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Molecular and Cellular Biology, October 2001, p. 7089-7096, Vol. 21, No. 20
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.20.7089-7096.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Mitogen-Regulated RSK2-CBP Interaction Controls
Their Kinase and Acetylase Activities
Karine
Merienne,1
Solange
Pannetier,1
Annick
Harel-Bellan,2 and
Paolo
Sassone-Corsi1,*
Institut de Génétique et de
Biologie Moléculaire et Cellulaire, CNRS, INSERM,
Université Louis Pasteur, 67404 Illkirch,
Strasbourg,1 and CNRS UPR 9079,
94800 Villejuif,2 France
Received 16 April 2001/Returned for modification 7 June
2001/Accepted 11 July 2001
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ABSTRACT |
The protein kinase ribosomal S6 kinase 2 (RSK2) has been implicated
in phosphorylation of transcription factor CREB and histone H3 in
response to mitogenic stimulation by epidermal growth factor. Binding
of phospho-CREB to the coactivator CBP allows gene activation through
recruitment of the basal transcriptional machinery. Acetylation of H3
by histone acetyltransferase (HAT) activities, such as the one carried
by CBP, has been functionally coupled to H3 phosphorylation. While
various lines of evidence indicate that coupled histone acetylation and
phosphorylation may act in concert to induce chromatin remodeling
events facilitating gene activation, little is known about the coupling
of the two processes at the signaling level. Here we show that CBP and
RSK2 are associated in a complex in quiescent cells and that they
dissociate within a few minutes upon mitogenic stimulus. CBP
preferentially interacts with unphosphorylated RSK2 in a complex where
both RSK2 kinase activity and CBP acetylase activity are inhibited.
Dissociation is dependent on phosphorylation of RSK2 on Ser227 and
results in stimulation of both kinase and HAT activities. We propose a
model in which dynamic formation and dissociation of the CBP-RSK2
complex in response to mitogenic stimulation allow regulated
phosphorylation and acetylation of specific substrates, leading to
coordinated modulation of gene expression.
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INTRODUCTION |
The induction of
immediate-early response genes is the result of a number of coordinate
events operating at the levels of intracellular signaling and
transcriptional activation (33). Upon physiological
challenge by mitogens and hormones and the consequent triggering of
corresponding transduction pathways, various molecular events elicit
the activation of transcription factors and the recruiting of specific
coactivators (18, 30). Some coactivators also bear histone
acetylase (HAT) activity, directly linking transcriptional activation
to distinct chromatin modifications (7, 29, 46).
Various signaling routes converge on transcription factor CREB (cyclic
AMP-responsive binding protein) (4, 20, 44) and control
its function by modulating its phosphorylation state. Phosphorylation
at a single serine residue (Ser-133) works as a molecular switch, as it
dictates CREB's ability to interact with the coactivator CBP
(CREB-binding protein), a large protein with HAT activity (5, 13,
39) which mediates functional contacts with the basal
transcriptional machinery (34). Activation of CREB by
growth factors was shown to be Ras dependent and to involve the
mitogen-activated protein kinases (MAPKs) (27). Although a
number of kinases downstream of the MAPKs may be implicated (19), members of the p90rsk
(ribosomal S6 kinase [RSK]) family have been identified as
mitogen-responsive CREB kinases (21, 51). In particular,
both CREB phosphorylation and c-fos transcriptional
induction are drastically impaired in response to epidermal growth
factor (EGF) in human fibroblasts derived from Coffin-Lowry syndrome
patients (21), which carry mutations in the gene encoding
the RSK2 kinase (49). RSK2 is a member of the
p90rsk family, which includes four closely
related isoforms (23, 52). A conserved feature of all
p90rsk proteins is the presence of two
nonidentical kinase catalytic domains, the N-terminal domain being
responsible for the phosphorylation of several targets (6,
22). The activity of the N-terminal domain is regulated upon
direct MAPK activation of the C-terminal catalytic domain by the ERKs
and involvement of PDK1 (3-phosphoinositide-dependent protein kinase 1)
(17, 24, 25, 31, 41, 45, 53).
