From the Departments of Obstetrics and
Gynecology, and ** Biological Chemistry, University of
Michigan, Ann Arbor, Michigan 48109, and the § Division of
Molecular Medicine, Department of Medicine and Department of
Biochemistry and Molecular Biology, Oregon Health and Science
University, Portland, Oregon 97201
Received for publication, January 17, 2003, and in revised form, February 3, 2003
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ABSTRACT |
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The coactivator function of
cAMP-responsive element-binding protein (CREB)-binding protein (CBP) is
partly caused by its histone acetyltransferase activity.
However, it has become increasingly clear that CBP acetylates both
histones and non-histone proteins, many of which are transcription
factors. Here we investigate the role of CBP acetylase activity in
CREB-mediated gene expression. We show that CREB is acetylated within
the cell and that in vitro, CREB is acetylated by CBP, but
not by another acetylase, p300/CBP-associated factor. The acetylation
sites within CREB were mapped to three lysines within the CREB
activation domain. Although inhibition of histone deacetylase activity
results in an increase of CREB- or CBP-mediated gene expression,
mutation of all three putative acetylation sites in the CREB activation
domain markedly enhances the ability of CREB to activate a
cAMP-responsive element-dependent reporter gene.
Furthermore, these CREB lysine mutations do not increase interaction
with the CRE or CBP. These data suggest that the transactivation
potential of CREB may be modulated through acetylation by CBP. We
propose that in addition to its functions as a bridging molecule and
histone acetyltransferase, the ability of CBP to acetylate CREB may
play a key role in modulating CREB-mediated gene expression.
Cyclic AMP (cAMP) is a second messenger produced in cells in
response to neurotransmitters and hormones (1). Increases in cAMP
levels activate a cAMP-dependent protein kinase, protein kinase A (PKA),1 which in
turn phosphorylates transcription factors, resulting in activation of
gene transcription. The transcription factor CREB (CRE-binding protein)
is the best studied link between PKA activation and gene transcription.
CREB was originally described as a transcription factor that binds to
an 8-bp element known as cAMP-response element (CRE) in the
somatostatin gene promoter (2). This DNA element mediates transcription
in response to changes in cAMP levels. Subsequently, CREs were found in
promoters of other genes activated by cAMP (3). The critical step in cAMP-induced, CREB-mediated gene expression appears to be
phosphorylation of CREB by PKA at a single serine (Ser-133). CREB, when
phosphorylated at Ser-133, binds to a nuclear protein, CBP (4), and a
closely related protein p300 (5), a protein first identified through its ability to associate with E1A (6). p300/CBP has been shown to interact with many cellular proteins, many of which are
transcription factors, supporting the concept that these coactivators
may function more generally in signal integration (7).
Precisely how CBP affects gene transcription has not been resolved. One
model is that CBP links DNA-bound activators to the general
transcription machinery (8-11). In addition to its "bridging" function, p300/CBP (12) and its associated protein P/CAF (13) may
enhance gene transcription by remodeling chromatin through the
acetylation of histones. To date, several known transcriptional regulators are known to possess intrinsic histone
acetyltransferase activity: GCN5 and its homologs (14, 15),
P/CAF (13), p300/CBP (16), TAFII 250 (17), and the nuclear hormone
receptor coactivators, SRC-1 (18) and ACTR (19). The targets of histone
acetyltransferases are not restricted to histones, however (for a
review, see Ref. 20). Acetylation of general transcription factors such
as TFIIE and TFIIF (21) has also been demonstrated. Acetylation of
factors related to transcription can either have positive or negative effects on transcriptional regulation. For example, acetylation of
tumor suppressor p53 at Lys-373 and Lys-382 by CBP increases p53 DNA
binding (22-24); in contrast, CBP acetylation of a
Drosophila protein TCF at Lys-25 inhibits its interaction
with the coactivator Armadillo, resulting in reduction of gene
expression (25). These studies demonstrate that in some cases,
acetylation of histones by histone acetyltransferases may not be the
primary event in regulation of the activity of these transcription pathways.
Acetyltransferase activity is critically important for the coactivator
function of CBP (26, 27). The observation that CBP and p300 may
acetylate other non-histone proteins leads us to investigate whether
CBP could acetylate CREB and whether acetylation of CREB influenced its
activation function. Treatment with deacetylase inhibitors such as
trichostatin A (TSA) and butyrate has been shown to enhance
CREB-mediated gene transcription on a stably transfected CRE reporter
but not on a transiently transfected CRE reporter (28). In addition,
TSA treatment prolongs the phosphorylation of CREB after forskolin
stimulation, suggesting that acetylation plays a role in regulating
CREB function, perhaps at the level of regulating phosphorylation of
CREB. However, in contrast to these findings, we demonstrate here that
TSA treatment enhances a somatostatin reporter gene activity in a
transient transactivation assay. Furthermore, we show that CBP, but not
P/CAF, acetylates CREB in vitro and that CREB is acetylated
within the cell. We mapped the CBP-acetylated lysines to three of the
five lysines within the CREB activation domain. Substitution mutations
of the target lysines within the CREB activation domain which are
acetylated by CBP result in enhancement of CREB-mediated gene
expression. These results suggest that acetylation of CREB by CBP may
modulate CREB intrinsic activity as a transcriptional activator.
