MOLECULAR AND CELLULAR BIOLOGY, Apr. 2004, p. 3404–3414
0270-7306/04/$08.00?0 DOI: 10.1128/MCB.24.8.3404–3414.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 24, No. 8
Acetylation of ?-Catenin by p300 Regulates
Laurence Le ´vy,† Yu Wei, Charlotte Labalette, Yuanfei Wu,
Claire-Ange ´lique Renard, Marie Annick Buendia,
and Christine Neuveut*
Unite ´ d’Oncogene `se et Virologie Mole ´culaire (INSERM U579), Institut
Pasteur, 75015 Paris, France
Received 24 July 2003/Returned for modification 30 December 2003/Accepted 16 January 2004
Lysine acetylation modulates the activities of nonhistone regulatory proteins and plays a critical role in the
regulation of cellular gene transcription. In this study, we showed that the transcriptional coactivator p300
acetylated ?-catenin at lysine 345, located in arm repeat 6, in vitro and in vivo. Acetylation of this residue
increased the affinity of ?-catenin for Tcf4, and the cellular Tcf4-bound pool of ?-catenin was significantly
enriched in acetylated form. We demonstrated that the acetyltransferase activity of p300 was required for
efficient activation of transcription mediated by ?-catenin/Tcf4 and that the cooperation between p300 and
?-catenin was severely reduced by the K345R mutation, implying that acetylation of ?-catenin plays a part in
the coactivation of ?-catenin by p300. Interestingly, acetylation of ?-catenin had opposite, negative effects on
the binding of ?-catenin to the androgen receptor. Our data suggest that acetylation of ?-catenin in the arm
6 domain regulates ?-catenin transcriptional activity by differentially modulating its affinity for Tcf4 and the
androgen receptor. Thus, our results describe a new mechanism by which p300 might regulate ?-catenin
?-Catenin was originally described as a component of cell-
cell adhesion complexes, where it binds to E-cadherin. More
recently, ?-catenin was shown to be a key effector of the Wnt
signaling pathway, which plays a pivotal role in growth and cell
fate at early and late developmental stages (reviewed in refer-
ences 37, 38, and 49). In the absence of Wnt signals, the
cytosolic pool of ?-catenin is maintained at a low level by
targeted degradation in a multiprotein complex including the
suppressor adenomatous polyposis coli (APC), Axin, glycogen
synthase kinase 3, and casein kinase I ? (16, 30, 41, 52, 53).
Wnt activation abrogates the degradation of ?-catenin and
induces its accumulation and translocation into the nucleus,
where it binds one of the four members of the T-cell factor/
lymphoid enhancer factor (Tcf/Lef) family and activates tran-
scription of target genes (4, 23). Growing evidence has associ-
ated Wnt signaling with tumor development. Constitutive Wnt
signaling in cancer cells results mainly from genetic defects in
the N-terminal region of the ?-catenin gene itself or in the
APC or Axin gene, which induce in all cases the stabilization
and nuclear translocation of ?-catenin (reviewed in reference
Although it is well established that the formation of nuclear
?-catenin/Tcf complexes plays a pivotal role in the activation of
Wnt target genes, the fine mechanisms of transcriptional acti-
vation and regulation are still under investigation (5, 17). In
the absence of ?-catenin, the Tcf/Lef transcription factors act
as transcriptional repressors by recruiting proteins such as
Groucho/TLE, CtBP, and histone deacetylase (6–9, 28, 40).
Upon Wnt activation, the binding of ?-catenin to Tcf generates
a bipartite transcription factor, in which Tcf provides the DNA
binding domain and the C terminus of ?-catenin provides the
transactivation domain, therefore inducing a transcriptional
switch. Recent physical and biochemical studies of the ?-cate-
nin–Tcf interaction have provided detailed information on the
mode of ?-catenin recognition by Tcf. Binding regions have
been mapped to the N-terminal domain of Tcf/Lef and arma-
dillo (arm) repeats 3 to 8 of ?-catenin, with critical hot spots
within repeat 8 (46). The crystal structure of ?-catenin/Tcf
complexes further revealed that the core arm repeat domain of
?-catenin forms a superhelix of helices, providing a long, pos-
itively charged groove that engages the negatively charged
?-catenin binding domain of Tcf (13, 14, 39). These studies
outlined the importance of two critical lysine residues of
?-catenin, K312 and K435, called the charged buttons, located
in arm repeats 5 and 8.
Different aspects of the regulation of Tcf-dependent tran-
scription by ?-catenin have been unraveled. ?-Catenin might
recruit the basal transcription machinery via its interaction
with the TATA-binding protein and Pontin 52 (TIP 49) (3, 18).
?-Catenin has also been shown to interact with cellular factors
essential for its transcriptional activity, such as pygopus and
Lgs/BCl9, or with proteins involved in histone modification
and chromatin remodeling, such as CBP/p300 and Brahma/
Brg-1 (2, 20, 25, 33, 36, 43, 44). A crucial role for CBP/p300 in
?-catenin/Tcf activity has been demonstrated during Xenopus
embryogenesis and ?-catenin-associated transformation (43,
The mechanism by which CBP/p300 stimulate transcription
* Corresponding author. Mailing address: Unite ´ d’Oncogene `se et
Virologie Mole ´culaire (INSERM U579), De ´partement de Virologie,
Institut Pasteur, 28 Rue du Dr. Roux, 75724 Paris Cedex 15, France.