A critical set of observations have directly linked the activation of
mitogen-activated transduction pathways with histone modifications and
the consequent remodeling of chromatin (11, 36). In
particular, the rapid and transient mitogen-induced phosphorylation of
a serine residue (Ser10) in the tail of histone H3 has been coupled to
the transcriptional activation of the immediate-early response genes
(8, 15, 16). Interestingly, this event of phosphorylation
appears to be coupled to acetylation, as the efficiency of histone
acetyltransferases (HATs) to subsequently acetylate the nearby Lys14 is
drastically increased (12, 35). Thus, activation of gene
expression through chromatin remodeling is the result of multiple,
coordinated events.
As somewhat anticipated, there are indications that common signaling
pathways and effector kinases are utilized in the phosphorylation of
transcription factors and histone tails (11). Although the Ser10 site in the H3 tail is the likely target of various kinases (11, 16, 43), there is evidence that RSK2 is the kinase involved in response to EGF (43). We have been wondering
whether the coupling of phosphorylation and acetylation at the level of the histone substrates could possibly be paralleled by a physical and
functional interplay of the respective effectors, kinases and
acetylases. Here we show that CBP and RSK2 associate in a complex in
quiescent cells and that they dissociate within a few minutes upon
mitogenic stimulus. CBP preferentially interacts with unphosphorylated
RSK2 in a complex where both RSK2 kinase activity and CBP acetylase
activity are inhibited. We propose a model in which dynamic formation
and dissociation of the CBP-RSK2 complex in response to mitogenic
stimulation allow regulated phosphorylation and acetylation of specific substrates.
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MATERIALS AND METHODS |
Antibodies.
Monoclonal antibodies directed against RSK2
phosphorylated at Ser227 (P-S227) and Thr577 (P-T577) have been
described previously (37). Anti-CBP (A-22) and anti-RSK2
(E1 and C-19) antibodies were from Santa Cruz Biotechnology Inc.,
anti-HA antibody was from Boehringer-Mannheim, anti-ERK,
anti-phosphorylated ERK, anti-CREB, and anti-phosphorylated CREB
antibodies were from New England Biolabs, and anti-H3 and
anti-phosphorylated H3 antibodies were from Upsate Biotechnology.
Preimmune sera were used in control experiments.
Transient transfections and stimulation of cells.
Expression
vectors encoding hemagglutinin (HA)-tagged human RSK2 or mutated
versions of RSK2, S227A and T577A, were described previously
(37), as well as the expression vector encoding murine wild-type CBP (1). The expression vector encoding PDK1 was a gift from G. Thomas (Basel, Switzerland). Transient transfections were performed by the phosphate calcium method. COS-1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 5% fetal
calf serum (FCS) and plated at 30% confluence before transfection. After transfection (24 h), cells were serum deprived for an additional 48 h and, when required, treated with PD98059, EGF, tetradecanoyl phorbol acetate (TPA), or UV light as previously described
(37).
Proteins.
Cells were washed once with ice-cold
phosphate-buffered saline (PBS) and resuspended in hypotonic buffer
(HB; 20 mM HEPES, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol [DTT],
0.2% NP-40, a cocktail of protease inhibitors, 20 mM sodium
fluorure, and 1 mM sodium orthovanadate) (42).
Cells were centrifuged for 20 s, giving supernatant (S0) and
pellet (P0). S0 was collected and supplemented to 120 mM NaCl and 10%
glycerol. Lysates were centrifuged at 13,000 × g for
20 min at 4°C, giving the cytoplasmic extract. P0 was resuspended
with HB supplemented to 420 mM NaCl and 20% glycerol, rocked for 30 min at 4°C, and centrifuged at 13,000 × g for 30 min
at 4°C. This fraction represented the nuclear extract. Whole-cell
extracts were prepared as described (53).
Western analysis.
