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© 2004 Nature Publishing Group http://www.nature.com/natstructmolbiol
ARTICLES
Regulation of the p300 HAT domain via a novel
activation loop
Paul R Thompson1,8, Dongxia Wang1, Ling Wang1, Marcella Fulco2, Natalia Pediconi3, Dianzheng Zhang4,
Woojin An5, Qingyuan Ge6, Robert G Roeder5, Jiemin Wong4, Massimo Levrero3,7, Vittorio Sartorelli2,
Robert J Cotter1 & Philip A Cole1
The transcriptional coactivator p300 is a histone acetyltransferase (HAT) whose function is critical for regulating gene expression
in mammalian cells. However, the molecular events that regulate p300 HAT activity are poorly understood. We evaluated
autoacetylation of the p300 HAT protein domain to determine its function. Using expressed protein ligation, the p300 HAT
protein domain was generated in hypoacetylated form and found to have reduced catalytic activity. This basal catalytic rate
was stimulated by autoacetylation of several key lysine sites within an apparent activation loop motif. This post-translational
modification and catalytic regulation of p300 HAT activity is conceptually analogous to the activation of most protein kinases
by autophosphorylation. We therefore propose that this autoregulatory loop could influence the impact of p300 on a wide
variety of signaling and transcriptional events.
The determination that the transcriptional coactivator p300 and its
paralog CBP have HAT activity1,2 has substantially increased our
understanding of their roles in the activation of gene expression in a
variety of pathways3. Furthermore, p300 and CBP have been suggested
to participate in a number of disease processes including several forms
of cancer, Huntington’s disease, HIV and cardiac hypertrophy4–10.
Regulation of both p300 and CBP has so far been known to occur primarily through the recruitment of various protein targets via one of
their many adaptor domains3,11. However, other mechanisms of regulation probably exist, because p300 and CBP undergo a variety of
covalent modifications, including sumolation, methylation and phosphorylation3,12–16. Furthermore, p300 has an E4 ligase activity17. In
addition, p300 and CBP have long been known to autoacetylate1,18,
although a role for this activity has not previously been demonstrated.
Earlier enzymologic studies of p300 using full-length p300 recombinant protein were carried out to provide a preliminary analysis of its
kinetic mechanism and substrate selectivity19. These studies showed
that p300 has a ping-pong kinetic mechanism and that product release
is probably at least partially rate determining. Using a series of different peptide substrates, preferred amino acid sequences around a targeted lysine were identified. Although these studies are informative,
their value is somewhat limited owing to the complexity and posttranslational heterogeneity of full-length p300.
Unlike those of several other HATs20,21, the detailed catalytic properties of the p300 and CBP HAT domains have not been well established. The study of these enzymes has been limited partly by the
difficulty of overexpressing them as recombinant proteins in
Escherichia coli. For example, others and we have found that GST-p300
fusion proteins express and purify with very low yields22. Here we
report a new method to express the p300 HAT domain and its use to
obtain insights into the domain’s catalytic properties and mechanism
of regulation.
RESULTS
Recombinant p300 HAT domain is heavily acetylated
We considered that the p300 HAT domain might be toxic to E. coli
because of indiscriminant acetylation of host proteins and therefore
we coexpressed the histone deacetylase (HDAC) Sir2 on a separate
plasmid. This substantially improved the production of the p300 HAT
domain (residues 1195–1673), and we obtained 0.5 mg of purified
protein from 12 l of E. coli culture.
This recombinant protein was further subjected to domain mapping using tryptic digestion (Fig. 1a), which revealed proteolytically
sensitive sites around residues 1284 and 1550, allowing us to designate
the domain architecture (Fig. 1b). Although a p300 HAT domain fragment (residues 1284–1673) prepared in the presence of Sir2 showed
1Department
of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. 2Muscle Gene
Expression Group, Laboratory of Muscle Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases Intramural Research Program, National
Institutes of Health, 50 South Drive, Room 1146, Bethesda, Maryland 20892-8024, USA. 3Laboratory of Gene Expression, Fondazione Andrea Cesalpino, University
of Rome La Sapienza, Policlinico Umberto I, Viale del Policlinico 155, 00161 Rome, Italy. 4Department of Molecular and Cellular Biology, Baylor College of
Medicine, One Baylor Plaza, Houston, Texas 77030, USA. 5Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, New York
10021, USA. 6Cell Signaling Technology, Beverly, Massachusetts 01915, USA. 7Department of Molecular Oncogenesis, Regina Elena Cancer Institute, Via delle
Messi d’Oro 156, 00158 Rome, Italy. 8Current address: Department of Chemistry and Biochemistry, University of South Carolina, 631 Sumter Street, Columbia,
South Carolina 29208, USA. Correspondence should be addressed to P.A.C. ([email protected]).
Published online 7 March 2004; doi:10.1038/nsmb740
308
VOLUME 11 NUMBER 4 APRIL 2004 NATURE STRUCTURAL & MOLECULAR BIOLOGY
ARTICLES
a
Lane
kDa
97
1
c
2
100
90
Amino acids
70
1284–1673
© 2004 Nature Publishing Group http://www.nature.com/natstructmolbiol
45
80
1195–1673
1284–1555
% intensity
66
47,968
60
50
40
Figure 1 Purification and partial proteolysis of p300(1195–1673) HAT
domain. (a) Lane 1, p300(1195–1673) HAT domain after purification on
cobalt chelation and Mono Q columns. Lane 2, fragments generated after
partial proteolysis of the p300(1195–1673) HAT domain with limiting
amounts of trypsin (0.33 µg ml–1). The fragments (arrows) were N-terminally
sequenced after transfer to PVDF. The residues comprising the major
fragments are designated at right. (b) Proposed domain architecture for the
p300(1284–1673) HAT domain based on partial proteolysis experiments.
(c) MALDI-TOF mass spectra of p300(1284–1673) purified HAT domain
after coexpression with Sir2A. The observed mass (47,968 Da) is 432 Da
greater than the expected mass (47,536 Da).
