From the Center for Cardiovascular Research and Department of Medicine, University of Rochester, Rochester, New York 14642
Received for publication, August 19, 2002, and in revised form, March 3, 2003
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ABSTRACT |
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p90 ribosomal S6 kinase 1 (RSK1) is a
serine/threonine kinase that is activated by extracellular
signal-related kinases 1/2 and phosphoinositide-dependent
protein kinase 1 upon mitogen stimulation. Under basal conditions, RSK1
is located in the cytosol and upon stimulation, RSK1 translocates to
the plasma membrane where it is fully activated. The ability of RSK1 to
bind the adapter protein 14-3-3 The p90 ribosomal S6 kinase
(RSK)1 family members have a
role in mitogen-activated cell growth and proliferation,
differentiation, and cell survival. RSK is a serine/threonine kinase
that is a substrate of ERK1/2 and lies downstream of the Raf/MEK/ERK
protein cascade (1). Recent studies have provided insight into
mechanisms responsible for activation of RSK1. RSK1 has two distinct
domains, both of which are catalytically functional. Phosphorylation of RSK1 by ERK on the carboxyl-terminal catalytic loop activates the RSK1
carboxyl-terminal kinase domain. This induces a conformational change
abrogating the negative effect of this domain, allowing activation of
the amino-terminal kinase domain by PDK1 (2). Cellular localization is
thought to be important in the regulation of RSK activity. Upon mitogen
stimulation RSK undergoes a rapid and transient localization to the
plasma membrane, which requires ERK docking and phosphorylation. At the
plasma membrane PDK1 inputs as well as ERK- and PDK1-independent events
lead to full activation of RSK (3). RSK can then phosphorylate its
substrates, which include the Na+/H+ exchanger
NHE1 isoform at the plasma membrane, nuclear transcription factors, and
transcriptional coactivator proteins (1, 4).
14-3-3 proteins are a family of 30-kDa acidic proteins that interact
with a wide variety of cellular proteins including protein kinases,
receptor proteins, enzymes, structural and cytoskeletal proteins, and
small G-proteins (5-8). The predominant 14-3-3-binding motif involves
a phosphoserine, but interaction can involve other varied motifs and
may not require phosphorylation (9-11). Because 14-3-3 proteins exist
as dimers, they can simultaneously bind multiple phosphorylation sites
on the same protein (12, 13) or function as scaffolds to form
protein-protein interactions (5-8, 14). Other roles of 14-3-3 proteins
include regulation of interacting proteins activity, subcellular
localization, and/or stability. For example, 14-3-3 proteins are known
to act as a scaffold for several proteins in the mitogen-activated
protein kinase cascades, including MEKK1, 2, and 3 (15), and to
regulate the kinase activity of Raf (16).
Work in our laboratory has shown that NHE1 is activated by
mitogens through phosphorylation of serine 703 by RSK (4).
Subsequently, 14-3-3 DNA Constructs and Mutagenesis--
RSK1 and 14-3-3 In Vitro Translation of Full-length RSK1--
Full-length rat
p90RSK1 under control of the T7 promoter in pcDNA3.1/Myc-His was
transcribed and translated in vitro using the
TNT T7-coupled reticulocyte lysate system (Promega). The
nascent protein was labeled using Transcend biotin-lysyl-tRNA
(Promega). Briefly, 40 µl of TNT Quick Master Mix, 1 µl
of methionine (1 mM), 1 µg of template DNA, and 1 µl of
biotin-lysyl-tRNA were mixed in a final volume of 50 µl, incubated
for 90 min at 30 °C, and immediately used for the binding assays.
Cell Culture--
PS127A cells (Chinese hamster lung fibroblasts
that overexpress NHE1) were a gift from Dr. J. Pouyssegur (University
of Nice, Nice, France). PS127A, NIH3T3, Cos7, and HEK293 cells were
grown in Dulbecco's modified Eagle's medium supplemented with 25 mM NaHCO3, 10 mM HEPES, pH 7.4, 50 IU/ml penicillin, 50 µg/ml streptomycin, 10% fetal bovine serum
(FBS) in a 5% CO2, 95% O2 incubator at 37 °C. The cells were serum-starved (0% FBS) overnight prior to the experiments.
Preparation of Cell Lysates--
The cell monolayers were rinsed
with ice-cold phosphate-buffered saline (150 mM NaCl, 20 mM Na2PO4, pH 7.4) and then scraped in 1 ml of phosphate-buffered saline. After a brief centrifugation, the
cells were solubilized in 1 ml of cell lysis buffer (10 mM HEPES, pH 7.4, 50 mM sodium pyrophosphate, 50 mM NaF, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1 mM Na3VO4,
0.5% Triton X-100 plus 1:1000 protease inhibitor mixture (Sigma)). The
cells were sonicated for 20 s (in the case of pull-down studies)
or needle-homogenized (in the case of coimmunoprecipitations), agitated
on a rotating rocker at 4 °C for 30 min, and centrifuged at
12,000 × g for 30 min to remove insoluble cellular
debris. For some pull-down experiments, phosphatase treatment of cell
lysates was performed by lysing cells in CIAP buffer (20 mM
Tris, pH 8, 150 mM NaCl, 1 mM
MgCl2, 1 mM dithiothreitol, 0.5% Triton X-100,
plus 1:1000 protease inhibitor mixture). The lysates were treated with
or without 200 units of calf intestinal alkaline phosphatase (Promega)
at 37 °C for 1 h. Where indicated the inhibitors
Na3VO4 (1 mM) and NaF (50 mM) were added.