Expression Vectors--
The construction of Rc/RSV-FLAG-CREB341
was described by Kwok et al. (8). pcDNA3-FLAG-CREB was
subcloned from the HindIII and XbaI fragments of
Rc/RSV-FLAG-CREB. Rc/RSV-FLAG-CREB lysine mutants were generated by
site-directed mutagenesis (Stratagene). pET23b CREB His6 WT
and its mutants were generated using PCR, and the PCR fragments were
subcloned into pET23b in-frame with six copies of histidine at the
carboxyl terminus. GAL4-CREB1-283 was constructed by fusing the GAL4
DNA binding domain(1-147) to the amino terminus of CREB1-283.
GAL4-CBP CBD(451-682) was described by Kwok et al. (29).
VP16-CREB341 and its lysine mutants were constructed by fusing CREB341
to the carboxyl terminus of the activation domain of VP16. All
sequences were confirmed by sequencing.
Recombinant Proteins--
The procedure to generate purified CBP
with two copies of FLAG tag (CBP 2XFLAG) and FLAG-P/CAF was described
by Kashanchi et al. (30). Baculovirus expressing FLAG-P/CAF
was obtained from Rich Maurer. His-tagged CREB341 protein, the CREB
activation domain (CREB1-283), and its lysine mutants were produced in
bacteria and purified using nickel-nitrilotriacetic acid resin as
described by the manufacturer (Qiagen).
F9 Cell Transactivation Assay--
The F9 cell transactivation
assay was described by Kwok et al. (8). F9 cells were plated
at 0.15 × 106 cells/60-mm plate. DNA was transfected
using calcium phosphate precipitation (Invitrogen). Rc/RSV vector was
used to normalize the total DNA used for each sample. Procedures to
determine chloramphenicol acetyltransferase (CAT) and luciferase
activities were described by Kwok et al. (8).
Cell Labeling--
COS-7 cells were seeded at 0.7 × 106 cells/100-mm plate and were maintained in 10% fetal
bovine serum (Invitrogen) in Dulbecco's modified Eagle's medium. A
day later, the cells were transfected with 15 µg of
pcDNA3-FLAG-CREB WT using calcium phosphate precipitation (Invitrogen). Control cells were transfected with pcDNA3 alone. Two
days later, the cells were incubated with 1 mCi/ml sodium [3H]acetate (2.5Ci/mmol) (ICN) for 1 h at 37 °C
in 5% CO2. The labeled proteins were then subjected to
immunoprecipitation as described by Kwok et al. (29) using
FLAG-M2 antibodies (Sigma). The precipitated proteins were separated by
10% SDS-PAGE, enhanced with Amplify (Amersham Biosciences), dried, and
exposed to x-ray film (Kodak) at Phosphorylation of CREB by PKA--
Purified CREB341 proteins
were phosphorylated by recombinant catalytic subunit of PKA in the
presence of ATP as described by Kwok et al. (8, 29).
Acetylation of CREB by CBP--
Purified CREB341 wild-type (WT),
CREB1-283, or its lysine mutant proteins (0.5 µM) were
acetylated in the presence of purified full-length 50 nM
FLAG-tagged CBP and 14C-acetyl-CoA (Amersham Biosciences)
(60 mCi/mmol, final concentration of acetyl-CoA, 50 µM in 20 µl). The reaction buffer contained 10 mM Tris (pH 7.6), 150 mM NaCl, 1 mM
EDTA, 1 mM dithiothreitol, 10 mM sodium
butyrate, and 5% glycerol. The reaction was carried out at 30 °C
for 1 h. After acetylation, the acetylated proteins were resolved
by SDS-PAGE; the gels were stained with Coomassie Blue and destained by
acetic acid/methanol, dried, and exposed to a Bio-Rad
phosphorimaging screen. The 14C signal was detected
using a Bio-Rad FX phosphorimager.
Acetylation of Peptides by CBP and P/CAF--
CREB
peptides and histone H3(7-22) peptide were synthesized either by Sigma
Genosys or by the Protein Core Facility at the University of Michigan.
All the peptides were purified by high performance liquid
chromatography to >95% purity. Individual peptides (0.5 µg)
were incubated with purified 0.1 µg of CBP or 0.1 µg of P/CAF in
the presence of [3H]acetyl-CoA (Amersham Biosciences)
(5.4 Ci/mmol acetyl-CoA, 2.3 µM final concentration) in a
20-µl reaction. The reaction buffer was the same as described above.
The reaction was carried out at 30 °C for 1 h and then spotted
on a P81 filter paper (Whatman), dried for 5 min, and washed three
times with 0.1% phosphoric acid. The filter paper was air dried and
counted using liquid scintillation counting.
