From the Howard Hughes Medical Institute and the Markey Center for Cell Signaling, Department of Medicine and Pharmacology, University of Virginia, Charlottesville, Virginia 22908
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ABSTRACT |
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Glutathione S-transferase
(GST)-fusion proteins containing the carboxyl-terminal tails of three
p90 ribosomal S6 kinase (RSK) isozymes (RSK1, RSK2, and RSK3)
interacted with extracellular signal-regulated kinase (ERK) but not
c-Jun-NH2-kinase (JNK) or p38 mitogen-activated
protein kinase (MAPK). Within the carboxyl-terminal residues of the RSK
isozymes is a region of high conservation corresponding to residues
722LAQRRVRKLPSTTL735 in RSK1. Truncation of
the carboxyl-terminal 9 residues,
727VRKLPSTTL735, completely eliminated the
interaction of the GST-RSK1 fusion protein with purified recombinant
ERK2, whereas the truncation of residues
731PSTTL735 had no effect on the interaction
with purified ERK2. ERK1 and ERK2 co-immunoprecipitated with
hemagglutinin-tagged wild type RSK2 (HA-RSK2) in BHK cell cytosol.
However, ERK did not co-immunoprecipitate with
HA-RSK2(1-729), a mutant missing the carboxyl-terminal 11 amino acids, similar to the minimal truncation that eliminated in
vitro interaction of ERK with the GST-RSK1 fusion protein. Kinase
activity of HA-RSK2 increased 6-fold in response to insulin.
HA-RSK2(1-729) had a similar basal kinase activity to that
of HA-RSK2 but was not affected by insulin treatment.
Immunoprecipitated HA-RSK2 and HA-RSK2(1-729) could be
activated to the same extent in vitro by active ERK2, demonstrating that HA-RSK2(1-729) was properly folded.
These data suggest that the conserved region of the RSK isozymes
(722LAQRRVRKL730 of RSK1) provides for a
specific ERK docking site approximately 150 amino acids
carboxyl-terminal to the nearest identified ERK phosphorylation site
(Thr573). Complex formation between RSK and ERK is
essential for the activation of RSK by ERK in vivo.
Comparison of the docking site of RSK with the carboxyl-terminal tails
of other MAPK-activated kinases reveals putative docking sites within
each of these MAPK-targeted kinases. The number and placement of lysine
and arginine residues within the conserved region correlate with
specificity for activation by ERK and p38 MAPKs in
vivo.
Mitogen-activated protein kinases
(MAPKs)1 transduce signals
from the cell surface to the nucleus, altering the activity and subcellular localization of transcription factors. ERK, JNK, and p38
MAPKs lie in distinct signaling pathways that are activated by distinct
stimuli. Whereas the minimal consensus phosphorylation sequence of
these proline-directed kinases would suggest promiscuous phosphorylation of many proteins, the kinases play an integral role in
the cellular growth machinery; therefore, substrate specificity must be
tightly regulated. It is becoming clear that the substrate specificity
of MAPKs with regard to transcription factors involves high affinity
binding of MAPK to sequences within the substrate that are distinct
from the consensus phosphorylation sequence (1, 2). Such an interaction
has been described for JNK and the transcription factors c-Jun and
activating transcription factor (ATF-2) (3-5). Recently, a sequence
within Elk-1 was shown to contain overlapping but distinct interaction
sites for ERK and JNK (6). Specific targeting interactions between
MAPKs and substrates may not be limited to transcription factors. One
possible ERK substrate for which targeting interactions might occur is p90 ribosomal S6 protein kinase (RSK). RSK is phosphorylated and activated by ERK in vitro (7), and inhibition of the ERK
pathway with the mitogen-activated protein/ERK kinase-specific
inhibitor PD98059 prevents in vivo activation of RSK (8,
9).
RSK is unusual in that it contains two distinct kinase catalytic
domains within a single polypeptide chain (10). In vivo ERK
phosphorylation sites within RSK have been identified (11, 12) (Fig.
