p53-responsive pG13-Luc reporter (400 ng) was cotransfected into p53-null Jurkat T cells
with the p53 expression plasmid (pC53-SN3) (400 ng), as indicated. Forskolin (20 ␮M) and/or H-89 (5 ␮M) was added (⫹) or not added (⫺) to the indicated
transfection reactions. (B) Forskolin had no effect on transfected p53 protein levels. Western blot analysis of p53 derived from cells transfected with pC53-SN3 (400
ng), in the absence (⫺) and presence (⫹) of forskolin (20 ␮M). (C) A CREB-responsive promoter responds appropriately to forskolin and H-89. The reporter plasmid
CRE-Luc (1 ␮g) (24) was transfected in Jurkat T cells in the presence (⫹) or absence (⫺) of forskolin (20 ␮M) and/or H-89 (5 ␮M) as indicated. (D) Forskolin activates
the p53-responsive Bax promoter. The Bax-Luc reporter plasmid (400 ng) was cotransfected in Jurkat T-cells with the p53 expression plasmid (pC53-SN3) (400 ng) in
the presence (⫹) or absence (⫺) of forskolin (20 ␮M), as indicated. (E) The amino terminus of p53 is sufficient for forskolin responsiveness. The Gal4 reporter plasmid
(pGalTK-Luc) (400 ng) was cotransfected in Jurkat T cells with the Gal4-p53 expression plasmids carrying either full-length p53 (pGal4-wtp53) or the amino terminus
of p53 (aa 1 to 52) (pGal4-p53N) (800 ng each) (12). Reactions were either cotransfected with the catalytic subunit of PKA (100 ng) or treated with forskolin (20 ␮M)
as indicated. Reported values for each transient-transfection experiment are the average luminescence ⫹ the standard error (error bar) from one experiment performed
in duplicate. Each experiment was performed at least twice.
the transcriptional mechanisms that govern p53 function (5, 16,
26, 32, 36). In this study, we demonstrate the convergence of
the PKA signaling pathway on p53 coactivator recruitment,
delineating a novel mechanism of p53 coactivator utilization.
We report the unexpected discovery that PKA phosphorylation
of CREB at Ser133 results in p53-dependent, indirect tethering
of CBP to p53-responsive genes.
Our studies support a model where the formation of a ternary complex, containing p53, pCREB, and CBP, facilitates
transcriptional activation of p53 target genes. In the ternary
complex, pCREB serves as a molecular bridge between p53
and CBP. Since the complex is phosphate dependent, pKID
likely makes the primary, and possibly exclusive, contacts with
KIX (31). Concomitantly, the bZIP domain interacts with residues located within the amino terminus of p53, without detectably interacting with the DNA. This interaction leaves the
DNA binding domain of p53 available, allowing p53 to deliver
the entire coactivator-containing complex to the promoters of
p53-responsive genes. A schematic illustrating these interactions is shown in Fig. 8.
Our experiments support the idea that the ternary complex
is strongly stabilized by a direct interaction between p53 and
CREB. The possibility that p53 also contacts KIX in this complex cannot be excluded; however, our evidence supports the
CREB-p53 interaction as the primary mode of p53 stabilization in the ternary complex. Specifically, we show that the
VOL. 20, 2000
pCREB MEDIATES p53 TRANSCRIPTIONAL ACTIVITY
4857
FIG. 7. (A) Forskolin induces expression of the p21 gene. Shown is the northern blot analysis of total RNA (15 ␮g) isolated from untreated or 20 ␮M
forskolin-treated MCF7 cells. RNA was hybridized with a p21-specific probe or a GAPDH-specific probe as a loading control. Specific transcripts are indicated. An
RNA kilobase ladder and 18S and 28S rRNAs are shown at left. (B) CREB phosphorylation correlates with p21 expression. Western blot analysis of pCREB
(anti-phosphoserine 133-CREB) (upper panel), p21 (gift from J. Wade Harper) (middle panel), and p53 (DO-1) (lower panel) was performed following a time course
of 20 ␮M forskolin treatment of MCF7 cells. Protein standards (in kilodaltons) are shown at left.
amino terminus of p53, which carries the activation domain of
the protein, is involved in direct interaction with CREB. We
show that an antibody against the amino terminus of p53
blocks complex formation between p53 and CREB, and we
supply evidence showing that the amino-terminal 52 aa of p53
are sufficient for mediating cAMP responsiveness in vivo. Since
we demonstrate that the p53 double point mutant (L22QW23S) is fully active for interaction with CREB in vitro, these
experiments imply that other residues within the amino terminus of p53 must participate in the interaction. Therefore, when
p53 is in complex with CREB, it may be unavailable for direct
coactivator interactions, as previous studies have shown that
the activation domain of p53 directly contacts CBP (16, 36).