Inhibition of Deacetylase Activity Enhances CREB-mediated Gene
Expression in a Transient Transfection Assay--
To investigate the
role of CREB acetylation in gene activation, we tested whether
inhibition of deacetylases (resulting in increases in acetylation)
would affect CREB-mediated gene expression. It has been reported that
TSA treatment enhances CREB-mediated gene transcription of a stably
transfected CRE reporter but not of a transient transfected CRE
reporter (28). Although the status of packaging of transiently
transfected DNA into chromatin is controversial, some have argued that
transiently expressed DNA is not arranged in regular chromatin arrays
(31). We demonstrate, however, that expression of a transiently
transfected somatostatin CRE-CAT (SRIF-CAT) reporter in F9 cells
is augmented by TSA in a dose-dependent manner (Fig.
1). These results suggest that, at least
in these F9 cells, either the transiently transfected SRIF-CRE reporter
assembles into a TSA-sensitive chromatin, or, alternatively,
acetylation of non-histone proteins may determine activity of the
SRIF-CAT reporter in this context.
CBP Acetylates CREB within Its Activation Domain--
To
investigate whether the acetyltransferase activity of CBP, apart from
its role as a histone-acetylating enzyme, plays a role in PKA-activated
gene expression, we tested whether CBP acetylates CREB. Using
recombinant purified full-length CBP and purified CREB protein, we show
that in vitro both CREB and PKA-phosphorylated CREB are
acetylated by CBP (Fig. 2B,
upper panel). Because in our experiments the efficiency of
PKA phosphorylation of CREB is close to 95% (data not shown), the
possibility that CBP acetylates a subpopulation of nonphosphorylated
CREB is minimal.
CREB belongs to the leucine zipper family of transcription factors
consisting of separable activation domain (AD) (1-283) and
DNA-binding/dimerization (bZIP) domain (284-341) (32, 33). Of 15 potential lysine acetylation sites in CREB, 5 lysines are located
within the CREB-AD and 10 lysines within the bZIP domain. Interestingly, when we mutated all 5 lysines (Fig. 2A;
Lys-91, Lys-94, Lys-123, Lys-136, Lys-155) to alanine within the
CREB-AD of full-length CREB (CREB 5K/A), we found that acetylation by CBP was markedly diminished compared with that of CREB WT (Fig. 2B). These results suggest that the major CBP acetylation
sites within CREB are located within the activation domain of CREB and that lysines within the bZIP domain of CREB are not the primary targets
of CBP acetylation in the context of the full-length protein.
To map the CBP acetylation sites within the CREB-AD, purified
bacterially expressed WT CREB1-283 protein and the mutant proteins containing single lysine residue were used in the in vitro
acetylation assays. Single lysine mutants were generated by mutation of
4 of the 5 lysines to alanine. As a negative control, all five lysines were mutated to alanine (5K/A). Single lysine mutant proteins and WT
CREB-AD(1-283) were incubated in vitro with recombinant CBP
and [14C]acetyl-CoA. In these experiments, CBP
preferentially acetylates Lys-91 and Lys-136, and to a lesser extent,
Lys-94 (Fig. 2C).
Although CBP acetylates CREB in vitro, CREB could also be a
substrate of an alternative acetylase within the cell. Thus we also
tested whether P/CAF could acetylate peptides corresponding to each of
the potential CREB acetylation sites (sequences of individual peptide
are shown in Fig. 2A). The results shown in Fig.
2D indicate that although CBP acetylates CREB peptides
containing Lys-91/Lys-94 and Lys-136, these peptides are not substrates
for P/CAF-dependent acetylation. As a positive control,
both CBP and P/CAF acetylate a histone H3(7-22) peptide. These results
indicate that CREB-AD is a substrate for CBP, but not P/CAF, in
vitro and potentially within the cell.
CREB Is Acetylated within the Cell--
To determine whether CREB
is acetylated within the cell, we transfected COS-7 cells with a
FLAG-CREB expression vector and then labeled the cells with sodium
[3H]acetate. Labeled FLAG-CREB proteins were
immunoprecipitated using the FLAG-M2 monoclonal antibody, separated by
SDS-PAGE, and visualized by fluorography. The results shown in Fig.
3A demonstrate that CREB is
acetylated in COS-7 cells. To confirm that incorporation of sodium
[3H]acetate corresponded to bona fide
acetylation of CREB in vivo, FLAG-CREB was
immunoprecipitated from transfected COS-7 cells and subjected to
Western blotting with an acetyllysine (anti-Ac-Lys)-specific monoclonal
antibody (4G12, Upstate Biotechnology) (Fig. 3B).
Substitution Mutation of Acetylation Sites Enhances the
Transactivation Potential of CREB--
We next asked what role the
acetylated lysines played in the transactivation potential of CREB. For
these experiments, we individually mutated Lys-91, Lys-94, and Lys-136
to alanine and tested the transcriptional activity of these CREB lysine
mutants for activation of the cAMP-responsive SRIF-CAT reporter. As
controls, we also mutated Lys-123 and Lys-155, which are not acetylated by CBP, to alanine.
