From the Department of Molecular Pharmacology and
Experimental Therapeutics, the § Mayo Graduate School, the
¶ Department of Biochemistry and Molecular Biology, and the
Molecular Neuroscience Program, Mayo Clinic and Foundation,
Rochester, Minnesota 55905
Received for publication, July 27, 2000, and in revised form, September 29, 2000
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ABSTRACT |
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Calmodulin Kinase II (CamKII) inhibits the
transcription of many CRE-dependent genes, but the
mechanism of dominant transcriptional inhibition is unknown. Here we
show that phosphorylation of serine 142 in CREB by CamKII leads to
dissociation of the CREB dimer without impeding DNA binding capacity.
CamKII-modified CREB binds to DNA efficiently as a monomer; however,
monomeric CREB is unable to recruit the CREB-binding protein (CBP) even
when phosphorylated at serine 133. Thus, CamKII confers a dominant
inhibitory effect on transcription by preventing dimerization of CREB,
and this mechanism may account for the attenuation of gene expression.
It is well established that both Ca2+-calmodulin
and cAMP-PKA1 signals are
involved in neuronal gene expression that underlie plasticity (1-7).
However, the mechanism by which multiple signals coordinate neuronal
transcription is not well understood. Both calcium and cAMP can
activate genes containing conserved CREs (8-14). CRE-binding proteins
including CREB-1 and ATF1 are substrates for both PKA and
calcium-dependent kinases such as nuclear CamKIV (15-19).
These kinases activate genes through their ability to phosphorylate
CREB-1 at serine 133, a modification that is known to increase the
affinity of CREB-binding protein and transcription (20-24).
However, elevation of calcium also stimulates
Ca2+/calmodulin-dependent protein kinase type II,
which is by far the most abundant kinase in the neuron (25, 26). CamKII
inhibits transcription of many CRE-dependent reporter
genes, and CamKII inhibition of transcription dominates stimulatory
effects of PKA (27) or CamKIV (15-19). CamKII modifies CREB at both
serine 133 and 142 (14, 16-19). Because modification at serine 133 is
stimulatory, inhibition is thought to involve serine 142 although
cooperative interactions with serine 133 have not been excluded
(15-19). However, the mechanism by which CamKII inhibits
CRE-dependent transcription and attenuates the CamKIV or
PKA response is not understood.
Plasmids and Transfection--
The pSomCAT (pss70CAT (9)) and
the DynCAT reporter genes have been previously reported (27-29). CAT
refers to the bacterial chloramphenicol acetyl transferase gene.
Mammalian expression vectors for CREB proteins were previously
constructed by modifying a vector containing a RSV promoter and a
histidine fusion of the entire rat CREB
(pET22b(+)His6-CREB) (29, 43). Mutant forms of CREB lacking
serine phosphorylation sites were generated by overlap polymerase chain
reaction using primers which contained a T Protein Expression, Phosphorylation, and
Purification--
The pET22b(+) vectors that express
His6-CREB, His6-CREB-S133A,
His6-CREB-S142A, or His6-CREB-S133A/S142A
fusion proteins were transformed into Escherichia coli
strain BL21(DE3), and protein was expressed and purified as described
previously (29). His6-CREB in the text is referred to as
CREB. CREB or mutant CREB was incubated with CaMKII (New England
BioLabs) at a ratio of 1 unit of enzyme/pmol protein at 37 °C for
1 h in the presence of 1.0 mM ATP, 2.0 mM CaCl2, and 2.4 µM calmodulin in reaction
buffer (20.0 mM Tris, pH 7.5, 10.0 mM
MgCl2, 0.50 mM dithiothreitol, 0.10 mM Na2EDTA). Phosphorylation of CREB or its
mutants by PKA has been previously described (29, 43). The efficiency
of CREB phosphorylation was quantified by phosphorimaging.
Gel Mobility Shift--
Gel mobility assays have been previously
described (29, 43, 44). The CRE binding templates were a 27-bp
somatostatin CRE (5'-AGAGATTGCCTGAC-GTCAGAGAGCTAG-3') and a 23-bp
dynorphin CRE3 (5'-GTGGCTGCTGCG-TCAGAGCATGA-3'). Added protein ranged
from 20.0 to 150.0 pmol and was incubated with 25.0 pmol of
32P-endlabeled probe.
