A Potential Role for Extracellular Signal-regulated Kinases
in Prostaglandin F2
-induced Protein Synthesis in
Smooth Muscle Cells*
Gadiparthi N.
Rao
,
Nageswara R.
Madamanchi,
Manjiri
Lele,
Laxmisilpa
Gadiparthi,
Anne-Claude
Gingras§,
Thomas E.
Eling¶, and
Nahum
Sonenberg§
From the Division of Cardiology, University of Texas Medical
Branch, Galveston, Texas 77555, the § Department of
Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada,
and ¶ Laboratory of Molecular Carcinogenesis, NIEHS, National
Institutes of Health,
Research Triangle Park, North Carolina 27709
 |
ABSTRACT |
To understand the mechanisms of prostaglandin
F2
(PGF2
)-induced protein synthesis
in vascular smooth muscle cells (VSMC), we have studied its effect on
two major signal transduction pathways: mitogen-activated protein
kinases and phosphatidylinositol 3-kinase (PI3-kinase) and their
downstream targets ribosomal protein S6 kinase (p70S6k) and
eukaryotic initiation factor eIF4E and its regulator 4E-BP1. PGF2
induced the activities of extracellular
signal-regulated kinase 2 (ERK2) and Jun N-terminal kinase 1 (JNK1)
groups of mitogen-activated protein kinases, PI3-kinase, and
p70S6k in a time-dependent manner in
growth-arrested VSMC. PGF2
also induced eIF4E and 4E-BP1
phosphorylation, global protein synthesis, and basic fibroblast growth
factor-2 (bFGF-2) expression in VSMC. Whereas inhibition of PI3-kinase
by wortmannin completely blocked the p70S6k activation, it
only partially decreased the ERK2 activity, and had no significant
effect on global protein synthesis and bFGF-2 expression induced by
PGF2
. Rapamycin, a potent inhibitor of
p70S6k, also failed to prevent PGF2
-induced
global protein synthesis and bFGF-2 expression, although it partially
decreased ERK2 activity. In contrast, inhibition of ERK2 activity by PD
098059 led to a significant loss of PGF2
-induced eIF4E
and 4E-BP1 phosphorylation, global protein synthesis, and bFGF-2
expression. PGF2
-induced phosphorylation of eIF4E and
4E-BP1 was also found to be sensitive to inhibition by both wortmannin
and rapamycin. These findings demonstrate that 1)
PI3-kinase-dependent and independent mechanisms appear to
be involved in PGF2
-induced activation of ERK2; 2)
PGF2
-induced eIF4E and 4E-BP1 phosphorylation appear to be mediated by both ERK-dependent and
PI3-kinase-dependent rapamycin-sensitive mechanisms; and 3)
ERK-dependent eIF4E phosphorylation but not PI3-kinase-dependent p70S6k activation
correlates with PGF2
-induced global protein synthesis and bFGF-2 expression in VSMC.
 |
INTRODUCTION |
Translational control plays an important role in regulation of
gene expression (1-4). Several kinase cascades are implicated in the
regulation of protein synthesis (5-7). One of the events associated
with protein synthesis is the phosphorylation of ribosomal protein S6
(8). The serine/threonine kinase, p70S6k, phosphorylates
ribosomal protein S6 (8, 9). Several studies have reported that
phosphatidylinositol 3-kinase
(PI3-kinase)1 plays a role in
the activation of p70S6k in response to a variety of
mitogens (10-12). Another event that is critical in the regulation of
protein synthesis is the binding of mRNA to ribosomes, which is
facilitated by a translation initiation complex, eIF4F (1-4). eIF4F
consists of three polypeptides as follows: eIF4A, an RNA helicase;
eIF4E, the cap-binding protein; and eIF4G, a bridging protein for eIF4A
and eIF4E (7, 13). The binding of eIF4F to mRNA is catalyzed by its
smallest subunit eIF4E via interaction with the cap structure present
in the 5' of the eukaryotic mRNAs (1, 3). eIF4F in combination with another initiation factor, eIF4B, is thought to unwind the mRNA secondary structure thereby rendering it capable of binding to ribosomes (3). Because of its lowest abundance and its critical role in
mRNA binding to ribosomes, eIF4E is considered to be a regulator of
protein synthesis (1, 3). In fact, eIF4E activity has been reported to
be regulated by several kinase cascades including protein kinase C and
Mnk1, a downstream target of MAPKs (14-16). In addition, eIF4E
activity is negatively regulated by its binding proteins, 4E-BP1 and -2 (17-20).
