Acetylation of CREB by CBP enhances CREB-dependent transcription
Qing Lu1, Amanda E. Hutchins1, Colleen M. Doyle1, James R. Lundblad2,and Roland P.S. Kwok 1 *.
1 Departments of Obstetrics and Gynecology, and Biological Chemistry, University of Michigan,
Ann Arbor, Michigan, 48109; 2 Division of Molecular Medicine, Department of Medicine, andDepartment of Biochemistry and Molecular Biology, Oregon Health & Science University,
Portland, Oregon 9720
*Author to whom correspondence should be addressed
Roland P.S. Kwok, Ph.D.Departments of Obstetrics and Gynecology, and Biological Chemistry,
University of Michigan
6428 Medical Science Building 11301 E. Catherine Street,
Ann Arbor, MI, 48109Tel: 734-615-1384
Fax: 734-936-8617
Email: [email protected]
Running Title: CREB acetylation by the coactivator CBP.
Key Words: CREB, CBP, p300, acetylation, P/CAF
Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on February 20, 2003 as Manuscript M300546200 by guest on July 9, 2018
http://ww
w.jbc.org/
Dow
nloaded from
2
Summary
The co-activator function of CREB-binding protein (CBP) is partly due to its histone
acetyltransferase activity. However it has become increasingly clear that CBP acetylates bothhistones 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 anotheracetylase, p300/CBP associated factor (P/CAF). The acetylation sites within CREB were
mapped to three lysines within the CREB-activation domain. While inhibition of histonedeacetylase 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 CRE-dependent reporter gene. Furthermore, these CREB lysinemutations 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 abilityof CBP to acetylate CREB may play a key role in modulating CREB-mediated gene expression.
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
3
Introduction
Cyclic adenosine monophosphate (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, PKA, 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 describedas a transcription factor that binds to an 8 base-pair element known as cAMP-response element
(CRE) in the somatostatin gene promoter (2). This DNA element mediates transcription inresponse 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 (serine-133).CREB, when phosphorylated at serine-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). CBP/p300 has been shown to interact with many cellular proteins, many of which aretranscription factors, supporting the concept that these co-activators 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 toits “bridging” function, CBP/p300 (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 (HAT) activity: GCN5 and its homologues (14) (15), P/CAF (13), CBP/p300
(16), TAFII 250 (17), and the nuclear hormone receptor co-activators, SRC-1 (18) and ACTR(19). The targets of HATs are not restricted to histones, however (for a review, see (20)).
Acetylation of general transcription factors, such as TFIIE, and TFIIF (21), has also beendemonstrated. Acetylation of factors related to transcription can either have positive or
negative effects on transcriptional regulation. For example, acetylation of tumor suppressor
p53 at lysines-373 and -382 by CBP increases p53 DNA binding (22-24); in contrast, CBPacetylation of a Drosophila protein TCF at lysine-25 inhibits its interaction with the co-
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
4
activator Armadillo, resulting in reduction of gene expression (25). These studies demonstrate
that, in some cases, acetylation of histones by HATs may not be the primary event inregulation of the activity of these transcription pathways.
Acetyltransferase activity is critically important for the co-activator 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 CREBinfluenced its activation function. Treatment with deacetylase inhibitors, such as Trichostatin
A (TSA) and butyrate, has been shown to enhance CREB-mediated gene transcription on astably 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 ofregulating 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 thetarget lysines within the CREB activation domain that are acetylated by CBP result in
enhancement of CREB-mediated gene expression. These results suggest that acetylation of
CREB by CBP may modulate CREB’s intrinsic activity as a transcriptional activator.
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
5
Materials and Methods
Expression Vectors The construction of Rc/RSV-FLAG-CREB341 was described in
Kwok et al. (8). pcDNA3-FLAG-CREB was subcloned from the HindIII and Xba1 fragment ofRc/RSV-FLAG-CREB. Rc/RSV-FLAG-CREB lysine mutants were generated by site-directed
mutagenesis (Stratagene). pET-23b CREB 6XHis WT and its mutants were generated using
PCR, and the PCR fragments were subcloned into pET23b in frame with six copies of histidineat the carboxyterminus. GAL4-CREB 1-283 was constructed by fusing the GAL4 DNA binding
domain (1-147) to the amino terminus of CREB 1-283. GAL4-CBP CBD (451-682) wasdescribed in Kwok et al. (29). VP16–CREB 341 and its lysine mutants were constructed by
fusing CREB 341 to the carboxy 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 in Kashanchi et al. (30). Baculovirusexpressing 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, andpurified using Ni/NTA resin as described by the manufacturer (Qiagen).
F9 cell transactivation assay The F9 cell transactivation assay was described in KwoK etal. (8). F9 cells were plated at 0.15X106 cells per 60mm plate. DNA was transfected using
calcium phosphate precipitation (Gibco). Rc/RSV vector was used to normalize total DNA usedfor each sample. Procedures to determine CAT and luciferase activities were described in Kwok
et al. (8).