1A). Two of these sites are essential for activation of RSK:
1) Ser363 in the linker between the two kinase domains, and
2) Thr573 in the activation loop of the carboxyl-terminal
kinase domain (12).2
Physiological substrates for the three mammalian isozymes of RSK (RSK1,
RSK2, and RSK3), which are encoded by separate genes (13), are
currently under investigation. RSK has been shown to phosphorylate the
cAMP response element-binding protein (14, 15), c-fos (16, 17), and the
estrogen receptor (18), suggesting that RSK as well as ERK plays a role
in transcriptional regulation.
Whereas studies describing the existence of an interaction between ERK
and RSK have been reported (19-21), analyses of the site(s) of
interaction and the in vivo significance of the interaction are essential. Examination of the RSK isozymes for the ability to
interact with ERK revealed that ERK co-immunoprecipitated with RSK2 and
RSK3, but not RSK1 (21). The site of interaction between RSK and ERK
was localized to the carboxyl-terminal 44 amino acids of RSK3 (21).
MAPK-interacting kinase (Mnk) was recently identified as a
serine/threonine kinase with sequence similarity to RSK that is
phosphorylated and activated by MAPK (22, 23). Mnk1 co-purified with
(22) and was phosphorylated by both ERK and p38 MAPK (22, 23), whereas
Mnk2 interacted specifically with ERK (22). Truncation of the
carboxyl-terminal 90 amino acids (residues 334-424) of Mnk1 eliminated
phosphorylation and activation by ERK (23). Thus, the site of
interaction between MAPK and the substrate kinases RSK and Mnk appears
to lie within the carboxyl-terminal regions of the substrate.
In the present studies, interaction between MAPK and three isozymes of
RSK was examined. An ERK docking site critical to this interaction was
identified, and the role of the ERK/RSK complex was assessed in
vivo.
Materials--
Dulbecco's modified Eagle's medium (11965) and
fetal calf serum were purchased from Life Technologies, Inc. Insulin
(Humulin®) was obtained from Eli Lilly and Co.
Phorbol-12,13-dibutyrate (PDB; P1269) was purchased from Sigma.
ImmunoPure® protein A/G-agarose beads (20421) were from Pierce.
Antibodies were obtained from the following sources: monoclonal
anti-ERK antibody (M12320), Transduction Laboratories; polyclonal
anti-ERK antibody (06-182), Upstate Biotechnology; anti-p38 MAPK
antibody (SC-535, C-20) and anti-JNK antibody (SC-474, C-17), Santa
Cruz Biotechnology, Inc.; polyclonal anti-HA antibody (PRB-101P),
Berkley Antibody Co.; monoclonal anti-HA antibody (12CA5), the
University of Virginia Lymphocyte Culture Center; and anti-mouse
IgG:horseradish peroxidase-linked antibody (NA931) and anti-rabbit
IgG:horseradish peroxidase-linked antibody (NA934), Amersham Life
Science. COS-1 cells (African green monkey kidney cells, SV40
transformed; ATCC CRL-1650) and BHK-21 (C-13) cells (hamster kidney
cells; ATCC CCL-1) were purchased from American Type Culture
Collection. Glutathione-Sepharose 4B beads (17-0756) were from
Pharmacia. Ribosomal S6 peptide (RRRLSSLRA, residues 231-239) was
synthesized at the University of Virginia Biomolecular Research Facility.