Vascular smooth muscle cell (VSMC) growth exhibits characteristic
features of several proliferative cardiovascular diseases such as
atherosclerosis and restenosis (21). Both receptor tyrosine kinase and
G protein-coupled receptor (GPCR) agonists can induce VSMC growth
(22-26). Of interest, receptor tyrosine kinase agonists such as
platelet-derived growth factor and fibroblast growth factor (FGF) cause
a hyperplastic effect in these cells, whereas G protein-coupled receptor agonists such as angiotensin II (ang II), endothelin, and
PGF2
often induce a hypertrophic effect (21-26). All
three of the latter agonists mediate their effects via
Gq-coupled receptors whose activation is characterized by
increases in phospholipase C activity and intracellular
Ca2+ mobilization (27-30). The hypertrophic effect of ang
II has been reported to be mediated by p70S6k and ERKs (31,
32). Although PGF2
, a cyclooxygenase metabolite of
arachidonic acid that is produced in a variety of cells in response to
various stimuli including oxidant stress, has been shown to be a potent
hypertrophic agonist for cardiac myocytes and VSMC, the mechanisms
underlying this effect are largely unclear. The purpose of the present
study is to investigate the signaling events that are evoked in
response to PGF2
leading to increased protein synthesis
in VSMC. We found that PGF2
activates ERK2 and JNK1
groups of MAPKs, PI3-kinase, and p70S6k and induces
phosphorylation of the translation regulators eIF4E and 4E-BP1 in VSMC.
We also show a correlation between PGF2
-induced ERKs
activation, eIF4E and 4E-BP1 phosphorylation, global protein synthesis,
and bFGF-2 expression. In addition, the present findings suggest that
ERK-dependent and PI3-kinase-dependent
rapamycin-sensitive mechanisms modulate PGF2
-induced
eIF4E and 4E-BP1 phosphorylation in VSMC.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Aprotinin, ATP, bovine myelin basic protein,
dibutyryl cyclic AMP (cAMP), EGTA,
-glycerophosphate, leupeptin,
phenylmethylsulfonyl fluoride (PMSF), phosphatidylinositol, sodium
deoxycholate, sodium fluoride, sodium orthovanadate, and sodium
pyrophosphate were obtained from Sigma. Prostaglandin F2
was from Cayman Chemical Co. (Ann Arbor, MI). Silica gel 60A thin layer
chromatography plates and P81 phosphocellulose filter paper were
purchased from Whatman. Anti-ERK2 (sc-154), anti-JNK1 (sc-474),
anti-p70S6k (sc-230), and anti-FGF-2 (sc-79) rabbit
polyclonal antibodies and GST-c-Jun-(1-79) (sc-4113) recombinant
protein were bought from Santa Cruz Biotechnology (Santa Cruz, CA).
Anti-PI3-kinase rabbit polyclonal antibody (06-195) was obtained from
Upstate Biotechnology Inc. (Lake Placid, NY). Anti-phospho-p38 MAPK
antibodies and PD 098059 were provided by New England Biolabs (Beverly,
MA). Rapamycin and wortmannin were from Calbiochem.
[35S]Methionine (1000 Ci/mmol),
[32P]orthophosphoric acid (8500 Ci/mmol),
[
-32P]ATP (8000 Ci/mmol), and
[methyl-3H]thymidine (70 Ci/mmol) were
obtained from NEN Life Science Products.