Protein extracts were resolved by standard
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Samples were electroblotted onto Protan nitrocellulose (Schleicher and
Schuell). Membranes were incubated in Tris-buffered saline-1% low-fat
milk overnight at 4°C with specific antibodies. Immunocomplexes were revealed by chemiluminescence with anti-mouse, anti-rabbit, or anti-goat immunoglobulin antibodies.
Kinase and HAT assays.
Equal amounts of total nuclear
extracts adjusted to 120 mM NaCl were incubated with anti-RSK2 or
anti-CBP antibodies overnight at 4°C and with protein G- or
A-Sepharose for an additional 30 min. Immunoprecipitates were washed
three times in HB supplemented to 120 mM NaCl, protease inhibitors, 1 mM sodium orthovanadate, and 0.5% NP-40, followed by one wash with
kinase assay buffer (20 mM MOPS [morpholinepropanesulfonic acid, pH
7.2], 25 mM 
-glycerol phosphate, 5 mM EGTA, 1 mM sodium
orthovanadate, and 1 mM DTT) or one wash with acetylation buffer (50 mM
Tris [pH 8.0], 10% glycerol, 10 mM sodium butyrate, 1 mM DTT, 100 mM
Pefabloc), depending on whether immunoprecipitates were subjected to
kinase or HAT assays. Kinase assays were performed using the standard
S6 kinase assay (Upstate Biotechnology), while HAT assays were done as
described (5), using a mix of histones (Sigma) or an H3
tail peptide corresponding to the first 24 amino acids.
Protein-protein association studies.
Production in bacteria
of glutathione S-transferase (GST)-CBP(1-1098) and
GST-CBP(1098-1877) proteins has been described (2). For
purification, bacteria were centrifuged and lysed in 150 mM NaCl-1 mM
DTT-5 mM EDTA-25% sucrose-50 mM Tris [pH 7.5] supplemented with
protease inhibitors. Lysates were sonicated for 3 min at 4°C, and
bacterial debris was removed by centrifugation at 16,000 × g for 30 min. Lysates were loaded onto glutathione-Sepharose beads overnight with COS protein whole-cell extracts containing ectopic
RSK2 or not. This incubation was followed by three washes with buffer I
(5 mM EDTA, 250 mM NaCl, 50 mM Tris, pH 7.5), three washes with buffer
II (5 mM EDTA, 120 mM NaCl, 50 mM Tris, pH 7.5), and one wash with
acetylation buffer.
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RESULTS |
Mitogen-regulated association of RSK2 and CBP.
To investigate
the possible interplay between phosphorylation and acetylation, we
tested whether CBP could associate with RSK2. COS cells were
cotransfected with expression vectors encoding RSK2 and CBP proteins,
and immunoprecipitations were performed on nuclear extracts using
either anti-CBP or anti-RSK2 antibodies. In both cases, RSK2 and CBP
were readily coprecipitated (Fig. 1A). We
tested whether stimulation by EGF could modulate the CBP-RSK2 interaction. Interestingly, association of RSK2 with CBP was detected when cells remained in a quiescent state, i.e., in a medium deprived of
serum. In contrast, nearly no association of RSK2 and CBP was detected
immediately (10 min) following stimulation of cells with EGF (Fig. 1A).
This indicated that dissociation of the RSK2-CBP complex could be
phosphorylation dependent.
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FIG. 1.
