The recent identification of a co-activator that binds to
phosphorylated CREB (
)has provided new insights into the
mechanisms that mediate transcriptional responses to cAMP(1) .
The co-activator, which is designated the CREB binding protein (CBP),
interacts with CREB after CREB is phosphorylated at
Ser
(2) . Previous studies have shown that
phosphorylation of CREB at Ser
is crucial for
cAMP-activated transcription(3) . An essential role for CBP in
mediating transcriptional responses to PKA was recently demonstrated in
intact cells by microinjection of neutralizing antibodies to
CBP(4) . Interestingly, CBP itself can function as a
transcriptional activator protein. When fused to a heterologous DNA
binding domain, so that it is brought to a promoter in a
phosphorylation-independent fashion, CBP is a strong activator of
transcription(1) . It has also been shown that CBP interacts
with the basal transcription factor, TFIIB(2) . This suggests a
simple model in which phosphorylation of CREB activates transcription
by recruiting CBP, which then interacts with basal transcription
factors.
In the present paper we have identified a point mutation
that greatly reduces the ability of CREB to mediate cAMP-stimulated
transcription. The discovery of this inactivating mutation is based on
recent studies that demonstrated that phosphorylation of Ser
of CREB by Ca
/calmodulin-dependent protein
kinase II blocks the ability of cAMP to activate CREB(5) . The
observation that Ser
can function as a negative
regulatory site led us to examine amino acid replacements of this
residue. We find that replacement of Ser
with Asp mimics
the effects of phosphorylation and greatly reduces the ability of CREB
to mediate cAMP-stimulated transcription. We have tested the ability of
CBP to interact with this mutant form of CREB. Surprisingly, CBP
interacts with the mutant protein in a manner that is indistinguishable
from the wild type protein.
MATERIALS AND METHODS
Cell Culture and Transfections
F9 and PC12 cells
were transfected by calcium phosphate precipitation as described
previously (5) . Typically cells received 5 µg of the
5xGAL4-TATA-luciferase indicator DNA, 2 µg of an expression vector
encoding the GAL4 DNA binding domain fused to the transcriptional
activation domain of CREB (GAL4-CREB
), and 5
µg of a PKA expression vector. Cells were collected and lysates
prepared 48 h (F9 cells) or 24 h (PC12 cells) after transfection. The
protein concentration of lysates was determined(6) , and
luciferase activities were measured using a constant amount of protein
as described elsewhere(7) . Each experiment included three
separate transfections for each group, and the experiments have been
repeated three to five times. The GAL4CREB
expression vector was prepared by polymerase chain amplification
of the CREB coding sequence by adding an upstream BamHI site
and a downstream XbaI site and substitution of the truncated
CREB sequence for the complete coding sequence in the previously
described GAL4-CREB vector(8) . A luciferase reporter gene
containing five GAL4 binding sites has been described
previously(5) . The CREB coding sequence was altered so that
Ser
was replaced with aspartate, glutamate, or lysine by
oligonucleotide-directed mutagenesis(9) . The mutations were
confirmed by nucleotide sequence analysis(10) .
Western Blot Analysis of the Expression of Transiently
Transfected CREB Mutants
COS-1 cells were transfected with 6
µg of either pBSSK(-) or GAL4-CREB expression vector per
60-mm dish, and cell lysates were prepared as described(5) .
Protein concentrations of cell lysates were determined(6) , and
40 µg of total protein from each sample was separated on a 12%
polyacrylamide denaturing gel. After electroblotting to a
polyvinylidene difluoride membrane, the filter was blocked and
incubated with a 1:5000 dilution of a polyclonal antiserum to
CREB(8) . The immune complexes were visualized using the
ImmunoSelect Photoblot chemiluminescent system (Life Technologies,
Inc.) following the manufacturer's protocol.