30
20
10
31
0
45,000
55,000
Mass
(m/z)
b
NH 3+
(1284)
Lys1554
CO2 –
(1673)
HAT activity and expressed fairly well, its observed molecular mass
was ∼430 AMU greater than the calculated one on the basis of sequence
(Fig. 1c). We suspected that this was due to extensive autoacetylation
of this domain, which was highly reactive upon immunoblotting with
an antibody to acetyl-lysine and showed substantial heterogeneity by
ion exchange chromatography analysis, suggestive of multiple charge
states (data not shown). Because the additional mass due to a single
acetyl modification is 42 AMU, the net average mass increase is consistent with about ten acetylations of this domain.
Generation of hypoacetylated p300 HAT domain
While attempting to define the boundaries necessary for efficient protein expression and catalytic activity, we prepared a series of p300 constructs (Fig. 2a)that showed that although the fragment comprising
residues 1284–1655 allowed very high soluble protein expression, it
was nearly devoid of catalytic activity (>1,000-fold reduction) and
could readily be purified in a hypoacetylated form. Thus, we used
expressed protein ligation23,24, which generates semisynthetic proteins
by a chemoselective reaction between a recombinant protein fragment
containing a C-terminal thioester and a synthetic peptide containing
an N-terminal cysteine, to obtain active p300 protein in a hypoacetylated state (Fig. 2b). The p300(1287–1652) construct was ligated to a
14-residue peptide, and the catalytically active semisynthetic protein
ss-p300-HAT was generated at 5 mg l–1 from E. coli cell culture after
purification (Fig. 2c). For high-efficiency ligations, a M1652G mutant
was used, as the methionine at this position inhibits the ligation reaction. Notably, this domain showed the predicted molecular mass by
MALDI-TOF mass spectrometry, suggesting minimal modification by
acetylation (Fig. 2d). Based on mass spectrometry analysis, we estimate that fewer than two acetylation events are present in ss-p300HAT, and we refer to the protein as ‘hypoacetylated’ enzyme.
p300 HAT domain activation by loop autoacetylation
Detailed kinetic analysis of ss-p300-HAT revealed that its Vmax/Km
(V/K) was approximately four-fold reduced as compared with that of
fully recombinant protein (p300(1287–1666), r-p300-HAT) generated
in the presence of Sir2 (Table 1 and Fig. 2e). Although this rate reduction might be related to damage during expressed protein ligation, we
considered that the level of acetylation itself could modulate such
activity. In this regard, we measured the activity of ss-p300-HAT and
r-p300-HAT after allowing autoacetylation to take place. We confirmed using mass spectrometry analysis that hyperacetylation was
occurring during this period. Notably, this treatment stimulated both
domains, the recombinant by 4-fold and the semisynthetic by 11-fold
(Fig. 3a), and the hyperacetylated domains showed acetyltransferase
activity within 30% of each other. Hyperacetylation of the p300 HAT
domain primarily led to a decrease in Km for both acetyl-CoA and the
peptide substrate (Table 1). Incubation with an inactive CoA analog
(desulfo-CoA) did not activate ss-p300-HAT; this refutes a stimulation
model involving allosteric binding of acetyl-CoA. Taken together,
these data support the notion that autoacetylation of the p300 HAT
domain can regulate its catalytic activity.
To further analyze these effects, we determined acetylation sites
within the p300 HAT domain (recombinant p300 HAT domain
Table 1 Kinetic parameters of recombinant and semisynthetic
p300 proteins
Km (µM)
kcat (s–1)
V/K (M–1 s–1)
H4-15
40 ± 8
0.26 ± 0.01
6,600
–
Acetyl-CoA
45 ± 3
0.29 ± 0.01
6,500
–
Substrate
(V/K) /
(V/K)
r-p300-HAT domain
Hyperacetylated r-p300-HAT domain
H4-15
51 ± 25
0.82 ± 0.12
16,000
2.5
Acetyl-CoA
39 ± 11
0.98 ± 0.06
25,000
3.9
H4-15
162 ± 77
0.26 ± 0.05
1,600
–
Acetyl-CoA
275 ± 63
0.43 ± 0.04
1,600
–
ss-p300-HAT domain
Hyperacetylated ss-p300-HAT domain
H4-15
50 ± 13
0.53 ± 0.04
11,000
7
Acetyl-CoA
31 ± 5
0.52 ± 0.05
17,000
11
V/K is the Vmax/Km(acetyl-CoA) or Vmax/Km(H4-15). The far right-hand column shows the
relative V/K for the acetylated versus the unacetylated p300 protein. Standard errors
of these values are shown. The steady-state kinetic parameters for the H4-15 peptide
and acetyl-CoA were determined in the presence of acetyl-CoA (2 mM) and the H4-15
peptide (400 µM), respectively. For cases in which complete saturation (substrate
concentrations >10 Km) was not reached, these parameters should be regarded as
apparent Km and kcat values rather than absolute Km or kcat measurements.
NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 11 NUMBER 4 APRIL 2004
309
a
d 100
Soluble Active
1673
1673
1673
1660
1655
Yes
Yes
No
Yes
Yes
Yes
Yes
ND
Yes
No
SH
b
O
Inactive p300
Intein
N
CBD
O
Inactive p300
Intein
S
NH
R-SH
2
a
b
3.5 x 104
2.5 x 10
K1473
K1499
4
V/K 2.0 x 104
(M –1s–1)
1.5 x 104
K1337
11
K1542
K1546
K1549
K1550
K1551
K1554
K1555
K1558
K1560
c
Lane
1
e
2
Intensity (%)
Peptide
+ H 2N
Hypoacetylated active p300
100
90
80
70
60
50
40
30
20
10
0
7,000
6,000
5,000
kDa
97
66
45
a
44,019
b
37,200 49,800
Mass
(m/z)
b
42,402
37,200
49,800
Mass
(m/z)
V/K 4,000
(M – 1s–1) 3,000
31
2,000
1,000
0
21.5
14.5
r-p300- ss-p300HAT
HAT
mutation of several of the other lysines affected basal catalysis
(Supplementary Table 1 online), these mutant proteins were fully activated upon hyperacetylation. For example, K1546R behaves similarly
to unmodified ss-p300-HAT. Note that the important acetylated
residues all fall near the proteolytically sensitive loop (Figs. 1b and 3b).