CBP Binding Assay--
A carboxyl-terminal truncation of the CBP
fusion protein (His6-CBP-(1-682)) was expressed in
E. coli strain BL21(DE3) using a
His6-mCBP-(1-682)pET15b vector (29). The bacterial extract was prepared as described (29, 43) and was stored at Sedimentation Equilibrium--
Sedimentation equilibrium
measurements were performed on an Optima XL-A equipped with UV-vis
detection system (Beckman Instruments) as described previously (29,
45). For DNA/protein complexes, His6-CREB and
P-His6-CREB were incubated with 1.0-2.5 µM
somatostatin CRE (5'-GCCTCCTTGGCTGACG-TCAGAGAGAG-3') or dynorphin CRE3
(5'-GTGGCTGCTGCGTCAGAGCATGA-3') that was endlabeled with fluorescein as
described previously (29). Complexes were formed after incubation of
protein with DNA overnight at room temperature. Samples were analyzed
only using double sector cells to increase the column length. Each
sample was analyzed at multiple rotor speeds (between 8,000 and 20,000 rpm) and at multiple concentrations. Experiments were carried out at
20 °C in an ANTi60 rotor until equilibrium was achieved. Equilibrium distributions were detected by scanning the absorbance across the cell
at 494 nm, the maximum absorbance wavelength for fluorescein. The use
of fluorescein tag on the DNA allows analysis of DNA/protein complexes
without interference from free protein or DNA as long as the DNA is
fully bound. Neither His6-CREB nor P-His6-CREB
absorb at 494 mm. Boundaries were analyzed as described above using
relevant partial specific volumes. The partial specific volume
( CamKII inhibits transcription of many CRE-dependent
reporter genes, and inhibition is independent of the CRE symmetry or
CRE number (Fig. 1). In contrast to
either CamKIV or PKA, constitutively active CamKII represses expression
of CRE-containing CAT reporter constructs when co-transfected in
mammalian cells (Fig. 1A). CamKII mediates the dominant
inhibitory effect because overexpression of an inactive form of CamKII
abrogates inhibition of either the SomCAT or DynCAT reporter genes
(data not shown). SomCAT and DynCAT expression are inhibited by
co-expression of CamKII with either CamKIV (not shown) or PKA (Fig.
1A), to the same extent as CamKII alone (Fig. 1A).
Therefore, CamKII produces a dominant inhibitory effect that overcomes
stimulation by either CamKIV or PKA (Fig. 1A).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
G point mutation in the
first residue of the serine codon (TCT), altering it to an alanine
codon (GCT). All plasmids were verified by sequencing. Transfections
utilized either human neuroblastoma (SK-N-MC) cells or African green
monkey kidney cells (CV-1) cultured according to supplier's (ATCC)
instructions. Transient transfection protocols utilized calcium
phosphate as has been described previously (29, 43). Cells in each
plate received 5.0 µg of CAT-reporter plasmid and 5.0 µg of MCV-PKA
(28), RSV-CaMKII-(1-290) (15), or RSV-CaMKIV-(1-313) (15).
70 °C. CBP
binding was analyzed on immobilized CREB/DNA complexes. DynCRE3 oligonucleotides were synthesized with a biotin label
(5'ABTABTGTGGCTGCTG-CGTCAGAGCATGA-3') (Mayo core
facility) and bound to streptavidin-agarose columns. DNA was added to
the column and incubated overnight at 4 °C with gentle rocking to
allow maximal DNA binding, after which the beads were washed
extensively with binding buffer to repack the column. CREB·DNA
complexes were formed by the addition of native or mutant CREB to the
immobilized DNA in the column. For all experiments, CREB was added to
obtain a constant subsaturated protein/DNA ratio (between 0.3 and 0.4)
to ensure complete binding of all dimeric or monomeric CREB. Finally,
CBP was added at a ratio of 1 CBP molecule per CREB·DNA complex and
incubated in a similar manner as for CREB. CBP or CREB proteins were
eluted in 20.0 mM Tris, 1.0 M KCl, 1.0 mM dithiothreitol, 1.0 mM MgCl2,
0.5 mM EDTA, 10.0% glycerol, pH 6.8. Separated proteins
were transferred to nitrocellulose membrane (Micron Separations Inc)
and detected with antibodies specific for CREB or CBP (1:500 dilution
of a rabbit anti-CBP polyclonal antibody, CBP (A22)) (Santa Cruz
Biotechnology). The relative amount of His6-CREB and
His6-CBP-(1-682) were evaluated by antibody detection (29)
of an equal volume of eluate obtained under identical column
conditions. Each experiment was repeated at least four times.