Cell Culture--
VSMC were isolated from the thoracic aortae of
200-300-g male Sprague-Dawley rats by enzymatic dissociation as
described earlier (33). Cells were grown in Dulbecco's modified
Eagles's medium supplemented with 10% (v/v) heat-inactivated calf
serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cultures
were maintained at 37 °C in a humidified 95% air, 5%
CO2 atmosphere. Cells were growth-arrested by incubating in
Dulbecco's modified Eagles's medium containing 0.1% calf serum for
72 h. Growth-arrested VSMC were treated with known concentrations
of PGF2
for the indicated periods or were left
untreated. To study the effect of the inhibitors of various kinases on
PGF2
-induced VSMC responses, cells were pretreated for
30 min with the inhibitors prior to stimulation with
PGF2
.
Western Blot Analysis--
After appropriate treatments, medium
was aspirated, and cells were rinsed with cold phosphate-buffered
saline (PBS) and frozen immediately in liquid nitrogen. Two
hundred-fifty microliters of lysis buffer (PBS, 1% Nonidet P-40, 0.5%
sodium deoxycholate, 0.1% SDS, 100 µg/ml PMSF, 100 µg/ml
aprotinin, 1 µg/ml leupeptin, 20 mM
-glycerophosphate,
2 mM sodium fluoride, 2 mM sodium
pyrophosphate, and 1 mM sodium orthovanadate
(Na3VO4)) was added to the frozen monolayers,
thawed on ice for 15 min, and scraped into 1.5-ml Eppendorf tubes. The
cell lysates were cleared by centrifugation at 12,000 rpm for 20 min at
4 °C. Protein content of the supernatants was determined using
Bradford reagent from Bio-Rad. Cell lysates containing equal amounts of
protein were resolved by electrophoresis on 0.1% SDS-10%
polyacrylamide gels. The protein was transferred electrophoretically to
a nitrocellulose membrane (Hybond, Amersham Pharmacia Biotech). After
blocking in 10 mM Tris-Cl buffer, pH 8.0, containing 150 mM sodium chloride, 0.1% Tween 20, and 5% (w/v) non-fat
dry milk, the membrane was treated with appropriate primary antibodies
followed by incubation with appropriate peroxidase-conjugated secondary
antibodies. The antigen-antibody complexes were detected using
chemiluminescence reagent kit (Amersham Pharmacia Biotech).
MAPK Assays--
After appropriate treatments, cells were washed
with cold PBS and solubilized on ice for 15 min in lysis buffer
containing 20 mM Hepes, pH 7.4, 2 mM EGTA, 1 mM dithiothreitol, 50 mM
-glycerophosphate, 1% Triton X-100, 10 units/ml aprotinin, 2 µM leupeptin,
2 mM Na3VO4, and 400 µM PMSF. The cell lysates normalized for protein were immunoprecipitated by incubating with anti-ERK2 and anti-JNK1 antibodies for ERK2 and JNK1 assays, respectively, for 2 h
followed by addition of 40 µl of 50% (w/v) protein A-Sepharose beads
for an additional hour. The beads were washed three times with lysis buffer, three times with wash buffer (100 mM Tris-Cl, pH
7.6, 500 mM lithium chloride, 0.1% Triton X-100 and 1 mM dithiothreitol), and three times with kinase buffer
(12.5 mM Mops, pH 7.5, 12.5 mM
-glycerophosphate, 7.5 mM MgCl2, 2 mM EGTA, 0.5 mM sodium fluoride, and 0.5 mM Na3VO4). The ERK2 activity
present in the immunoprecipitates was determined by resuspension in 30 µl of kinase buffer containing 5 µg of myelin basic protein, 20 µM ATP, and 1 µCi of [
-32P]ATP and
incubation at 30 °C for 20 min. For JNK1 assay, incubation with the
kinase buffer was the same as that for ERK2 assay except that 1 µg of
GST-c-Jun-(1-79) was used instead of myelin basic protein (34). The
reactions were stopped by adding 20 µl of 4× Laemmli sample buffer
and heating the samples at 95 °C for 5 min. The samples were
analyzed by electrophoresis on 0.1% SDS-12% polyacrylamide gels. The
dried gel was exposed to X-Omat AR x-ray film with an intensifying
screen for 1 to 4 h at
80 °C and developed. For determination
of p38 MAPK activity, an equal amount of protein from control and test
samples was analyzed by Western blotting for phosphorylated p38 MAPK
using its phosphospecific antibodies.