2.5 x 104
2.0 x 104
V/K
(M –1s–1)
1.5 x 104
1.0 x 10
Hypoacetylated
Hyperacetylated
11
10
4
6
1.0 x 104
K1637
R
S
O
c
3.0 x 104
4
O
Inactive p300
After
ligation
Before
ligation
CBD
SH
isolated from the E. coli Sir2 expression system) using the mass
spectrometric method of post-source decay (PSD) analysis25, which
provided fragmentation information for the analyzed peptides on a
MALDI-TOF mass spectrometer. Peptides generated after tryptic
digestion were identified unambiguously with PSD spectra
(see Supplementary Fig. 1 online), and coverage represented 82% of
the amino acid sequence of the p300 HAT domain. Notably, 13 of 37
lysine residues were found to be acetylated. Many of the acetylated
residues clustered in a region determined to be a flexible loop by
protease sensitivity studies (Fig. 3b).
To further examine the significance of these modifications, sitedirected mutagenesis and expressed protein ligation were carried out
to generate the corresponding mutant lysine-arginine proteins corresponding to all of the acetylated lysine residues. Four of these mutant
proteins (K1499R, K1549R, K1554R, and K1558R K1560R ss-p300HAT) were defective in activation by acetylation (Fig. 3c and Table 1).
K1549R and K1558R K1560R ss-p300-HAT showed 20–40% inhibition upon hyperacetylation whereas K1499R and K1554R showed
diminished activation upon hyperacetylation. Because K1499R
showed unusually large Km increases, a K1499A mutant was also generated. Notably, K1499A ss-p300-HAT showed enhanced basal activity
but no stimulation upon hyperacetylation. Thus, Lys1499 may be
important in maintaining the depressed basal activity and this
effect can be partially overcome by side chain truncation. Although
90
80
70
60
50
40
30
20
10
0
Intensity (%)
1284
1290
1296
1284
1284
O
Figure 2 Expression and purification of semisynthetic p300 HAT domain by
expressed protein ligation. (a) Constructs generated to identify the minimal
p300 HAT domain. The columns at right indicate whether the protein was
soluble, active or both. The constructs described here were coexpressed
with Sir2 and purified analogously to the p300(1195–1673) HAT domain.
(b) Synthesis and purification of the ss-p300-HAT domain using the
expressed protein ligation technology. (c) Purification gel of unligated (lane 1)
and ligated (lane 2) ss-p300 HAT domain. A single protein band with an
apparent molecular mass of 44 kDa was observed. (d) MALDI mass spectra
for ligated (top; peak a, 44,006 Da calculated; 44,019 Da observed) and
unligated (bottom; peak b, 42,420 Da calculated; 42,402 Da observed)
ss-p300-HAT domain. A small amount of unligated protein (peak b) is present
after the ligation reaction. (e) Comparison of the V/K (Vmax/Km(acetyl-CoA))
values (see Table 1) for ss-p300-HAT domain and the corresponding
construct, recombinant p300 HAT domain (r-p300-HAT), obtained by
coexpression with the histone deacetylase Sir2.
O
© 2004 Nature Publishing Group http://www.nature.com/natstructmolbiol
ARTICLES
0.5 x 104
7
1
0.5 x 104
0.8
0.6
0
0
r-p300HAT
ss-p300HAT
ss-p300- K 1499R K1499A K1546R K1549R K1554R K1558R
K1560R
HAT
Hypoacetylated
Hyperacetylated
Figure 3 Autoacetylation of p300. (a) Autoacetylation of p300 stimulates its HAT activity. A comparison of the V/K (Vmax/Km(acetyl-CoA)) values (see Table 1)
for both the Sir2 coexpressed r-p300-HAT domain (lane 1, hypoacetylated; lane 2, hyperacetylated) and ss-p300 HAT domain (lane 3, hypoacetylated; lane
4, hyperacetylated). The hyperacetylated forms of both enzymes were generated by preincubation with acetyl-CoA for 2 h. (b) Sites of acetylation in the p300
HAT domain. Note the clustering of sites in the flexible loop region. Sites of acetylation that help to regulate the HAT activity of p300 are bold. (c) Mutational
analysis of acetylated residues.
310
VOLUME 11 NUMBER 4 APRIL 2004 NATURE STRUCTURAL & MOLECULAR BIOLOGY
ARTICLES
© 2004 Nature Publishing Group http://www.nature.com/natstructmolbiol
a
hp300
hCBP
mCBP
Nej
CBP-1
1499-KELPYFEGDFWPNVLEESIKELEQEEEERKR----E-1530
1535-KELPYFEGDFWPNVLEESIKELEQEEEERKK----E-1566
1530-KELPYFEGDFWPNVLEESIKELEQEEEERKK----E-1561
2227-AELPYFEGDFWPNVLEESIKELDQEEEEKRKQAEAA-2262
1326-TQLPYFEGDFWPNVIEDCIREASNEEAQRKV----K-1357
:************:*:.*:* .:** :::
hp300
hCBP
mCBP
Nej
CBP-1
1531-ENTSN------ESTDVTKGDSKNAKKKNNKKTSKNK-1560
1567-ESTAAS-----ETTEGSQGDSKNAKKKNNKKTNKNK-1597
1562-ESTAAS-----ETPEGSQGDSKNAKKKNNKKTNKNK-1592
2263-EAAAAANLFSIEENEVS-GDGKKKGQKKAKKSNKSK-2297
1358-EDDDDG-----EDADGGLGGGDSGKKKSSKNKKNNL-1388
*
* :
*.... :*. *:..:.
hp300
hCBP
mCBP
Nej
CBP-1
1561-SSLSRGNKKKPGMPNVSNDLSQKLYATMEKHKEVFF-1596
1598-SSISRANKKKPSMPNVSNDLSQKLYATMEKHKEVFF-1633
1592-SSISRANKKKPSMPNVSNDLSQKLYATMEKHKEVFF-1627
2298-AAQ-RKNSKKSNEHQSGNDLSTKIYATMEKHKEVFF-2332
1389-KKNAKMNKKKAG-SITGNEVADKLYSQFEKHKEVFF-1423
: *.**..