o) of the fluorescein-labeled CRE-containing
oligonucleotides were calculated as described previously (29, 45). The
o of the DNA/protein complex was 0.695 ml/g for a
His6-CREB dimer and 0.679 ml/g for a His6-CREB
monomer. For fitting, data from multiple rotor speeds and multiple
concentrations were fit simultaneously to obtain a best value using a
nonlinear Levenberg-Marquardt fitting routine incorporated into a macro
using Kaleidograph (29) or using NONLIN (46). Fitting from both
routines yielded similar results. Molecular weights for one or two
components were obtained from the best fit of the data using nonlinear
Levenberg-Marquardt fitting routine (47) according to the
following,
where A is the absorbance, ra is the
distance of meniscus to the axis of the rotor, r is the distance of
each point on the boundary to the rotor axis,
(Eq. 1)
is the angular speed,
o is the partial specific volume of the component, and
M is the molecular weight of the component, which is
obtained from the fitting. Under subsaturating conditions, fitting was
performed assuming a two component system in which one component was
fixed to be the free DNA.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
CamKII confers a dominant inhibitory effect
on CREB-dependent gene expression. A,
effects of kinases on the transcription of CRE-containing genes.
Som represents the promoter region of the somatostatin gene
and contains only one symmetric CRE site. Dyn represents the
promoter region of the rat dynorphin gene and contains four asymmetric
(non-palindromic) CRE sites. In each experiment, CV-1 cells were
transfected with 5 µg of pss70CAT (Som) or 2.0pCAT
(Dyn) (26), 5 µg of MT-PKA (catalytic subunit),
RSV-CaMKIV-(1-313), and RSV-CaMKII-(1-290), as indicated in CV-1
cells. An aliquot of 5 µg of each indicated plasmid was used. The (+)
indicates the presence of the transfected plasmid whereas ( )
indicates absence. B, schematic of dynorphin gene promoter
(top) and dynorphin-CAT reporter genes (22). Relative
activity is calculated by the ratio of CAT activity in the presence of
CamKII to CAT activity in the absence of CamKII. Transfection
conditions are as in A. C, the inhibitory effect
of CaMKII on somatostatin activation depends on CREB binding to the
CRE. Transfections are as in A; 5 µg of RSV-expression
plasmids for CREB or K304E were utilized. Native refers to native
CREB-1 protein; K304E refers to a CREB-1 mutant that contains a lysine
to glutamic acid change and cannot bind to the CRE (26). D,
CamKII-mediated repression of the rat prodynorphin promoter requires
CREB binding. CREB is native CREB; K304E is CREB containing a lysine to
glutamic acid base change in CREB. Fold inhibition is the ratio of CREB
inhibition in the presence of CamKII to the absence of CamKII.
Transfections are as in C; 5 µg of RSV-expression plasmids for CREB
or K304E were co-transfected with CamKII-(1-290) and 2.0pCAT
(Dyn). When CREB cannot bind, CamKII relieves inhibition of
expression driven by the rat prodynorphin promoter.
CREB-1 is the major factor that mediates transcriptional regulation of both somatostatin (27) and prodynorphin by PKA (28). For both Dyn (Fig. 1B) and Som (Fig. 1C) constructs, we find that the inhibitory effect of CamKII is mediated by CREB and occurs at CRE elements required for stimulation by CamKIV or PKA. CRE-3 is the major site for CREB-dependent regulation of prodynorphin (28). In the presence of CamKII, DynCAT expression is inhibited relative to control (Fig. 1B). However, deletion or point mutations that abolish CREB-1 binding at the CRE-3 of prodynorphin (28) also relieve CamKII-dependent inhibition (Fig. 1B) relative to control. A mutant CREB (CREB-K304E (28)) that cannot bind to the CRE but is a substrate for CamKII phosphorylation relieves CamKII-dependent inhibition of either SomCAT (Fig. 1C) or DynCAT (Fig. 1D) promoters. Thus, gene activation by either CamKIV or PKA and inhibition by CamKII appear to involve modification of the same CREB·CRE complex.
CamKII modifies CREB-1 at two positions, serine 142 and serine 133 (Fig. 2A). In CV-1 cells,
transcription of SomCAT is inhibited by expression of CamKII (Fig.
2B). To determine the relationship between CREB
phosphorylation and CaMKII-mediated inhibition, we constructed and
expressed native and three mutant forms of CREB. Each mutant contains a
serine to an alanine change at either or both phosphorylation sites
(Fig. 2A). In the absence of kinase, native CREB,
S142A, and S142A/S133A displayed similar activity (Fig.