Vector Construction--
Expression vectors were generously
provided by the following: pMT2.HA-RSK1 (rat), pMT2.HA-RSK2 (mouse),
and pMT2.HA-RSK3 (human), Dr. Christian Bjørbæk (Harvard Medical
School, Boston, MA); pEThis.MEK1/ERK2 (active ERK2) and
pGexKG.SapK Cell Culture and Transfection--
BHK-21 (C-13) cells were
grown in a 37 °C humidified atmosphere containing 10%
CO2 in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin (DMEM/5% FCS/PS). The cells were plated (20-40%
confluence) on 150-mm dishes 16-20 h before transfection. The cells
were transfected with 10 µg of CsCl-banded DNA (pK3H.RSK,
pK3H.RSK2(1-729), or pK3H) per 150-mm dish as described
for transfection of 100-mm dishes in the Calcium Phosphate ProFection®
System manual (Promega). At 48-72 h after transfection, the cells were
serum-starved for 1-1.5 h in DMEM/PS, followed by treatment with 94 nM insulin (or vehicle) for 12 min at 37 °C. Three
150-mm plates/condition were washed six times with cold
phosphate-buffered saline (PBS), and the cells were scraped into lysis
Buffer A (10 mM Tris base, pH 7.5, 150 mM
NaCl2, 1% Triton X-100, 0.5% Nonidet P-40, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 10 µg/ml each of leupeptin and aprotinin, 1 mM phenylmethylsulfonyl fluoride, 200 µM
sodium orthovanadate, and 1 µM microcystin-LR) for immune
complex kinase assays or lysis Buffer B (50 mM Tris base,
pH 8, 150 mM NaCl2, 1% Nonidet P-40, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 10 µg/ml each of leupeptin and aprotinin, 1 mM phenylmethylsulfonyl fluoride, 200 µM
sodium orthovanadate, and 1 µM microcystin-LR) for
co-immunoprecipitation experiments. The cells were lysed by incubation
on ice for 20 min, and the supernatant was clarified by centrifugation
at 12,000 × g for 10 min for immune complex kinase
assays or at 100,000 × g for
co-immunoprecipitation.
Preparation of COS Cell Cytosol and Purified ERK--
Confluent
COS-1 cells growing in 10% fetal calf serum were treated in the
absence or presence of 2 µM PDB for 15 min at 37 °C.
The plates were washed with cold PBS, and cells were scraped into D-PBS
(2.7 mM KCl, 1.1 mM
KH2PO4, 138 mM NaCl, and 8.1 mM Na2HPO4, pH 7.4). Cells were
pelleted, and an equal volume of lysis Buffer C (50 mM
HEPES, pH 7.4, 1% Nonidet P-40, 0.5% Triton X-100, 10% glycerol, 150 mM NaCl, 1 mM Na3VO4, 2 µM microcystin LR, 2 mM phenylmethylsulfonyl
fluoride, and 1 CompleteTM protease inhibitor mixture
tablet (Boehringer Mannheim)/50 ml) was added to the cell pellet. The
cell pellet was resuspended, and cells were lysed by sonication. The
lysate was centrifuged at 12,000 × g for 10 min in a
microcentrifuge. Cytosol was frozen in liquid nitrogen and stored at
Incubation of GST-RSK Carboxyl-Terminal Proteins with Cytosol or
Purified ERK--
Glutathione-Sepharose 4B beads (25 µl bed volume)
were copiously washed with PBS. The washed beads were rotated with 30 µg of GST or GST-fusion protein in 25 mM HEPES, pH 7.4, 1 mM EDTA, and 1 mM dithiothreitol for 1 h
at 4 °C. The washed GST-bound beads were incubated with COS-1 cell
cytosol (200 µl; 17 mg/ml total protein) or 100 nM
purified ERK2 in lysis Buffer C for 1.5 h at 4 °C with gentle
mixing. The beads were pelleted by centrifugation, and the supernatant
was removed and saved. The beads were washed three times with 750 µl
of cold PBS. SDS-sample buffer was added to the washed beads and
supernatant, and the samples were boiled for 5 min and processed for
Western analysis. Unless otherwise indicated, monoclonal antibody
(M12320) was used for immunoblotting ERK.
Immunoprecipitation and Immune Complex Kinase Assay--
BHK
cell cytosol (1 ml) from cells expressing HA-RSK2 or
HA-RSK2(1-729) treated in the absence or presence of 94 nM insulin for 12 min was pre-cleared by incubation for
1 h with immobilized protein A/G-agarose beads (20 µl bed
volume). Beads were pelleted by centrifugation, and the supernatant was
removed. For co-immunoprecipitation, the supernatant was incubated with
30 µg of monoclonal anti-HA antibody (12CA5) at 4 °C for 1 h,
followed by incubation of the supernatant with anti-mouse
IgG:horseradish peroxidase-linked for 30 min on ice. Immobilized
protein A/G-agarose beads (20 µl bed volume) were incubated
with the supernatant for 30 min on a Nutator rocker. The beads were
pelleted by centrifugation, and the supernatant was removed and saved.
The beads were washed once with 500 µl of lysis Buffer B and twice
with 500 µl of cold PBS. SDS-sample buffer was added to the beads and
the supernatant, and the samples were boiled for 5 min and processed
for Western analysis with polyclonal anti-HA antibody and polyclonal
anti-ERK antibody.