As we and others have demonstrated previously (8, 9), CREB WT activates
the SRIF-CAT reporter in a PKA- and dose-dependent manner
(Fig. 4A, black
squares). The dose-response curves of CREB with single lysine
mutations (K91A, K94A, and K136A) were similar, with a slight increase
in activity relative to that of CREB WT (Fig. 4A). In the
absence of the catalytic subunit of PKA, there were no differences
between the activities of the CREB WT and the single lysine mutants
(Fig. 4B). At the plateau level of activation (3.6 µg of
CREB), there is a slight, but not significant, increase in
transactivation by K91A, K94A, and K136A (Fig. 4C). Fig.
4D demonstrates similar levels of CREB expression in the
samples used in Fig. 4C. These results suggest that single
mutation of the putative CBP acetylation sites has no significant
effect on the transactivation potential of CREB.
Because in vitro mapping experiments indicated that CBP
acetylates as many as 3 lysine residues in the activation domain of CREB, we next tested whether multiple mutations involving Lys-91, Lys-94, and Lys-136 would affect CREB-mediated gene expression. We
first generated CREB double lysine mutants and tested their ability to
enhance transcription. With cotransfection of the catalytic subunit of
PKA, the CREB double lysine mutants, K91A/K94A, K91A/K136A, and
K94A/K136A, significantly enhance CRE-dependent
transcription in a dose-dependent manner (Fig.
5A) and have a higher plateau level of activation (3.6 µg of CREB expression vector level) (Fig. 5C). Interestingly, the transactivation activity of the
double lysine mutants involving two of the three acetylated lysines
(Lys-91, Lys-94, and Lys-136) shows increases in basal,
non-PKA-dependent transactivation (Fig. 5, B and
C). These results prompted us to test whether mutation
involving all 3 lysines would achieve maximum enhancement of gene
transcription. The results shown in Fig. 6, A and C, indicate
that although the expression of each mutant is similar at the 3.6 µg
of transfected CREB expression vector (Fig. 6D), the
transactivation potential of the triple lysine mutant (K91A/K94A/K136A)
is 4 times higher than that of CREB WT. In contrast, the
transactivation potentials of mutant K91A/K94A/K123A (Fig. 6,
A and C) and mutant K91A/K94A/K155A (Fig.
6C) are no different from that of the double lysine mutant
(K91A/K94A), which is about 2.5 times the CREB WT. The transactivation
potential of CREB lacking all 5 lysines within the CREB-AD is the same
as that of the triple lysine mutant (K91A/K94A/K136A) (Fig.
6C), suggesting that mutation of Lys-123 and/or Lys-155,
which are not acetylated by CBP (Fig. 2), have no additional effect on
CREB-mediated gene activation. As shown in Fig. 5, B and
C, the basal activity (without PKA) of the double and triple
lysine mutants is higher compared with the CREB WT (Fig. 6,
B and C).
Because a Lys Lysine Acetylation Sites within the Activation Domain of CREB Are
Not Required for Its Interaction with the CRE or CBP--
One
explanation of the observed enhancement of CREB-mediated gene
expression is that the CREB lysine mutants may have enhanced interaction with CRE. To test this model, we fused the CREB-AD to the
DNA binding domain of the activator GAL4 and tested the ability of
GAL4-CREB-AD and its lysine mutants to activate a GAL4 UAS-dependent-CAT reporter (5XGAL4-CAT). We reasoned that
fusion of the CREB activation domain to a heterologous DNA binding
domain would distinguish between mutation-dependent
alterations in intrinsic transactivation potential from influences on
the DNA binding activity of CREB. However, the results shown in Fig.
8 demonstrate that mutation of these
lysines alters the intrinsic activity of CREB in the absence of the
CREB DNA binding domain. Like the augmentation of the activity of the
triple lysine mutant in the context of full-length CREB, GAL4-CREB-AD
K91A/K94A/K136A shows higher activity than GAL4-CREB-AD K91A/K94A,
GAL4-CREB-AD K91A, or GAL4-CREB-AD WT. These results suggest that
lysine mutations within CREB-AD do not alter the ability of CREB to
interact with the CRE but rather alter AD function.
Lysine mutations within the CREB-AD may also enhance the
transactivation function of CREB by enhancing its interaction with CBP.
To address this issue, we asked whether increasing CBP expression would
enhance the transcriptional activity of CREB lysine mutants as one
would expect if these lysines influenced the interaction of CREB with
CBP. We have shown previously that in F9 cells, coexpression of CBP
enhances CREB-mediated gene expression (8). However, the results shown
in Fig. 9, A and B,
do not support this hypothesis. In the absence of coexpressed CBP, the
transactivation activity of the CREB lysine mutants is increased in a
PKA-dependent manner. Cotransfection of CBP enhances CREB
WT as well as its lysine mutants in a dose-dependent and
parallel manner, suggesting that mutation of the lysine acetylation
sites within the CREB-AD does not affect the interaction of CREB with
CBP. In the absence of coexpressed catalytic subunit of PKA, CBP has no
effect on the transactivation activity of CREB WT or of its lysine
mutants (Fig. 9B).