2A, Native CREB, Kinase). In the presence of CamKII,
SomCAT expression is inhibited by expression of either native CREB or the S133A mutant, both of which can be phosphorylated at serine 142 (Fig. 2B, Native versus A133). Relative to native CREB,
inhibition of SomCAT is relieved in the presence of the S142A and
S133A/S142A mutants, neither of which can be phosphorylated at serine
142 (Fig. 2B, Native versus A142 and
A133A142). Because inhibition is not significantly different
between native CREB and the CREB-S133A mutant, the data indicate that
inhibition is not a cooperative effect of modification at both sites.
Rather, the inhibitory effect of CamKII requires only CREB
phosphorylation at Ser-142. Interestingly, we never see expression of
SomCAT with S142A in the presence of kinase equal to native CREB in the
presence of the kinase. However, there is endogenous CREB in the cells.
Therefore, we expect that in some cases the S142A mutant will dimerize
with a native CREB molecule that contains an intact 142 site hence
lowering expression of the reporter.
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We next examined why phosphorylation of CREB at 142 is inhibitory. Phosphorylation at serine 142 by CamKII might be preferred and might negatively influence the extent of phosphorylation at serine 133. In this case, the inability to efficiently modify CREB at 133 may explain the inhibitory effect. To test whether the serine 142 site is preferred, we evaluated the efficiency of CREB phosphorylation by CamKII at each site. Native or mutant CREB was incubated with CamKII or PKA in the presence of [32P]ATP and the extent of phosphorylation was measured in vitro (Fig. 2C). We find that CaMKII alone efficiently modifies native CREB at both 133 and 142. CREB contains twice as much incorporated label after CamKII phosphorylation as after PKA phosphorylation. Modification of either S133A or S142A by CamKII occurs equally and about half as well as for the native CREB (Fig. 2C). In all cases, mutant CREB proteins are equally expressed and are appropriate substrates for phosphorylation. Mutant S142A cannot be phosphorylated on serine 142 and is phosphorylated to the same extent by CamKII as is native CREB by PKA. Additionally, S133A is not a substrate for PKA, and the double mutant is not a substrate for PKA or CamKII (Fig. 2C). Finally, CREB modification at the inhibitory site 142 occurs at an equal rate as for the stimulatory site at 133 (Fig. 2D). Thus, inhibition by CamKII does not occur because serine 133 is slowly modified. We conclude that CamKII displays no site preference and modifies either the 133 or the 142 site to the same extent.
Using these mutants, we also confirmed that CamKII modification of CREB at these two sites is not cooperative. Phosphorylation of CREB by PKA at serine 133 does not prevent or influence modification at serine 142 by CamKII (Fig. 2C). This is evident from the fact that incorporated label at site 142 in native CREB by CamKII is roughly the same before or after phosphorylation at 133. This means that CamKII can fully modify CREB even after phosphorylation by PKA or CamKIV (Fig. 2C). Similarly, mixing CamKII-modified S133A and native CREB does not prevent efficient phosphorylation at serine 133 (not shown). Thus, CamKII efficiently phosphorylates either site independent of modification at the other. We conclude that CamKII-mediated inhibition can prevent stimulation by PKA or CamKIV, but PKA or CamKIV modification does not prevent a subsequent inhibitory modification at 142. Phosphorylation of CREB at serine 142 by CamKII is necessary and sufficient for the dominant inhibitory effect of CamKII on transcription. However, differential phosphorylation does not explain inhibition.
CamKII-modified CREB might inhibit transcription by reducing binding of
CREB at the CRE site. We find, however, that CamKII does not decrease
and, in fact, modestly increases binding of purified CREB to either the
SomCAT (Fig. 3A) or DynCAT
CREs (Fig. 3B). Because binding of CREB is required for
activity of the somatostatin promoter, it is unlikely that
transcriptional attenuation by CamKII is caused by increasing DNA
binding activity there.
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Instead, we find that CamKII-mediated phosphorylation of serine 142 attenuates dimerization of full-length CREB (Fig. 3, C and
D). The self-association state of CREB is measured by
sedimentation equilibrium using fluorescein-labeled DNA templates (Fig.