Phone: 33-145 68 88 51. Fax: 33-145 68 89 43. E-mail: cneuveut
† Present address: Laboratory of Developmental Signalling, Cancer
Research United Kingdom London Research Institute, London
WC2A 3PX, United Kingdom.
is likely multifactorial (reviewed in references 12 and 27). CBP/
p300 can contribute to the formation of a multiprotein activa-
tion complex bridging various factors to the general transcrip-
tion machinery. In addition, CBP/p300 possess intrinsic histone
acetyltransferase (HAT) activity, and histone acetylation reg-
ulates promoter activity by relieving chromatin-dependent re-
pression. More recently, CBP/p300 have been shown to acet-
ylate a growing number of nonhistone proteins, notably
transcription factors such as p53, E2F, HMG I(Y), HNF-4, and
human immunodeficiency virus Tat (15, 22, 31, 34, 42). Acet-
ylation of these factors may affect different biological func-
tions, including DNA binding affinity, transcriptional activity,
stability, and subcellular localization. Recent studies have re-
ported acetylation of ?-catenin at lysine 49 and acetylation of
the Caenorhabditis elegans Tcf/Lef homolog POP-1 at three
neighboring residues (K185, 187, and 188), suggesting that
acetylation might also be involved in the transcriptional activity
of ?-catenin–Tcf complexes (11, 50).
In this study, we showed that ?-catenin is acetylated in vivo
and in vitro by p300. We found that lysine K345, located in arm
repeat 6, is an acetyl acceptor and that acetylation of K345
increases the affinity of ?-catenin for Tcf4. We further dem-
onstrated that acetylation of K345 specifically enhanced the
coactivator function of ?-catenin in a Tcf-dependent manner.
Our data thus identify a new mechanism implicated in the
regulation of ?-catenin transcriptional activity and in the acti-
vation of Wnt-responsive genes.
MATERIALS AND METHODS
Cell culture and transfection. HeLa, 293, and SW480 cells were maintained in
Dulbecco’s modified Eagle’s medium with 10% fetal calf serum. Cells were
transiently transfected with ?-catenin, p300, and Tcf4 vectors as indicated in the
figure legends with the Lipofectamine reagent (Invitrogen). Total amounts of
transfected DNA were kept constant by addition of empty pcDNA3 vector. All
transfection experiments were repeated at least three times in duplicate. For
luciferase assays, cells were lysed and assayed for luciferase activity 48 h post-
transfection. Because p300 was found to activate transcription of the thymidine
kinase–?-galactosidase plasmid used to normalize luciferase activity for trans-
fection efficiency, it could not be used for normalization; the results were con-
firmed by multiple independent assays.
Antibodies. Monoclonal antibodies against ?-catenin were purchased from
Transduction Laboratories. Antihemagglutinin (anti-HA), anti-Axin, and anti-
APC antibodies were from Babco, Santa Cruz, and Oncogene Research Prod-
ucts, respectively. Monoclonal anti-acetyl-lysine antibodies were from Cell Sig-
naling Technology and Upstate Biotechnology.
Plasmids. The ?-catenin expression vector pCMV-T41A?-catenin, carrying a
Myc-tagged dominant stable ?-catenin mutated at residue 41 (threonine to
alanine), has been described previously (47). Mutations of lysine residue K345
and/or K49 to R or A were generated in this construct with the QuickChange XL
site-directed mutagenesis kit (Stratagene) and verified by sequencing. Glutathi-
one S-transferase (GST)-arm 1-12 and deletion mutants were generated by
cloning PCR-amplified fragments of the ?-catenin armadillo domain in pGEX-
5X-1 (Amersham Biosciences). GST-arm 1-12 K354R and GST-arm 1-12 K345R
were generated by site-directed mutagenesis (Stratagene). The HA-Tcf 4 con-
struct and reporter plasmid pTOP-FLASH were kindly provided by H. Clevers
and have been described previously (23). The androgen receptor (AR) expres-
sion vector was provided by G. Castona, and expression vectors for wild-type
p300 and the p300 ?HAT mutant deleted of amino acids 1413 to 1721 were
provided by Y. Nakatami. GST p300 HAT (a gift of S. Emiliani) was generated
by cloning a PCR-amplified fragment of p300 (positions 1195 to 1810) into the
Recombinant proteins and in vitro binding assays. GST-p300 HAT and GST-
arm proteins were expressed in Escherichia coli BL21 and purified by glutathi-
one-Sepharose affinity (Sigma) according to standard protocols. Tcf4 and AR
proteins were in vitro translated in the presence of [35S]methionine with the
TNT-coupled reticulocyte lysate system (Promega). For the in vitro binding assay
and competition experiments,35S-labeled Tcf4 or AR was mixed with GST or
GST-arm 1-12 bound to Sepharose beads, in the absence or presence of com-
petitor peptide (337LWTTSRVLKVLSVCSSN353), acetylated (Ac-arm 6 pep-
tide) or not (arm 6 peptide) at K345 for 2 h at 4°C in binding buffer (20 mM
HEPES [pH 7.9], 200 mM NaCl, 1 mM MgCl2, 0.5% NP-40, 1 mM dithiothreitol,
and 1 mg of bovine serum albumin/ml). The beads were then washed six times
with binding buffer, and bound proteins were analyzed by sodium dodecyl sul-
fate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography.
Acetyltransferase assays. For in vivo acetylation analysis, 293 cells were
washed in phosphate-buffered saline at 24 h posttransfection, and incubated in
methionine- and cysteine-free Dulbecco’s modified Eagle’s medium supple-
mented with 2% fetal bovine serum for 1 h at 37°C. Cells were then metabolically
labeled with [3H]sodium acetate at 1 mCi/ml for 1 h at 37°C. Whole-cell extracts
were prepared in lysis buffer (300 mM NaCl, 50 mM Tris [pH 7.5], 0.5% Triton
X-100, 1 mM dithiothreitol) and protease inhibitor cocktail (Life Technologies),
immunoprecipitated with monoclonal ?-catenin antibody, and analyzed on
SDS–8% PAGE. Gels were fixed in 30% methanol and 10% acetic acid, soaked
in Amplify (Amersham), dried, and exposed to X-ray film.