In Vitro Binding Assay for the Interaction of CREB with
CBP
The coding sequence for amino acids 462-661 of CBP (1) was cloned in-frame to the biotin carboxylase carrier
protein (BCCP) in the PinPoint Xa-1 bacterial expression vector
(Promega). Biotinylated BCCP-CBP fusion protein was expressed in Escherichia coli strain JM109 and purified by affinity
chromatography using a monomeric avidin column following the
supplier's protocol. Recombinant CREB (5) was
phosphorylated with 330 nM purified recombinant catalytic
subunit of the cAMP-dependent protein kinase or 300 nM recombinant Ca
/calmodulin-dependent protein
kinase II
(11) in the presence of
[
-
P]ATP using previously described reaction
conditions(5) . After phosphorylation, the reaction mixtures
were diluted in binding buffer (20 mM Tris, pH 7.4, 50 mM NaCl, 1 mM dithiothreitol, and 0.5% nonfat dry milk) and
then chromatographed on Sephadex G-50 (Sigma) columns to remove free
ATP. To prepare immobilized CBP, 40 µg of biotinylated BCCP-CBP
were incubated with 100 µl (packed volume) of streptavidin-agarose
beads (Life Technologies, Inc.) for 2 h at room temperature with
rotation and then washed extensively with the binding buffer. The
binding assays were performed in duplicate 80-µl reactions
containing binding buffer and 10 µl (packed volume) of BCCP-CBP
bound to streptavidin-agarose beads and varying amounts of
phosphorylated CREB. After incubation for 2 h at room temperature with
rotation, the reaction mixtures were transferred to disposable
mini-columns (Kontes) attached to a vacuum manifold. Each column was
washed quickly with 3 ml of binding buffer, and radioactivity bound to
the column was determined.
In Vivo Analysis of the Interaction of CREB with
CBP
A mammalian version of the yeast two-hybrid screen (12) was used to detect the in vivo interaction of
CREB and CBP. To prepare a fusion of the VP16 activation domain and the
CREB activation domain, the coding sequence for residues 412-490
of VP16 (13) was placed in-frame with an initiation codon. The
coding sequence for the transcriptional activation domain of wild type
or mutant CREB (amino acids 1-283) was amplified by polymerase
chain reaction and subcloned in-frame at the 3` end of the VP16
activation domain and then subcloned into the pcDNA3 mammalian
expression vector (Invitrogen). For the mammalian two-hybrid assay, F9
cells were transfected with 5 µg of
5xGAL4-TATA-luciferase(5) , 5 µg of pGAL-CBP3(1) ,
5 µg of a wild type or mutant expression plasmid for VP16-CREB, and
either 5 µg of pBSSK(-) or 5 µg of an expression vector
for the catalytic
subunit of PKA(14) . Cells were
collected and luciferase activities determined as described above. Each
experiment included three separate transfections for each group, and
the experiments have been repeated three to five times.
RESULTS AND DISCUSSION
Replacement of Ser
of CREB with Asp
Reduces PKA-activated Transcription
Our recent studies of
Ca
regulation of CREB activity led to the
identification of a phosphorylation site within the CREB activation
domain that negatively regulates CREB activity. These studies
demonstrated that when Ser
is phosphorylated by
Ca
/calmodulin-dependent protein kinase II, the
ability of PKA to activate CREB is greatly reduced(5) . To
further assess the functional role of Ser
, we replaced
this residue with aspartate or glutamate, negatively charged amino
acids that might mimic the effects of phosphorylating
Ser
. Ser
was also replaced with lysine to
determine if simply placing a charged residue at this position has an
effect on CREB activity. The CREB mutants were tested for their ability
to mediate PKA-stimulated transcription in a transient transfection
assay utilizing GAL4-CREB fusion proteins in both F9 and PC12 cells (Fig. 1). A GAL4-CREB
fusion protein was
studied in order to eliminate interference from other endogenous
proteins that bind to CREs (8, 15, 16, 17) . In both PC12 and
F9 cells, the GAL4-CREB fusion was very strongly activated by PKA
(please note that for many of the samples, the control values are
difficult to distinguish from the x axis). The replacement of
Ser
with Asp very sharply reduced responses to PKA.