To confirm the results shown in Fig. 9, A and B,
we performed a mammalian two-hybrid assay using a GAL4 DNA binding
domain fusion with the CREB binding domain of CBP (GAL4-CBP CBD) and CREB WT and its lysine mutants fused to the carboxyl terminus of the
activation domain of VP16. The results shown in Fig. 9, C
and D, suggest that VP16 CREB WT and its lysine mutants
activate GAL4-CBP CBD in a dose-dependent manner with the
cotransfection of the catalytic subunit of PKA. As a control, CREB M1,
in which the PKA phosphorylation site (Ser-133) is mutated to alanine, does not activate the GAL4-CBP CBD. Without the cotransfection of the
catalytic subunit of PKA, VP16 CREB WT as well as its lysine mutants do
not activate GAL4-CBP CBD (Fig. 9D).
It has become increasingly clear that in addition to its bridging
function and its histone acetylase activity, p300/CBP regulates the
activity of transcription factors and other nuclear proteins by
acetylation (20). Although a previous report demonstrated that
inhibition of deacetylases enhances CREB-mediated transcription from a
stably transfected reporter but not from a transiently transfected
reporter (28), our results show that TSA treatment significantly
enhances the SRIF-CAT reporter activity in transiently transfected
cells. The differences between our results and this previous study may
be the result of differences in the cell lines used: we used F9 cells
in contrast to the NIH 3T3 cell line D5 used in their study.
Nevertheless, our results suggest that acetylation of
non-histone proteins may be responsible for the activation of
CREB-mediated expression. We find that CREB is acetylated at 3 lysines
within its activation domain by both CBP and p300. Mutation of these
lysines significantly enhances CREB-mediated gene expression, suggesting that regardless of the acetylation state of chromatin proteins (transiently transfected templates versus stably
transfected templates), acetylation of CREB augments the
transactivation potential of CREB. We propose a model in which when
CREB is activated by PKA phosphorylation, it recruits p300/CBP, and
p300/CBP in turn acetylates CREB.
p300/CBP Specifically Acetylates the Activation Domain of
CREB--
In this study we demonstrate that CREB is specifically
acetylated by CBP and p300, but not by P/CAF. Bannister et
al. (34) have suggested that a glycine or a serine residue
immediately before the acetylated lysine is important for CBP
acetylation. However, the sequences surrounding the 5 lysines within
the CREB-AD do not fit this pattern (Fig. 2A). Thompson
et al. (35) reported that a positively charged residue
(either lysine or arginine) at either the
Nevertheless, the sequence surrounding Lys-136 does not fit the CBP
consensus acetylation sequence described by either Bannister et
al. (34) or Thompson et al. (35). Moreover, the levels of acetylation by CBP of Lys-91, Lys-94, and Lys-136 differ: Lys-136 has the highest and Lys-94 the lowest level in vitro (Fig.
2). The lower acetylation level of Lys-91 and Lys-94 may be because the
CREB1-283 mutant that contains only Lys-91 has a K94A(+4 lysine) mutation, which disrupts the putative consensus acetylation site described by Thompson et al. Likewise, the CREB1-283 mutant
that contains only Lys-94 has a K91A mutation ( Acetylation Alters the Activity of the CREB Transactivation
Domain--
In the simplest model, the mechanism by which CREB
acetylation might augment CREB activation of gene expression is that
these lysines participate in restraining the conformation of the CREB molecule, allowing CREB to enhance gene expression by increasing its 1)
interaction with CRE, 2) interaction with CBP, 3) sensitivity to
phosphorylation by PKA, and 4) prolonging the dephosphorylation rate of
CREB.
Several transcription factors, when acetylated by CBP, increase their
binding to DNA (20). CREB has been shown to bind to the CRE with high
affinity, but the role of phosphorylation in regulating DNA association
remains controversial (40-42). Studies have shown that Tax-1, a human
T-cell leukemia virus type 1 protein, facilitates the binding of CREB
to the Tax-response element (which is also a CRE) by a direct
interaction with CREB and flanking DNA (43, 44). These studies suggest
that the binding of CREB to CRE may be altered by other proteins. It is
possible that lysine mutation within the CREB-AD may allow CREB to
interact with other proteins, resulting in an increase in interaction
with CRE. Our results however indicate that CREB acetylation alters
transactivation potential independent of the CREB DNA binding domain
and interaction with the CRE (Fig. 8).
CREB acetylation or mutation of these lysines may enhance the
interaction with CBP. Recent studies have shown that the recruitment of
CBP to CREB is a critical factor in CREB-activated gene expression. Cardinaux et al. (45) produced a constitutively active CREB by substituting the CREB kinase-inducible domain with the
CBP-interacting sequence of SREBP (DIEDML) (46). This chimeric CREB
protein activates the somatostatin CRE reporter independently of PKA by constitutively binding CBP. Mutation of Tyr-134 to phenylalanine (Y134F) increases the phosphorylation of CREB by PKA and permits CREB
to interact with CBP in the absence of PKA in vivo (47). Conversely, Shaywitz et al. (48) have shown that altering 1 amino acid (L607F) within the CREB binding domain of CBP increases the
binding strength of the kinase-inducible domain of CREB, even in the
absence of PKA phosphorylation. These results suggest that the
transactivation potential of CREB is a function of the interaction between CREB and CBP. However, using the F9 transactivation assay (Fig.