.*::: *:*: :********
K1473
b
c
K1499
K1337
3.5 x 10 4
3.0 x 10 4
K1555
K1558
K1560
K1637
d
Figure 4 A proteolytically sensitive loop region that regulates p300 HAT
activity. (a) Alignment of loop sequences from human p300. The sequences
for several p300 homologs, corresponding to the HAT domain of human
p300(1284–1673), were aligned using ClustalW35. Only the sequences of
the proteolytically sensitive loop region are shown. The sequences used for
this alignment are human p300 (hp300; GenBank entry NP_001420),
human CBP (hCBP; entry NP_004371), murine CBP (mCBP; entry
AAL87531), D. melanogaster p300 homolog Nej (Nej; entry NP_524642),
and C. elegans p300 homolog CBP-1 (CBP-1; entry NP_499160). The sites
of acetylation within this region, and the conservation of these residues
between p300 homologs, are highlighted in bold. (b) The domain structure
of the loop deletion mutant. (c) A comparison of the V/K (Vmax/Km(acetyl-CoA))
values for both ss-p300 HAT domain (lane 1, hypoacetylated; lane 2,
hyperacetylated) and the loop deletion mutant (lane 3, hypoacetylated;
lane 4, hyperacetylated) in the hypo- and hyperacetylated states.
(d) Acetylation of synthetic loop peptide by hyperacetylated ss-p300-HAT.
The concentrations of the enzyme and peptide substrate were 200 nM
and 1 mM, respectively.
2.5 x 10 4
0.9
V/K 2.0 x 10 4
(M –1s–1)
1.5 x 10 4
11
1.0 x 10 4
0.14
0.5 x 10 4
0.12
0
0.10
0.08
ss-p300- Loop
HAT deletion
v
(s–1) 0.06
Hypoacetylated
Hyperacetylated
0.04
0.02
0
500
1,000 1,500
Acetyl-CoA
( µM)
2,000
2,500
Mechanistic analysis of the p300 activation loop
Sequence comparison of this proteolytically sensitive loop region
(residues 1520–1560) of the p300 HAT domain shows that it is well
conserved in human CBP and other mammalian homologs but has
reduced sequence similarity to CBP from C. elegans and Drosophila
melanogaster, whereas the surrounding sequences are more highly
conserved (Fig. 4a). The catalytically important acetylation sites at
positions 1499, 1549, 1558 and 1560 are conserved in mouse and
human CBP, whereas there is some divergence at these positions in
C. elegans and D. melanogaster. In mammals, the sequence (residues
1520–1560) is noteworthy for containing large clusters of positive and
negative charges. Given this unusual sequence and its reduced conservation, we hypothesized that this loop region could be deleted from
the architectural framework, leaving behind stable protein (Fig. 4b).
Indeed, we removed amino acids 1523–1554 from the p300 HAT
domain and used expressed protein ligation to express this loop deletion mutant, which was catalytically active. Moreover, kinetic measurements showed that it behaved as a constitutively active HAT,
showing essentially no change upon hyperacetylation (Fig. 4c).
Taken together, these data support the notion that the p300
sequence in the vicinity of residues 1520–1560 serves as an autoinhibitory loop within the p300 HAT domain. Although we do not yet
have a detailed structural understanding of the basis for inhibition, we
considered a model in which this loop serves as a pseudosubstrate.
Previous studies on p300 have shown that multiple positive charges in
peptide sequences lead to efficient substrates for p300 HAT19. Thus,
this loop region may sit within the p300 enzyme active site, preventing
turnover. Because the p300 HAT domain behaves as a monomer (on
the basis of gel filtration studies), such an interaction would be predicted to be intramolecular. Upon acetylation by itself or another HAT,
this loop would be dislodged, allowing for increased activity.
Consistent with this idea, stimulation by hyperacetylation decreases
the Km for acetyl-CoA and peptide substrate rather than increasing the
kcat (Table 1). However, the Km values do not necessarily refer directly
to Kd values, because p300 has a ping-pong kinetic mechanism in
which product release partially determines the reaction rate19. To further investigate the model of autoinhibition, the peptide composed of
residues 1523–1554 was synthesized and examined as a p300 substrate.
As predicted based on its cluster of positive residues, this synthetic
loop peptide could undergo efficient acetylation catalyzed by hyperacetylated ss-p300-HAT domain (for acetyl-CoA, Km = 60 ± 9 µM;
kcat = 0.140 ± 0.004 s–1; Vmax/Km(acetyl-CoA) = 2,300 M–1 s–1, Fig. 4d).
These studies suggest that p300 can autoacetylate in an intermolecular
fashion, but do not rule out the possibility that intramolecular
autoacetylation is possible. Regardless, these findings support the
notion that in the context of the p300 HAT domain, this peptide loop
serves as an intramolecular ‘pseudosubstrate,’ preventing external
entry of histones and other proteins from the active site.