Cell labeling Cos-7 cells were seeded at 0.7X106 cells per 100mm plate and were
maintained in 10% fetal bovine serum (Gibco) in DMEM. A day later, the cells were transfectedwith 15µg of pcDNA3 FLAG-CREB WT using calcium phosphate precipitation (Gibco).
Control cells were transfected with pcDNA3 alone. Two days later, the cells were incubated with
1mCi/ml [3H]-sodium acetate (2.5Ci/mmol) (ICN) for 1 hour at 370C/5% CO2. The labeledproteins were then subjected to immunoprecipitation as described in Kwok et al. (29) using
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
6
FLAG-M2 antibodies (Sigma). The precipitated proteins were separated by 10% SDS-PAGE,
enhanced with Amplify (Amersham), dried, and exposed to X-ray film (Kodak) at –700C for 4weeks.
Phosphorylation of CREB by PKA Purified CREB341 proteins were phosphorylated
by recombinant catalytic subunit of PKA in the presence of ATP as described in Kwok et al.
(8,29).
Acetylation of CREB by CBP Purified CREB341 wildtype (WT), CREB 1-283, or itslysine mutant proteins (0.5mM) were acetylated in the presence of purified full-length FLAG-
tagged CBP (50nM) and 14C-acetyl coenzyme A (Amersham) (60mCi/mmol, finalconcentration of acetyl coenzyme A 50mM in 20ml). The reaction buffer contained 10mM
Tris (pH 7.6), 150mM NaCl, 1mM EDTA, 1mM DTT, 10mM sodium butyrate, and 5%
glycerol. The reaction was carried out at 300C for 1 hour. After acetylation, the acetylatedproteins 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 HPLC to >95% purity. Individual
peptides (0.5mg) were incubated with purified CBP (0.1mg) or P/CAF (0.1mg) in the presence
of [3H]-acetyl-coenzyme A (Amersham) (5.4 Ci/mmol acetyl-coenzyme A, 2.3mM final
concentration) in a 20ml reaction. The reaction buffer was the same as described above. The
reaction was carried out at 300C for 1 hour, and then spotted on a P81 filter paper(Whatman), dried for 5 minutes and washed 3 times with 0.1% phosphoric acid. The filter
paper was air-dried and counted using liquid scintillation counting.
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
7
Results
Inhibition of deacetylase activity enhances CREB-mediated gene expression in a transienttransfection 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-mediatedgene 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). While the status of packaging of transiently transfected DNA into chromatin iscontroversial, some have argued that transiently expressed DNA is not arranged in regular
chromatin arrays (31). We demonstrate, however, that expression of a transiently tranfectedsomatostatin CRE-CAT (SRIF-CAT) reporter in F9 cells is augmented by TSA in a dose-
dependent manner (Figure 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 (P-CREB) are acetylated by
CBP (Figure 2B, upper panel). Since in our experiments the efficiency of PKA phosphorylationof CREB is close to 95% (data not shown), the possibility that CBP acetylates a subpopulation of
non-phosphorylated 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, five lysines are located within theCREB-AD and ten lysines within the bZIP domain. Interestingly, when we mutated all five
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
8
lysines (Figure 2A; K91, K94, K123, K136, K155) to alanine within the CREB-AD of full length
CREB (CREB 5K/A), we found acetylation by CBP was markedly diminished compared to thatof CREB WT (Figure 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 bZIPdomain of CREB are not the primary targets of CBP acetylation in the context of the full-length
protein.
In order to map the CBP acetylation sites within the CREB-AD, purified bacterially
expressed wildtype CREB 1-283 protein and the mutant proteins containing single lysine residuewere used in the in vitro acetylation assays. Single lysine mutants were generated by mutation of
four of the five lysines to alanine. As a negative control, all five lysines were mutated to alanine
(5K/A). Single-lysine mutant proteins and wildtype CREB-AD (1-283) were incubated in vitro
with recombinant CBP and [14C]-acetyl-CoA. In these experiments, CBP preferentially acetylates
lysine-91 and lysine-136, and to a lesser extent, lysine-94 (Figure 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 peptidescorresponding to each of the potential CREB-acetylation site (sequences of individual peptide
are shown in Figure 2A). The results shown in Figure 2D indicate that whereas CBP acetylates
CREB peptides containing K91/K94 and K136, 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 andpotentially within the cell.
CREB is acetylated within the cell
To determine whether CREB is acetylated within the cell, we transfected Cos-7 cells witha FLAG-CREB (F-CREB) expression vector, and then labeled the cells with [3H]-sodium
acetate. Labeled F-CREB proteins were immunoprecipitated using the FLAG-M2 monoclonal
antibody, separated by SDS-PAGE, and visualized by fluorography. The results shown in Figure3A demonstrate that CREB is acetylated in Cos7 cells. To confirm that incorporation of [3H]-
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
9
sodium acetate corresponded to bona fide acetylation of CREB in vivo, F-CREB was
immunoprecipitated from transfected Cos-7 cells, and subjected to western blotting with anacetyl-lysine (anti-ac-K)-specific monoclonal antibody (4G12, Upstate Biotechnology) (Figure
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 K91, K94, and K136 to alanine, andtested the transcriptional activity of these CREB lysine mutants for activation of the cAMP-
responsive SRIF-CAT reporter. As controls, we also mutated K123 and K155, which are not
acetylated by CBP, to alanine.