Stimulation of cells with EGF induces RSK2-CBP complex
dissociation. (A) Coimmunoprecipitation of RSK2 and CBP ectopically
expressed in COS-1 cells. Transfected cells were serum deprived for
48 h following transfection and then treated or not for 10 min
with EGF. Nuclear extracts were prepared and immunoprecipitated with
anti-CBP A-22 (left) and anti-RSK2 C-19 (right) antibodies. Detection
of RSK2 in anti-CBP immunoprecipitates was performed using anti-RSK2 E1
antibody (left), whereas anti-CBP A-22 antibody was used to reveal the
presence of CBP in anti-RSK2 immunoprecipitates. Similar levels of
ectopic CBP and RSK2 were detected in nuclear extracts prepared from
cells transfected with CBP or with both CBP and RSK2 expression
vectors. Detection of ERKs with anti-ERK antibody shows that equivalent
amounts of total proteins were present in nuclear extracts
immunoprecipitated with anti-CBP (left) or anti-RSK2 (right)
antibodies. (B) The kinetics of association of RSK2 and CBP inversely
correlates to the phosphorylation status of RSK2. COS-1 cells were
stimulated with EGF for various times (10, 30, and 60 min). Association
of RSK2 with CBP was followed by detecting the presence of RSK2 in
nuclear proteins immunoprecipitated with the anti-CBP antibody. In
parallel, the phosphorylation status of RSK2 was evaluated from nuclear
extracts, using anti-phospho-RSK2 antibodies P-TS227 and P-T577. On the
left panel, RSK2 was ectopically expressed, while on the right panel
endogenous RSK2 and CBP were subjected to coimmunoprecipitation and
Western analysis.
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Maximal phosphorylation and activation of RSK proteins are known to
occur 5 to 30 min following mitogenic stimulation (
10,
21). To test the kinetics of RSK2 phosphorylation and
association
with CBP, we determined a time course after EGF
stimulation. CBP-RSK2
association was tested in parallel with the
levels of RSK2 phosphorylation
using two anti-phospho-RSK monoclonal
antibodies (Fig.
1B). These
antibodies, P-S227 and P-T577, are directed
against two critical
activation sites of RSK2 located within the
activation loops of
the N-terminal and C-terminal kinase domains,
respectively,(
37).
Dissociation of RSK2 from CBP was
maximal 10 to 30 min after stimulation,
concomitant with a drastic
increase in RSK2 phosphorylation. As
soon as RSK2 phosphorylation
decreased, association with CBP increased
(compare 30- and 60-min
points in Fig.
1B). In conclusion, association
of RSK2 with CBP
correlates inversely with RSK2
phosphorylation.
Analogous results were obtained by analyzing the endogenous RSK2 and
CBP proteins. RSK2 was present in anti-CBP immunoprecipitates
prepared
from NIH 3T3 (Fig.
1B, right panel), COS, and HEK 293
cells (not shown)
following the same kinetics after EGF stimulation
as in transfected
cells. In additional experiments we have found
that the other forms of
p90
rsk, RSK1 and RSK3, as well as the related
kinase MSK1 are also found
in anti-CBP immunoprecipitates after ectopic
expression. In particular,
paralleling what we observed here with RSK2,
dissociation of the
MSK1-CBP complex is concomitant with an increase in
MSK1 phosphorylation
(not shown). Our results confirm and extend
previous observations
showing CBP interaction with
p90
rsk (
38), and reveal that
phosphorylation of RSK2 is a critical
event regulating RSK2-CBP complex
formation.
Phorbol esters and UV light regulate RSK2-CBP association.
The
results described above would predict that stimulation of cells with
any RSK-activating factor should induce dissociation of the RSK2-CBP
complex. Since RSK proteins are phosphorylated and activated not only
by growth factor stimulation, but also by phorbol esters and stress
factors (10, 17, 37), we tested whether treatment of cells
with TPA and UV light also affected the association of RSK2 and CBP
(Fig. 2). Both treatments induced dissociation of the complex within minutes following stimulation to an
extent comparable to dissociation induced by EGF. Importantly, dissociation again correlated with an increase in RSK2 phosphorylation, as revealed by the P-T577 antibody.
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FIG. 2.
Stimulation of cells with TPA and UV leads to RSK2-CBP
complex dissociation. COS-1 cells were cotransfected with RSK2 and CBP
and treated or not with TPA for 15 min, UV light for 30 min, or EGF for
10 min. Induction of RSK2 phosphorylation following treatment was
determined using the P-T577 antibody. Anti-phospho-CREB antibody
(P-CREB) was also used to show the effectiveness of the various
treatments.
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Phosphorylation of RSK2 at Ser227 controls association with
CBP.