9, A and B) and the mammalian two-hybrid assay
(Fig. 9, C and D), we show that CREB lysine
mutation does not affect the ability of CREB to interact with CBP,
suggesting that, although one of the acetylated lysines is located
within the kinase-inducible domain, a region of CREB that is necessary
for the interaction of CREB with CBP, these lysines do not play a
substantial role in the interaction between CREB and CBP.
The observation that Lys-136 is acetylated by CBP is intriguing because
of its proximity to the PKA phosphorylation site, a site that is
conserved in ATF-1 (49) and CREM (50), both of which are activated by
phosphorylation. The basic residues surrounding the PKA phosphoacceptor
site are important for recognition by PKA. Arginines at What Is the Role of Acetylation in Modulating the Transactivation
Potential of CREB?--
An important factor regulating
CREB-dependent transactivation is the duration of the
phosphorylation of CREB. Studies have shown that after cAMP
stimulation, the transcriptional response follows so-called
"burst-attenuation" kinetics with maximum rates 30-60 min after
stimulation followed by a gradual attenuation phase that may last as
long as several hours (52). The attenuation phase is not dependent on a
loss of PKA activity but rather results from dephosphorylation of CREB
(52, 53). Inhibition of phosphatases prolongs the attenuation phase,
resulting in the increase in PKA-stimulated gene expression (52-55).
How dephosphorylation of CREB is regulated is not clear, however.
Michael et al. (28) demonstrated that inhibition of
deacetylase activity, without affecting phosphatase activity, prolongs
the phosphorylation of CREB after forskolin stimulation. These results
suggest that acetylation of components of the PKA signaling pathway may
regulate the dephosphorylation of CREB. Our results are consistent with
this observation.
Collectively, our results demonstrate that in addition to acting as a
bridging factor as well as a histone acetyltransferase, CBP may
regulate the intrinsic transactivation potential of CREB by directly
acetylating the activation domain of CREB. The precise mechanism of
this alteration in CREB activity is unclear. The three acetylated
lysines within the activation domain are important for the
transactivation function of CREB. Mutation of these lysines to either
alanine or arginine was equally effective in enhancing the activity of
CREB, suggesting that a lysine residue at this position rather than a
charged residue per se restrains the activity of CREB. We
postulate that acetylation, like mutation, increases the activity of
CREB, perhaps through an alteration in the structure of CREB to a more
active conformation. The structural changes induced by acetylation may
prolong CREB phosphorylation by diminishing phosphatase-dependent attenuation of CREB activity, either
by directly interfering with recruitment of phosphatases or by altering phosphatase recognition of CREB as a substrate.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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70 °C for 4 weeks.
RESULTS
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ABSTRACT
INTRODUCTION
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Fig. 1.
Inhibition of deacetylases enhances CREB- and
CBP-dependent transactivation. F9 cells were
transfected with a SRIF-CRE CAT reporter, RSV-luciferase, and 2.4 µg
of FLAG-CREB, RSV-cPKA (the catalytic subunit of PKA), and Rc/RSV-CBP.
Various concentrations of trichostatin A were used as indicated for
18 h. The results are expressed as the mean ± S.E.
(n = 3) relative CAT activity after correcting for
transfection efficiency with luciferase activity.
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Fig. 2.
CBP acetylates Lys-91, Lys-94, and Lys-136
within the CREB activation domain. A, sequences of CREB
peptides that contain lysine within the activation domain of CREB.
B, CREB is acetylated by CBP in vitro.
Acetylation was carried out with purified CBP, CREB341, or
PKA-phosphorylated CREB (P-CREB) and CREB with five Lys Ala mutations within the activation domain (5K/A) in the presence of
[14C]acetyl-CoA. Controls did not include CBP. The
proteins were separated by SDS-PAGE, dried, and exposed to a
PhosphorImager screen. C, purified CREB1-283 proteins were
used in the in vitro acetylation assay. CREB1-283 proteins
(0.5 µg) were incubated with or without 0.1 µg of purified CBP and
[14C]acetyl-CoA at 30 °C for 1 h. The proteins
were separated by SDS-PAGE, stained with Coomassie Blue, destained,
dried, and exposed to a PhosphorImager screen. CREB1-283 (5K/A)
protein has all 5 lysines mutated to alanine
(K91A/K94A/K123A/K136A/K155A). The rest of the CREB1-283 lysine
mutants have all lysines but 1 (as indicated) mutated to alanine. A
scan of the Coomassie stain of the CREB1-283 proteins used in the
assay is shown in bottom panel of C. D, CREB peptides or histone H3 peptide was incubated with
CBP or P/CAF as indicated in the presence of
[3H]acetyl-CoA. The acetylated peptides were precipitated
on P81 filter paper and counted for 3H activity by liquid
scintillation counting. Results are expressed as the mean ± S.E.