3C and Ref. 29). The use of fluorescein labels allows
detection of the protein/DNA complex using visible light without
interference from the free protein (29). Under conditions of
transcriptional activity, native CREB is a dimer and activity is
enhanced by phosphorylation at serine 133 (Fig. 3D,
Native kinase and +CamKIV/PKA). Transcription is also supported by both CamKII-modified S142A mutant and the S133A/S142A double mutant (Fig. 3D). Neither mutant can be
phosphorylated at serine 142 and both are dimers on DNA. In contrast,
transcription is inhibited when CamKII modifies the native or S133A
mutant, both of which are phosphorylated at position 142. It has been clearly demonstrated that unphosphorylated CREB or PKA-modified CREB
binds to DNA as a dimer (9, 29, 30, 31). However, after modification by
CamKII, CREB binds DNA efficiently as a monomer, but monomer binding is
not associated with transcriptional stimulation (Figs. 3,
D-F, 1A, and 2B). When serine 142 is
phosphorylated, the monomer-dimer equilibrium is shifted far toward
monomer because dimer assembly is prevented even under high
concentrations of CREB (Fig. 3, E and F). For the
native protein, transcription is inhibited and dimerization is
prevented even when serine 133 is modified (Fig. 3D,
Native +CamKII). Modification of CREB at serine 142 prevents
CREB dimer assembly at the CRE. If modification at serine 142 also
causes previously bound dimers to dissociate, then loss of dimerization
might explain the dominant inhibitory effect of CamKII on
transcription. Indeed, bound CREB dimers dissociate after CamKII
modification before or after serine 133 modification by PKA (Fig.
3G). We conclude that CamKII inhibits transcription by
preventing CREB dimerization on DNA.
It is well documented that CREB phosphorylation at serine 133 stimulates transcription by increasing the binding affinity of CBP
(20-24, 32). In contrast, protein-protein association of a 59-amino
acid KID domain peptide in CREB with the KIX domain peptide
fragment in CBP is inhibited by phosphorylation at serine 142 (20). We,
therefore, asked whether phosphorylation at 142 and loss of CREB
dimerization inhibit transcription by weakening CBP binding to
full-length CREB bound to DNA. Full-length native or mutant CREB was
bound (ratio of 0.4) to immobilized biotin-labeled dynorphin CRE3.
Purified CBP was added last to the CREB·DNA complex, and CBP binding
was evaluated. CBP binds modestly to unphosphorylated CREB, and binding
is significantly enhanced by PKA modification at serine 133 (Fig.
4). Under these conditions, CREB is a
dimer (Fig. 4). In contrast, CBP binding is not enhanced on native CREB after CaMKII modification even though serine 133 is modified (Fig. 4A). CBP binding is also not enhanced for the S133A mutant
after modification by CamKII. Under these conditions, native CREB and the S133A mutant are monomers (Fig. 4). In all CBP binding experiments, the amount of CREB substrate is similar (Fig. 4, far right
panel). Thus, monomers of CREB efficiently bind to DNA when
modified by CamKII, but CBP is unable to bind to monomeric CREB even
when serine 133 is phosphorylated. In other words, phosphorylation at
Ser-133 is necessary but not sufficient for recruiting CBP to the
CREB·CRE complex. CREB dimerization is also required.
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DISCUSSION |
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The mechanism by which CamKII inhibits CREB-dependent reporter activity is not well understood. We show here that phosphorylation of CREB at serine 142 prevents dimerization and uncouples CBP binding. Many studies have shown that CREB is a dimer in its unphosphorylated state and when modified by PKA (9, 29-31). Therefore, our results suggest a model in which CREB dimerization acts as a transcriptional switch that operates through phosphorylation and allows a distinct response depending on the kinase. The data reveal several features that may be important with respect to CREB interactions in vivo.
First, CamKII-induced loss of CBP association and CREB dimerization occurs when CREB is bound to DNA. Therefore, these events can directly lead to transcriptional inhibition. It had previously been shown that casein kinase II modification of a 59-amino acid region of the CREB KID domain inhibited protein-protein association with a KIX domain peptide of CBP (20). Phosphorylation at serine 142 likely disrupts interaction with a Tyr-650 that is an essential residue for the KID/KIK binding interface and the hydrophobic interface (20, 33). However, casein kinase II modifies at least four other sites within the KID domain. Heretofore, it was not known whether CBP dissociation occurred in the full-length CREB protein, whether dissociation occurred when CREB was bound to DNA, or whether dissociation involved CREB dimerization. Our results not only confirm that CREB modification at serine 142 is sufficient to account for the negative effects on transcription but also reveal that CREB dimerization is involved.