For the immune complex kinase assay, 1 µg of polyclonal anti-HA
antibody was added to 500 µl of pre-cleared BHK cell cytosol (Fig.
5A) or 10 µg of polyclonal anti-HA antibody were added to 1 ml of pre-cleared BHK cell cytosol (Fig. 5B) and incubated
for 1 h. Immobilized protein A/G-agarose beads (20 µl bed
volume) were incubated with the supernatant for 12 h. The beads
were pelleted by centrifugation, and the supernatant was removed and
saved. The beads were washed once with 500 µl of lysis Buffer A, once with 500 µl of cold PBS, and twice with 500 µl of kinase wash buffer (75 mM National Center for Biotechnology Information Accession Numbers
for Protein Sequences--
The accession numbers for protein sequences
are as follows: rat RSK1, 2117822 (DBSOURCE PIR, A53300); mouse RSK2,
125691 (DBSOURCE SWISS-PROT, P18654); human RSK3, 2117823 (DBSOURCE PIR, A57459); mouse Mnk1, 1929059 (DBSOURCE European Molecular Biology
Laboratory, Y11091); mouse Mnk2, 1929061 (DBSOURCE European Molecular
Biology Laboratory, Y11092); human MAPKAPK-2, 1346538 (DBSOURCE
SWISS-PROT, P49137); human MAPKAPK-3, 1256005 (DBSOURCE GenBankTM,
U43784); human PRAK, 3133291 (DBSOURCE GenBankTM, AF032437); human
MSK1, 3411157 (DBSOURCE GenBankTM, AF074393); and mouse MSK2, 3786406 (DBSOURCE GenBankTM, AF074714).
Interaction of GST-RSK Carboxyl Termini with MAPK in
Cytosol--
GST-fusion proteins of the carboxyl termini of three RSK
isozymes were constructed to examine the interaction between RSK and
MAPK. Bacterially expressed GST-RSK1(672-735),
GST-RSK2(676-740), and GST-RSK3(669-733)
contain the amino acid sequence (~60 residues) from the end of the
carboxyl-terminal kinase domain catalytic core to the carboxyl-terminal residue (Fig. 1B).
GST-RSK-bound glutathione beads were incubated with cytosol from
nonstarved COS-1 cells treated in the presence or absence of 2 µM PDB. Washed beads were examined for the presence of
ERK, JNK, and p38 MAPKs (Fig. 2).
Immunoblotting revealed the presence of ERK from nontreated cells and
active ERK from PDB-treated cells with each of the GST-RSK fusion
proteins. Neither JNK nor p38 MAPK was found to be associated with the
GST-RSK fusion proteins above the levels observed with GST alone. Under
similar conditions, using purified recombinant kinase-defective ERK2
(K52R ERK; 100 nM), kinase-defective JNK3 (100 nM to 1 µM), and wild type p38 MAPK
(100-500 nM), only ERK2 bound to the GST-RSK fusion
proteins at levels above that bound to GST alone (data not shown).
Thus, the carboxyl-terminal 60 amino acids of the various RSK isozymes specifically interact with ERK. Zhao et al. (21) reported
that ERK does not co-immunoprecipitate with HA-RSK1. However,
activation of the three RSK isozymes is blocked by the inhibition of
mitogen-activated protein/ERK kinase (21). Thus, the GST-RSK1
interaction with ERK demonstrated here is consistent with the presence
of an ERK-binding motif in the carboxyl-terminal tail of all three
RSKs. The suggestion that ERK might bind to HA-RSK1 with less affinity
than to HA-RSK2 or HA-RSK3 (21) is not excluded.
Truncation of GST-RSK1(672-735) and Identification of
Residues Critical to ERK Interaction--
To delineate the sequences
important for ERK interaction, further truncations of the identified
region of RSK1 were performed. An amino-terminal deletion to residue
716 (GST-RSK1(716-735)) containing the carboxyl-terminal
20 amino acids, a region that is highly conserved between RSK and Mnk
(Fig. 1B), was examined for interaction with ERK.
GST-RSK1(716-735) was determined to be sufficient for
interacting with K52R ERK2 (Fig.