In vitro acetylation assays were performed with a GST fusion protein contain-
ing the catalytic domain of p300 or with PCAF or p300 proteins produced and
purified from a baculovirus overexpression system (35); 1 ?g of GST-arm protein
was incubated with PCAF or GST-p300 at 30°C for 1 h in 20 ?l of reaction buffer
(50 mM Tris-HCl [pH 8.0], 1 mM dithiothreitol, 0.1 mM EDTA, 50 mM KCl, 5%
glycerol, 10 ?M sodium butyrate, protease inhibitor cocktail) and [14C]acetyl-
coenzyme A (1 ?Ci/reaction; Amersham). Proteins were resolved on SDS-
PAGE, and gels were treated as described above.
FIG. 1. ?-Catenin is acetylated in vivo. (A) 293 cells were transfected with T41A-?-catenin (5 ?g) either alone or together with 5 ?g of p300
or the empty vector. After metabolic labeling with3H-labeled sodium acetate, proteins were immunoprecipitated (IP) with ?-catenin antibodies
and resolved on SDS-PAGE. The gel was then exposed to X-ray film (top panel). Immunoprecipitated proteins were analyzed by Western blotting
(WB) with anti-?-catenin antibodies (bottom panel). (B) Endogenous ?-catenin was immunoprecipitated from 293 or SW480 cells with anti-?-
catenin antibodies. Control immunoprecipitation was performed with an anti-Flag antibody. The samples were resolved by SDS-PAGE, and
acetylation was assessed with anti-acetyl-lysine antibodies (upper panel). The amount of ?-catenin in the immunoprecipitates was determined with
anti-?-catenin antibodies (bottom panel).
VOL. 24, 2004 ACETYLATION OF ?-CATENIN3405
FIG. 2. ?-Catenin is acetylated at lysine residue K345 by p300. (A) Arm domain 6 of ?-catenin is acetylated in vitro by p300. (Left panel)
Schematic representation of GST-arm repeat constructs used in the in vitro acetylation assay. GST–full-length armadillo construct (GST-arm 1-12)
or serial deletion constructs were incubated with GST-p300 recombinant protein and [14C]acetyl-coenzyme A. Reaction products were separated
on SDS–10% PAGE. The gel was Coomassie blue-stained (bottom right panel) and then dried and exposed to X-ray film (upper right panel).
(B) K345 is an acetyl acceptor in vitro. The amino acid sequence of the arm 6 domain contains two lysines (upper panel). GST-arm 1-12 (wt),
GST-arm 1-12 K345R, or GST-arm 1-12 K354R was used for in vitro acetylation assays with GST-p300 recombinant protein. Reaction products
were analyzed by SDS-PAGE and autoradiography. (C) In vitro acetylation assays with PCAF recombinant protein revealed acetylated PCAF and
histone H3 but no acetylated form of ?-catenin. (D) Lysine 345 is acetylated in vivo. HeLa cells were transfected with T41A-?-catenin or
T41A-?-catenin K345R. Proteins were immunoprecipitated with ?-catenin antibodies and resolved on SDS-PAGE. Blots were hybridized either
with anti-acetyl-lysine antibodies or with anti-?-catenin antibodies. WB, Western blotting.
3406LE´VY ET AL.MOL. CELL. BIOL.
Immunoprecipitation and Western blotting. Cells were lysed either in buffer A
(150 mM NaCl, 50 mM Tris-HCl [pH 7.4], 0.5% [vol/vol] Nonidet P-40, and
protease inhibitor cocktail) or in buffer B (300 mM NaCl, 50 mM Tris [pH 7.5],
0.5% Triton X-100, and protease inhibitor cocktail) as indicated and quickly
frozen on dry ice. Lysates were thawed, centrifuged at 14,000 rpm for 10 min, and
cleared by incubation with 30 ml of a protein A-protein G mixture. Antibodies
were incubated with 50 ?l of 50% (vol/vol) protein A-protein G-Sepharose for
2 h and added to the lysates. After overnight incubation, the beads were exten-
sively washed with the same buffer, and bound proteins were resolved by SDS-
For Western blot analysis, proteins were transferred to a nitrocellulose mem-
brane and probed with the indicated antibody for 1 h. Reactive proteins were
developed with secondary antibodies conjugated to alkaline phosphatase and
visualized with chemiluminescence according to the manufacturer’s protocol
(Tropix). For quantitative measurements, gels were analyzed with a video acqui-
sition system (Intelligent Dark Box II; Fuji) and Image Gauge software (Fuji).
EMSA. For the electrophoretic mobility shift assay (EMSA), in vitro-trans-
lated Tcf 4 was incubated with GST-arm 1-12 in binding buffer (20 mM HEPES,
pH 7.9, 75 mM NaCl, 1 mM dithiothreitol, 2 mM MgCl2, 10% glycerol) with 1
?g of poly(dI · dC) for 10 min at room temperature. Then [?32P]dCTP-labeled
Tcf probe (105cpm) was added, and the mixture was incubated for an additional
20 min at room temperature. The DNA probes used were complementary pairs
of synthetic oligonucleotides with overhangs containing the consensus Tcf site
(Tcf1, 5?-AGCTGGTAAGATCAAAGGG-3?, and Tcf2, 5?-TGCCGCCCTTTG
ATCTTACC-3?; the Tcf site is italic). For competition assays, increasing amounts
of peptide were incubated for 5 min at room temperature with in vitro-translated
Tcf4 prior to addition of arm 1-12. DNA-protein complexes were separated on
a 4% acrylamide gels. Gels were dried and either exposed to X-ray films or
visualized and quantified with a PhosphorImager (Storm Imaging System; Mo-
lecular Dynamics) as indicated in the figure legends.