Since p53 binds to DNA as a tetramer, it is possible that
individual monomers participate in a combination of separable
contacts with CREB and/or various domains of CBP, generating a highly complex regulatory response.
The identification of this unique ternary complex also has
implications for CREB-regulated transcription, as the binding
of the bZIP domain to p53 would likely abolish the DNA
binding properties of the protein. Therefore, under conditions
of p53 activation, pCREB may be diverted from CRE-responsive genes to p53-responsive genes. Although the physiological
circumstances leading to costimulation of these pathways is not
known, there is evidence that UV irradiation induces CREB
phosphorylation at Ser133 (19); thus, pCREB and p53 may
synergize in response to a UV-induced signal transduction
pathway. More importantly, the PKA pathway of p53-activated
transcription may represent an incomplete picture of this new
model of p53 transcription function, as a number of other
kinases, including AKT and pp90rsk, phosphorylate CREB at
Ser133 (3, 8, 13, 37).
Our observation that the bZIP domain of CREB participates in the interaction with p53 raises the question of whether
additional bZIP-containing transcription factors are competent for p53 binding. Evidence supporting this idea comes from
the co-IP assay (Fig. 4D), where we detected up to four ATF/
CREB immuno-reacting family members. It is likely that one
of these proteins was the cellular transcription factor ATF-1,
which shares a high degree of sequence similarity with CREB
(and cross-reacts with the anti-CREB antibody). It is also possible that additional bZIP proteins, outside of the ATF/CREB
family, may also bind to p53, thus expanding the repertoire
of cellular proteins that may partner with p53 and regulate
p53-dependent transcriptional activation. These might include
components of the AP-1 complex, c-fos and c-jun, and the
c/EBP family of transcription factors—each in complex with a
coactivator. If this prediction is correct, it would suggest that
the p53-pCREB interaction characterized herein may represent a prototype of a much more general mechanism that
exerts precise control of p53 transcription function.
FIG. 8. Model showing the ternary complex, containing CBP, pCREB, and
p53, assembled on a p53-responsive promoter.
4858
GIEBLER ET AL.
The observations reported in this study are very reminiscent
of a recent study showing that the bZIP transcription factor
CHOP participates in transcriptional activation via proteinprotein interactions with the AP-1 complex (35). CHOP appears to be recruited to preexisting, DNA-bound AP-1 complexes through a direct tethering mechanism. Similar to the
case with CREB, the bZIP domain of CHOP is required, and
it appears to be directly involved in protein-protein interaction
with the AP-1 complex. The bZIP protein ATF-6 has also been
shown to function in a similarly unusual fashion (39). ATF-6
interacts directly with the activation domain of serum response
factor, contributing to serum stimulation of the c-fos promoter.
Although the bZIP has not been directly implicated in the
ATF-6/serum response factor protein-protein interaction, it is
provocative that so many bZIP proteins appear to be involved
in this unusual tethering mechanism.
Together, the emerging evidence suggests that transcription
factors may function in unexpected ways, assembling vertically,
as well as horizontally, on target promoters, and exerting multilayered transcriptional responses. The interplay between the
interacting transcription factors and their associated coactivators may serve to precisely modulate responses to external
stimuli, impacting decisions that control cellular differentiation, oncogenesis, and programmed cell death.
MOL. CELL. BIOL.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
ACKNOWLEDGMENTS
H.A.G. and I.L. contributed equally to this work.
We are grateful to C.-Z. Giam for providing the GST-CREB-His6
expression plasmid, Tom Shenk for providing the Gal4 DBD-p53 expression system, Xinbin Chen for the p53-inducible cell line, J. Wade
Harper for the anti-p21 antibody, and Anne Brauweiler for construction of the CREB deletion mutants.
This work was supported by INSERM (I.L.) and NIH CA80002.
25.
REFERENCES
1. Adler, V., M. R. Pincus, T. Minamoto, S. Y. Fuchs, M. J. Bluth, P. W.
Brandt-Rauf, F. K. Friedman, R. C. Robinson, J. M. Chen, X. W. Wang,
C. C. Harris, and Z. Ronai. 1997. Conformation-dependent phosphorylation
of p53. Proc. Natl. Acad. Sci. USA 94:1686–1691.
2. Akimaru, H., D. X. Hou, and S. Ishii. 1997. Drosophila CBP is required for
dorsal-dependent twist gene expression. Nat. Genet. 17:211–214.