3A). To localize the residues
within this 20-amino acid sequence that were critical to ERK
interaction, truncations of the 63-amino acid GST-RSK1 fusion protein
(GST-RSK1(672-735)) were used instead of the 20-amino acid
GST-RSK1 fusion protein (GST-RSK1(716-735)) for two
reasons: 1) using a peptide smaller than 20 amino acids fused to GST
might preclude ERK interaction simply due to steric interference by GST
rather than elimination of critical residues, and 2) although the
carboxyl-terminal 20 residues were sufficient for interaction with ERK,
additional residues might also be involved. As seen in Fig.
3B, deletion of the carboxyl-terminal 5 amino acids
(GST-RSK(672-730)) had no effect on the ability of the
fusion protein to bind to K52R ERK2 compared with that of
GST-RSK1(672-735). Removal of the carboxyl-terminal 20 amino acids (GST-RSK1(672-715)) or deletion of the
carboxyl-terminal 9 amino acids (GST-RSK1(672-726)), which
bisects the conserved region (722LAQRRVRKL730
of RSK1) of the RSK isozymes, completely eliminated the interaction of
the fusion proteins with K52R ERK2 (Fig. 3B). Because
alteration of the conserved region of RSK was deleterious to the
interaction of GST-RSK1 with ERK, it is likely that a critical
combination of residues within this conserved region acts as the
docking site for ERK.
In Vivo Examination of RSK/ERK Interaction--
To examine whether
the sequence critical to in vitro interaction between
GST-RSK and ERK was important for interaction within the context of the
full-length kinase in vivo, the interaction between ERK and
HA-tagged wild type RSK (HA-RSK2) or mutant RSK2 (HA-RSK2(1-729)) in which the carboxyl-terminal 11 amino
acids are deleted was examined in BHK cells.
HA-RSK2(1-729) approximates the minimal truncation of
GST-RSK1 (GST-RSK1(672-726)) that eliminates in
vitro interaction with ERK. As seen in Fig. 4, ERK1 and ERK2 co-immunoprecipitated
with HA-RSK2 but did not co-immunoprecipitate with
HA-RSK2(1-729) above the levels observed in
immunoprecipitates from cells transfected with the empty vector. Thus,
the ERK docking site required for in vitro interaction is
also essential for in vivo interaction of RSK and ERK.
To determine whether deletion of the ERK docking site altered the
in vivo activation of RSK, the kinase activity of HA-RSK2 or
HA-RSK2(1-729) immunoprecipitated from
serum-deprived BHK cells incubated in the presence and absence
of insulin was measured using the ribosomal S6 peptide as
substrate. The activity of HA-RSK2 was increased ~6-fold by
insulin treatment (Fig. 5A).
Basal kinase activity of HA-RSK2(1-729) was similar to
that of HA-RSK2; however, insulin treatment had no effect on the
kinase activity of HARSK2(1-729) (Fig.
5A). These results demonstrate that although all identified ERK phosphorylation sites are intact in HA-RSK2(1-729),
the enzyme remains inactive under physiological conditions that stimulate HA-RSK2. Thus, the formation of a complex between ERK and the
carboxyl-terminal tail of RSK is absolutely essential for efficient ERK
activation of RSK in the living cell. Although the ERK docking site has
been removed from HA-RSK(1-729), ERK should have
sufficient affinity for the consensus phosphorylation sequence to
phosphorylate and activate RSK in vitro when present at
supraphysiological concentrations. HA-RSK2 and
HA-RSK2(1-729) were immunoprecipitated from serum-deprived
BHK cells and incubated in the presence or absence of purified
recombinant active ERK2. Active ERK2 increased the kinase activity of
HA-RSK2(1-729) ~6-fold, which is identical to the levels
of activation observed with HA-RSK2 (Fig. 5B),
indicating that HA-RSK2(1-729) is a functional enzyme.
Additionally, truncation of the ERK docking site did not affect the
ability of ERK to induce a mobility shift on SDS-PAGE as has been
reported for wild type RSK (Ref. 21; Fig. 5B).