Second, CREB when modified by CamKII binds to DNA as a monomer. This was surprising because early studies using model leucine zipper and DNA binding domains suggested that dimerization was essential for CREB binding (34). Recently, however, detailed spectroscopic and kinetic studies using full-length proteins have revealed that CREB monomers sequentially assemble into a dimer on DNA (29, 35). Monomeric forms of CREB have also been reported to activate transcription (36). Sequential monomer binding is also observed for other bZip peptides including GCN4 and BLH (37). We have used sedimentation equilibrium to calculate the mass of the CREB·DNA complex. The analysis conclusively demonstrates that CamKII-modified CREB is a monomer when bound to DNA. This is in contrast to dimer binding of either unphosphorylated or PKA-modified CREB within the same experiment (Fig. 3D). Sedimentation equilibrium can be used reliably to evaluate monomer or dimer states in proteins because the calculated mass is independent of either the shape or the charge of the complex (38). Gel shift analysis also confirms that CREB remains bound to DNA when modified by CamKII. However, gel shift analysis cannot unambiguously confirm a monomer-dimer complex, especially when there has been a change in the phosphorylation state. It is well documented that proteins of very different mass can migrate at the same position on gels because of charge and shape effects (39, 40). This is because gel mobility in an electric field is proportional to the ratio of the charge to the frictional coefficient (37), both of which are altered by CREB phosphorylation (20). Indeed, the slower mobility of the CamKII-modified CREB·DNA complex relative to the native CREB·DNA complex implies that conformational change in CREB has occurred because the addition of two phosphate groups should enhance mobility if charge were the only factor. Although monomeric CREB can bind DNA, a CamKII-modified monomer cannot sustain productive interactions with CBP and the transcription machinery.
Finally, loss of CREB dimerization and CBP binding after
phosphorylation by CamKII suggests a mechanism for the dominant
negative effect on transcription at some genes as well as activation of others. CREB dimerization serves as a molecular switch that integrates calcium and cAMP signals and coordinates gene expression (Fig. 5). PKA or CamKIV can stimulate
expression of CRE-dependent genes by CREB phosphorylation
at serine 133. Under these conditions, CREB exists as a dimer on DNA
and successfully attracts CBP to the target gene. However, in the
presence of CamKII or other kinases that may modify CREB at serine 142, CREB dimerization is prevented and CREB is no longer able to compete
for CBP binding (Fig. 5). Phosphorylation at serine 142 prevents CREB
dimerization on DNA before or after binding. Because dimer dissociation
can also occur before or after modification of Ser-133 by another
kinase, phosphorylation of CREB at Ser-142 produces a dominant
inhibitory effect. CamKII is highly concentrated in the dendrites where
it constitutes the major protein of post-synaptic structures (25, 26,
41, 42). Because kinases often translocate slowly from synapse to
nucleus, the timing may allow a window of opportunity for gene
expression to occur before attenuation by CREB modification at serine
142. Thus, kinase modification of CREB at serine 142 can program gene expression by shifting the CREB dimer-monomer equilibrium.
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Whereas CamKII widely inhibits expression of reporter genes, a role for
CamKII in transcriptional inhibition in vivo has not been
clearly demonstrated. Therefore, regulation in vivo is
likely to be more complex then is suggested by a simple reporter
system. However, the ability of serine 142 phosphorylation to regulate CREB dimerization may also explain gene activation by CamKII. For gene
activation, it is possible that modification at serine 142 and CREB
dimer dissociation allow the formation of new heterodimers that restore
affinity for CBP. Phosphorylation of CREB provides a mechanism for
regulation of transcription because either the dimer or monomer can
receive signals that influence the choice of partner. Differential
phosphorylation may control selection of a new dimerization partner
in vivo through a monomer intermediate. These effects are
likely to play a role in response specificity and may broadly apply to
other kinases. The ability to control CREB dimerization and CBP binding
by phosphorylation at 142 provides a common mechanism by which signals
from diverse pathways can be integrated to control diverse function and
neuronal plasticity.
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ACKNOWLEDGEMENTS |
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We thank Drs. R. H. Goodman and R. A. Maurer for generously providing His6-CBP-(1-682)pET15b, RSV-CamKII1-290 and RSV-CamKIV1-313 plasmids. We also thank R. Janknecht, C. Spiro, R. Dyer, J. Trushina, and G. Goellner for scientific discussion and reading of the manuscript.
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FOOTNOTES |
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* This work was supported by the Mayo Foundation, National Institutes of Health Grants DK 43694-01A2 and MH-56207 and National Science Foundation Grant IBN 9728120 (to C. T. M.).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.