RSK proteins are composed of two unrelated kinase domains
connected by a regulatory linker region (17, 31, 32).
Mitogenic stimulation induces activation of the Ras-MEK-ERK cascade,
which in turn leads to activation of the C-terminal kinase domain of RSK2 through direct phosphorylation of Thr577 by ERKs (14, 17, 48, 53). This results in autophosphorylation of Ser386, a residue located in the linker region, and recruitment of PDK1 to this
site (24, 29). PDK1 then phosphorylates Ser227, resulting in activation of the N-terminal kinase domain, which is involved in
substrate phosphorylation (6, 22, 31, 41). We wished to
establish which phosphorylation event in RSK2 modulates its capacity to
interact with CBP.
In cells stimulated with EGF but concomitantly treated with the
MEK1/2-specific inhibitor PD98059 (
40), the CBP-RSK2
dissociation
is impaired (Fig.
3A). Thus,
block of ERK signaling, which results
in mostly dephosphorylated RSKs,
elicits a stable RSK2-CBP complex.
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FIG. 3.
Dissociation of RSK2-CBP complex is induced by
phosphorylation of serine 227 within the activation loop of the
N-terminal kinase domain of RSK2. (A) Unphosphorylated RSK2 binds to
CBP. COS-1 cells were cotransfected with wild-type RSK2 and CBP and
left untreated, treated with EGF, treated with EGF and PD98059, or
cotransfected with PDK1. Phosphorylation levels of RSK2 were detected
using both P-S227 and P-T577 antibodies, while activation of the
MAPK/ERK pathway was evaluated using an anti-phospho-ERK antibody
(P-ERK). (B) Preferential association of CBP with the N-terminal domain
of RSK2 (amino acids 1 to 350). The lower panel shows that comparable
levels of the truncated RSK2 proteins were used. (C) Association of CBP
with the RSK2 mutant S227A is not affected by stimulation of cells with
EGF. COS-1 cells were transfected with wild-type RSK2, mutant S227A,
and mutant T577A and stimulated or not with EGF for 10 min.
Phosphorylation levels of wild-type and mutated versions of RSK2 were
detected using P-S227 and P-T577 antibodies.
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In order to selectively phosphorylate the N-terminal kinase domain of
RSK2, we ectopically expressed PDK1, which is a constitutively
active
kinase in serum-deprived cells (
3) (Fig.
3A). This
resulted
in powerful Ser227 phosphorylation but nearly no Thr577
phosphorylation,
as revealed by the anti-phospho-RSK2 specific
antibodies. The
effect of PDK1 on Ser227 phosphorylation disrupts
CBP-RSK2 association,
as it results in a threefold activation of RSK2
even in cells
deprived of serum (Fig.
3A and data not shown). These
results
were confirmed by the use of truncated RSK2 proteins in which
the two catalytic domains were separated (Fig.
3B). Only the
N-terminally
truncated protein RSK2(1-350) bound
CBP.
These results were further supported by coimmunoprecipitation assays of
CBP with RSK2 mutants (Fig.
3C). To selectively prevent
phosphorylation
of Ser227 or Thr577, we used two modified versions
of RSK2, mutants
S227A and T577A, respectively. Mutant T557A only
weakly associated with
CBP in unstimulated cells, while EGF stimulation
resulted in
dissociation of the complex. In both cases, phosphorylation
at Ser227
of mutant T577A was higher than that of wild-type RSK2
in basal
conditions. Therefore, the importance of phosphorylation
at Ser227 in
decreasing the association with CBP is confirmed.
In contrast to mutant
T577A, mutant S227A associated with CBP
when cells were both serum
deprived and EGF stimulated. Interestingly,
mutation of Ser227 to
alanine did not preclude phosphorylation
of Thr577. Indeed, Thr577 of
mutant S227A was normally phosphorylated
in response to EGF stimulation
and, compared to wild-type RSK2,
was even hyperphosphorylated in cells
deprived of serum. These
data indicated that dissociation of RSK2 from
CBP did not require
phosphorylation of Thr577, while phosphorylation at
Ser227 is
a prerequisite for dissociation of the
complex.