Several MAPK-activated protein kinases (MAPKAPKs) have been identified
for both ERK (RSK and Mnk) and p38 MAPK (MAPKAP-2, MAPKAP-3, RSKb, and
PRAK; Refs. 27-32). The most recently described MAPKAPKs are MSK1 (33)
and RSKb (31); both have two kinase domains, like RSK1-3, and are
novel RSKs. Additional members of each group are likely to be
discovered, including a group so far undetected, activated by JNK. All
identified MAPKAPKs have sequence similarities in their catalytic
domains to calmodulin-activated protein kinases, residing in RSKs in
the carboxyl-terminal kinase domain.
Sequence alignment of the ERK docking site of RSK1
(722LAQRRVRKL) with the carboxyl-terminal tails of Mnk1/2,
MAPKAPK-2, MAPKAPK-3, RSKb, PRAK, and MSK1 reveals a likely docking
site within each of these MAPK-targeted kinases (Fig.
6). The order of alignment is with
respect to the number of contiguous basic amino acids within the
proposed docking sites (i.e. two for RSK1, and five for
MAPKAPK-3). This order of alignment also allows for grouping of the
kinases into ERK-specific and p38 MAPK-specific kinases, as well as a
group that is regulated by both ERK and p38 MAPKs.
RSK1, RSK2, RSK3, and Mnk2 are activated specifically by ERK
(Fig. 6). The alignment for this group suggests that the sequence LAQRRXXXXL/I (X, any amino acid) may be important
for the specific binding and activation of these MAPKAPKs by ERK.
MAPKAPK-2 and MAPKAPK-3 have been extensively studied and have been
found to be activated specifically by p38 MAPKs in vivo
(27-30). PRAK (the human homolog of mouse MAPKAPK-5) is also specific
for p38 MAPK in vivo (32). RSKb (identical to MSK2) was
identified in a two-hybrid screen with p38
Thus, comparisons of MAPK specificity with residues aligned in
the conserved regions correlate the number of contiguous basic amino
acids (K/R) with MAPK specificity. ERK-specific MAPKAPKs have two
contiguous K/Rs, ERK/p38 MAPK-specific MAPKAPKs have three and four
contiguous K/Rs, and p38 MAPK-specific MAPKAPKs have four and five
contiguous K/Rs. A strong inference is that the number and position of
basic residues within the context of the secondary structure (predicted
to be helical) may be important determinants of specificity. Mutational
analyses of these motifs and flanking sequences are warranted to assess
the effect on ERK and p38 MAPK specificity. The basic residues in the
putative MAPK docking sites are clustered and may also define nuclear
localization sequences (34).
Specific docking sites for MAPKs in the MAPKAPKs are likely to play at
least two roles in signaling. As demonstrated herein, the targeting
motif for ERK is required for RSK2 activation in vivo. Thus,
complex formation of MAPKs with the carboxyl-terminal tails of MAPKAPKs
is likely to be a general feature that is important for the specific
regulation of these MAPK-targeted kinases by MAPKs in vivo.
In addition, docking sites may enable co-localization of a specific
MAPK with a specific MAPKAPK in complexes where they collaborate to
regulate transcriptional and translational machinery.
INTRODUCTION
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Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
(K>A) (JNK3), Dr. Melanie H. Cobb (University of
Texas Southwestern Medical Center, Dallas, TX); and pET14b.p38 WT
(mouse), Dr. Jiahuai Han (The Scripps Research Institute, LaJolla, CA).
SapK
(K>A) was subsequently subcloned into pET28a. The pK3H
expression vector encoding an amino-terminal triple hemagglutinin (HA1)
tag was kindly provided by Dr. Ian Macara (University of Virginia,
Charlottesville, VA) (24). pGEX2T bacterial expression constructs
encoding the carboxyl-terminal tail residues of RSK1, RSK2, and RSK3
were created by polymerase chain reaction amplification of the 3' end
of each RSK. pK3H.RSK2 was generated by polymerase chain reaction
amplification of pMT2.HA-RSK2 (mouse) cDNA and subcloning into a
unique BamHI site in pK3H. PK3H.RSK2(1-729),
which encodes a RSK2 (mouse) mutant in which the carboxyl-terminal 11 amino acids are deleted, was created by ligating annealed
oligonucleotides (encoding a Bpu1102I site and a stop codon)
to pK3H.RSK2 linearized with Bpu1102I. The sequences were
verified by automated sequencing. Oligonucleotides used are available
on request. Purification of GST-fusion proteins was performed according
to a protocol supplied for the GST Gene Fusion System (Pharmacia Biotech).