?-Catenin is acetylated by p300 on lysine 345. It has been
shown that the CBP/p300 acetyltransferases can bind ?-catenin
and activate Tcf-dependent gene expression (20, 33, 43, 44).
Because a growing number of nonhistone regulatory proteins
are covalently modified by acetylation (reviewed in references
1 and 24), we investigated whether ?-catenin is acetylated in
vivo. To this end, we used either 293 cells transfected with a
FIG. 3. K345 of ?-catenin is important for ?-catenin/Tcf4 binding. (A) HeLa cells were cotransfected with either T41A-?-catenin, T41A-?-
catenin K345R, or T41A-?-catenin K345A (5 ?g) and with Tcf4-HA expression vector (2.5 ?g). Proteins were extracted in lysis buffer A. Following
coimmunoprecipitation (IP) with anti-?-catenin antibodies, proteins were resolved on SDS-PAGE, and Tcf4 was detected by Western blotting
(WB) with anti-HA antibodies. The amounts of ?-catenin in precipitates and Tcf4 in total cell lysates were shown by Western blotting with
anti-?-catenin and anti-HA antibodies. (B) HeLa cells were cotransfected with the TOPFLASH reporter (0.1 ?g) and T41A-?-catenin or the
indicated K345 mutant (0.2 ?g) or the empty vector. Luciferase activities were assayed 48 h after transfection. The basal activity of the TOPFLASH
plasmid cotransfected with empty vector was arbitrarily set at 1, and other values were calculated accordingly. The data shown are the averages
of three separate experiments done in duplicate. (Bottom) Expression levels of the different constructs determined by Western blotting with
anti-?-catenin antibodies. (C) HeLa cells were transfected with T41A-?-catenin or the corresponding K345R or K345A mutant. Cellular extracts
were prepared in buffer B, and coimmunoprecipitation assays were performed with anti-?-catenin monoclonal antibodies. Immunoprecipitates
were resolved on SDS-PAGE, and blots were hybridized with either APC (left) or Axin (right panel) antibodies. Immunoprecipitated ?-catenin
levels were shown by Western blotting with anti-?-catenin antibodies.
VOL. 24, 2004 ACETYLATION OF ?-CATENIN 3407
stable dominant form of ?-catenin (T41A-?-catenin) or
SW480 cells in which wild-type ?-catenin is constitutively ac-
tive owing to defective APC. 293 cells were transfected with
T41A-?-catenin alone or in combination with p300, and met-
abolically labeled with3H-labeled sodium acetate. After im-
munoprecipitation of ?-catenin with a specific monoclonal an-
tibody, proteins were separated on SDS-polyacrylamide gels.
Acetylation of T41A-?-catenin was detected by the incorpora-
tion of radioactive acetate, and overexpression of p300 in-
creased the amount of the acetylated form but not the total
amount of ?-catenin protein (Fig. 1A, lanes 3 to 4). Although
endogenous ?-catenin was efficiently immunoprecipitated in
nontransfected 293 cells, the acetylated form was almost un-
detectable after autoradiography (Fig. 1A, lanes 1 to 2). By
contrast, in SW480 colon carcinoma cells, immunoprecipita-
tion of endogenous wild-type ?-catenin followed by Western
blot analysis with anti-acetyl-lysine antibodies confirmed that
endogenous ?-catenin was acetylated (Fig. 1B). Moreover, a
faint signal corresponding to endogenous acetylated ?-catenin
was also detected in 293 cells with anti-acetyl-lysine antibodies
(Fig. 1B), suggesting that anti-acetyl-lysine Western blotting
might be more sensitive for detecting ?-catenin acetylation
than in vivo labeling.
We next sought to determine which individual lysine resi-
dues of ?-catenin were acetylated. For this study, we focused
on ?-catenin domains involved in its transcriptional activity,
particularly on the armadillo repeat domain, composed of 12
copies of a 42-amino-acid sequence (arm repeat), which is
involved in the interaction of ?-catenin with Tcf/Lef. Bacteri-
ally expressed GST-arm 1-12, carrying the full-length armadillo
domain, and serial deletion constructs were incubated with
purified recombinant p300 HAT in the presence of [14C]acetyl-
coenzyme A, and acetylated proteins were detected by auto-
radiography. Our results show that the armadillo domain of
?-catenin was acetylated in vitro by p300 and that acetylation
was strongly reduced upon deletion of the arm 6 repeat (Fig.
2A), suggesting that acetylation sites may reside within this
The arm 6 repeat contains two lysine residues (Fig. 2B), and
mutant arm 1-12 constructs in which K345 or K354 was
changed to arginine were subjected to in vitro acetylation as-
says. Substitution of K345 but not K354 to arginine eliminated
acetylation of arm 1-12 by p300 (Fig. 2B), demonstrating that
K345 is the main site of acetylation by p300 within the arm
domain. Coomassie staining allowed us to verify that similar
amounts of GST fusion proteins were used in the acetylation
reactions (Fig. 2B, bottom panel). In contrast, recombinant
CBP/p300-associated factor (PCAF) failed to acetylate GST-
arm 1-12 in vitro, although it was able to acetylate histone H3
(Fig. 2C), showing that acetylation of ?-catenin is achieved
specifically by p300.