Cellular analysis of the p300 activation loop
To explore the relevance of these in vitro findings to the action of p300
in vivo, we did a series of additional experiments in cellular systems. To
determine whether any of the loop acetylation sites were actually
acetylated on endogenous protein, we used an acetylated heptapeptide
as an antigen to generate a polyclonal antibody directed at the 1499
acetylation site designed to recognize only the 1499-acetylated form of
p300 (anti-Ac-K1499-p300). This antibody interacts undetectably
with K1499R ss-p300 HAT domain and a hypoacetylated ss-p300 HAT
domain (Fig. 5a, lanes 1, 3 and 4), but strongly with hyperacetylated
ss-p300 HAT domain (Fig. 5a, lane 2). This antibody was then tested
with U2OS cells and found to stain a protein band that comigrates
with p300 (Fig. 5b, lanes 7 and 10). We confirmed that anti-Ac-K1499p300 recognizes p300 by immunoprecipitation with an antibody to
p300, which led to enhanced staining with anti-Ac-K1499-p300,
whereas control antibody did not (Fig. 5b, lanes 2, 3, 5, 6, 8, 9, 11 and
12). Moreover, the relative intensity of this band, as compared with
that of total p300, is enhanced markedly after treatment of cells with a
cocktail of HDAC inhibitors (Fig. 5b, compare lanes 10 and 12 with
NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 11 NUMBER 4 APRIL 2004
311
ARTICLES
Anti-p300
Control
Input
Control
Anti-p300
Input
p3
00
∆
sfe
cte
d
p3
00
W
T
No
ntr
an
sfe
cte
d
p3
p3
312
an
ntr
No
p3
00
∆
T
W
00
p3
© 2004 Nature Publishing Group http://www.nature.com/natstructmolbiol
00
00
∆
W
T
lanes 7 and 9). We conclude that the loop a
b
– HDAC
+ HDAC
1499 site of p300 is partially acetylated in vivo
inhib itor s
inhib itor s
Lane 1 2
3 4
and subject to modulation by HDACs. We
Anti-Ac-K1499-p300-HAT
have also demonstrated that hyperacetylated
ss-p300 HAT domain can be largely deacetyCoomassie-p300-HAT
lated by the action of recombinant Sir2
in vitro (P.R.T. and P.A.C., unpublished data). c
Anti-p 300
In the context of a cellular environment,
1
2 3
4
5 6
the p300 loop deletion protein based on our
studies is predicted to show enhanced acetylkDa
transferase reactivity. Full-length wild-type
Anti-Ac-p 300
250
p300 and p300 loop deletion (p300∆) con146
7 8
9
10 11 12
structs were overexpressed in 293 cells and the
98
pattern of staining of the cellular lysates with
64
antibodies to acetyl-lysine was explored. As
d
can be seen, several proteins of high molecular mass showed enhanced staining in the
– – + – + Doxo
presence of p300∆ as compared with wildWB anti-Ac-Lys
type p300 with antibodies to acetyl-lysine, in
IP anti-HA
WB anti-HA
Na butyrate – + – + – +
the presence and absence of the nonspecific
WB:
anti-Ac-lysines
HDAC inhibitor sodium butyrate (Fig. 5c).
HA-p73a
20
Although the identity of these acetylated tar15
get proteins has not been determined, these
10
results suggest that the presence of a p300 reg5
ulatory acetylation loop can suppress the
0
acetylation of a variety of cellular proteins.
+
+
+
+ HA-p73
Anti-FLAG
–
–
The tumor suppressor protein p73 is a sub+
+ Doxo
–
– p300WT
+
+
strate of p300 HAT activity during apoptosis
Tubulin
–
–
26
+
+
p300
secondary to DNA damage by doxorubicin .
– + – + – +
Na
butyrate
Doxorubicin stimulates p300-mediated
e
16
acetylation of p73 (ref. 26). We therefore eval- Figure 5 Role of p300 autoacetylation in vivo.
14
uated whether the p300∆ protein versus the (a) Immunoblot of anti-acetyl-K1499-p300 with
Luciferase 12
semisynthetic
p300
recombinant
HAT
domain
wild-type protein might mediate enhanced
activity 10
8
p73 acetylation in 293 cells. Notably, we proteins: hypoacetylated ss-p300-HAT (lane 1),
x 10 6
6
found increased basal and doxorubicin- hyperacetylated ss-p300-HAT (lane 2),
4
hypoacetylated K1499R ss-p300-HAT (lane 3),
stimulated acetylation of p73 by p300∆ as hyperacetylated K1499R ss-p300-HAT (lane 4).
2
0
compared with wild-type p300 (Fig. 5d). Immunoblot using anti-acetyl-K1499-p300 (top)
– +
R1881 (50 nM) – + – +
These results further confirm that the loop is and Coomassie-stained SDS-PAGE loading control
AR (10 ng) + + + +
+ +
involved in inhibiting the HAT activity of gel (bottom). (b) 1499-acetylated p300 is
_
p300WT p300∆
p300 in vivo in the acetylation of a well- present in anti-p300 immunoprecipitates. Cell
lysates were prepared from U2OS cells and
established substrate.
immunoprecipitated with anti-p300 and control antibody (p63). (c) The p300 loop deletion mutant
To study the involvement of the p300 activa- (p300∆), as compared with wild-type p300 (p300WT), shows an in vivo enhanced acetyltransferase
tion loop in p300’s role as a transcriptional activity toward multiple and discrete cellular polypeptides (top) in transiently transfected 293 cells.
coactivator, we examined the effect of tran- Control experiments (bottom) indicate that the difference in the acetyltransferase activity of wild type
siently transfected p300∆ on androgen- and loop deletion p300 mutant cannot be ascribed to different expression levels or protein stability.
androgen receptor–mediated transcriptional (d) HA-p73α acetylation by p300 is enhanced by activation loop deletion. 293 cells were transiently
enhancement of an MMTV-luciferase reporter transfected with p73α and p300 constructs as indicated in the presence or absence of doxorubicin.