Immunoprecipitations and Pull-downs--
For
coimmunoprecipitation studies, HEK293 cells were cotransfected with
FLAG-tagged RSK1 and Xpress-tagged 14-3-3
Immunoprecipitates and pull-downs were then washed four times with 1 ml
of cell lysis buffer before the addition of Laemmli sample buffer.
After heating at 95 °C for 3 min, the proteins were resolved on
SDS-PAGE and transferred to nitrocellulose membranes for Western
analysis. Immunoblotting was performed with anti-Xpress antibody
(Invitrogen), M2 anti-FLAG antibody (Sigma), anti-RSK1 antibody (Santa
Cruz), anti-14-3-3 Preparation of Membrane Fractions--
The cell
monolayers were rinsed and scraped in 1 ml of ice-cold
phosphate-buffered saline. After a brief centrifugation the cells were
resuspended in 750 µl of membrane fractionation buffer (25 mM Tris-HCl, pH 7.4, 5 mM EGTA, 5 mM EDTA, 100 mM NaF, 5 mM dithiothreitol, and 1:1000 protease inhibitor mixture). The lysates were prepared by needle homogenization and nuclei, and unbroken cells
were pelleted at 850 × g for 10 min. Equal amounts of
protein in postnuclear supernatants were centrifuged at 100,000 × g for 1 h to obtain soluble fractions and total
membrane pellets. The soluble fraction was removed, and the membrane
pellets were solubilized in 250 µl of buffer containing 1% Triton
X-100, sonicated, and rocked for 1 h at 4 °C and analyzed by
SDS-PAGE as described above.
RSK1 Kinase Assays--
Twenty milliunits of recombinant RSK1
(Upstate Biotechnology Inc.) was incubated in 30 µl of kinase
buffer (30 mM HEPES, pH 7.5, 100 mM NaCl, 10 mM MgCl2, 10 mM MnCl2,
and 0.2 mg/ml bovine serum albumin) overnight with 10 µg of
GST-14-3-3 CREB Reporter Gene Assays--
HEK293 cells were plated at
2 × 105 cells/well in 24-well plates 24 h prior
to transfection. The cells were transfected with different expression
plasmids together with the activator plasmid for CREB (pFA2-CREB) and
the reporter gene for luciferase (pFR-Luc) (Stratagene). A plasmid
expressing the enzyme Renilla luciferase was used as an
internal control (pRL-TK; Promega). After 24 h, the cells were
serum-starved for 24 h and then stimulated as indicated. Firefly
and Renilla luciferase activities were measured using a Dual
Luciferase Reporter System (Promega) with a Wallac 1420 luminometer.
The data are expressed as firefly luciferase normalized by
Renilla luciferase activity.
Immunocytochemistry and Confocal Microscopy--
Cos7 cells
seeded on glass coverslips were transfected with pCMV-FLAG-RSK1 using
LipofectAMINE 2000. After 24 h the cells were serum-starved for a
further 12 h and treated with 20% FBS. The cells were fixed in
3% paraformaldehyde, permeabilized with 0.2% Triton X-100, and
blocked in phosphate-buffered saline with 5% FBS, 0.2% bovine serum
albumin. The cells were stained with anti-FLAG antibody to visualize
RSK1 and anti-14-3-3 RSK1 Binds 14-3-3
To determine whether the interaction between 14-3-3 The Interaction between RSK and 14-3-3
Binding to 14-3-3 proteins is usually mediated via a
RSXpSXP or RXXXpSXP
consensus motif (12, 13). RSK1 contains a number of putative
14-3-3 Serum Stimulation Reduces the RSK1-14-3-3 Subcellular Colocalization of RSK1 and 14-3-3--
14-3-3 proteins
function as scaffolding proteins and have been shown to affect the
subcellular localization of interacting proteins (reviewed in Refs. 7
and 14). For example, 14-3-3 can sequester proteins in the cytosol
and/or target them to the plasma membrane. RSK1 has previously been
shown to be located in the cytosol under basal conditions and to
translocate to the plasma membrane after stimulation where it becomes
fully activated (3). Therefore, we determined the subcellular
distribution of 14-3-3
To demonstrate colocalization of RSK1 and 14-3-3 14-3-3
To assess the functional significance of the RSK-14-3-3 binding
in vivo, we compared the kinase activity of wild type RSK1 to the non-14-3-3
The increased CREB activity in cells transfected with RSK1S154A
versus those transfected with RSK1WT combined with the
finding that 14-3-3
Because full activation of RSK requires plasma membrane
translocation (3), the increased activity of RSK1S154A suggested an
increase in the amount of this mutant at the plasma membrane compared
with RSK1WT. Therefore, we performed subcellular fractionation in
HEK293 cells overexpressing RSK1WT or RSK1S154A. The amount of
RSK1S154A in the membrane fraction under basal conditions was increased
by 28.3 ± 2.9% (p < 0.05) as compared with
RSK1WT, whereas upon serum stimulation the relative amounts of RSK1WT
and mutant in the membrane fraction were similar (Fig.