To confirm that K345 is an acetyl acceptor in vivo, HeLa
cells were transfected with either the T41A-?-catenin or T41A-
?-catenin K345R expression vector. ?-Catenin proteins were
immunoprecipitated with specific antibodies, and acetylated
?-catenin was detected with anti-acetyl-lysine antibodies. Fig-
ure 2D shows that acetylation of the K345R ?-catenin mutant
was markedly decreased.
FIG. 4. Acetylation of K345 increases the affinity of ?-catenin for Tcf4. (A) Recombinant GST-arm 1-12 or GST-arm 1-12 K345R or histone
H3 was incubated with recombinant p300 protein in the presence or absence of [14C]acetyl-coenzyme A. Acetylated proteins were analyzed by
SDS-PAGE and autoradiography. Coomassie blue staining confirmed that equivalent amounts of proteins were loaded. (B) For EMSA, recom-
binant proteins were eluted from the beads, and identical amounts of proteins were incubated with in vitro-translated Tcf4 and32P-labeled Tcf
probe. Reactions were resolved on 4% acrylamide gels (left panel). The relative intensities of ?-catenin/Tcf4/DNA complexes were quantified with
a PhosphorImager and are graphically depicted in the right-hand panel.
3408 LE´VY ET AL.MOL. CELL. BIOL.
Identification of K345 as a residue important for ?-catenin/
Tcf4 interaction. Having identified K345 as an acetyl acceptor,
we sought to determine the role of K345 in ?-catenin function.
Notably, arm 6 is included in the domain involved in ?-catenin
interaction with Tcf/Lef (46), and this interaction is a critical
step for targeting ?-catenin to specific promoters and recruit-
ment of transcriptional coactivators. We therefore assessed
whether mutation of K345 would impair the ?-catenin–Tcf4
interaction. Two ?-catenin mutants in which K345 was
changed to arginine (T41A-?-catenin K345R) or alanine
(T41A-?-catenin K345A) were transfected into HeLa cells,
either alone or together with HA-Tcf4. Cell lysates were im-
munoprecipitated with ?-catenin antibodies, followed by im-
munoblotting analysis with anti-HA antibodies. Figure 3A
shows that mutation of K345 to alanine abolished Tcf4 binding,
whereas the K345R mutant was still able to bind Tcf4. The loss
of binding of the K345A mutant ?-catenin to Tcf4 was con-
firmed by immunoprecipitating the cell lysates with an anti-HA
antibody followed by Western blot analysis with anti-?-catenin
antibodies (data not shown).
Transfection experiments with the TOPFLASH luciferase
reporter containing Tcf/Lef consensus binding sites showed
that the K345R mutant activated transcription as efficiently as
wild-type ?-catenin, while the K345A mutant, which is defec-
tive for Tcf4 binding, was less efficient (Fig. 3B). In addition,
immunofluorescence studies showed normal nuclear localiza-
tion of K345A mutant ?-catenin, indicating that impaired bind-
ing to Tcf4 and transcriptional activity were not due to a defect
in nuclear accumulation (data not shown). Collectively, these
results indicate that K345 is involved in the binding of ?-cate-
nin to Tcf4.
Since ?-catenin arm repeats 3 to 8 have been found to be
involved in the interaction with other cellular partners such as
APC and Axin, we investigated whether mutation of K345
would impair these interactions. HeLa cells were transfected
with T41A-?-catenin or T41-?-catenin K345A or K345R, and
cell lysates were immunoprecipitated with ?-catenin antibodies
and analyzed by Western blotting with anti-APC or anti-Axin
antibodies. Both wild-type and mutant ?-catenins were still
able to interact with endogenous APC (Fig. 3C, left panel) and
Axin (Fig. 3C, right panel). These results are in agreement with
previous data showing that K345A mutant ?-catenin was effi-
ciently degraded by ectopic expression of conductin or APC
Acetylation of K345 increases the affinity of ?-catenin for
Tcf4. The finding that K345 is important for ?-catenin–Tcf4
binding prompted us to examine the functional effect of acet-
ylation on the ?-catenin–Tcf4 interaction by EMSA. GST-arm
1-12 or GST-arm 1-12 K345R was incubated in acetylation
buffer with recombinant p300 protein in the presence or ab-
sence of [14C]acetyl-coenzyme A. Recombinant proteins were
either analyzed by SDS-PAGE and autoradiography (Fig. 4A)
or incubated with in vitro-translated Tcf4 protein and
labeled synthetic double-stranded DNA containing the consen-
FIG. 5. Acetylated arm 6 peptide competes for ?-catenin/Tcf binding more efficiently than its nonacetylated counterpart. Acetylated or
nonacetylated arm 6 peptide at increasing concentrations was incubated with in vitro-translated Tcf4. GST-arm 1-12 and32P-labeled Tcf probe
were added to the reaction for EMSA analysis, followed by autoradiography. The relative intensities of the ?-catenin/Tcf4/DNA complex in the
corresponding lanes were quantified by the Syngene Photo Image System (Syngene, Cambridge, United Kingdom) and are shown in bar graphs
in the bottom panel.