Additional experiments confirmed these results. In particular,
transfection of cells with truncated versions of RSK2 showed
that the
C-terminal kinase domain of RSK2 did not bind CBP (not
shown). In
contrast, the N-terminal kinase domain of RSK2 interacted
with CBP when
Ser227 was not phosphorylated (not shown). Collectively,
these data
suggested that phosphorylation of Ser227 in the N-terminal
kinase
domain of RSK2 is necessary and sufficient to induce dissociation
of
the RSK2-CBP complex. As the mechanism of activation of RSK2
by
mitogens implies that phosphorylation of Ser227 depends on
phosphorylation of Thr577 (
17,
24), both phosphorylation
events
should be associated with complex dissociation. This is indeed
what was observed (Fig.
1B).
Association of CBP inhibits kinase activity of RSK2.
Our data
show that RSK2 and CBP preferentially associate when RSK2 is inactive,
suggesting that complex formation might constitute a mechanism to
impair RSK2 kinase activity. To test this possibility, we transfected
COS cells with RSK2 alone or with CBP and determined the in vitro RSK2
kinase activity on anti-RSK2 immunoprecipitates (Fig.
4A). The presence of ectopic CBP in
nuclear extracts reduced RSK2 kinase activity by about fourfold when
cells were deprived of serum. A weaker reduction of RSK2 kinase
activity (twofold) was also observed in EGF-stimulated cells, likely
due to an incomplete phosphorylation of the RSK2 pool. We also directly
evaluated the phosphorylation levels of endogenous CREB and histone H3,
two physiological targets of RSK2 (21, 43, 51) (Fig. 4B).
Western analysis using anti-phospho-CREB and anti-phospho-H3 antibodies showed that basal phosphorylation levels of both CREB and H3 were impaired by CBP coexpression. In EGF-stimulated cells, overexpression of RSK2 induced high phosphorylation levels of CREB and H3,
irrespective of the presence of ectopic CBP. Thus, altogether, these
results indicated that binding of CBP to RSK2 impaired its kinase
activity.
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FIG. 4.
RSK2-CBP complex formation inhibits RSK2 kinase
activity. (A) RSK2 kinase activity is inhibited by the presence of
ectopic CBP. COS-1 cells transfected with RSK2 or with both RSK2 and
CBP and stimulated or not with EGF for 10 min. Nuclear extracts were
prepared and immunoprecipitated (IP) with anti-RSK2 C-19 antibody, and
an in vitro kinase assay was performed using the S6 peptide as the
substrate. The total levels of RSK2 protein were verified by Western
analysis. (B) Basal phosphorylation levels of CREB and histone H3 are
downregulated in the presence of ectopic CBP. COS-1 cells were treated
as in panel A, and the phosphorylation status of endogenous CREB and
histone H3, two targets of RSK2, was detected using phospho-CREB and
phospho-H3 antibodies. Western analysis was performed on similar
amounts of total proteins, as shown by using anti-CREB antibody. In
addition, equivalent amounts of ectopic RSK2 were present in cells
overexpressing CBP or not.
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Association with RSK2 inhibits HAT activity of CBP.
The
general coactivator CBP enhances transcription of various genes by at
least two molecular mechanisms: first, by bridging various
transcription factors, such as CREB, with the basal transcriptional machinery, and second, CBP has a potent HAT activity which links transcription to chromatin remodeling (26). The HAT domain
of CBP is adjacent to the C/H3 region (39), which
corresponds to the RSK binding site (38). Interestingly,
binding of the adenovirus E1A protein with this same region modulates
HAT activity, probably by inducing local conformational changes
(9, 28). We asked whether binding of RSK2 to CBP could
also regulate its HAT activity. To test this, we transfected cells with
CBP alone or with RSK2 and prepared anti-CBP immunoprecipitates from
nuclear extracts. The in vitro HAT activity present in the
immunoprecipitates was then assessed using an H3 peptide corresponding
to the N-terminal tail of histone H3 (Fig.