As we and others have previously demonstrated (8,9), CREB WT activates the SRIF-CAT
reporter in a PKA- and dose-dependent manner (Figure 4A, closed-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 (Figure 4A). In the absence of thecatalytic subunit of PKA, there were no differences between the activities of the CREB WT and
the single-lysine mutants (Figure 4B). At the plateau level of activation (3.6mg CREB), there is a
slight, but not significant, increase in transactivation by K91A, K94A, and K136A (Figure 4C).
Figure 4D demonstrates similar levels of CREB expression in the samples used in Figure 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 involvingK91, K94 and K136 would affect CREB-mediated gene expression. We first generated CREB
double-lysine mutants and tested their ability to enhance transcription. With co-transfection ofthe catalytic subunit of PKA, the CREB double-lysine mutants, K91/94A, K91/136A, and
K94/136A, significantly enhance CRE-dependent transcription in a dose-dependent manner
(Figure 5A) and have a higher plateau level of activation (3.6ug of CREB expression vectors
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
10
level) (Figure 5C). Interestingly, the transactivation activity of the double-lysine mutants
involving two of the three acetylated lysines (K91, K94, and K136) show increases in basal, non-PKA-dependent transactivation (Figures 5B and 5C). These results prompted us to test whether
mutation involving all three lysines would achieve maximum enhancement of gene transcription.The results shown in Figure 6A and 6C indicate that while the expression of each mutant is
similar at the 3.6mg of transfected CREB expression vector (Figure 6D), the transactivation
potential of the triple-lysine mutant (K91/94/136A) is 4-times higher than that of CREB WT. In
contrast, the transactivation potentials of mutant K91/94/123A (Figure 6A and 6C) and mutantK91/94/155A (Figure 6C) are no different from that of the double-lysine mutant (K91/94A),
which is about 2.5-times of the CREB WT. The transactivation potential of CREB lacking all
five lysines within the CREB-AD is the same as that of the triple-lysine mutant (K91/94/136A)(Figure 6C), suggesting that mutation of K123 and/or K155, which are not acetylated by CBP
(Figure 2), have no additional effect on CREB-mediated gene activation. As shown in Figures5B and 5C, the basal activity (without PKA) of the double- and triple-lysine mutants is higher
compared to the CREB WT (Figures 6B and 6C).
Because mutation of a lysine to an alanine (K/A) neutralizes the positive charge of lysine,
it is possible that increases in transactivation caused by K/A mutation is due to an alteration incharge. To address this issue, we mutated each lysine within the CREB-AD to arginine (K/R)
and tested the transactivation activity of these mutants for activation of the SRIF-CAT reporter.
The results shown in Figure 6 indicate that the double-lysine (K91/94R) and triple-lysine(K91/94/136R) mutants enhance the activity of CREB in a dose-dependent manner (Figure 7A)
and also at plateau expression levels (Figure 7C). Thus, the pattern of transactivation of CREBK/R mutants is similar to that described for the CREB K/A mutants (Figures 6). These data
indicate that lysine per se, not the charge, determines the function of CREB.
Lysine acetylation sites within the activation domain of CREB are not required for itsinteraction 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
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
11
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 intrinsictransactivation potential from influences on the DNA binding activity of CREB. However, the
results shown in Figure 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 activityof the triple-lysine mutant in the context of full-length CREB, GAL4-CREB-AD K91/94/136A
shows higher activity than GAL4-CREB-AD K91/94A, 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 activation domain 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 wouldexpect if these lysines influenced the interaction of CREB with CBP. We have previously shown
that, in F9 cells, co-expression of CBP enhances CREB-mediated gene expression (8). However,the results shown in Figure 9 A and B do not support this hypothesis. In the absence of co-
expressed CBP, the transactivation activity of the CREB lysine mutants is increased in a PKA-
dependent manner. Co-transfection of CBP enhances CREB WT as well as its lysine mutants in adose-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 co-expressed catalytic subunit of PKA, CBP has no effect on the transactivation activity of CREB
WT or of its lysine mutants (Figure 9B).
To confirm the results shown in Figures 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 carboxy terminus of the
activation domain of VP16. The results shown in Figures 9 C and D suggest that VP-16 CREB
WT and its lysine mutants activate GAL4-CBP CBD in a dose-dependent manner with the co-transfection of the catalytic subunit of PKA. As a control, CREB M1, in which the PKA
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
12
phosphorylation site (serine-133) is mutated to alanine, does not activate the GAL4-CBP CBD.