VOL. 24, 2004ACETYLATION OF ?-CATENIN3409
sus Tcf binding motif. EMSA analysis showed that acetylation
of arm 1-12 increased its affinity for Tcf4 in a dose-dependent
manner (Fig. 4B, compare lanes 3 to 5 with lanes 6 to 8). As
previously observed in coimmunoprecipitation assays, arm
1-12 K345R was still able to bind Tcf4, but the binding was not
increased when arm 1-12 K345R was incubated with active
p300 protein (Fig. 4B, lanes 9 to 14). Moreover, competition
experiments with increasing concentrations of a synthetic pep-
tide containing acetylated K345 (ac-arm 6 peptide) or the
corresponding nonacetylated peptide (arm 6 peptide) showed
that the ac-arm 6 peptide was more effective than its nonace-
tylated counterpart in competing with the binding of arm 1-12
with Tcf4 (Fig. 5). Taken together, our results demonstrate
that acetylation of ?-catenin at K345 increases its affinity for
Acetylated ?-catenin is preferentially associated with Tcf4
in vivo. To determine whether acetylation of ?-catenin at K345
increases its affinity for Tcf4 in vivo, we investigated whether
the pool of acetylated ?-catenin was preferentially associated
with Tcf4 compared with nonacetylated ?-catenin. 293 cells
were transfected with an HA-Tcf4 expression vector or the
empty vector, and immunoprecipitation was first carried out
with an anti-HA antibody. Bound ?-catenin was then eluted
from the beads in 1% SDS. In a second step, both the eluate
and the supernatant obtained from the first immunoprecipita-
tion were immunoprecipitated with ?-catenin-specific antibod-
ies. Western blot analysis with anti-acetyl-lysine antibodies or
anti-?-catenin antibodies and quantification with a video ac-
quisition system (Fuji) showed that the ratio of acetylated
?-catenin to total ?-catenin was increased by 10-fold in the
Tcf4-bound fraction compared to the results with the unbound
fraction (Fig. 6). These data clearly indicate that acetylated
?-catenin is preferentially found in Tcf-containing complexes
in vivo and lend support to the notion that coactivation of
?-catenin/Tcf complexes by p300 is mediated in part by the
acetylation of ?-catenin.
HAT activity of p300 stimulates ?-catenin transactivation
potential. Since acetylation was found to increase the affinity of
?-catenin for Tcf4, we next investigated whether acetylation of
?-catenin by p300 regulates its transcriptional activity. 293 cells
were transfected with the TOPFLASH luciferase reporter
alone or with HA-Tcf4, T41A-?-catenin, T41A-?-catenin
K345R, p300, or p300 ?HAT, alone or in combination. As
reported previously (20, 43, 44), p300 significantly enhanced
?-catenin transcriptional activity (10-fold increase over that
with ?-catenin/Tcf4) (Fig. 7A). The acetyltransferase activity
of p300 contributed to this activation because cotransfection
with p300 ?HAT had little effect on ?-catenin/Tcf4 activity.
Moreover, the T41A-?-catenin K345R mutant was less effi-
ciently coactivated by p300 (fivefold increase in T41A-?-cate-
nin K345R activity) and p300 ?HAT could no longer activate
this mutant. To rule out a nonspecific effect of p300, we
checked the expression levels of ?-catenin and Tcf4 in the
presence of p300 and observed similar levels (Fig. 7A, bottom
panels). We also verified that p300 and the p300 ?HAT mutant
were expressed at comparable levels. These results suggest that
coactivation of ?-catenin/Tcf by p300 is mediated, at least in
part, by acetylation of the ?-catenin K345 residue.
?-Catenin has been shown to be acetylated at K49 by p300
(50). It was therefore of interest to determine whether acety-
lation of K49 could also be involved in the cooperation be-
tween p300 and ?-catenin. To this end, we cotransfected
?-catenin mutants containing either a single mutation at K49
or K345 or the double mutation into 293 cells and tested
whether the transcriptional activity of these mutants was af-
fected by p300 in a TOPFLASH reporter assay. Our results
show that while the K49R mutant was fully activated by p300
(13.3-fold), activation of the double mutant K49/345R by p300
was significantly reduced (5.9-fold) (Fig. 7B). We verified by
Western blotting that all the ?-catenin mutants were expressed
at similar levels (Fig. 7B, lower panel). Taken together, our
data suggest that acetylation of K345 by p300 contributes to a
major extent to the increase in ?-catenin transcriptional activ-
ity by p300. Our results are in agreement with a previous report
showing that mutation of ?-catenin at K49 does not impair
TOPFLASH transactivation or modulate ?-catenin’s interac-
tion with Tcf (50).
Acetylation of ?-catenin K345 does not affect the affinity of
?-catenin for the AR. It has been shown recently that ?-catenin
can interact with the AR and activate transcription in a ligand-
FIG. 6. Acetylated ?-catenin associates preferentially with Tcf4.
293 cells were transfected with 5 ?g of HA-Tcf4 or empty vector.
Proteins were extracted in lysis buffer B, and in a first step, Tcf4 and
bound proteins were coimmunoprecipitated with anti-HA antibodies.
Both the immunoprecipitated (IP) fraction and the supernatant were
recovered and used for a second round of immunoprecipitation with
anti-?-catenin antibodies. Proteins were resolved on SDS-PAGE and
analyzed by Western blotting (WB) with anti-acetyl-lysine antibodies
(lanes 1 to 4) or anti-?-catenin antibodies (lanes 5 to 8). The ratios of
acetylated ?-catenin to total ?-catenin were measured with Image
Gauge software (Fuji) and are presented in graphic form. Tcf4 levels
in the immunoprecipitates were determined by anti-HA Western blot-
ting (lanes 9 and 10). Analysis of cell lysates by Western blotting with
antiactin antibodies confirmed that equivalent amounts of proteins
were used for immunoprecipitation assays (bottom).