Immunoprecipitated p73 was analyzed with a polyclonal anti-acetyl-lysine (top, anti-Ac-Lys) or anti-HA
system in HeLa cells. p300∆ more robustly
(bottom). Densitometric quantification (graph) was based on the average of four runs and standard
coactivates gene expression as compared with errors are shown. (e) The p300∆ mutant, as compared with p300WT, has an increased coactivator
wild-type p300 under these conditions activity toward androgen receptor (AR). HeLa cells were transfected with a MMTV-LTR-driven luciferase
(Fig. 5e), with an 80% net increase of p300∆ reporter36 and expression constructs for AR and p300WT or p300∆, and treated with or without AR
versus wild-type p300 as compared with non- agonist R1881 as indicated. The luciferase assays were carried out in triplicate and repeated twice;
p300 transfected cells. This transcriptional error bars represent the s.e.m.
stimulation depends on the presence of androgen, consistent with the role of p300 as a
coactivator. This result links constitutive HAT activity to enhanced the p300 HAT domain that can be modified by autoacetylation to
enhance its HAT activity. Future work is necessary to further dissect
stimulation of gene expression for a physiologically relevant reporter.
the contributions of this form of regulation at specific promoters
and p300-dependent pathways in normal and disease states. AntiDISCUSSION
By using expressed protein ligation in combination with several other Ac-K1499-p300 should be useful in this regard. Although acetylation
biochemical strategies, we have identified a novel inhibitory loop in of the p300 HAT domain was self-catalyzed in this study, we cannot
VOLUME 11 NUMBER 4 APRIL 2004 NATURE STRUCTURAL & MOLECULAR BIOLOGY
© 2004 Nature Publishing Group http://www.nature.com/natstructmolbiol
ARTICLES
rule out the possibility that other cellular acetyltransferases such as
PCAF or GCN5 could also modulate the activity of p300 and CBP.
Additionally, any of the 13 acetylation sites mapped here by MS may
contribute to modulating protein-protein interactions. For example,
site-specific acetylation of the p300 HAT domain could influence its
interaction with other domains in the full-length protein or even
to modulate its interactions with other cellular proteins. With regard
to the latter possibility, Twist, Inhat, PCNA and E1A interact with the
p300 HAT domain and/or inhibit its HAT activity18,27,28. Therefore, it
will be worthwhile to determine whether such interactions are affected
by p300 acetylation. Finally, the development of HAT inhibitors as
potential therapeutics29,30 may be influenced by p300 drug screens
that use a heterogeneous enzyme mixture carrying different acetylation states.
A noteworthy technical advance described in this study is the use of
expressed protein ligation to generate the p300 HAT domain in underacetylated form. Because a wide array of enzymes autocatalyze protein
post-translational modifications, this approach could be applied, in
addition to acetyltransferases, to several kinases, methyltransferases,
glycosyltransferases and other self-modifying proteins to allow preparation of homogeneous proteins.
The p300 HAT regulatory scheme proposed here is reminiscent of a
strategy used by many members of the protein kinase superfamily, in
which autoinhibitory loops often suppress kinase activity until phosphorylated31,32. Modulation of HAT activity by acetyl-lysine-bromo
domain interactions has been proposed, similar to the way tyrosine
kinases can be regulated by SH2 domains33. The results described here
provide the first demonstration to our knowledge of an autocatalytic
reversible switch in a nonkinase enzyme, an apparent example of convergent evolution. The related regulatory strategies used by p300, CBP
and protein kinases establish another parallel between these two distinct classes of enzymes that catalyze reversible post-translational
modifications in the context of cellular signaling.
METHODS
Chemicals, peptides and DNA constructs. TRIZMA, ampicillin, chloramphenicol, kanamycin A, 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) and BSA
were obtained from Sigma. CoCl2 and IPTG were purchased from Fisher.
Chelating Sepharose fast flow was obtained from Amersham Biosciences and
chitin beads from New England BioLabs. Acetyl-CoA and [14C]acetyl-CoA
were obtained from Amersham Biosciences and NEN Life Sciences Products,
respectively. Oligonucleotide primers were purchased from Integrated DNA
Technologies. Synthetic peptides were synthesized by solid-phase peptide synthesis using the Fmoc strategy on a Rainin PS3 machine. All peptides were purified by reverse-phase HPLC, and electrospray mass spectrometry was done to
confirm their correct structures. DNA constructs were generated using standard subcloning techniques and QuikChange mutagenesis and the sequences
were confirmed by DNA sequencing.
Purification of bacterial expressed p300 HAT domain. See Supplementary
Methods online for a detailed methodology. In brief, coexpression of
p300(1195–1673) and Sir2 was done by cotransforming constructs bearing
these genes into codon plus E. coli BL21(DE3)-RIL competent cells. Kanamycin
A (50 µg ml–1 final) and ampicillin (100 µg ml–1 final) were used for antibiotic
selection of the p300 and GST-Sir2 expression constructs, respectively. Cells
(6 × 2 l) bearing the p300 and GST-Sir2 expression constructs were grown at
37 °C to an A600 of 0.6, at which point protein expression was induced by the
addition of IPTG (0.5 mM final concentration). Cells were grown at 22 °C for
an additional 16 h and were then harvested by centrifugation (5,000g), resuspended in lysis buffer and lysed by two passages through a French pressure cell.
The lysates were then cleared by centrifugation (20,000g). The cleared cell lysate
was then applied to a 25 ml (2.6 × 10 cm) Q Sepharose column (Amersham
Biosciences) and the flowthrough from this column was applied to a cobalt column. Bound protein was eluted with a stepwise gradient of imidazole; fractions
obtained in this manner were analyzed by 15% (w/v) SDS-PAGE, and fractions
containing recombinant p300 HAT domain were pooled and concentrated.
Concentrated protein was dialyzed and further purified using an analytical
Mono Q (HR 5/5) strong anion exchange column. Bound proteins were eluted
with a linear gradient of NaCl from 50 to 1,000 mM. Fractions containing
recombinant p300 were pooled and concentrated to ∼1 mg ml–1.