Without the co-transfection of the catalytic subunit of PKA, VP-16 CREB WT as well as itslysine mutants does not activate GAL4-CBP CBD (Figure 9D).
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
13
Discussion
It has become increasingly clear that, in addition to its bridging function and its histone
acetylase activity, CBP/p300 regulates the activity of transcription factors and other nuclearproteins by acetylation (20). While 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 enhancesthe SRIF-CAT reporter activity in transiently transfected cells. The differences between our
results and this previous study may be due to the differences in the cell lines used: we used F9cells 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 three lysines within its activation domain by bothCBP 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 thetransactivation potential of CREB. We propose a model in which when CREB is activated by
PKA phosphorylation, it recruits CBP/p300, and CBP/p300 in turn acetylates CREB.
CBP/p300 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. have suggested that a glycine or a serine residue immediatelybefore the acetylated lysine is important for CBP acetylation (34). However, the sequences
surrounding the five lysines within the CREB-AD do not fit this pattern (Figure 2 A). Thompson
et al., reported that a positively charged residue (either lysine or arginine) at either the -3 or + 4position relative to the acetylated-lysine is required for CBP acetylation (35). While not all
CBP-acetylation sites fit this profile, the sequences surrounding two of the five lysines (K91 andK94) within the CREB-AD fit the pattern described by Thompson et al. (35) (Figure 2A).
Furthermore, K91 and K94 locate within the a-peptide region of the CREB molecule, and in
some isoforms of CREB, such as CREBD (also known as CREB327), this region is deleted by
alternative splicing (36,37). Studies have shown that CREB341 and CREB327 are uniformly
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
14
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 thatCREB327 may act as an inhibitor of CREB 341 (39). In our experiments, mutation of K122
(equivalent to K136 of CREB341), like that of CREB341, markedly increases CREB327’sability to activate the SRIF-CRE CAT reporter in the F9 transactivation assay (data not shown).
Nevertheless, the sequence surrounding K136 does not fit the CBP consensus acetylationsequence described by either Bannister et al. (34) or Thompson et al. (35). Moreover, the levels
of acetylation by CBP of K91, K94, and K136 differ: K136 has the highest and K94 the lowestlevel in vitro (Figure 2). The lower acetylation level of K91 and K94 may be due to the fact that
CREB 1-283 mutant that contains only K91 has a mutation of K94 (+4 lysine) to alanine, which
disrupts the putative consensus acetylation site described by Thompson et al. Likewise, CREB 1-283 mutant that contains only K94 has a mutation of K91 (-3 lysine) to alanine. Determination of
the true relative degree of acetylation by CBP awaits more detailed kinetic measurements.
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) interactionwith 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 ofphosphorylation in regulating DNA association remains controversial, (40-42). Studies have
shown that Tax-1, a Human T-cell Leukemia Virus Type 1 (HTLV-1) protein, facilitates thebinding of CREB to the Tax-response element (TRE, 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 mayallow CREB to interact with other proteins, resulting in an increase in interaction with CRE. Our
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
15
results however indicate that CREB acetylation alters transactivation potential independent of the
CREB DNA binding domain and interaction with the CRE (Figure 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 KID with the CBP-interacting sequence of SREBP (DIEDML) (46). Thischimeric CREB protein activates the somatostatin CRE-reporter independently of PKA by
constitutively binding CBP. Mutation of tyrosine-134 to phenylalanine (Y134F) increases thephosphorylation of CREB by PKA, and permits CREB to interact with CBP in the absence of
PKA in vivo (47) Conversely, Shaywitz et al. have shown that altering one amino acid (L607F)
within the CREB-binding domain of CBP increases the binding strength of the kinase inducibledomain (KID) of CREB, even in the absence of PKA phosphorylation (48). 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 (Figures 9A and B) and the mammalian two-hybrid assay (Figures 9C 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 locatedwithin the KID, 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 K136 is acetylated by CBP is intriguing due to its proximity to the
PKA-phosphorylation site, a site which is conserved in ATF-1 (49) and CREM (50), both ofwhich are activated by phosphorylation. The basic residues surrounding the PKA-
phosphoacceptor site are important for recognition by PKA. Arginines at –3 and –2 positions
relative to the phosphorylation site are preferred for phosphorylation by PKA (51). However, thenecessity for basic residues carboxy terminal to the PKA-phosphorylation site is unclear. Du et al
(47) demonstrated that simultaneous mutation of arginine-135 and lysine-136 to glutamineconverts 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 arginine-135 and lysine-136 are
required since the contribution of each residue was not individually tested. Nevertheless, theseresults suggest that acetylation of lysine-136 may affect the phosphorylation of CREB by PKA.
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
16
In the absence of co-expressed catalytic subunit of PKA, CREB-lysine mutants show a slight
increase in basal activity, perhaps due to an increase in its affinity for PKA as suggested by Du etal. (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 inFigure 9D do not support this model. Our results suggest that lysines 91, 94, and 136 restrain the
transactivation potential of CREB in a manner separable from effects on phosphorylation, CBP
binding or interaction with DNA.