70 °C. Kinase-defective ERK2 (K52R ERK2) and active ERK2 were
prepared as described previously (25, 26).
-glycerolphosphate, pH 7.4, 3.75 mM EGTA, 150 µM Na3VO4, 1.5 mM dithiothreitol, 6 µM protein kinase A inhibitor, and 30 mM
MgCl2). S6 kinase mix (50 µl; 75 mM
-glycerolphosphate, pH 7.4, 3.75 mM EGTA, 150 µM Na3VO4, 1.5 mM
dithiothreitol, 6 µM protein kinase A inhibitor, 30 mM MgCl2, 300 µM ATP
([
-32P]ATP),and 150 µM S6 peptide) was
added to the beads and incubated at 30 °C for 20 min. Portions of
the reaction mixture were spotted onto P81 phosphocellulose and washed
with 75 mM phosphoric acid. Phosphate incorporation into
peptide substrate was determined by counting the P81 papers in the
presence of the scintillation mixture. SDS-sample buffer was added to
the remainder of the reaction mixtures. The samples were boiled and
processed for Western analysis with polyclonal anti-HA antibody.
RESULTS AND DISCUSSION
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Fig. 1.
Schematic of RSK1 and sequence alignment of
the GST-RSK fusion proteins with mouse Mnk1 and Mnk2. A,
schematic of RSK1 depicting the two kinase domains, the phosphorylation
sites2 identified by Dalby et al. (12), and the
carboxyl-terminal 20 amino acids. B, alignment of the
GST-RSK fusion proteins with mouse Mnk. Asterisks (*) mark identical
residues between the RSK and Mnk isozymes. A conserved region between
RSK isozymes is highlighted.
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Fig. 2.
Interaction of GST-RSK carboxyl termini with
MAPK in cytosol. GST-RSK fusion protein or GST bound to
glutathione beads was incubated with COS-1 cell cytosol as described
under "Experimental Procedures." Cells used to determine the
presence of active ERK were treated with 2 µM PDB.
Supernatant and washed beads were subjected to Western analysis.
Although the anti-ERK antibody (M12320) recognizes both ERK1 and ERK2,
ERK2 is the predominant isozyme in COS cells, and immunodetection of
ERK1 was not always observed in the cytosol from untreated cells.
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Fig. 3.
Identification of residues that are critical
to interaction with ERK. GST-RSK(716-735)
(A) or truncations of GST-RSK1(672-735)
(B) or GST alone bound to glutathione beads were incubated
with purified recombinant kinase-defective K52R ERK2 as described
under "Experimental Procedures." Washed beads were subjected to
Western analysis. Truncations of GST-RSK1(672-735) are
described in B. The conserved region of RSK isozymes is
indicated.
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Fig. 4.
In vivo interaction of RSK2 and
ERK. HA-RSK2 and HA-RSK2(1-729) from BHK cells
treated in the absence and presence of 94 nM insulin were
immunoprecipitated with monoclonal anti-HA antibody (12CA5). The
immunoprecipitates were subjected to Western analysis with polyclonal
anti-HA and polyclonal anti-ERK antibodies (see "Experimental
Procedures").
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Fig. 5.
Activation of RSK2 in vitro and
in vivo. A, HA-RSK2 and HA-RSK2(1-729)
were immunoprecipitated from the cytosol of serum-deprived BHK cells
treated in the absence (CONTROL) or presence
(INSULIN-TREATED) of 94 nM insulin. The kinase
activity of immunoprecipitates from cells transfected with the empty
vector has been subtracted. Activity is presented as mean ± S.D.
*, means are significantly different (Student's paired t
test, p = 0.002). The means of control and
insulin-treated HA-RSK2(1-729) activity are not
significantly different (Student's paired t test,
p = 0.16 (n = 5)). The
insert is a representative immunoblot showing an equivalent
quantity of enzyme immunoprecipitated from control cells expressing
HA-RSK2 (WT ) or HA-RSK2(1-729)
(1-729
) and insulin-treated cells expressing HA-RSK2
(WT+) or HA-RSK2(1-729) (1-729+).