9, A and B).
The major finding of the present study is that the
serine/threonine kinase RSK1 is a 14-3-3-interacting protein whose
activity is negatively regulated by 14-3-3. The association between RSK and 14-3-3 14-3-3 proteins have previously been shown to bind mainly through the
motifs RSXpSXP and
RXXXpSXP, where pS denotes phosphoserine and
X is any amino acid; however, there are strong preferences for particular amino acids over others in the X positions
(12, 13). To locate binding sites in RSK1 we made eight serine to alanine mutations at close matches to these consensus motifs
(RLS154KEV, RLGS307GP, RDS363PGI,
RGFS380FV, RDPS457EE,
REAS513FV, RIS630S631GK, and
RKLPS732TT). Only mutation of serine 154 greatly diminished
binding of RSK1 to 14-3-3 The activation of RSK is dependent on a combination of both
phosphorylation and plasma membrane localization events. ERK
phosphorylates RSK1 within the carboxyl-terminal domain, and the
phosphoinositide dependent kinase PDK1 phosphorylates RSK1 within the
NH2-terminal domain (1, 2). Richards et al.
(3) demonstrated recently that stimulated RSK1 transiently
associates with the plasma membrane before accumulating in the nucleus.
They also showed that the activity of a kinase inactive RSK1 mutant
lacking the ERK docking site could be restored by addition of a
membrane-targeting myristoylation site to the level of myristoylated
wild type RSK1. This suggests that ERK has a role in escorting RSK to a
plasma membrane-associated complex, although the exact mechanism of
localization of RSK remains unclear (3).
14-3-3 proteins have been shown to play an important role in the
subcellular distribution of a variety of signaling proteins. They
appear to have the ability to both sequester proteins in the cytosol
(for example protein kinase C Upon activation by serum, a portion of both RSK1 and 14-3-3 In this study, incubation of RSK1 with GST-14-3-3 These results suggest that 14-3-3 plays a role in maintaining RSK1
signaling fidelity. Under basal conditions it may prevent activation by
weak ERK signals. It is possible that RSK1 is held in the cytoplasm in
an inactive conformation by 14-3-3 In summary, we demonstrate for the first time that RSK1 is a
14-3-3-binding protein and suggest a functional role of the
14-3-3 was investigated because RSK1
contains several putative 14-3-3-binding motifs. We demonstrate that
RSK1 specifically and directly binds 14-3-3
. This interaction was
dependent on phosphorylation of serine 154 within the motif RLSKEV of
RSK1. Binding of RSK1 to 14-3-3
was maximal under basal conditions
and decreased significantly upon mitogen stimulation. After 5 min of
serum stimulation, a portion of 14-3-3
and RSK1 translocated to the
membrane fraction, and immunofluorescence studies demonstrated
colocalization of RSK1 and 14-3-3
at the plasma membrane in
vivo. Incubation of recombinant RSK1 with 14-3-3
decreased
RSK1 kinase activity by ~50%. Mutation of RSK1 serine 154 increased
both basal and serum-stimulated RSK activity. In addition, the
epidermal growth factor response of RSK1S154A was enhanced compared
with wild type RSK. The amount of RSK1S154A was significantly increased
in the membrane fraction under basal conditions. Increased
phosphorylation of two sites essential for RSK1 kinase activity
(Ser380 and Ser363) in RSK1S154A compared with
RSK1 wild type, demonstrated that 14-3-3 interferes with RSK1
phosphorylation. These data suggest that 14-3-3
binding negatively
regulates RSK1 activity to maintain signal specificity and that
association/dissociation of the 14-3-3
-RSK1 complex is likely to be
important for mitogen-mediated RSK1 activation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
binds to this site and prevents
dephosphorylation of serine 703 (17). In the present study we
investigated whether RSK1 was also a binding partner for 14-3-3
because it contains a number of putative 14-3-3 interaction motifs. We
found that 14-3-3
specifically interacts with RSK1 in a
phosphorylation-dependent manner, and the site of
interaction was identified as serine 154. RSK-14-3-3
binding is
maximal under quiescent conditions and upon mitogen stimulation; both
RSK and 14-3-3
translocate to the plasma membrane where they
presumably dissociate because binding to 14-3-3
is decreased.
Furthermore, 14-3-3
binding is inhibitory to RSK activity, and
RSK1S154A, which does not interact with 14-3-3, has increased activity
both basally and upon mitogen stimulation. This study suggests an
important role for 14-3-3
in the negative regulation of RSK kinase activity.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
point
mutations were created using the QuikChange site-directed mutagenesis
kit (Stratagene) following the manufacturer's protocol. GST-14-3-3
was a gift from A. J. Muslin (Washington University School of
Medicine). 14-3-3
constructs were subcloned into
pcDNA3.1Xpress/His vector (Invitrogen), and rat RSK1 was subcloned
into pCMV-FLAG (Stratagene) or pcDNA3.1/Myc-His (Invitrogen). All
of the constructs and mutants were verified by DNA sequencing.