3. Andrisani, O. M. 1999. CREB-mediated transcriptional control. Crit. Rev.
Eukaryot. Gene Expr. 9:19–32.
4. Arias, J., A. S. Alberts, P. Brindle, F. X. Claret, T. Smeal, M. Karin, J.
Feramisco, and M. Montminy. 1994. Activation of cAMP and mitogen responsive genes relies on a common nuclear factor. Nature 370:226–229.
5. Avantaggiati, M. L., V. Ogryzko, K. Gardner, A. Giordano, A. S. Levine, and
K. Kelly. 1997. Recruitment of p300/CBP in p53-dependent signal pathways.
Cell 89:1175–1184.
6. Baker, S. J., S. Markowitz, E. R. Fearon, J. K. Willson, and B. Vogelstein.
1990. Suppression of human colorectal carcinoma cell growth by wild-type
p53. Science 249:912–915.
7. Bannister, A. J., and T. Kouzarides. 1996. The CBP co-activator is a histone
acetyltransferase. Nature 384:641–643.
8. Bonni, A., A. Brunet, A. West, S. R. Datta, M. A. Takasu, and M. E.
Greenberg. 1999. Cell survival promoted by the Ras-MAPK singalling pathway by transcription-dependent and -independent mechanisms. Science 286:
1358–1362.
9. Cereseto, A., F. Diella, J. C. Mulloy, A. Cara, P. Michieli, R. Grassman, G.
Franchini, and M. E. Klotman. 1996. p53 functional impairment and high
p21waf1/cip1 expression in human T-cell lymphotropic/leukemia virus typeI-transformed cells. Blood 88:1551–1560.
10. Chen, X., L. J. Ko, L. Jayaraman, and C. Prives. 1996. p53 levels, functional
domains, and DNA damage determine the extent of the apoptotic response
of tumor cells. Genes Dev. 10:2438–2451.
11. Chrivia, J. C., R. P. S. Kwok, N. Lamb, M. Hagiwara, M. R. Montminy, and
R. H. Goodman. 1993. Phosphorylated CREB binds specifically to the nuclear protein CBP. Nature 365:855–859.
12. Dobner, T., N. Horikoshi, S. Rubenwolf, and T. Shenk. 1996. Blockage by
adenovirus E4orf6 of transcriptional activation by the p53 tumor suppressor.
Science 272:1470–1473.
13. Du, K., and M. Montminy. 1998. CREB is a regulatory target for the protein
kinase Akt/PKB. J. Biol. Chem. 273:32377–32379.
14. Gartenhaus, R. B., and P. Wang. 1995. Functional inactivation of wild-type
28.
26.
27.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
p53 protein correlates with loss of IL-2 dependence in HTLV-I transformed
human T lymphocytes. Leukemia 9:2082–2086.
Giebler, H. A., J. E. Loring, K. Van Orden, M. A. Colgin, J. E. Garrus, K. E.
Escudero, A. Brauweiler, and J. K. Nyborg. 1997. Anchoring of CREB
binding protein to the human T-cell leukemia virus type-1 promoter: a
molecular mechanism of Tax transactivation. Mol. Cell. Biol. 17:5156–5164.
Gu, W., X. L. Shi, and R. G. Roeder. 1997. Synergistic activation of transcription by CBP and p53. Nature 387:819–823.
Harris, N., E. Brill, O. Shohat, M. Prokocimer, D. Wolf, N. Arai, and V.
Rotter. 1986. Molecular basis for heterogeneity of the human p53 protein.
Mol. Cell. Biol. 6:4650–4656.
Harrod, R., Y. Tang, C. Nicot, H. S. Lu, A. Vassilev, Y. Nakatani, and C.-Z.
Giam. 1998. An exposed KID-like domain in human T-cell lymphotropic
virus type 1 Tax is responsible for the recruitment of coactivators CBP/p300.
Mol. Cell. Biol. 18:5052–5061.
Iordanov, M., K. Bender, T. Ade, W. Schmid, C. Sachsenmaier, K. Engel, M.
Gaestel, H. J. Rahmsdorf, and P. Herrlich. 1997. CREB is activated by UVC
through a p38/HOG-1-dependent protein kinase. EMBO J. 16:1009–1022.
Kato, Y., Y. Shi, and X. He. 1999. Neutralization of the Xenopus embryo by
inhibition of p300/CREB-binding protein function. J. Neurosci. 19:9364–
9373.
Kern, S. E., J. A. Pietenpol, S. Thiagalingam, A. Seymour, K. W. Kinzler, and
B. Vogelstein. 1992. Oncogenic forms of p53 inhibit p53-regulated gene
expression. Science 256:827–830.