** To whom correspondence should be addressed: Dept. of Molecular Pharmacology and Experimental Therapeutics, Mayo Foundation, 200 First St., S. W., Rochester, MN 55905. Tel.: 505-284-1597; Fax: 505-284-9111; E-mail: mcmurray.cynthia@mayo.edu.
Published, JBC Papers in Press, September 29, 2000, DOI 10.1074/jbc.M006727200
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ABBREVIATIONS |
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The abbreviations used are: PKA, cAMP-dependent protein kinase; CRE, cAMP responsive enhancer; CREB-1, cAMP response element-binding protein; CamKIV, calmodulin-dependent kinase type IV; CBP, CREB-binding protein; CamKII, Ca2+/calmodulin-dependent protein kinase type II; CAT, chloramphenicol acetyl transferase; Som, somatostatin; Dyn, prodynorphin; RSV, Rous sarcoma virus.
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REFERENCES |
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1. | Buonomano, D. V., and Merzenich, M. M. (1998) Neuroscience 21, 149-186[CrossRef] |
2. | Xia, Z., and Storm, D. R. (1997) Curr. Opin. Neurobiol. 7, 391-396[CrossRef][Medline] [Order article via Infotrieve] |
3. | Abel, T., Nguyen, P. V., Barad, M., Deuel, T. A., Kandel, E. R., and Bourtchouladze, R. (1997) Cell 88, 615-626[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Lisman, J.,
Malenka, R. C.,
Nicoll, R. A.,
and Malinow, R.
(1997)
Science
276,
2001-2003 |
5. |
Blitzer, R. D.,
Connor, J. H.,
Brown, G. P.,
Wong, T.,
Shenolikar, S.,
Iyengar, R.,
and Landau, E. M.
(1998)
Science
280,
1940-1943 |
6. | Silva, A. J., Kogan, J. H., Frankland, P. W., and Kida, S. (1998) Annu. Rev. Neurosci. 21, 127-148[CrossRef][Medline] [Order article via Infotrieve] |
7. | Iyengar, R. (1996) Science 271, 461-463 |
8. | Hardingham, G. E., Chawla, S., Johnson, C. M., and Bading, H. (1998) Nature 385, 260-265 |
9. | Montminy, M. (1997) Annu. Rev. Biochem. 66, 807-822[CrossRef][Medline] [Order article via Infotrieve] |
10. | Hagiwara, M., Shimomura, A., Yoshida, K., and Imak, J. (1996) Adv. Pharmacol. 36, 277-285[Medline] [Order article via Infotrieve] |
11. | Hagiwara, M., Brindle, P., Harootunian, A., Armstrong, R., Rivier, J., Vale, W., Tsien, R., and Montminy, M. R. (1993) Mol. Cell. Biol. 13, 4852-4859[Abstract] |
12. | Dash, P. K., Karl, K. A., Colicos, M. A., Prywes, R., and Kandel, E. R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5061-5065[Abstract] |
13. | Sheng, M., Thompson, M. A., and Greenberg, M. E. (1991) Science 252, 1427-1430 |
14. | Sheng, M., McFadden, G., and Greenberg, M. E. (1990) Neuron 4, 571-582[Medline] [Order article via Infotrieve] |
15. |
Sun, P.,
Lou, L.,
and Maurer, R. A.
(1996)
J. Biol. Chem.
271,
3066-3073 |
16. |
Shimomura, A.,
Ogawa, Y.,
Kitan, T.,
Fujisawa, H.,
and Hagiwara, M.
(1996)
J. Biol. Chem.
271,
17957-17960 |
17. | Sun, P., Enslen, H., Myung, P. S., and Maurer, R. A. (1994) Genes Dev. 8, 2527-2539 |
18. |
Enslen, H.,
Sun, P.,
Brickey, D.,
Soderling, S. H.,
Klamo, E.,
and Soderling, T. R.
(1994)
J. Biol. Chem.
269,
15520-15527 |
19. | Matthews, R. P., Guthrie, C. R., Wailes, L. M., Zhao, X., Means, A. R., and McKnight, G. S. (1994) Mol. Cell. Biol. 14, 6107-6116[Abstract] |
20. | Parker, D., Jhala, U. S., Radhakrishnan, I., Yaffe, M. B., Reyes, C., Shulman, A. I., Cantley, L. C., Wright, P. E., and Montminy, M. (1998) Mol. Cell 2, 353-359[Medline] [Order article via Infotrieve] |
21. |
Chawla, S.,
Hardingham, G. E.,
Quinn, D. R.,
and Bading, H.