. Immunoprecipitations were
carried out by preclearing cell lysates with protein A/G-agarose (Santa
Cruz) for 1 h followed by incubation with anti-Xpress antibody for
3 h and with protein A/G-agarose for a further 1 h or M2
anti-FLAG-agarose beads for 4 h. Pull-downs were performed by
incubating 10 µg of GST-14-3-3
or GST-14-3-3
K49Q (produced as
described previously (17)) bound to glutathione-Sepharose overnight at
4 °C.
antibody (Santa Cruz), or anti-NHE1 antibody
(Chemicon), anti-phospho-RSK1 (Ser363) (Upstate
Biotechnology), and anti-phospho-RSK (Ser380) (which was a
gift from Dr. C. Chrestensen, University of Virginia). Immunoreactive
bands were detected with horseradish peroxidase-conjugated secondary
antibodies (Amersham Biosciences) and enhanced chemiluminescence.
or GST-14-3-3
K49A. To initiate the kinase reaction, 10 µg of S6 peptide, 7.5 µM ATP, and 5 µCi of
[
-32P]ATP (Amersham Biosciences) were added, and the
reaction was incubated for 10 min at 30 °C. The reaction was
terminated by spotting 25 µl of the reaction onto P81
phosphocellulose filter paper. The filters were washed five times in
0.75% phosphoric acid and one time in acetone, and radioactive
incorporation was determined by Cerenkov counting.
antibody and then incubated in secondary
antibodies (Vector). The images were acquired using an Olympus IX70
Fluoview confocal microscope.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
in Vitro and in Vivo--
Previously our
laboratory has demonstrated a direct interaction between the
Na+/H+ exchanger isoform NHE1 and 14-3-3
at
serine 703, the site of NHE1 phosphorylation by RSK (17). Because
phosphorylation by RSK on serine 703 is required for binding of the
scaffolding protein 14-3-3 and RSK1 contains several putative
14-3-3-binding sites similar to the consensus 14-3-3-binding motif
RSXpSXP, we investigated whether 14-3-3
could
interact with RSK1. Indeed, we found that RSK1 from extracts of PS127
fibroblasts was able to bind 14-3-3
in vitro (Fig.
1A). Previously, lysine 49 has
been shown to be essential for interaction with 14-3-3-binding partners
Bcr, Raf, and Cbl via a phosphoserine type interaction (18, 19).
Mutation of lysine 49 (K49A) in 14-3-3
abolished NHE1 interaction
with 14-3-3 (17). This mutation also abolished binding of RSK1 to 14-3-3
. In addition, GST alone was unable to bind to RSK1 (Fig. 1A).
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Fig. 1.
14-3-3 binds to RSK1
in vitro and in vivo.
A, PS127 cells were lysed, and a pull-down assay was
performed with GST-14-3-3, GST-14-3-3 K49Q, or GST beads. B,
biotin-labeled RSK1 was synthesized by in vitro translation
and incubated with GST-14-3-3
or GST-14-3-3
K49Q. Proteins bound
to beads were subjected to SDS-PAGE and blotted with horseradish
peroxidase-streptavidin. C, HEK293 cells were transfected
with FLAG-RSK1 and Xpress-14-3-3
. Cells transfected with empty
vector were used as a control. The cell lysates (lanes 1 and
2) were immunoprecipitated (IP) with anti-Xpress
antibody (lanes 3 and 4) or M2 anti-FLAG-agarose
beads (lanes 5 and 6) and immunoblotted for
FLAG-RSK1 and Xpress-14-3-3
.
and RSK1 was
direct, full-length RSK was synthesized by in vitro
translation, and its ability to bind to GST-14-3-3
was determined.
In vitro translated RSK bound avidly to GST-14-3-3
but
bound weakly to GST-14-3-3
K49A (Fig. 1B). To demonstrate
an in vivo interaction between RSK1 and 14-3-3
in
mammalian cells, HEK293 cells were transiently transfected with
FLAG-RSK1 and Xpress-14-3-3
. Xpress-14-3-3
immunoprecipitates
were prepared, and their ability to interact with RSK1 was examined by
Western blot. As shown in Fig. 1C (middle panels), 14-3-3
interacted with RSK1 in vivo. The
reverse immunoprecipitation (Fig. 1C, right
panels) demonstrates that FLAG-RSK1 can also coprecipitate 14-3-3
.
Requires
Phosphorylation--
Many interactions with 14-3-3 are dependent on
binding partner phosphorylation. To determine whether this is also the
case for the RSK1-14-3-3
interaction, PS127 cell lysates were
incubated with alkaline phosphatase for 1 h at 37 °C before
GST-14-3-3
pull-down. As shown in Fig.
2A, phosphatase treatment
(CIAP) inhibited the binding of RSK to 14-3-3
in the absence but not
the presence of phosphatase inhibitors. Hence dephosphorylation of RSK1
abolished its ability to bind 14-3-3
. In support of this conclusion,
treatment of intact cells with the broad spectrum serine/threonine
protein kinase inhibitor staurosporine also decreased the binding of
RSK1 to GST-14-3-3
in vitro (Fig. 2B).
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Fig. 2.
