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: rkwok@umich.edu

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

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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.

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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-

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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.

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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

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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.

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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

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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]-

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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

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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

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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

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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).

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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

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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

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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.

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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

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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.

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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

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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.

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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.

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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).

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Lu et al., Figure 1

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Lu et al., Figure 2

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B.

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K91/K94 VQTVQSSCKDLKELFSQTQK123 VDSVTDSQKRREILSRRK136 REILSRRPSYRKILNDLSSDAK155 GVPRIEEEKSEEETSA

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Lu et al., Figure 2 D

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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. 

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