PI3-Kinase Assay--
After appropriate treatments, cells were
lysed in 1 ml of lysis buffer (20 mM Hepes, pH 7.4, 2 mM EGTA, 50 mM
-glycerophosphate, 1 mM dithiothreitol, 1 mM
Na3VO4, 1% Triton X-100, 10% glycerol, 2 µM leupeptin, 10 units/ml aprotinin, and 400 µM PMSF) for 20 min on ice. The cell lysates were cleared
by centrifugation at 12,000 rpm for 15 min at 4 °C. Five hundred
micrograms of protein from control and each treatment was
immunoprecipitated with 5 µl of anti-PI3-kinase antibodies for 2 h at 4 °C, followed by incubation with 40 µl of 50% (w/v) protein
A-Sepharose beads for an additional hour. The immunoprecipitates were
washed three times with lysis buffer, three times with wash buffer, and
three times with TNF buffer (10 mM Tris-Cl, pH 7.4, 10 mM NaCl, 1 mM EDTA, and 10 µM
Na3VO4). The kinase activity was measured by
resuspending the immunoprecipitates in 30 µl of TNF buffer and
incubating with 10 µl of 2 mg/ml phosphatidylinositol, 10 µl of 100 mM MgCl2, 2 µl of 100 mM ATP, and
20 µCi of [
-32P]ATP for 10 min at 22 °C. The
reaction was terminated by addition of 20 µl of 5 N HCl
and 200 µl of chloroform:methanol (1:1) mix. The aqueous and organic
phases were separated by centrifugation at 2000 rpm for 10 min. The
organic phase containing the phosphoinositol phosphates was spotted
onto silica gel 60A TLC plate coated with 1% potassium oxalate and
developed in a solvent system consisting of
chloroform:methanol:water:ammonium hydroxide (90:70:14.6:5.4). The TLC
plate was exposed to X-Omat AR x-ray film for 4-6 h at
80 °C and developed.
Phosphorylation Assay--
Growth-arrested VSMC were
metabolically labeled with 300 µCi/ml
[32P]orthophosphoric acid for 4 h at 37 °C and
subjected to appropriate treatments. After treatments, medium was
aspirated, and cells were rinsed with cold PBS and lysed in 500 µl of
lysis buffer (50 mM Tris-Cl, pH 7.5, 150 mM
NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM EGTA, 80 mM
-glycerophosphate, 50 mM sodium fluoride, 2 mM sodium pyrophosphate,
1 mM Na3VO4, and 0.5 mM
PMSF) for 20 min on ice. The cell lysates were collected into 1.5-ml
Eppendorf tubes and cleared by centrifugation at 12,000 rpm for 20 min
at 4 °C. Cell lysates containing equal amounts of protein from each
condition were incubated with 5 µl of eIF4E or 4E-BP1 polyclonal
antibodies (35, 36) for 2 h on ice with gentle rocking. Forty
microliters of 10% (w/v) protein A-Sepharose beads were added, and
incubation was continued for another 2 h. The beads were collected
by centrifugation at 4000 rpm for 2 min at 4 °C and washed five
times with cold lysis buffer and once with cold PBS. The beads were
heated in 40 µl of Laemmli sample buffer at 95 °C for 5 min. The
proteins were resolved by electrophoresis on 0.1% SDS-10%
polyacrylamide gel. The gel was dried and exposed to X-Omat AR x-ray
film with an intensifying screen for 2-4 h at
80 °C and developed.
Protein and DNA Synthesis--
Growth-arrested VSMC were treated
with the indicated concentrations of PGF2
for the
indicated times or were left untreated. Protein and DNA syntheses were
measured by labeling cells with 1 µCi/ml
[35S]methionine and 1 µCi/ml
[3H]thymidine, respectively. After labeling, cells were
washed with cold PBS, trypsinized, and collected by centrifugation. The
cell pellet was suspended in cold 10% (w/v) trichloroacetic acid and vortexed vigorously to lyse cells. After standing on ice for 20 min,
the mixture was passed through a glass fiber filter (GF/C, Whatman).
The filter was washed once with cold 5% trichloroacetic acid and once
with cold 70% (v/v) ethanol. The filter was dried, placed in a liquid
scintillation vial containing the mixture, and the radioactivity
measured in a liquid scintillation counter (LS 3801, Beckman).