Phosphorylation of RSK1 is required for the
binding of 14-3-3 . A, PS127
cell lysates were incubated with or without CIAP and then pulled down
with GST-14-3-3
beads. Lanes 1 and 4, control;
lanes 2 and 5, 200 units of CIAP; lanes
3 and 6, 200 units of CIAP plus phosphatase inhibitors
(Inh.). Proteins bound to beads were subjected to SDS-PAGE
and immunoblotted with anti-RSK1 antibody. B, HEK293 cells
transfected with FLAG-RSK1 were treated with 1 µM
staurosporine (Stauro) for 1 h before lysis and
pull-down with GST-14-3-3
. Proteins bound to beads were subjected to
SDS-PAGE and immunoblotted with anti-FLAG antibody.
binding sites as shown in Fig.
3A
(RLS154KEV, RLGS307GP, RDS363PGI,
RGFS380FV, RDPS457EE, REAS513FV,
RIS630S631GK, and RKLPS732TT). RSK1
constructs with indicated serine to alanine point mutations were
transiently transfected into HEK293 cells, and the possible involvement
of these motifs in the RSK1-14-3-3
interaction was investigated
using GST-14-3-3
pull-down and coimmunoprecipitation assays.
Pull-down experiments demonstrated that mutation of S154A within the
motif RLS(154)KEV strongly reduced RSK1-14-3-3
interaction, whereas
the other mutations had little or no effect (Fig. 3B). Coimmunoprecipitation confirmed that RSK1S154A no longer bound to
14-3-3
in vivo (Fig. 3C). These data suggest
that RSK1 interacts with 14-3-3
through phosphorylated serine
154.
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Fig. 3.
Association of RSK1 mutants with
14-3-3 . A, sequence of rat
RSK1 showing potential 14-3-3
-binding motifs underlined.
B, HEK293 cell lysates transfected with RSK1
containing serine to alanine mutants of potential
14-3-3
-binding motifs were pulled down with GST-14-3-3
beads.
Proteins bound to beads were subjected to SDS-PAGE and immunoblotted
with anti-RSK1 antibody. C, HEK293 cells were transfected
with Xpress-14-3-3
, and FLAG-RSK1 or FLAG-RSK1S154A. Cells
transfected with empty vector were used as a control. The cell lysates
were immunoprecipitated (IP) with anti-Xpress antibody and
immunoblotted for FLAG-RSK1 (upper panel) or
Xpress-14-3-3
(middle panel). The lower panel
shows equal expression of the RSK constructs.
Interaction--
Because serum stimulates RSK1 activity, which then
activates NHE1 through phosphorylation of serine 703 (increasing the
NHE1-14-3-3 interaction) (4, 17), we investigated the effect of serum on the RSK1-14-3-3
interaction. PS127 fibroblasts were stimulated with 20% serum, and RSK association with GST-14-3-3
was determined in a pull-down assay. Binding of RSK1 to 14-3-3
was decreased by
34.6 ± 7.5% in cells stimulated for 5 min, 36.2 ± 4.1% at
10 min, and 28.2 ± 6.5% at 20 min (p < 0.05;
Fig. 4, A and B).
This effect was also observed in HEK293 cells and Cos7 cells
overexpressing exogenous RSK1 (data not shown). Preincubating PS127
cells for 30 min with the MEK1 inhibitor PD98059 (30 µM),
which prevents agonist-stimulated ERK1/2 and RSK activation, reversed
this inhibition (Fig. 4, A and B). As a control,
NHE1 interaction with GST-14-3-3
was verified. Binding of
GST-14-3-3
to NHE1 was stimulated by serum, and this stimulation was
significantly inhibited by PD98059 treatment (Fig. 4C,
especially at 10 and 20 min).
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Fig. 4.
Serum stimulation inhibits the interaction
between RSK1 and 14-3-3 . PS127 cells were
stimulated with 20% FBS for 0, 5, 10, and 20 min with and without
pretreatment for 1 h with 30 µM PD98059, and the
cell lysates were pulled down with GST-14-3-3
beads. A,
proteins bound to beads were subjected to SDS-PAGE and immunoblotted
with anti-RSK1 antibody. Relative binding of RSK1 to 14-3-3
is shown
as mean ± S.E. of five experiments (bottom panel).
B, densitometric analysis of relative binding of RSK1 to
14-3-3 interaction after serum stimulation. The values are normalized
to the 0 time point, which was set at 100%. The results are expressed
as the means ± S.E. *, p < 0.05 compared with
control (n = 5). C, proteins bound to
GST-14-3-3
beads were subjected to SDS-PAGE and immunoblotted with
anti-NHE1 antibody.
and RSK1 before and after serum stimulation
using subcellular fractionation in PS127 fibroblasts. In the absence of
serum, RSK1 and 14-3-3
were predominantly localized in the cytosol.
After stimulation with 20% fetal calf serum for 5 min, there was a
significant translocation of both 14-3-3
and RSK1 to the membrane
fraction (Fig. 5A) In
contrast, the distribution of actin was unchanged after fetal calf
serum treatment (Fig. 5A). This translocation also occurred
with endogenous RSK and 14-3-3
in NIH3T3 cells and in HEK293 cells
expressing exogenous RSK (data not shown).