Purification of semisynthetic p300 HAT domain. See Supplementary Methods
online for a detailed methodology. In brief, semisynthetic protein was readily
expressed and purified as a fusion with the VMA intein-chitin-binding
domain. E. coli BL21(DE3)-RIL cells bearing the M1652G mutation were
grown to an A600 of 0.45, at which point the incubator temperature was reduced
to 16 °C and the media allowed to cool. After 15 min, protein expression was
induced by the addition of IPTG to a final concentration of 1 mM. Cells were
then grown overnight for 16 h at 16 °C, harvested by centrifugation, resuspended in intein lysis buffer and lysed by two passages through a French pressure cell. The lysate was cleared by centrifugation and applied to a 5-ml chitin
column. After extensive washing, excess buffer was drained and the C-terminal
peptide containing the N-terminal cysteine (15 mg), corresponding to residues
1653–1666 of p300, was added to the chitin column to initiate the ligation reaction. The reaction was incubated for 16 h at room temperature, at which point
ligated protein was eluted from the column. Fractions containing ss-p300-HAT
domain, as determined by SDS-PAGE analysis, were pooled, concentrated and
dialyzed. Concentrated and dialyzed protein was then applied to a Mono S HR
5/5 (Amersham Biosciences) strong cation exchange column, and ss-p300HAT domain was eluted using linear gradients of NaCl (50 to 1,000 mM).
Purified protein was concentrated to ∼5 mg ml–1, dialyzed against 20 mM
HEPES, pH 7.9, 50 mM NaCl, 2 mM DTT, and 10% (v/v) glycerol, and then
flash-frozen in liquid N2 and stored at –80 °C. Protein concentrations were
determined by Bradford assay (BioRad) and BSA was used as a standard. Mass
spectra for all of the proteins described in this study were obtained at the Johns
Hopkins Mass Spectrometry Facility (Baltimore).
Amino acid sequencing of p300 HAT domain. Bacterially expressed p300
HAT domain was sequenced by PSD analysis. In brief, the purified protein
was digested by trypsin at 37 °C for 18 h. The digested peptides were separated by reverse-phase HPLC, and fractions were subjected to mass spectrometry and PSD analysis on an AXIMA-CFR mass spectrometer. PSD spectra
were obtained from most of the precursor ions and the spectra were
interpreted manually. Some of the peptides were further analyzed by digestion with aminopeptidase, carboxypeptidase, trypsin or endoproteinase
aspartate-N. See Supplementary Note and Supplementary Figure 1 online
for more information.
Proteolysis studies. Partial proteolysis of p300(1195–1673) HAT domain was
observed upon incubation with limiting amounts of trypsin. Specifically, 18 µg
of p300(1195–1673) HAT domain, in a final volume of 30 µl (25 mM Tris-HCl,
pH 8.0), was incubated alone or in the presence of trypsin at a final concentration of 0.33 µg ml–1 and the reactions allowed to proceed for 45 min on ice. For
N-terminal sequencing, the proteolysis products were separated by 15% (w/v)
SDS-PAGE, transferred to polyvinylidene fluoride, and the major proteolytic
fragments excised for N-terminal sequencing at the Sequencing and Synthesis
Facility, Johns Hopkins School of Medicine (Baltimore).
HAT assay. A rapid and nonradioactive HAT assay that measures the production of CoASH by its facile reaction with DTNB was developed and validated by
comparison to the previously described radioactive assay. Prior to assaying,
enzyme was diluted with freshly prepared reaction buffer (50 mM HEPES,
pH 7.9, 50 µg ml–1 bovine serum albumin, and 0.1 mM EDTA) containing
1 mM DTT and pre-incubated for 2 min at 30 °C. The enzyme was then stored
on ice and was stable for several h. Fixed concentrations of acetyl-CoA (2 mM)
and the H4-15 peptide (400 µM) were used to measure the kcat and Km parameters for peptides and acetyl-CoA, respectively. Individual reactions (150 µl total
volume) were initiated with enzyme (3 µl; 80–160 nM final concentration) and
allowed to proceed for 0–15 min. Enzyme activity was quenched by the addition of 300 µl of quench buffer (3.2 M guanidinium-HCl, 100 mM sodium
phosphate dibasic pH 6.8). To measure CoASH production, 50 µl of DTNB
NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 11 NUMBER 4 APRIL 2004
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(2 mM final, dissolved in 100 mM sodium phosphate dibasic pH 6.8 and
10 mM EDTA) was added to the quenched reactions and the absorbance at
412 nm determined. Thiophenolate production was quantified assuming
ε = 13.7 × 103 M–1 cm–1 (ref. 34). Background absorbances were determined
and subtracted from the absorbance determined for individual reactions.
Assays were performed in duplicate at 30 °C, enzyme activity was linear for at
least 15 min, and turnover of the limiting substrate did not exceed 10%. Initial
rates were fitted by nonlinear least-fit squares to
© 2004 Nature Publishing Group http://www.nature.com/natstructmolbiol
v = Vm[S] / (Km + [S])
(1)
using the Kaleidagraph version 3.5 (http://www.kaleidagraph.com). Rates
obtained between duplicate runs were typically within 20% of each other.
Autoacetylation of p300 HAT domain. The p300 HAT domain was diluted into
buffer (10 µM final) containing 50 mM HEPES pH 7.9, 50 µg ml–1 bovine
serum albumin, 0.1 mM EDTA, 1 mM DTT. Acetyl-CoA was added to a final
concentration of 125 µM and the autoacetylation reactions incubated for 2 h at
30 °C. Autoacetylated p300 HAT domain was stored at 4 °C and was stable for
several days.