B, HA-RSK2 and HA-RSK2(1-729) were
immunoprecipitated from the cytosol of serum-deprived BHK cells. Immune
complexes were incubated for 15 min at 30° C in the absence or
presence of 1.5 µM active ERK2. After incubation, the
kinase activity of the immunoprecipitates was measured. Activity is
presented as mean ± S.D. (n = 3). The kinase
activity of immunoprecipitates from cells transfected with the empty
vector and incubated in the absence or presence of active ERK2 has been
subtracted. The insert demonstrates the equivalent quantity
of immunoprecipitated enzyme as well as the SDS-PAGE mobility shift
induced by the incubation of HA-RSK2 (WT+) and
HA-RSK2(1-729) (1-729+) in the presence of
active ERK2 compared with the incubation of HA-RSK2 and
HA-RSK2(1-729) in the absence of active ERK2
(WT
and 1-729
, respectively).
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Fig. 6.
Sequence alignment of ERK- and p38
MAPK-interacting kinases. Alignment of the ERK docking sequence of
RSK (conserved region) with ERK- and p38 MAPK-activated
kinases. Conserved residues are highlighted in solid boxes,
and nonconserved basic amino acids within the putative docking sites
are highlighted in dashed boxes. For consensus sequences:
X, any amino acid; and , any hydrophobic amino acid (with
the exception of MAPKAPK-3). National Center for Biotechnology
Information accession numbers for protein sequences are as follows: rat
RSK1, 2117822 (PIR, A53300); mouse RSK2, 125691 (SWISS-PROT, P18654);
human RSK3, 2117823 (PIR, A57459); mouse Mnk1, 1929059 (European
Molecular Biology Laboratory, Y11091); mouse Mnk2, 1929061 (European Molecular Biology Laboratory, Y11092); human MAPKAPK-2,
1346538 (SWISS-PROT, P49137); human MAPKAPK-3, 1256005 (GenBankTM,
U43784); human PRAK, 3133291 (GenBankTM, AF032437); human MSK1,
3411157 (GenBankTM, AF074393); mouse MSK2, 3786406 (GenBankTM,
AF074714).
MAPK and is activated
predominantly by p38 MAPK and more weakly by ERK both in
vitro and in cultured cells (31). Thus, alignment of the conserved
region of these MAPKAPKs correlates the sequence
L
(K/R)(K/R)(K/R)(K/R)XXXX (
, hydrophobic amino acid;
X, any amino acid) with p38 MAPK specificity. A consensus cannot be suggested for those MAPKAPKs activated by both ERK and p38
MAPK (Mnk1 and MSK1; Refs. 22 and 33), because there are too few
aligned sequences.
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ACKNOWLEDGEMENTS |
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We thank Drs. David L. Brautigan, Deborah A. Lannigan, and Ian G. Macara for productive discussions during the course of this study, and we thank Dr. Deborah A. Lannigan for critical review of the manuscript. We are indebted to Corky Harrison for excellent administrative support and to E. Daniel Hershey and Tiffany A. Freed for technical expertise.
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FOOTNOTES |
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* This work was supported by Howard Hughes Medical Institute and National Institutes of Health Grant DK41077.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Box 577, Howard Hughes
Medical Institute, University of Virginia Health Sciences Center,
Charlottesville, VA 22908-0001. Fax: 804-924-9659; E-mail: tws7w{at}virginia.edu.
The abbreviations used are: MAPK, mitogen-activated protein kinase; GST, glutathione S-transferase; RSK, ribosomal S6 kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun-NH2-kinase; HA, hemagglutinin; HA-RSK2, HA-tagged wild type RSK2; MAPKAPK, MAPK-activated protein kinase; Mnk, MAPK-interacting kinase; PDB, phorbol-12,13-dibutyrate; PBS, phosphate-buffered saline.
2 Dalby et al. (12) report an extra codon for a glutamine at position 157 in the pMT2 construct encoding rat RSK1. Numbering for the phosphorylation sites of RSK1 in the present studies is in agreement with the published sequence of rat RSK1 (35).
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REFERENCES |
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