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 transcriptionalresponse follows so-called “burst-attenuation” kinetics with maximum rates 30 to 60 minutes
after stimulation followed by a gradual attenuation phase which may last as long as several hours
(52). The attenuation phase is not dependent on a loss of PKA activity, but rather results fromdephosphorylation of CREB (52,53). Inhibition of phosphatases prolongs the attenuation phase,
resulting in the increase in PKA-stimulated gene expression (52-55). How dephosphorylation ofCREB is regulated is not clear, however. Michael et al. demonstrated that inhibition of
deacetylase activity, without affecting phosphatase activity, prolongs the phosphorylation of
CREB after forskolin stimulation (28). These results suggest that acetylation of components ofthe 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 CREBby 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 areimportant 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. Wepostulate that acetylation, like mutation, increases the activity of CREB, perhaps through an
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
17
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.
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
18
Figure Legends
Figure 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 mg FLAG-
CREB, RSV-cPKA (the catalytic subunit of PKA) and Rc/RSV-CBP. Various concentrations of
Trichostatin A (TSA) were used as indicated for 18 hours. The results are expressed as
Mean(±SEM) (N=3) relative CAT activity after correcting for transfection efficiency with
luciferase activity.
Figure 2. CBP acetylates K91, K94, and K136 within the CREB activation domain. (A) The
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 5 lysine-to-alanine mutations withinthe activation domain (5K/A) in the presence of 14C acetyl-coenzyme A. Controls did not include
CBP. The proteins were separated by SDS-PAGE, dried, and exposed to a phosphorimager
screen. Purified CREB 1-283 proteins were used in the in vitro acetylation assay. CREB1-283proteins (0.5mg) were incubated with or without 0.1mg of purified CBP and 14C-acetyl-coenzyme
A at 30°C for 1 hour. The proteins were separated by SDS-PAGE, stained with Coomassie Blue,
destained, dried, and exposed to a phosphorimager screeen. (C) The CREB 1-283 (5K/A)
protein has all five lysines (K91/94/123/136/155) mutated to alanine. The rest of the CREB 1-
283 lysine mutants have all lysines but one (as indicated) mutated to alanine. A scan of theCoomossie stain of the CREB 1-283 proteins used in the assay is shown in lower panel of the
figure. (D) CREB peptides or Histone H3 peptide was incubated with CBP or P/CAF asindicated in the presence of [3H]acetyl-coenzyme A. The acetylated peptides were precipitated
on P81 filter paper and counted for [3H] activity by liquid scintillation counting. Results are
expressed as Mean(±SEM)(N=3).
Figure 3. CREB is acetylated by CBP in cells. (A) Cos7 cells were either transfected with
pcDNA3 vectors alone (control) or with pcDNA3 FLAG-CREB (F-CREB). The cells werelabeled with 3H-sodium acetate, and whole cell extracts were immunoprecipitated using FLAG
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
19
M2 antibodies. The precipitates were then separated by SDS-PAGE, fixed, enhanced using
Amplify (Amersham), dried, and exposed to X-ray film. (B) Cos7 cells were either transfectedwith pcDNA3 vectors alone (control) or with pcDNA3 FLAG-CREB 341. The cell extracts were
immunoprecipitated using FLAG M2 antibodies. The precipitates were separated by SDS-PAGE.The separated proteins were transferred to a PVDF membrane and probed with FLAG M2
antibodies and an anti-acetyl-lysine antibody.
Figure 4. Single lysine mutations within the CREB activation domain have no effect onCREB-mediated gene activation in F9 cells. F9 cells were transfected with the SRIF-CRECAT reporter, RSV-luciferase, and varying amounts (1.2, 2.4, 3.6, or 4.8mg) of either Rc/RSV-
FLAG-CREB WT or Rc/RSV-F-CREB lysine mutants, with (A) or without (B) RSV-cPKA (thecatalytic 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 thanthree times with similar results. The results shown are a representation of one experiment. In (C),
3.6mg of Rc/RSV F-CREB or its lysine mutants were used. The data are expressed as
Mean(±SEM)(N=3). Dark and white bars represent with or without the co-transfection of RSV-
cPKA, respectively. (D) Expression of each CREB protein is shown. Equal amounts of proteinwere 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 b-tubulin antibodies
Figure 5. Double Mutations of K91, K94, or K136 to alanine enhance CREB-mediated geneactivation in F9 cells. The format of the experiments is identical to that described in Figure 3. In
(C), the data are expressed as Mean (±SEM)(N=3). * represents statistically significant (Tukeytest) at P≤0.01 compared with CREB WT.
Figure 6. Triple lysine mutations within CREB enhance CREB-mediated gene expression.The format of the experiments is identical to that described in Figure 3. In (C), the data are
expressed as Mean relative CAT activity (±SEM)(N=3). * represents statistically significant(Tukey test) at P≤0.01 compared with CREB WT.