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Fig. 5.
After serum stimulation RSK1 and
14-3-3 translocate to the membrane
fraction. Postnuclear supernatants of PS127 cells were separated
into cytosol and membrane fractions. Total, cytosol, and membrane
fractions were subjected to SDS-PAGE and immunoblotted as shown. Note
that four times more of the membrane fraction (v/v) was loaded as
compared with cytosol and total fraction.
in intact cells,
Cos7 cells were transfected with FLAG-RSK1, and RSK1 and 14-3-3
were
visualized by immunofluoresence staining with anti-FLAG antibody and
anti-14-3-3
, antibody respectively (Fig.
6). In unstimulated Cos7 cells, RSK1 and
14-3-3
appeared diffusely distributed in the cytoplasm with
significant colocalization (Fig. 6A). In cells stimulated
with 20% fetal calf serum for 5 min, both 14-3-3
and RSK1
translocated to the periphery of the cells (Fig. 6B). Overlaying the two images demonstrated significant colocalization, which was greatest at the plasma membrane (Fig. 6B).
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Fig. 6.
Subcellular colocalization of
14-3-3 and RSK1. Cos7 cells plated
on glass coverslips were transfected with FLAG-RSK1. After 36 h,
the cells were either left untreated (Control) or stimulated
with 20% FBS for 5 min. The cells were then fixed, permeabilized, and
stained with anti-14-3-3
antibody (red) or anti-FLAG
antibody for RSK1 (green). A, control Cos7 cells
stained for 14-3-3
(left panel) or FLAG-RSK1
(middle panel) and overlaying of the two images (right
panel). B, serum-stimulated Cos7 cells stained for
14-3-3
(left panel) or FLAG-RSK1 (middle
panel) and overlaying of the two images (right panel).
The cells were visualized with a Olympus IX70 Fluoview confocal
microscope. Bar, 10 µm.
Inhibits the Kinase Activity of RSK1--
It is possible
that RSK1 binding to 14-3-3 could either increase or decrease kinase
activity based on previous reports (reviewed in Ref. 14). Because
overexpressing a dominant negative 14-3-3 construct in cells would
inhibit the Raf-MEK1-ERK pathway (20) and therefore decrease RSK kinase
activity, it was not satisfactory to perform this experiment.
Therefore, we determined the effect of incubating active recombinant
RSK1 with 14-3-3
in vitro. Recombinant RSK1 was
preincubated with GST alone, GST-14-3-3
K49Q, or GST-14-3-3
for
4 h, and then a kinase assay was performed using S6 peptide as the
substrate. The activity of RSK1 in the presence of GST-14-3-3
was
significantly reduced by ~50% as compared with GST alone or GST-14-3-3
K49Q (Fig. 7A).
This finding suggests that binding of RSK1 to 14-3-3 may suppress RSK1
activity in vivo.
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Fig. 7.
14-3-3 inhibits RSK1
kinase activity through interaction at S154. A,
recombinant RSK1 was incubated overnight with 10 µg of GST alone,
GST-14-3-3
K49Q, or GST-14-3-3
at 4 °C. Kinase activity was
determined using S6 peptide as the substrate. The data are expressed as
relative radioactivity (cpm) incorporated into S6 peptide. The results
are the means ± S.E. *, p < 0.05. B,
HEK293 cells were transfected with the activator plasmid for CREB
(pFA2-CREB), the reporter gene for luciferase (pFR-Luc), and
Renilla plasmid (pRL-TK). The cells were also transfected
with either RSK1 wild type or RSK1 S154A mutant. After 24 h of
serum starvation, the cells were stimulated with 20% serum for 6 h. CREB activity was measured by assaying for firefly luciferase
values. Renilla luciferase activity was measured as an
internal control, and the results are presented as relative luciferase
activity. The results are the means ± S.E. *, p < 0.05 (n = 4). C, CREB activity was
assayed as described in B at increasing doses of EGF. The
results are the means ± S.E. (n = 3-6).
-binding RSK1S154A mutant using a luciferase reporter assay system to measure the activity of CREB. CREB is activated by cAMP-dependent protein kinase and by all three
members of the RSK family (RSK1-3) in cells stimulated by activators
of the Ras/MEK/ERK1/2 cascade (21-23). As shown in Fig. 7B,
CREB activity in cells expressing RSK1 wild type (RSK1WT) was increased
after serum stimulation. However, in cells overexpressing RSKS154A, there was an 80% increase in CREB basal activity and a 114% increase in serum-stimulated activity relative to RSK1WT. This suggests that
14-3-3 binding to RSKS154 inhibits RSK activity both basally and after
activation with serum. We next investigated the effect of the RSK1S154A
mutation on EGF-stimulated CREB activity. The cells were transfected
with RSK1WT or RSK1S154A and stimulated for 6 h with different
concentrations of EGF, after which CREB activity was measured. In cells
transfected with RSK1S154A, the EGF dose-response curve was
significantly shifted to the left compared with cells expressing RSK1WT
(EC50 of 1 ng/ml versus 6 ng/ml; Fig.