3410 LE´VY ET AL.MOL. CELL. BIOL.
dependent fashion (45). Arm repeat 6 of ?-catenin was found
to be involved in binding with the AR, and the AR was shown
to compete with Tcf for ?-catenin binding (10, 51). Therefore,
we wondered if acetylation of K345 would also increase the
binding of ?-catenin to the AR. To this end, we performed
competition experiments. GST-arm 1-12 was immobilized on
agarose beads and incubated with either in vitro-translated
Tcf4 (Fig. 8, upper panels) or in vitro-translated AR (Fig. 8,
lower panels) in the absence or in the presence of increasing
concentrations of nonacetylated or acetylated arm 6 peptide.
Consistent with the EMSA results (see Fig. 5), the acetylated
arm 6 peptide was a better competitor of ?-catenin/Tcf4 bind-
ing than the nonacetylated peptide. By contrast, the nonacety-
lated peptide seemed more efficient in competing the ?-cate-
nin and AR binding than the acetylated peptide. Thus,
acetylation of K345 might specifically increase the affinity of
?-catenin for Tcf4, while acetylation seems to decrease the
affinity of ?-catenin for the AR.
Activation of Wnt target genes by ?-catenin/Tcf is tightly
controlled by a complex network of regulatory events, includ-
ing interactions with various cellular partners and different
covalent modifications of ?-catenin and Tcf4 (17). Notably, the
role of CBP and p300 as coactivators of ?-catenin is well
established, and their recruitment to Tcf-dependent promoters
plays a crucial role during development and cell transforma-
tion (20, 43). In this study, we have shown that ?-catenin is
acetylated by p300 in vivo and in vitro and identified ?-catenin
residue K345 as a target site for acetylation. Substitution of
K345 greatly reduced but did not totally eliminate the acety-
lation signal in vivo. These results are consistent with a recent
report showing that CBP acetylates residue K49, located in the
N-terminal domain of ?-catenin (50). In both studies, PCAF
was unable to acetylate ?-catenin, indicating that ?-catenin
acetylation is specifically achieved by CBP/p300. Whether acet-
ylation modulates ?-catenin functions is therefore an impor-
K345 is located in arm repeat 6, a region implicated in
?-catenin interaction with a variety of cellular partners. Our
mutagenesis studies indicate that K345 is not significantly in-
volved in ?-catenin interaction with APC or Axin 1, but it was
found to be critical for Tcf4 binding. Interestingly, mutation to
alanine abolished the interaction with Tcf4 but mutation to
arginine had no effect, probably because the positively charged
arginine can maintain the interaction with the negatively
charged residues of Tcf4. This interpretation is supported by a
recent structural analysis of the ?-catenin/Tcf complex, sug-
gesting that charged residues around ?-catenin K312, includ-
ing K345, might interact with the negatively charged region of
Tcf4 extending from E23 to E29 and facilitate the initial an-
FIG. 7. Cooperation between p300 and ?-catenin is severely re-
duced by K345R mutation. (A) 293 cells were cotransfected with 0.1
?g of TOPFLASH reporter plasmid and either empty pCDNA3 vector
or expression vectors for HA-Tcf4 (0.1 ?g), T41A-?-catenin (0.25 ?g),
T41A-?-catenin K345R (0.25 mg), p300 (1.5 ?g), and/or p300 ?HAT
(1.5 ?g) as indicated. Luciferase activities were determined 48 h post-
transfection. The basal TOPFLASH activity was set at 1, and data
shown are the averages of three independent experiments performed
in duplicate. WB, Western blotting. (Bottom panel) The expression
levels of ?-catenin, Tcf4, p300, and p300 ?HAT were revealed by
Western blotting with anti-?-catenin, anti-HA, and anti-Flag antibod-
ies, respectively. (B) 293 cells were transfected with TOPFLASH and
HA-Tcf4 (0.1 ?g) and with either empty pCDNA3 vector or expression
vectors for T41A-?-catenin or the indicated lysine mutants (0.25 ?g),
in combination or not with p300 or p300 ?HAT (1.5 ?g) as indicated.
For each ?-catenin mutant, TOPFLASH activity in cells cotransfected
with Tcf4 and empty vector was arbitrarily set at 1. Data shown rep-
resent the activation by p300 or p300 ?HAT for each ?-catenin mu-
tant. Values are the averages of three independent experiments per-
formed in duplicate. The expression levels of the different ?-catenin
constructs in the presence of the coactivator are shown in the insets
VOL. 24, 2004 ACETYLATION OF ?-CATENIN3411
chorage of Tcf4 to ?-catenin (13). The finding that the ?-cate-
nin mutant K345R was still able to bind Tcf4 suggests that
acetylation does not serve merely to neutralize a positive
charge but may create a novel interface facilitating the binding
of ?-catenin to Tcf4. Similarly, acetylation has been shown to
increase the DNA binding potential of E2F1, although argi-
nine mutants retained the ability to bind DNA (31). The role
of K345 in the ?-catenin–Tcf4 interaction was further con-
firmed by the finding that the capacity of the K345A and
K345R mutants to bind Tcf4 was strictly correlated with their
ability to transactivate the TOPFLASH reporter (Fig. 3B).