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
20
Figure 7. Mutation of K91, K94, and K136 to arginine enhances CREB-mediated geneactivation in F9 cells. The format of the experiments is identical to that described in Figure 3. In(C), the data are expressed as Mean relative CAT activity (±SEM)(N=3). * represents
statistically significant (Tukey test) at P≤0.01 compared with CREB WT.
Figure 8. GAL4-CREB 1-283 lysine mutants enhance GAL4-CREB-mediated geneexpression. F9 cells were transfected with a 5XGAL4-CAT reporter, RSV-luciferase, and eitherRc/RSV GAL4-CREB WT or its lysine mutants. In (A), 0.125, 0.25, 0.5, or 1mg of expression
vectors of GAL4-CREB 1-283 or its lysine mutants was used. RSV-cPKA were co-transfected.
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. Theresults shown are a representation of one experiment. In (B), 1mg of the expression vector of
GAL4-CREB 1-283 or its lysine mutants was used. Dark and white bars represent with or
without the cotransfection of the RSV-cPKA, respectively. The results were expressed as Meanrelative CAT activity (±SEM)(N=3). * represents statistically significant (Tukey test) at P≤0.01
compared with GAL4-CREBWT.
Figure 9. CREB lysine mutations do not affect the interaction with CBP. In (A) and (B), F9cells were transfected with the SRIF-CAT reporter, RSV-luciferase, Rc/RSV FLAG-CREB or its
lysine mutants (3.6mg), with (A) or without (B) the co-transfection 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 experimentswere 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(0.5mg), with (C) or without (D) the co-transfection of the catalytic subunit of PKA, and various
amounts of Rc/RSV VP16 CREB 341 or its lysine mutants, as indicated. Results are expressed as
Mean relative CAT activity after correcting for transfection efficiency with luciferase activity,
and the experiments were repeated more than three times with similar results.
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
21
Acknowledgement
We would like to thank the technical support of Madeleine Pham. We are grateful to Dr. Sarah
Smolik for comments on this manuscript. This work was supported by funding from anAmerican Cancer Society Research Grant to R.P.S.K., and by Public Health Services grants
DK051732 and DK060133 from the NIDDK to J.R.L. J.R.L. is a Scholar of the Mallinckrodt
Foundation. This work supported in part by National Institute of Diabetes and Digestive andKidney Diseases of the National Institutes of Health (#5P60DK-20572).
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
22
References
1. Mayr, B., and Montminy, M. (2001) Nat Rev Mol Cell Biol 2, 599-609.2. Montminy, M. R., Sevarino, K. A., Wagner, J. A., Mandel, G., and Goodman, R. H.
(1986) Proc Natl Acad Sci U S A 83, 6682-66863. Montminy, M. (1997) Annu Rev Biochem 66, 807-8224. Chrivia, J. C., Kwok, R. P., Lamb, N., Hagiwara, M., Montminy, M. R., and Goodman,
R. H. (1993) Nature 365, 855-8595. Eckner, R., Ewen, M. E., Newsome, D., Gerdes, M., DeCaprio, J. A., Lawrence, J. B.,
and Livingston, D. M. (1994) Genes Dev 8, 869-8846. Moran, E. (1993) Curr Opin Genet Dev 3, 63-707. Goodman, R. H., and Smolik, S. (2000) Genes Dev 14, 1553-15778. Kwok, R. P., Lundblad, J. R., Chrivia, J. C., Richards, J. P., Bachinger, H. P., Brennan,
R. G., Roberts, S. G., Green, M. R., and Goodman, R. H. (1994) Nature 370, 223-2269. Swope, D. L., Mueller, C. L., and Chrivia, J. C. (1996) J Biol Chem 271, 28138-2814510. Bisotto, S., Minorgan, S., and Rehfuss, R. P. (1996) J Biol Chem 271, 17746-1775011. Kee, B. L., Arias, J., and Montminy, M. R. (1996) J Biol Chem 271, 2373-237512. Ogryzko, V. V., Schiltz, R. L., Russanova, V., Howard, B. H., and Nakatani, Y. (1996)
Cell 87, 953-95913. Yang, X. J., Ogryzko, V. V., Nishikawa, J., Howard, B. H., and Nakatani, Y. (1996)
Nature 382, 319-32414. Candau, R., Moore, P. A., Wang, L., Barlev, N., Ying, C. Y., Rosen, C. A., and Berger,
S. L. (1996) Mol Cell Biol 16, 593-60215. Xu, W., Edmondson, D. G., and Roth, S. Y. (1998) Mol Cell Biol 18, 5659-566916. Bannister, A. J., and Kouzarides, T. (1996) Nature 384, 641-64317. Mizzen, C. A., Yang, X. J., Kokubo, T., Brownell, J. E., Bannister, A. J., Owen-Hughes,
T., Workman, J., Wang, L., Berger, S. L., Kouzarides, T., Nakatani, Y., and Allis, C. D.(1996) Cell 87, 1261-1270
18. Spencer, T. E., Jenster, G., Burcin, M. M., Allis, C. D., Zhou, J., Mizzen, C. A.,McKenna, N. J., Onate, S. A., Tsai, S. Y., Tsai, M. J., and O'Malley, B. W. (1997) Nature389, 194-198
19. Chen, H., Lin, R. J., Schiltz, R. L., Chakravarti, D., Nash, A., Nagy, L., Privalsky, M. L.,Nakatani, Y., and Evans, R. M. (1997) Cell 90, 569-580
20. Sterner, D. E., and Berger, S. L. (2000) Microbiol Mol Biol Rev 64, 435-45921. Imhof, A., Yang, X. J., Ogryzko, V. V., Nakatani, Y., Wolffe, A. P., and Ge, H. (1997)
Curr Biol 7, 689-69222. Gu, W., and Roeder, R. G. (1997) Cell 90, 595-60623. Liu, L., Scolnick, D. M., Trievel, R. C., Zhang, H. B., Marmorstein, R., Halazonetis, T.