7C), demonstrating that 14-3-3
binding inhibits EGF-stimulated RSK1 activity and thus CREB activation.
and RSK1 interact maximally under basal
conditions suggest that 14-3-3
inhibits RSK1 by preventing its
activation rather than by interfering with RSK1-substrate interaction.
To verify this hypothesis we used phospho-specific antibodies targeting two phosphorylation sites on RSK1, Ser380 and
Ser363. Ser380 is autophosphorylated basally,
and it is further phosphorylated by the carboxyl-terminal RSK1 kinase
domain upon docking and activation by ERK (1, 2, 24, 25).
Ser363 phosphorylation is thought to occur subsequently,
after translocation of the RSK-ERK complex to the plasma membrane (3).
We found that in RSK1S154A constructs, phosphorylation of both
Ser380 and Ser363 was increased basally (by
27.3 ± 2.6% and 137.9 ± 10.6% respectively, n = 3) and after 5 min of serum stimulation (by
35.9 ± 3.9 and 28.1 ± 2.8%, respectively,
n = 3; Fig. 8,
A and B), compared with RSK1WT. These data
demonstrate that 14-3-3 inhibits the activation of RSK1.
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Fig. 8.
14-3-3 inhibits RSK1
kinase activity through inhibition of RSK1 phosphorylation. HEK293
cells were transfected with FLAG-RSK1 or FLAG-RSK1S154A. Cells
transfected with empty vector were used as a control. The cells were
treated with or without serum for 5 min and RSK1 constructs were
immunoprecipitated (IP) with anti-FLAG antibody.
A, immunoprecipitates were immunoblotted with p380 RSK
antibody. The lower panel shows equal expression of the RSK
constructs. B, immunoprecipitates were immunoblotted with
p363 RSK antibody. The lower panel shows equal expression of
the RSK constructs. The blots are representative of three similar
experiments.
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Fig. 9.
The amount of RSK1S154A in the membrane
fraction is increased under basal conditions as compared with
RSK1WT. A, postnuclear supernatants of HEK293 cells
transfected with RSK1WT or RSK1S154A were separated into cytosol and
membrane fractions. The total and membrane fractions were subjected to
SDS-PAGE and immunoblotted as shown. B, densitometric
analysis of relative amounts of RSK1 and RSK1S154A in the membrane and
total fractions basally (Control) and after serum
stimulation. The values are relative to RSK1WT at each condition, which
was set at 100%. The results are expressed as the means ± S.E.
*, p < 0.05 (n = 5).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was demonstrated by (i) the ability of a GST-14-3-3
fusion protein to bind RSK1 from cell lysates, (ii) direct binding of
in vitro translated RSK1 to GST-14-3-3
, and (iii)
coprecipitation of transfected 14-3-3
and RSK1. An important role
for phosphorylated serine was previously demonstrated using 14-3-3
mutated at lysine 49, which reduces the phosphoserine-mediated
interaction of binding partners Bcr, Raf, Cbl, and Bad but not the
phosphorylation-independent interaction with Bax (18, 19, 26). Mutation
of lysine 49 (K49Q) in 14-3-3
also abolished
phosphoserine-dependent interaction with NHE1 (17).
Association of 14-3-3
and RSK1 was dependent on a phosphoserine type
interaction based on three findings. First, 14-3-3
K49Q exhibited
greatly reduced binding to RSK. Second, binding was inhibited by
alkaline phosphatase treatment in vitro. Third, the
serine/threonine kinase inhibitor staurosporine decreased the
RSK1-14-3-3 interaction in vivo.
both in vitro and in
vivo, suggesting that phosphorylated serine 154 is the site of
14-3-3
interaction with RSK1. Interestingly, this motif lies within
a predicted coiled-coil domain of RSK (amino acids 151-173; analyzed
using Lupas's method at the EMBnet-CH website). Coiled-coil domains
are known to mediate protein-protein interactions (27) and have
previously been shown to be involved in the interaction of
-aminobutyric acid, type B receptors with 14-3-3 proteins (28).
; (29), dictoystelium myosin II heavy
chain protein kinase C (30), and Raf1 (16, 31)) and to cause
translocation to the plasma membrane (for example testicular protein
kinase 1) (32). Data in the present study suggest that 14-3-3
may
play a role in sequestering RSK1 in the cytosol under basal conditions.
RSK1-14-3-3
binding is maximal when cells are quiescent, and
significant colocalization can be seen using immunofluoresence.
translocate to the membrane fraction, and they partially colocalize in vivo at the plasma membrane. Serum also reduces 14-3-3
binding to RSK1 by 30-40%. These results suggest that 14-3-3
translocates with RSK1 to the plasma membrane, and once there the two
proteins dissociate. This dissociation may be regulated in two ways.