Reduced transactivation efficiency has also been demonstrated
previously for a series of ?-catenin mutants defective in Lef-1
binding (46). This effect was not apparently related to abnor-
mal subcellular localization of these ?-catenin mutants, since
exogenously overexpressed mutants accumulated in the nu-
cleus at the same rate as ?-catenin (data not shown). It is also
unlikely that substitution of lysine 345 might affect the stability
of ?-catenin, since it does not impair ?-catenin interaction with
APC and Axin 1, two major partners within the degradation
An important aspect of our results is that K345 acetylation
increases the affinity of ?-catenin for Tcf4. This conclusion is
supported by several lines of evidence (see Fig. 4 to 8). (i) In
the EMSA, p300 increased the binding of the wild-type ?-cate-
nin arm domain (arm 1-12) to Tcf4 but had no effect on the
corresponding K345R mutant. (ii) In competition assays, a
peptide containing acetylated K345 (ac-arm 6 peptide) dis-
turbed this interaction in a dose-dependent manner, more ef-
ficiently than the homologous nonacetylated peptide, and (iii)
comparable results were also obtained in GST pull-down ex-
periments. (iv) In coimmunoprecipitation assays, the acety-
lated fraction of endogenous ?-catenin was found to associate
preferentially with Tcf4. Moreover, the increased affinity of
acetylated ?-catenin for Tcf4 affected the transcriptional activ-
ity of the complex, since cooperation between p300 and ?-cate-
nin was significantly reduced upon alteration of lysine 345 of
?-catenin to arginine, although this mutant was still able to
bind Tcf4. However, the transactivating activity of ?-catenin/
Tcf complexes involves the recruitment of a large number of
cofactors through the ?-catenin arm domain (17, 48). There-
fore, we cannot completely rule out that acetylation of K345
could also influence the binding of such factors and thereby
participate in transcription regulation.
Importantly, we observed that HAT-deficient p300 had little
effect on ?-catenin-mediated activation of the TOPFLASH
reporter (Fig. 7A) and the natural ?-catenin-responsive inter-
leukin-8 promoter (data not shown), implying that the acetyl-
transferase activity of p300 was required for its coactivator
function. In addition, while p300 retained some capacity to
coactivate the K345R ?-catenin mutant, the HAT-deficient
p300 was unable to coactivate this mutant. These results
strongly suggest that recruitment of p300 by ?-catenin on Tcf-
dependent promoters fulfills different functions, as previously
described in different settings (26). Besides its ability to bridge
DNA-associated activators to the basal transcription machin-
ery, p300 might act by altering chromatin structure though
intrinsic HAT activity and modulate ?-catenin activity though
its factor acetyltransferase activity. Such functional duality has
been convincingly demonstrated for coactivation of HMGI(Y)
and p65 by CBP/p300 or PCAF, because in these cases, the
HAT activity stimulates transcription, but the factor acetyl-
transferase activity could serve the opposite function in turning
off transcription (21, 34). Alternatively, as proposed by Miller
and Moon (32), overexpressed mutant ?-catenin could com-
pete with endogenous ?-catenin for binding to the degradation
complex, leading to nuclear accumulation of both wild-type
and mutant proteins.
Although coactivation of ?-catenin by p300 is well estab-
lished (20, 29, 33, 43, 44), the contribution of HAT and factor
acetyltransferase activities has received little attention so far.
Recently, Hecht and collaborators reported that coactivation
of ?-catenin at the siamois promoter is independent of the
acetyltransferase activity of CBP/p300 (20). The reasons for
the apparent discrepancy between this observation and our
present data are unclear, but it might be explained by recent
results showing that different Tcf proteins such as Lef1 and
Tcf4E could perform specific, nonredundant functions at dif-
ferent natural ?-catenin-responsive promoters (19). It will be
of interest to determine whether the acetyltransferase activity
of p300 is needed for coactivation of ?-catenin in different
promoter contexts and whether acetylation of ?-catenin affects
its binding to different members of the Tcf family. Although we
have observed already that acetylation of K345 but not that of
K49 mediates, at least in part, the transcriptional coactivation
of ?-catenin/Tcf by p300, further research on the functional
effect of lysine 49 acetylation could determine whether it is
implicated in modulating the acetylation of lysine 345.
FIG. 8. Acetylation of lysine 345 specifically increases the affinity of ?-catenin for Tcf4. In vitro-translated Tcf4 (upper panels) and AR (lower
panels) were incubated with immobilized GST-arm 1-12 in the presence of increasing amounts of acetylated or nonacetylated arm 6 peptide. Bound
proteins were resolved on SDS-PAGE and analyzed by autoradiography.
3412 LE´VY ET AL.MOL. CELL. BIOL.
Besides their role in Tcf-dependent transcription, it has been
shown that ?-catenin and CBP/p300 are transcriptional coac-
tivators of the AR. Chesire and Isaacs (10) put forward the
notion of a reciprocal balance between the activation of AR-
and Tcf-related transcription by ?-catenin, which might be
involved in normal prostate development and in prostate tu-
mor progression. In this study, we showed that acetylation of
?-catenin at K345 specifically increases the affinity of ?-catenin
for Tcf4 but seems to decrease the affinity of ?-catenin for the
AR (Fig. 8). One might thus speculate that this posttransla-
tional modification of ?-catenin is involved in differential gene
activation through Tcf versus the AR.
In conclusion, the role of p300 in the regulation of Wnt
signaling appears to be complex. Thus, acetylation might par-
ticipate in the activation of specific sets of genes upon Wnt
We thank O. Bischof, K. T. Jeang, J. G. Judde, and R. Kiernan for
critical reading of the manuscript. We are grateful to P. Tiollais and A.
Dejean for constant interest in this work. We also thank G. Castoria,
H. Clevers, S. Emiliani, and Y. Nakatani for kindly providing the
constructs used in this study.
This work was supported in part by grant 4395 from the Association
pour la Recherche sur le Cancer (ARC). L.L. and Y.W. were funded
by an ARC fellowship. C.L. was funded by an ENS fellowship.
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