(n = 3).
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Fig. 3.
CREB is acetylated by CBP in cells.
A, COS-7 cells were either transfected with pcDNA3
vectors alone (control) or with pcDNA3 FLAG-CREB
(F-CREB). The cells were labeled with sodium
[3H]acetate, and whole cell extracts were
immunoprecipitated using FLAG-M2 antibodies. The precipitates were then
separated by SDS-PAGE, fixed, enhanced using Amplify (Amersham
Biosciences), dried, and exposed to x-ray film. B, COS-7
cells were either transfected with pcDNA3 vectors alone (control)
or with pcDNA3 FLAG-CREB341. The cell extracts were
immunoprecipitated using FLAG-M2 antibodies. The precipitates were
separated by SDS-PAGE. The separated proteins were transferred to a
polyvinylidene difluoride membrane and probed with FLAG-M2
antibodies and an anti-acetyllysine antibody (Ac-Lys-Ab).
View larger version (27K):
[in a new window]
Fig. 4.
Single lysine mutations within the CREB
activation domain have no effect on CREB-mediated gene activation in F9
cells. F9 cells were transfected with the SRIF-CRE CAT reporter,
RSV-luciferase, and varying amounts (1.2, 2.4, 3.6, or 4.8 µg) of
either Rc/RSV-FLAG-CREB WT or Rc/RSV-FLAG-CREB lysine mutants, with
(A) or without (B) RSV-cPKA (the catalytic
subunit of PKA). The results are expressed as CAT activity after
correcting for transfection efficiency with luciferase activity. In
A, the experiment was repeated more than three times with
similar results. The results shown are a representation of one
experiment. In C, 3.6 µg of Rc/RSV FLAG-CREB or its lysine
mutants was used. The data are expressed as the mean ± S.E.
(n = 3). Dark and white bars
represent with or without the cotransfection of RSV-cPKA, respectively.
D, expression of each CREB protein is shown. Equal amounts
of protein were used per lane from the extracts of the
experiments from C. The proteins were separated by 10%
SDS-PAGE and probed with FLAG M2 antibodies and anti -tubulin
antibodies.
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[in a new window]
Fig. 5.
Double mutations of Lys-91, Lys-94, or
Lys-136 to alanine enhance CREB-mediated gene activation in F9
cells. The format of the experiments is identical to that
described in Fig. 3. In C, the data are expressed as the
mean ± S.E. (n = 3). * represents statistical
significance (Tukey test) at p 0.01 compared
with CREB WT.
View larger version (24K):
[in a new window]
Fig. 6.
Triple lysine mutations within CREB enhance
CREB-mediated gene expression. The format of the experiments is
identical to that described in Fig. 3. In C, the data are
expressed as mean relative CAT activity ± S.E. (n = 3). * represents statistical significance (Tukey test) at
p 0.01 compared with CREB WT.
Ala mutation neutralizes the positive charge of
lysine, it is possible that increases in transactivation caused by a
Lys
Ala mutation is caused by an alteration in charge. To address
this issue, we mutated each lysine within the CREB-AD to arginine (Lys
Arg) and tested the transactivation activity of these mutants for
activation of the SRIF-CAT reporter. The results shown in Fig. 6
indicate that the double lysine (K91R/K94R) and triple-lysine
(K91R/K94R/K136R) mutants enhance the activity of CREB in a
dose-dependent manner (Fig.
7A) and also at plateau expression levels (Fig. 7C). Thus, the pattern of
transactivation of CREB Lys
Arg mutants is similar to that
described for the CREB Lys
Ala mutants (Fig. 6). These data
indicate that lysine per se, not the charge, determines the
function of CREB.
View larger version (21K):
[in a new window]
Fig. 7.
Mutation of Lys-91, Lys-94, and Lys-136 to
arginine enhances CREB-mediated gene activation in F9 cells. The
format of the experiments is identical to that described in Fig. 3. In
C, the data are expressed as mean relative CAT activity ± S.E. (n = 3). * represents statistical significance
(Tukey test) at p 0.01 compared with CREB
WT.
View larger version (15K):
[in a new window]
Fig. 8.
GAL4-CREB1-283 lysine mutants enhance
GAL4-CREB-mediated gene expression. F9 cells were transfected with
a 5XGAL4-CAT reporter, RSV-luciferase, and either Rc/RSV GAL4-CREB WT
or its lysine mutants. In A, 0.125, 0.25, 0.5, or 1 µg of
expression vectors of GAL4-CREB1-283 or its lysine mutants was used.
RSV-cPKA was cotransfected. The results are expressed as CAT activity
after correcting for transfection efficiency with luciferase activity.
In A, the experiment was repeated three times with similar
results. The results shown are a representation of one experiment. In
B, 1 µg of the expression vector of GAL4-CREB1-283 or its
lysine mutants was used. Dark and white bars
represent with or without the cotransfection of RSV-cPKA, respectively.