5A). Ectopically expressed RSK2 impaired CBP's HAT activity, although modestly. To further support these data,
a recombinant GST-CBP(1098-1877) construct, which contained both HAT
and C/H3 domains, was incubated with COS cells extracts in which
ectopic RSK2 was present. Reactions were then pulled down, and HAT
activities were assessed using the H3 peptide. As predicted, Western
analyses using anti-RSK2 antibody showed that GST-CBP(1098-1877)
interacted with RSK2 (not shown). The presence of RSK2 impaired
CBP(1098-1877) HAT activity in a dose-dependent manner (Fig. 5B).
Thus, we concluded that binding of RSK2 to CBP inhibited its HAT
activity.
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FIG. 5.
RSK2-CBP complex formation inhibits CBP HAT activity.
(A) CBP HAT activity is inhibited by ectopic expression of RSK2. COS-1
cells transfected with CBP or with both CBP and RSK2 were stimulated or
not with EGF. Nuclear extracts were immunoprecipitated (IP) with
anti-CBP antibody, and in vitro CBP HAT activity was assessed using an
N-terminal H3 peptide. The total levels of CBP protein were verified by
Western analysis. (B) The HAT activity of GST-CBP constructs containing
HAT and E1A domains is inhibited when incubated with RSK2. The
GST-CBP(1098-1877) construct was incubated with COS whole-cell
extracts in which ectopic RSK2 was not (no RSK2), slightly (1/10 RSK2),
or highly (RSK2) present. GST proteins were subsequently pulled down,
and the HAT activity was determined as in panel A using the H3 peptide.
The GST-CBP(1-1098) construct, which lacks the HAT domain, was used as
the negative control.
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DISCUSSION |
The induction of gene transcription is the result of multiple
signaling events acting in concert at the level of specific activators
and coactivators, as well of selected chromatin locations where
remodeling may occur following histone modifications (11, 34, 47,
50). The dynamic nature of these events is critical as it
dictates the programming of gene expression. Thus, deciphering the
functional interplays which operate among signaling components is an
important step towards understanding all cell responses. Here we have
shown that a growth factor-induced kinase, RSK2, physically associates
in a dynamic fashion with a transcriptional coactivator, CBP, which has
HAT activity. This finding underscores the possibility that
phosphorylation and acetylation of specific substrates may be regulated
by this interaction and thus that the parameters regulating the
interaction itself are essential to the control of downstream events.
The association between p90rsk and CBP was
described previously, but the signaling events regulating the
interaction were not explored in detail, also because the HAT activity
of CBP was not yet characterized at the time (38). Our
findings support a model in which formation of the RSK2-CBP complex is
phosphorylation dependent. In contrast to the well-characterized CREB-CBP interaction, where association is induced by CREB
phosphorylation at Ser133 (13), here we have shown that
phosphorylation and activation of RSK2 induce dissociation from CBP
(Fig. 1). Presumably, following the dissociation, RSK2 becomes
available to phosphorylate CREB in response to EGF (21,
51). At the same time CBP becomes available to interact with the
newly phosphorylated CREB. The dynamics of this tripartite regulation
fit the kinetics of early gene transcriptional activation well (Fig.
1). In addition to CREB, other potential substrates are likely to
benefit from the combined function of the activated RSK2 and CBP.
Indeed, the enzymatic activities carried by CBP and RSK2 could exert a
coordinate action at the level of the histone H3 tail, on which the two
major sites of modification, phosphorylation (Ser10) and acetylation
(Lys14), are closely spaced (12) (Fig.
6). In support of this view, it is
thought that the combined phosphorylation-acetylation of the H3 tail
constitutes an essential step in the local remodeling of chromatin
structure (11, 12, 35).
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FIG. 6.