D., and Berger, S. L. (1999) Mol Cell Biol 19, 1202-120924. Sakaguchi, K., Herrera, J. E., Saito, S., Miki, T., Bustin, M., Vassilev, A., Anderson, C.
W., and Appella, E. (1998) Genes Dev 12, 2831-284125. Waltzer, L., and Bienz, M. (1998) Nature 395, 521-52526. Ludlam, W. H., Taylor, M. H., Tanner, K. G., Denu, J. M., Goodman, R. H., and Smolik,
S. M. (2002) Mol Cell Biol 22, 3832-3841
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
23
27. Martinez-Balbas, M. A., Bannister, A. J., Martin, K., Haus-Seuffert, P., Meisterernst, M.,and Kouzarides, T. (1998) Embo J 17, 2886-2893
28. Michael, L. F., Asahara, H., Shulman, A. I., Kraus, W. L., and Montminy, M. (2000) MolCell Biol 20, 1596-1603
29. Kwok, R. P., Laurance, M. E., Lundblad, J. R., Goldman, P. S., Shih, H., Connor, L. M.,Marriott, S. J., and Goodman, R. H. (1996) Nature 380, 642-646
30. Kashanchi, F., Duvall, J. F., Kwok, R. P., Lundblad, J. R., Goodman, R. H., and Brady, J.N. (1998) J Biol Chem 273, 34646-34652
31. Smith, C. L., and Hager, G. L. (1997) J Biol Chem 272, 27493-2749632. Daniel, P. B., Walker, W. H., and Habener, J. F. (1998) Annu Rev Nutr 18, 353-38333. Shaywitz, A. J., and Greenberg, M. E. (1999) Annu Rev Biochem 68, 821-86134. Bannister, A. J., Miska, E. A., Gorlich, D., and Kouzarides, T. (2000) Curr Biol 10, 467-
47035. Thompson, P. R., Kurooka, H., Nakatani, Y., and Cole, P. A. (2001) J Biol Chem 276,
33721-3372936. Hoeffler, J. P., Meyer, T. E., Waeber, G., and Habener, J. F. (1990) Mol Endocrinol 4,
920-93037. Ruppert, S., Cole, T. J., Boshart, M., Schmid, E., and Schutz, G. (1992) Embo J 11, 1503-
151238. Berkowitz, L. A., and Gilman, M. Z. (1990) Proc Natl Acad Sci U S A 87, 5258-526239. Yamamoto, K. K., Gonzalez, G. A., Menzel, P., Rivier, J., and Montminy, M. R. (1990)
Cell 60, 611-61740. Nichols, M., Weih, F., Schmid, W., DeVack, C., Kowenz-Leutz, E., Luckow, B.,
Boshart, M., and Schutz, G. (1992) Embo J 11, 3337-334641. Wolfl, S., Martinez, C., and Majzoub, J. A. (1999) Mol Endocrinol 13, 659-66942. Richards, J. P., Bachinger, H. P., Goodman, R. H., and Brennan, R. G. (1996) J Biol
Chem 271, 13716-1372343. Lundblad, J. R., Kwok, R. P., Laurance, M. E., Huang, M. S., Richards, J. P., Brennan, R.
G., and Goodman, R. H. (1998) J Biol Chem 273, 19251-1925944. Lenzmeier, B. A., Giebler, H. A., and Nyborg, J. K. (1998) Mol Cell Biol 18, 721-73145. Cardinaux, J. R., Notis, J. C., Zhang, Q., Vo, N., Craig, J. C., Fass, D. M., Brennan, R.