Indeed, the Asp
mutation had almost as severe an effect
as mutation of the PKA phosphorylation site (Ser
replaced
with Ala). Interestingly, replacement of Ser
with Glu or
Lys had little or no effect on PKA-stimulated CREB activity. The
finding that replacement with Glu has little effect is very interesting
and demonstrates that it is not the simple placement of a negative
charge at position 142 that blocks activation of CREB. An approximately
1.5-Å difference in the positioning of the negative charge
completely changes the effect of the substitution.
Figure 1:
Replacement of
Ser
with Asp reduces activation of GAL4-CREB by PKA.
Either PC12 cells (A) or F9 cells (B) were
transfected with 2 µg of GAL4-CREB
fusion
constructs carrying different substitutions at Ser
or
Ser
, 5 µg of 5xGAL4-TATA-luciferase reporter, and 5
µg of either pBSSK(-) or an expression plasmid for the
catalytic subunit
of PKA (RSV-PKA). Nomenclature for the
mutant GAL4-CREB
vectors indicates the position
of the mutation within the CREB coding sequence followed by the
single-letter amino acid code for the mutant residue. Luciferase
activity was determined 24 or 48 h after transfection. Values are the
means ± S.E. of the mean for three separate
transfections.
It is possible
that replacement of Ser
with Asp results in poor
expression or decreased stability of the protein. To examine this
possibility, we compared the relative expression levels of GAL4-CREB
fusion proteins containing different substitutions at
Ser
. Immunoblotting analysis of extracts from COS-1 cells
transfected with expression vectors for wild type or mutant GAL4-CREB
revealed that all of the proteins are expressed at similar levels (Fig. 2). Therefore, the reduced PKA-stimulated activity of the
Asp replacement at Ser
is not due to low level expression
of the mutant. Similar results (although with a considerably weaker
signal) were obtained with F9 cells (data not shown).
Figure 2:
GAL4-CREB fusion proteins containing
different substitutions at Ser
are expressed at similar
levels in transfected cells. COS-1 cells were transfected with 6 µg
of pBSSK(-) or an expression vector for the indicated wild type
and mutant GAL4-CREB proteins. Nomenclature for the mutant GAL4-CREB
vectors indicates the position of the mutation within the CREB coding
sequence followed by the single-letter amino acid code for the mutant
residue. Two separate transfections were performed for each construct.
Cells were collected 24 h after transfection and lysed by sonication,
and 40 µg of protein from each sample were separated on a
denaturing polyacrylamide gel. The proteins were transferred to a
polyvinylidene difluoride membrane, and then GAL4-CREB fusion proteins
and endogenous CREB were visualized by immunostaining with a polyclonal
antiserum to CREB. The arrows indicate the migration of bands
corresponding to the GAL4-CREB fusion produced by the transfected
expression vector (upperarrow) and endogenous CREB (lowerarrow).