Schematic representation of the
phosphorylation-regulated association of CBP and RSK2. In quiescent
cells, the two proteins are found associated in a complex with low
kinase and HAT activities. In serum-starved cells, the basal
phosphorylation levels of two RSK2 substrates, CREB and histone H3, are
also low. Upon EGF mitogenic stimulation and consequent activation by
phosphorylation of RSK2 at Ser227, CBP and RSK2 dissociate. This
results in increased Ser133 CREB and Ser10 H3 phosphorylation
(21, 43, 51). The newly phosphorylated CREB associates
with CBP, whose HAT activity is also increased. The coordinated
activation of both kinase and HAT activities may converge at the
histone H3 tail, where concerted modifications of Ser10 and Lys14 have
been reported previously (12, 35). This possible scenario
does not take into account additional signaling routes which could
influence the function of the various components. While it is likely
that physical interactions among other kinases and HAT molecules will
be found, deciphering the regulatory pathways controlling their
functions is essential to the understanding of the molecular events
involved in activation of gene expression. Ac-K14, acetylated
residue K14; P-S10, phosphorylated residue S10.
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Earlier studies have reported that CBP and not specifically identified
members of the p90rsk family interact upon
activation of the Ras-dependent signaling pathway in PC12 cells
(38). The difference in the mitogen-regulated CBP-RSK2
association reported here may be related to the choice of the cell type
in which the analyses were performed. We have readily reproduced our
results in at least three different proliferating cells (NIH 3T3,
COS-1, and HEK 293), while PC12 cells were not used in our study. In
addition, the use of specific anti-phospho-RSK2 antibodies and of
single-amino-acid mutations (Ser227) powerfully validates the
regulatory scenario reported here (Fig. 6).
An important outcome of this study concerns the reciprocal regulation
of kinase and HAT activities exerted, respectively, by CBP and RSK2.
Indeed, at the time the two proteins are associated in a complex, i.e.,
before mitogenic stimulation, both RSK2 kinase and CBP HAT activities
are downregulated. Within a very few minutes after EGF stimulation and
consequent dissociation of the complex, both kinase and HAT activities
increase significantly. Therefore, these observations are crucial for
the understanding of how the two processes of phosphorylation and
acetylation are connected. A possible view of our findings is related
to the antagonistic function that the inactive RSK2 may exert by
associating with CBP, as we have shown that the interaction decreases
the HAT activity. We favor a model in which activation of the MAPK
signaling pathway and the consequent phosphorylation of RSK2 constitute
a switch, as they allow dissociation of the CBP-RSK2 complex and
activation of HAT function. Thus, activation of RSK2 by growth factors
may result in coordinated transcription activation and chromatin remodeling.
Our findings hint at the possibility that similar scenarios may exist
for other kinases and HAT molecules. It is indeed conceivable that
situations like the one described here may operate under the control of
various signaling pathways, specificity being provided by either the
distinct combinations in the association or the dynamic modifications
at various regulatory sites.
| |
ACKNOWLEDGMENTS |
We thank for help, discussions, and generous gifts of reagents
the following colleagues: A. Hanauer, C. D. Allis, P. Cheung, G. Thomas, M. Frödin, G. M. Fimia, S. Jacquot, M. Zéniou,
and J. L. Mandel.
This work was supported by grants from Centre National de la Recherche
Scientifique, Institut National de la Santé et de la Recherche
Médicale, Centre Hospitalier Universitaire Régional, Fondation de la Recherche Médicale, Association Française
contre les Myopathies, Université Louis Pasteur, Human Frontier
Science Program, Organon (Akzo/Nobel), and Association pour la
Recherche sur le Cancer.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institut de
Génétique et de Biologie Moléculaire et Cellulaire,
CNRS, INSERM, Université Louis Pasteur, 1, rue Laurent Fries,
67404 Illkirch, Strasbourg, France. Phone: 33 388 653410. Fax: 33 388 653246. E-mail: paolosc{at}igbmc.u-strasbg.fr.
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Molecular and Cellular Biology, October 2001, p. 7089-7096, Vol. 21, No. 20
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.20.7089-7096.2001
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Abstract
Introduction