G., and Goodman, R. H. (2000) Mol Cell Biol 20, 1546-155246. Oliner, J. D., Andresen, J. M., Hansen, S. K., Zhou, S., and Tjian, R. (1996) Genes Dev
10, 2903-291147. Du, K., Asahara, H., Jhala, U. S., Wagner, B. L., and Montminy, M. (2000) Mol Cell Biol
20, 4320-432748. Shaywitz, A. J., Dove, S. L., Kornhauser, J. M., Hochschild, A., and Greenberg, M. E.
(2000) Mol Cell Biol 20, 9409-942249. Liu, F., and Green, M. R. (1990) Cell 61, 1217-122450. Foulkes, N. S., Borrelli, E., and Sassone-Corsi, P. (1991) Cell 64, 739-74951. Songyang, Z., Blechner, S., Hoagland, N., Hoekstra, M. F., Piwnica-Worms, H., and
Cantley, L. C. (1994) Curr Biol 4, 973-98252. Hagiwara, M., Alberts, A., Brindle, P., Meinkoth, J., Feramisco, J., Deng, T., Karin, M.,
Shenolikar, S., and Montminy, M. (1992) Cell 70, 105-11353. Wadzinski, B. E., Wheat, W. H., Jaspers, S., Peruski, L. F., Jr., Lickteig, R. L., Johnson,
G. L., and Klemm, D. J. (1993) Mol Cell Biol 13, 2822-2834.
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
24
54. Alberts, A. S., Montminy, M., Shenolikar, S., and Feramisco, J. R. (1994) Mol Cell Biol14, 4398-4407
55. Bito, H., Deisseroth, K., and Tsien, R. W. (1996) Cell 87, 1203-1214.
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
25
Lu et al., Figure 1
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
26
Lu et al., Figure 2
A.
B.
C.
K91/K94 VQTVQSSCKDLKELFSQTQK123 VDSVTDSQKRREILSRRK136 REILSRRPSYRKILNDLSSDAK155 GVPRIEEEKSEEETSA
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
27
Lu et al., Figure 2 D
0
5000
10000
15000
20000
25000
30000
CBP
P/CAF+ + + + + +
+ + + + + +
K91/K94 K123 K136 K155 Histone H3
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
28
Lu et al. Figure 3
A.
B.
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
29
0
2
4
6
8
0 1 2 3 4 50
2
4
6
8
0 1 2 3 4 5
K94A
K136A
K155A
K123A
K91A
WT
0
1
2
3
4
5
6
B.A.
C. D.
CREB (µg)
Lu et al., Figure 4
CREB (µg)
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
30
0
1
2
3
4
0 1 2 3 4 50
1
2
3
4
0 1 2 3 4 5
0
2
4
6
8
10
CREB (µg) CREB (µg)
WT
K91/94A
K91/123A
K94/136A
K91/136A
K94A K123AK91A
Lu et al., Figure 5
C.
A. B.
**
***
*
WTK91/123A
K94/136A
K91/94AK91/136A
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
31
0
2
4
6
8
10
0 1 2 3 4 50
2
4
6
8
10
0 1 2 3 4 5
0
5
10
15
K91A
WT
K91/94AK91/94/123A
K91/94/136A
WT K91/94/136A
K91/94/123AK91/94AK91A
K91/94A
*
***
*
*
**
*
C.
B.
D.
A.
Lu et al., Figure 6
CREB (µg) CREB (µg)
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
32
0
1
2
3
4
5
6
0 1 2 3 4 50
1
2
3
4
5
6
0 1 2 3 4 5
K91/94R
K91R
K91/94/136R
WT
WT
K91RK91/94RK91/94/136R
CREB (µg) CREB (µg)
0
1
2
3
4
5
6
**
C.
B.
D.
A.
Lu et al., Figure 7
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
33
0
10
20
30
40
50
60
0 0.25 0.5 0.75 1
GAL-CREB 1-283 (µg)
WT
K91/94/136A
K91/94A
K91A
A.
B.
Lu et al., Figure 8
0
10
20
30
40
WT K91A K91/94A K91/94/136AControl
*
*
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
34
0
2
4
6
8
10
12
0 2 4 6 80
2
4
6
8
10
12
0 2 4 6 8
A.
0
10
20
30
40
50
0 25 50 75 1000
10
20
30
40
50
0 25 50 75 100
C. D.
B.
WT
K91A
K91/94A
K91/94/136A
CBP (µg) CBP (µg)
VP16 CREB 341 (ng) VP16 CREB 341 (ng)
VP16 CREB WT
VP16 CREB M1
K91AK91/94/136A
K91/94A
Lu et al., Figure 9
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
KwokQing Lu, Amanda E. Hutchins, Colleen M. Doyle, James R. Lundblad and Roland P. S.
Acetylation of CREB by CBP enhances CREB-dependent transcription
published online February 20, 2003J. Biol. Chem.
10.1074/jbc.M300546200Access the most updated version of this article at doi:
Alerts:
When a correction for this article is posted•
When this article is cited•
to choose from all of JBC's e-mail alertsClick here
by guest on July 9, 2018http://w
ww
.jbc.org/D
ownloaded from
Top Related