First, phosphatases are likely to play a role in dephosphorylating the RSK1-binding site (serine 154), and second, other 14-3-3
binding partners located at the plasma membrane may compete for 14-3-3
interaction. It is interesting to note that the
Na+/H+ exchanger NHE1 has previously been shown
to be a RSK substrate and that upon mitogen stimulation, 14-3-3
binds at the site of RSK phosphorylation (4, 17). Therefore, once
phosphorylated by RSK, NHE1 may be a competing binding protein for
14-3-3
at the membrane.
inhibited RSK
activity by 50%. It has been demonstrated that multiple other signaling proteins bind to 14-3-3 and that their activities are inhibited by 14-3-3. For example, 14-3-3 inhibits the activity of
phosphatidylinositol 3-kinase (33), protein kinase C isoforms
(29)
and µ (34), dictoystelium myosin II heavy chain protein kinase C
(30), and testicular protein kinase 1 (32). We have identified
Ser154 as a major 14-3-3-binding site and found that
RSK1-14-3-3 interaction is reduced in S154A mutants. Using the
activity of CREB, a transcription factor downstream of Raf (1, 22) as a
functional readout for RSK activity, we further demonstrated that the
S154A mutation increased basal and serum-stimulated RSK activity. In
addition when a dose-response curve for EGF-dependent CREB
stimulation was performed, the EC50 for RSK1S154A was
significantly lower than that of wild type RSK1. Increased
phosphorylation of two sites essential for RSK1 kinase activity
(Ser380 and Ser363) in the RSK1S154A mutant as
compared with RSK1WT both basally and after mitogen stimulation
suggests a role for 14-3-3
in the inhibition of RSK1 kinase activity
by interfering with its phosphorylation.
, thus blocking phosphorylation of
RSK1 activation sites. Because activation of RSK requires plasma
membrane localization, increased basal activity of the S154A mutant
correlates well with increased amounts in the plasma membrane compared
with RSK1WT. Therefore, 14-3-3 is not a major determinant of membrane
translocation of RSK1, although immunofluorescence and membrane
fractionation data suggest that the two proteins move in a complex to
the plasma membrane before dissociating. Rather, 14-3-3 is a modulator
of RSK1 kinase activity. At the plasma membrane, 14-3-3 may continue to
alter the kinetics of RSK1 activity until complete dissociation of the proteins. Binding to14-3-3
appears to mask phosphorylation sites in
RSK1 crucial for its activation, and it may also block the access of
substrates or ATP to the catalytic site of RSK1 or continue to maintain
RSK1 in a partially inactive conformation. In this way, 14-3-3
could
play an important role in the negative feedback regulation of active
RSK1. Our data suggest some similarities between the regulation of RSK1
and Raf by 14-3-3. There are two serine sites on Raf that bind to
14-3-3, serine 259 and serine 621 (12, 16, 35). The activity of the Raf
S259A mutant is increased basally and after EGF stimulation to a
greater extent than wild type Raf (16, 35-37), and the mutant has
increased amounts in the plasma membrane, suggesting 14-3-3 binding to
Ser259 antagonizes Raf activity by sequestering it in the
cytosol (38, 39).
/RSK1 interaction in vivo. Based on the model shown
in Fig. 10, we propose that RSK1 and
14-3-3
are bound in the cytosol, maintaining RSK1 in an inactive
conformation. Upon stimulation with mitogens, both 14-3-3
and RSK1
translocate to the plasma membrane. RSK1-14-3-3
complexes
dissociate at the plasma membrane, allowing full activation of RSK1.
Finally, RSK1 phosphorylates substrates such as Ser703 of
NHE1. This study demonstrates added complexity of the cellular regulation of RSK. Further understanding of the mechanisms of 14-3-3 regulation of RSK signaling will give insights into the role of RSK in
cell growth and proliferation. Because previous studies by our
laboratory have demonstrated increased RSK activity in cells and
tissues of hypertensive animals, it will be of interest to study the
potential role of 14-3-3 in this altered RSK regulation.
View larger version (15K):
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Fig. 10.
Model for the role of
14-3-3 in regulation of RSK1 function. In
unstimulated cells, RSK1 and 14-3-3
bind in cytosol, perhaps
maintaining RSK1 in an inactive conformation. On stimulation with
mitogens, both 14-3-3
and RSK1 translocate to the plasma membrane.
RSK 14-3-3
complexes then dissociate, allowing full activation of
RSK1. RSK1 then phosphorylates plasma membrane substrates such as
Ser703 of NHE1 and translocates to the nucleus to
phosphorylate nuclear substrates.
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FOOTNOTES |
---|
* This work was supported by Grants RO1 HL44721 and HL07949 from the National Institutes of Health (to B. C. B.).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.
Recipient of a fellowship from the Canadian Institutes of Health
Research. Present address: INSERM U541, Paris, France.
§ To whom correspondence should be addressed: University of Rochester, Center for Cardiovascular Research, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 585-273-1946; Fax: 585-273-1497; E-mail: Bradford_Berk@urmc.rochester.edu.
Published, JBC Papers in Press, March 4, 2003, DOI 10.1074/jbc.M208475200
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ABBREVIATIONS |
---|
The abbreviations used are: RSK, p90 ribosomal S6 kinase; CREB, cAMP response element-binding protein; CIAP, calf intestinal alkaline phosphatase, ERK, extracellular signal-related kinase; GST, glutathione S-transferase; MEK, mitogen-activated protein kinase/ERK kinase; NHE1, Na+/H+ exchanger isoform 1; PDK1, phosphoinositide-dependent protein kinase 1; EGF, epidermal growth factor; FBS, fetal bovine serum; RSK1WT, RSK1 wild type.
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