(1998)
Science
281,
1505-1509 |
22. | Goldman, P. S., Tran, V. K., and Goodman, R. H. (1997) Recent Prog. Hormone Res. 52, 103-120[Medline] [Order article via Infotrieve] |
23. | Chrivia, J. C., Kwok, R. P., Lamb, N., Hagiwara, M., Montminy, M. R., and Goodman, R. H. (1993) Nature 365, 855-859[CrossRef][Medline] [Order article via Infotrieve] |
24. | Kwok, R. P. S., 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, 223-226[CrossRef][Medline] [Order article via Infotrieve] |
25. | Tompa, P., and Friedrich, P. (1998) Trends Neurosci. 21, 97-102[CrossRef][Medline] [Order article via Infotrieve] |
26. | Deisseroth, K., Heist, E. K., and Tsien, R. W. (1998) Nature 392, 198-202[CrossRef][Medline] [Order article via Infotrieve] |
27. | Gonzalez, G. A., and Montminy, M. R. (1989) Cell 59, 675-680[Medline] [Order article via Infotrieve] |
28. | Collins-Hicok, J., Lin, L., Spiro, C., Laybourn, P. J., Tschumper, R., Rapacz, B., and McMurray, C. T. (1994) Mol. Cell. Biol. 14, 2837-2848[Abstract] |
29. |
Wu, X.,
Spiro, C.,
Owen, W. G.,
and McMurray, C. T.
(1998)
J. Biol. Chem.
273,
20820-20827 |
30. |
Richards, J. P.,
Bachinger, H. P.,
Goodman, R. H.,
and Brennan, R. G.
(1996)
J. Biol. Chem.
271,
13716-13723 |
31. | Loriaux, M. M., Rehfuss, R. P., Brennan, R. G., and Goodman, R. H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9046-9050[Abstract] |
32. | Lundblad, J. R., Kwok, R. P. S., Laurance, M. E., Harter, M. L., and Goodman, R. H. (1995) Nature 374, 85-88[CrossRef][Medline] [Order article via Infotrieve] |
33. | Radhakrishnan, I., Perez-Alvarado, G. C., Parker, D., Dyson, H. J., Montminy, M. R., and Wright, P. E. (1997) Cell 91, 741-752[Medline] [Order article via Infotrieve] |
34. | Dwarki, V. J., Montminy, M. R., and Verma, I. M. (1990) EMBO J. 9, 225-232[Abstract] |
35. | Metallo, S. J., and Schepartz, A. (1997) Nat. Struct. Biol. 4, 115-117[Medline] [Order article via Infotrieve] |
36. | Krajewski, W., and Lee, K. A. (1994) Mol. Cell. Biol. 14, 7204-7210[Abstract] |
37. |
Schepartz, A.,
and Metallo, S. J.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
11735-11739 |
38. | Van Holde, K. E. (1985) Physical Biochemistry , 2nd Ed. , Prentice-Hall, Inc., Englewood Cliffs, NJ |
39. | Ornstein, L. (1964) Annals N. Y. Acad. Sci. 121, 321-349 |
40. | Orr, M. D., Blakley, R. I., and Pangou, D. (1972) Anal. Biochem. 45, 68-85[Medline] [Order article via Infotrieve] |
41. | Kelly, P. T., McGuinness, T. L., and Greengard, P. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 945-949 |
42. | Kennedy, M. B., Bennett, M. K., and Erondu, N. E. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 7357-7361 |
43. |
Spiro, C.,
Bazett-Jones, D. P.,
Wu, X.,
and McMurray, C. T.
(1995)
J. Biol. Chem.
270,
27702-27710 |
44. | Spiro, C., Richards, J. P., Chandrasekaran, S., Brennan, R. G., and McMurray, C. T. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4606-4610[Abstract] |
45. | Zhang, F., Lemieux, S., Wu, X., St.-Armaud, D., McMurray, C. T., Major, F., and Anderson, D. (1998) Mol. Cell. 2, 141-147[CrossRef][Medline] [Order article via Infotrieve] |
46. | Johnson, M. L., Correia, J. J, Yphantis, D. A., and Halvorson, H. R. (1981) Biophys. J. 36, 575[Abstract] |
47. | Press, W. H., Teukolsky, S. A., Vettering, W. T., and Flannery, B. P. (1992) Numerical Recipes in C , pp. 656-706, Cambridge University Press, New York |