The results are expressed as mean relative CAT activity ± S.E.
(n = 3). * represents statistical significance (Tukey
test) at p 0.01 compared with GAL4-CREB WT.
View larger version (23K):
[in a new window]
Fig. 9.
CREB lysine mutations do not affect the
interaction with CBP. In A and B, F9 cells
were transfected with the SRIF-CAT reporter, RSV-luciferase, Rc/RSV
FLAG-CREB, or its lysine mutants (3.6 µg) with (A) or
without (B) the cotransfection of the catalytic subunit of
PKA and various amounts of Rc/RSV-CBP-HA-RK as indicated. Results are
expressed as CAT activity after correcting for transfection efficiency
with luciferase activity, and the experiments were repeated more than
three times with similar results. In C and D, F9
cells were transfected with a 5XGAL4-CAT reporter, RSV-luciferase, and
Rc/RSV GAL4-CBP 451-682 (0.5 µg), with C or without
D the cotransfection of the catalytic subunit of PKA, and
various amounts of Rc/RSV VP16 CREB341 or its lysine mutants, as
indicated. Results are expressed as the mean relative CAT activity
after correcting for transfection efficiency with luciferase activity,
and the experiments were repeated more than three times with similar
results.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
3 or +4 position relative
to the acetylated lysine is required for CBP acetylation. Although not
all CBP acetylation sites fit this profile, the sequences surrounding 2 of the 5 lysines (Lys-91 and Lys-94) within the CREB-AD fit the pattern
described by Thompson et al. (35) (Fig. 2A).
Furthermore, Lys-91 and Lys-94 locate within the
-peptide region of
the CREB molecule, and in some isoforms of CREB, such as CREB
(also
known as CREB327), this region is deleted by alternative splicing (36,
37). Studies have shown that CREB341 and CREB327 are uniformly
expressed in most tissues (38) and that CREB327, like CREB341, acts as
a transcriptional activator of cAMP-mediated gene expression (37, 38).
However, one study has suggested that CREB327 may act as an inhibitor
of CREB341 (39). In our experiments, mutation of Lys-122 (equivalent to
Lys-136 of CREB341), like that of CREB341, markedly increases the
ability of CREB327 to activate the SRIF-CRE CAT reporter in the F9
transactivation assay (data not shown).
3 lysine).
Determination of the true relative degree of acetylation by CBP awaits
more detailed kinetic measurements.
3 and
2
positions relative to the phosphorylation site are preferred for
phosphorylation by PKA (51). However, the necessity for basic residues
carboxyl-terminal to the PKA phosphorylation site is unclear. Du
et al. (47) demonstrated that simultaneous mutation of
Arg-135 and Lys-136 to glutamine converts CREB to a higher affinity
substrate for PKA, resulting in a constitutively active form of CREB.
However, it is not known whether mutations of both Arg-135 and Lys-136
are required because the contribution of each residue was not tested
individually. Nevertheless, these results suggest that acetylation of
Lys-136 may affect the phosphorylation of CREB by PKA. In the absence of the coexpressed catalytic subunit of PKA, CREB lysine mutants show a
slight increase in basal activity, perhaps because of an increase in
its affinity for PKA as suggested by Du et al. (47). If
mutation of these lysines increased phosphorylation, we would expect an
enhanced interaction of CREB with CBP in the mammalian two-hybrid
assay; however, results shown in Fig. 9D do not support this
model. Our results suggest that Lys-91, Lys-94, and Lys-136 restrain
the transactivation potential of CREB in a manner separable from
effects on phosphorylation, CBP binding, or interaction with DNA.
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ACKNOWLEDGEMENTS |
---|
We thank Madeleine Pham for technical support. We are grateful to Drs. Sarah Smolik and Julie Broadbent for comments on this manuscript.
![]() |
FOOTNOTES |
---|
* This work supported in part by NIDDK National Institutes of Health Grant 5P60DK-20572, Public Health Services NIDDK National Institutes of Health Grants DK051732 and DK060133 (to J. R. L.), and by an American Cancer Society research grant (to R. P. S. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Scholar of the Mallinckrodt Foundation.
To whom correspondence should be addressed: Dept. of
Obstetrics and Gynecology, and Biological Chemistry, University of
Michigan, 6428 Medical Science Bldg. 1, 1301 E. Catherine St., Ann
Arbor, MI 48109. E-mail: rkwok@umich.edu.
Published, JBC Papers in Press, February 20, 2003, DOI 10.1074/jbc.M300546200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PKA, cAMP-dependent protein kinase (protein kinase A); AD, activation domain; bZIP, DNA binding/dimerization domain; CAT, chloramphenicol acetyltransferase; CBD, CREB binding domain; CBP, CREB-binding protein; CRE, cAMP-responsive element; CREB, cAMP-responsive element-binding protein; CREB 5K/A, five lysine mutations within CREB; P/CAF, p300/CBP-associated factor; SRIF, somatotropin release inhibiting factor; RSV, Rous sarcoma virus; TSA, trichostatin A; WT, wild-type.
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