Antibody generation, immunoblotting and transfection studies. Polyclonal
anti-Ac- K1499-p300 was generated by immunizing rabbits with a synthetic
acetyl-Lys1499 heptapeptide (KLH coupled) corresponding to residues surrounding Lys1499 of human p300. Antibodies were purified by protein A and
peptide affinity chromatography (Cell Signaling Technology). For analysis of
p300 acetylation, U2OS (3 × 107) cells were cultured in DMEM containing 10%
(v/v) FBS and lysed at 4 °C by gentle vortexing in RIPA buffer (50 mM TrisHCl, pH 7.4, 0.5% (w/v) NP-40, 0.25% (w/v) Na-deoxycholate, 150 mM NaCl,
1 mM EDTA, Roche protease inhibitor cocktail). Lysates were clarified by centrifugation at 12,000g for 20 min and immunoprecipitated at 4 °C for 2 h with
anti-p300 (sc-584, Santa Cruz Biotechnology), followed by precipitation with
40 µl of protein A- and protein G-Sepharose (Sigma) overnight at 4 °C. After
four washes with PBS buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4,
1.8 mM KH2PO4, 2 mM EDTA, 0.2% (w/v) NP-40, Roche protease inhibitor
cocktail), the immunoprecipitates were electrophoresed in 10% (w/v) SDSPAGE, immunoblotted with anti-p300 or anti-Ac-K1499-p300, and detected
using ECL chemiluminescence (Amersham Biosciences). In cases where cells
were treated with HDAC inhibitors, 5 mM sodium butyrate, 10 mM nicotiamide and 100 ng ml–1 TSA were included 10 h before cell harvest. The p62
(TFIIH subunit) antibody was used as an immunoprecipitation control. Cells
were treated with HDAC inhibitors as indicated (lanes 4–6 and 10–12). Lanes 1
and 7 represent 5% of the extract used for the immunoprecipitation.
Immunoprecipitates were subjected to SDS-PAGE and immunoblotted with
anti-p300 (lanes 1–6) or anti-Ac-p300 (anti-Ac-K1499-p300) (lanes 7–12).
p300 is immunoprecipitated by anti-p300 in all cases but not by control antibody (p63), as shown in lanes 2, 5, 8 and 11. For experiments quantifying the
acetylation of total cell extracts, 293T cells were transfected with plasmids
expressing either epitope Flag-p300 wild type or Flag-p300 loop deletion
mutant. After 48 h, transfected cells were harvested and lysed in buffer containing 50 mM Tris-HCl, pH 8.0, 125 mM NaCl, 1 mM DTT, 5 mM MgCl2, 1 mM
EDTA, 10% (v/v) glycerol, and 0.1% (w/v) NP-40 supplemented with 1 mM
PMSF, protease inhibitor mix (Complete, Roche), 1 mM Na3VO4, 10 mM NaF.
When indicated, 10 mM sodium butyrate was added to the culture medium
12 h before harvesting. Whole-cell extracts were fractionated on 4–20% (w/v)
SDS-PAGE and electrotransferred onto a nitrocellulose membrane.
Immunoblotting was done with polyclonal anti-acetyl-lysine (Upstate 06-933),
monoclonal anti-Flag M2 (Sigma), and monoclonal anti-tubulin
(Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, Iowa,
USA). For the p73 studies, human embryonic kidney 293 cells were cultured in
DMEM plus antibiotics and 10% (v/v) heat-inactivated FBS. Transient transfections were done using the calcium phosphate precipitation method. The
pCDNA-HAp73α expression vector has been described26. Immunoblotting
and immunoprecipitation assays were done essentially as described26. Cells
were lysed in 50 mM Tris, pH 8, 120 mM NaCl and 0.5% (w/v) NP-40.
Immunoprecipitations were carried out by incubating 500–1,500 µg of total
cell lysate with an agarose-conjugated monoclonal antibody against HA (F7-
314
AC, Santa Cruz Biotechnology). Protein concentration was determined by the
BioRad dye-binding assay. The levels of cotransfected p300 were checked by
immunoblotting total cell lysates using a specific antibody against p300. The
relative acetylation levels were quantified by comparing the densitometric
intensity of each acetylated p73α band, after normalization for its expression
level, with the intensity of the normalized p73 acetylated band from untreated
293 cells cotransfected with the wild-type p300 expression vector. Standard
errors in Figure 5d were based on four independent runs. For the androgen
receptor experiments, HeLa cells were cultured in DMEM (Invitrogen) supplemented with 10% (v/v) FCS (Sigma). For transfection, HeLa cells were plated
at 2 × 105 cells per plate in six-well dishes and grown for 24 h to allow attachment. Cells were washed twice with 37 °C PBS and incubated with phenol-free
DMEM supplemented with 10% (v/v) charcoal-stripped FCS and penicillinstreptomycin. Cells were transfected 4–6 h later with 10 ng pCR3.1-AR, 100 ng
MMTV-luc reporter and 10 ng wild-type p300 or p300∆ mutant. Transfection
was carried out with lipofectamine (Invitrogen) according to the manufacturer’s instructions. At 24 h after transfection, the medium was aspirated and
replaced with phenol-free DMEM plus 10% (v/v) charcoal-stripped FCS and
penicillin-streptomycin supplemented with 50 nM of R1881 or ethanol (vehicle
control). At 24 h after R1881 (androgen) treatment, cells were lysed in lysis
buffer (Promega) and assayed for luciferase activity. Experiments were done at
two different times, in triplicate for each sample, and error bars represent s.e.m.
Consistent transfection efficiency and p300 protein expression in HeLa cells
were confirmed by western blots (data not shown).
ACKNOWLEDGMENTS
This work was supported by grants from the US National Institutes of Health to
P.A.C. and J.W. and from the Ellison Medical Foundation to P.A.C., by a Canadian
Institutes for Health Research postdoctoral fellowship to P.R.T., and by grants
from AIRC, MURST-Cofin and MURST-FIRB to M.L. W.A. was supported by a
National Research Fellowship Award. We thank C. Wolberger, M. Ott and J. Boeke
for helpful discussions and for reagents. We thank N. Rust for technical assistance.
We thank D. Leahy, R. Alani and J. Liu for comments on the manuscript.
Note: Supplementary information is available on the Nature Structural & Molecular
Biology website.
COMPETING INTERESTS STATEMENT
The authors declare competing financial interests (see the Nature Structural &
Molecular Biology website for details).
Received 22 December 2003; accepted 28 January 2004
Published online at http://www.nature.com/natstructmolbiol/
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