Ko, L. J., and C. Prives. 1996. p53: puzzle and paradigm. Genes Dev.
10:1054–1072.
Kwok, R. P. S., J. R. Lundblad, J. C. Chrivia, J. P. Richards, H. P. Bächinger, R. G. Brennan, S. G. E. Roberts, M. R. Green, and R. H. Goodman.
1994. Nuclear protein CBP is a coactivator for the transcription factor
CREB. Nature 370:223–226.
Lenzmeier, B. A., H. A. Giebler, and J. K. Nyborg. 1998. Human T-cell
leukemia virus type 1 Tax requires direct access to DNA for recruitment of
CREB binding protein to the viral promoter. Mol. Cell. Biol. 18:721–731.
Levine, A. J., M. E. Perry, A. Chang, A. Silver, D. Dittmer, and D. Welsh.
1994. The 1993 Walter Hubert Lecture: the role of the p53 tumour-suppressor gene in tumorigenesis. Br. J. Cancer 69:409–416.
Lill, N. L., S. R. Grossman, D. Ginsberg, J. DeCaprio, and D. M. Livingston.
1997. Binding and modulation of p53 by p300/CBP coactivators. Nature
387:823–827.
Mestas, S. P., and K. J. Lumb. 1999. Electrostatic contribution of phosphorylation to the stability of the CREB-CBP activator-coactivator complex. Nat.
Struct. Biol. 6:613–614.
Ogryzko, V. V., R. L. Schiltz, V. Russanove, B. H. Howard, and Y. Nakatani.
1996. The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell 87:953–959.
Parker, D., K. Ferreri, T. Nakajima, V. J. LaMorte, R. Evans, S. C. Koerber,
C. Hoeger, and M. R. Montminy. 1996. Phosphorylation of CREB at Ser-133
induces complex formation with CREB-binding protein via a direct mechanism. Mol. Cell. Biol. 16:694–703.
Pise-Masison, C. A., C. Kyeong-Sook, M. Radonovich, J. Dittmer, S.-J. Kim,
and J. N. Brady. 1998. Inhibition of p53 transactivation function by the
human T-cell lymphotropic virus type 1 Tax protein. J. Virol. 72:1165–1170.
Radhakrishnan, I., G. C. Perez-Alvarado, D. Parker, J. H. Dyson, M. R.
Montiminy, and P. Wright. 1997. Solution structure of the KIX domain of
CBP bound to the transactivation domain of CREB: a model for activator:
coactivator interaction. Cell 91:7441–7452.
Scolnick, D. M., N. H. Chehab, E. S. Stavridi, M. C. Lien, L. Caruso, E.
Moran, S. L. Berger, and T. D. Halazonetis. 1997. CREB-binding protein
and p300/CBP-associated factor are transcriptional coactivators of the p53
tumor suppressor protein. Cancer Res. 57:3693–3696.
Shi, Y., and C. A. Mello. 1998. CBP/p300 homolog specifies multiple differentiation pathways in Caenorhabditis elegans. Genes Dev. 12:943–955.
Shikama, N., J. Lyon, and N. B. La Thangue. 1997. The p300/CBP family:
integrating signals with transcription factors and chromatin. Trends Cell
Biol. 7:230–236.
Ubeda, M., M. Vallejo, and J. F. Habener. 1999. CHOP enhancement of
gene transcription by interactions with Jun/Fos AP-1 complex proteins. Mol.
Cell. Biol. 19:7589–7599.
Van Orden, K., H. A. Giebler, I. Lemasson, M. Gonzales, and J. K. Nyborg.
1999. Binding of p53 to the KIX domain of CREB binding protein. A
potential link to human T-cell leukemia virus, type I-associated leukemogenesis. J. Biol. Chem. 274:26321–26328.
Wang, J.-M., J.-R. Chao, W. Chen, M. L. Kuo, J. J.-Y. Yen, and H.-F.
Yang-Yen. 1999. The antiapoptotic gene mcl-1 is up-regulated by the phosphatidylinositol 3-kinase/Akt signaling pathway through a transcription factor complex containing CREB. Mol. Cell. Biol. 19:6195–6206.
Yao, T. P., S. P. Oh, M. Fuchs, N. D. Zhou, L. E. Chn’g, D. Newsome, R. T.
Bronson, E. Li, D. M. Livingston, and R. Eckner. 1998. Gene dosagedependent embryonic development and proliferation defects in mice lacking
the transcriptional integrator p300. Cell 93:361–372.
Zhu, C., F. E. Johansen, and R. Prywes. 1997. Interaction of ATF6 and
serum response factor. Mol. Cell. Biol. 17:4957–4966.