Replacement of Ser
with Asp Does Not
Interfere with Binding of CREB to CBP in Vitro
It is
possible that replacement of Ser
with Asp or
phosphorylation of Ser
blocks the interaction of CREB and
CBP. As binding of CBP to CREB appears to be essential for mediating
cAMP-stimulated transcription (4) decreased binding to CBP
would offer an explanation for the reduced activity of the Asp
replacement. This hypothesis is consistent with the observation
that Ser
is located within the region of CREB that is
required for interaction with CBP(1) . Since the Asp
replacement at Ser
greatly decreases, but does not
completely block, the activation of CREB by PKA, it is possible that
the effect of this mutation is a quantitative rather than a complete
block of CREB binding to CBP. Therefore, we developed an in vitro binding assay to quantitate the effect of the Asp replacement of
Ser
on the binding of CREB to CBP. The CREB binding
domain of CBP (amino acids 462-661) was expressed in E. coli as a fusion protein with a portion of the BCCP. The BCCP fragment
is biotinylated in E. coli(18) . Biotinylated
BCCP-CBP
was purified, coupled to
streptavidin-agarose, and tested for binding to
[
P]CREB (Fig. 3). When wild type CREB was
phosphorylated with PKA and [
-
P]ATP, the
radiolabeled CREB bound to BCCP-CBP
streptavidin-agarose in an apparently saturable fashion. Very
little binding to the streptavidin-agarose was observed in the absence
of BCCP-CBP
. Furthermore, although the
tissue-specific transcription factor, Pit-1, can be phosphorylated by
PKA(19) , phosphorylated Pit-1 did not bind to the immobilized
CBP (Fig. 3). Thus, this system offers a quantitative and highly
specific assay for analysis of the interaction of CREB with CBP. This
assay demonstrated similar concentration dependence for the binding of
CBP to wild type CREB or CREB in which Ser
was replaced
with Asp. We also found that phosphorylation of CREB with
Ca
/calmodulin-dependent protein kinase II had little
or no effect on binding to CBP (data not shown). The preceding
experiments tested the interaction of CREB with the 462-661
region of CBP. This region of CBP was used because it is sufficient for
PKA-dependent interactions with CREB (1) and because it has
been technically difficult to produce full-length CBP. However, it
remained possible that substitution of Ser
of CREB with
Asp might interfere with binding to full-length CBP. To address this
possibility we isolated full-length CBP from cultured cells by
immunoprecipitation, resolved the immunoprecipitated proteins by gel
electrophoresis, and transferred the proteins to a membrane. Binding of
P-CREB to the immobilized CBP was then tested using an
overlay assay, as described previously(1) . Autoradiographic
analysis of the overlay assay demonstrated similar binding of
full-length CBP to wild type CREB or CREB in which Ser
was replaced with Asp (data not shown). Thus despite the fact
that replacement of Ser
with Asp severely blunts
transcriptional activation, this mutation does not detectably interfere
with the interaction of CBP and CREB in vitro.
Figure 3:
Replacement of Ser
of CREB
with Asp does not interfere with the in vitro binding of CREB
to CBP. Biotinylated BCCP-CBP
was bound to
streptavidin beads. Either the BCCP-CBP beads (closedsymbols) or streptavidin beads alone (opensymbols) were incubated with wild type or mutant
[
P]CREB or [
P]Pit-1,
which had been previously radiolabeled by incubation with PKA and
[
P]ATP. Unbound proteins were removed by
filtration, and bound radioactivity was determined. For the mutant CREB
protein, the position of the mutation within the CREB coding sequence
is indicated followed by the single-letter amino acid code for the
mutant residue.
Analysis of the Interaction of CREB with CBP in Mammalian
Cells Using a Two-hybrid Assay
To further examine interactions
between CREB and CBP, we have developed an in vivo protein
interaction assay similar to the yeast two-hybrid system(12) .
As with the yeast two-hybrid system, the assay involves use of a
GAL4-responsive reporter gene and two fusion proteins that interact
with each other to form a functional transcription factor. One of the
fusion proteins contains the DNA binding domain of the yeast
transcription factor, GAL4, fused in-frame to the CREB binding domain
of CBP (amino acids 462-661). CBP
does
not contain a transcriptional activation domain(1) , and by
itself, the GAL4-CBP
fusion protein did not
activate expression of the reporter gene, either in the presence or
absence of PKA. The other fusion protein contains the VP16
transcriptional activation domain linked to the CREB transcriptional
activation domain (CREB
b-zip). As the VP16-CREB
b-zip fusion
protein does not contain a DNA binding domain, it also cannot activate
transcription. GAL4-CBP
and VP16-CREB
b-zip
should not form a competent transcription factor until PKA
phosphorylates VP16-CREB
b-zip and induces binding to
GAL4-CBP
. Neither GAL4-CBP
nor VP16-CREB
b-zip resulted in PKA-stimulated reporter gene
expression (Fig. 4). However, when both the
GAL4-CBP
and the wild type VP16-CREB
b-zip
expression vector were transfected with a PKA expression vector, a very
vigorous activation of the reporter gene was obtained. In several
different experiments PKA has yielded a 1000-6000-fold increase
in the activity of the two-hybrid reporter system. Replacement of
Ser
of CREB with Ala within the VP16-CREB
b-zip
fusion protein drastically reduced PKA-stimulated transcriptional
activation. These findings suggest that phosphorylation of CREB at
Ser
of the VP16-CREB
b-zip fusion protein leads to
interactions with GAL4-CBP
and results in very
strong transcriptional activation using this mammalian two-hybrid
system. These findings demonstrate that this system provides a very
sensitive method for detecting interactions of CREB and CBP in
vivo. It is important to note that this activation is very
dependent on phosphorylation by PKA, consistent with the fact that
there is no detectable interaction between CBP and unphosphorylated
CREB(2) . We have used this mammalian two-hybrid system to test
the effects of mutations at Ser
on interactions between
CREB and CBP in vivo. Replacement of Ser
with
Asp, Glu, or Lys had little or no effect on PKA-induced binding of CREB
and CBP, while replacement of Ser
with Ala greatly
decreased the ability of PKA to induce CREB-CBP binding. Therefore, in vivo, as well as in vitro, the Asp replacement at
Ser
did not interfere with CREB and CBP interaction.
Figure 4:
Replacement of Ser
of CREB
with Asp does not interfere with the in vivo binding of CREB
to CBP. The in vivo interaction of CREB and CBP was assessed
using a mammalian version of the two-hybrid assay. F9 cells were
transfected with 5 µg of the 5xGAL4-TATA-luciferase reporter gene,
5 µg of GAL4-CBP
expression vector, 5
µg of wild type or mutant expression vectors for a
VP16-CREB
b-zip fusion protein, and either 5 µg of
pBSSK(-) or 5 µg of an expression vector for the catalytic
subunit of PKA. The designation for the mutant VP16-CREB vectors
indicates the position of the mutation within the CREB coding sequence
followed by the single-letter amino acid code for the mutant residue.
-Fold activation was determined by dividing the luciferase activity for
each sample, which was co-transfected with the PKA catalytic subunit
expression vector, by the mean basal activity for that
group.
The present findings demonstrate that substitution of a negatively
charged residue, Asp, at Ser
of the transcriptional
activation domain of CREB greatly reduces PKA-mediated activation of
CREB. Although this mutation severely reduces PKA-mediated
transcriptional activation, the mutation does not interfere with the
interaction of CREB and CBP either in vitro or in
vivo. These finding are rather surprising in view of the
observation that CBP appears to function as a constitutive
transcriptional activator when brought to a promoter as a GAL4 fusion
protein(1) . Indeed, based on the constitutive activity of CBP
and the ability of CBP to interact with the basal transcription
apparatus, one might consider that CREB simply functions as a scaffold
that recruits CBP to the promoter in a PKA-dependent fashion. However,
the present findings suggest that such a simple model is probably not
correct and provide strong evidence that the binding of CBP to CREB is
not sufficient for transcriptional activation. If binding of CBP to
CREB is not sufficient for transcriptional activation, what essential
step in the PKA-mediated response is disrupted by replacement of
Ser
of CREB with Asp? It may be that this mutation
interferes with binding of an additional factor to CREB. This factor
could be another co-activator. Biochemical fractionation experiments
and in vitro transcription studies have demonstrated that at
least four separable co-activators may be required for maximal
responses to several different transcriptional
activators(20, 21, 22, 23) .
Replacement of Ser
with Asp might also interfere with the
interaction of CREB and the basal transcription apparatus. A recent
study demonstrated that CREB can interact with Drosophila TAF
110, a component of the TFIID
complex(24) . Alternatively, CBP may need to acquire a
particular conformation in order to communicate with the basal
transcriptional machinery and activate transcription. Asp replacement
of Ser
of CREB, although not reducing the affinity for
CBP, may alter the conformation of bound CBP and therefore block the
interaction of CBP with downstream factors. Further characterization of
the structure and function of factors required for transcriptional
responses to phosphorylated CREB should help resolve these two
alternatives.