All the experiments were repeated at least three times with similar
pattern of results. Statistical analysis was performed by Student's
t test. p values < 0.05 were considered to
be significant.
 |
RESULTS |
To test the hypertrophic effect of PGF2
in VSMC, we
first studied the dose-response effect of PGF2
on
protein synthesis. Growth-arrested VSMC were treated with and without
various concentrations of PGF2
for 24 h, and
protein synthesis was determined by [35S]methionine
incorporation. As shown in Fig.
1A, PGF2
induced protein synthesis in a dose-dependent manner with a
maximum effect (2-fold) at 1 µM. Increase in protein
synthesis by PGF2
occurred in a
time-dependent manner as well (Fig. 1B). Earlier
studies have reported that PGF2
causes mitogenesis in
some cell types (30). To find whether PGF2
-induced
increase in protein synthesis is due to stimulation of cell division,
we tested its effect on DNA synthesis. PGF2
had no
significant effect on VSMC DNA synthesis as determined by
[3H]thymidine incorporation (Fig. 1C). Serum
(10% v/v), which served as a positive control, stimulated VSMC DNA
synthesis by 2-fold as compared with control. These results clearly
indicate that PGF2
induces protein synthesis without an
effect on DNA synthesis in VSMC.

View larger version (43K):
[in this window]
[in a new window]
|
Fig. 1.
Effect of
PGF2 on protein and DNA
synthesis in VSMC. Growth-arrested VSMC were treated with and
without various concentrations of PGF2 for 24 h
(A) or with 1 µM PGF2 for
different times (B) and protein synthesis was measured by
[35S]methionine incorporation. Cells were labeled with 1 µCi/ml [35S]methionine for the last 12 h of the
24-h incubation period in the dose-response experiment and continuously
in the time course experiment. The control values shown in B
were that of VSMC labeled for 24 h in the absence of
PGF2 . No significant differences were observed between
this and earlier time point controls. To determine DNA synthesis,
growth-arrested VSMC were treated with and without 1 µM
PGF2 or 10% (v/v) calf serum for 24 h and labeled
with 1 µCi/ml [3H]thymidine for the last 12 h of
the 24-h incubation period (C). * p < 0.01 versus control.
|
|
To understand the signal transduction pathways of
PGF2
-induced protein synthesis, we first studied the
role of MAPKs. Growth-arrested VSMC were treated with and without 1 µM PGF2
for various times or with
different concentrations of PGF2
for 10 min, and the
MAPK activities were determined. ERK2 and JNK1 activities were measured
by immunocomplex kinase assay using myelin basic protein and
GST-c-Jun-(1-79) as substrates, respectively, whereas p38 MAPK
activation was determined by Western blotting using its phosphospecific
antibodies. PGF2
activated ERK2 and JNK1 in a time- and
dose-dependent manner (Fig.
2, A-D).
Activation of ERK2 by PGF2
was maximum (7-fold) at 10 min, and thereafter it declined gradually reaching almost basal levels
by 60 min (Fig. 2A). PGF2
also activated JNK1
with a peak activity (4-fold) at 10 min, which then persisted at least
for 60 min (Fig. 2B). Dose-response study showed that
PGF2
at 1 µM concentration was found to be
more potent in the activation of ERK2 (Fig. 2C), whereas
higher concentrations appear to be more effective in the activation of
JNK1 (Fig. 2D). PGF2
, however, had no
significant effect on the activation of p38 MAPK in VSMC (Fig.
2E). Ang II (100 nM), which served as a positive
control, activated p38 MAPK 3-fold as compared with untreated cells.
For comparative purposes, the PGF2
was used at 1 µM concentration.

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 2.
PGF2
activates ERK2 and JNK1 groups of MAPKs in a time- and
dose-dependent manner in VSMC. Growth-arrested VSMC
were treated with and without 1 µM PGF2
for the indicated times or with different concentrations of
PGF2 for 10 min, and cell lysates were prepared. Equal
amount of protein from each condition was immunoprecipitated with
anti-ERK2 or anti-JNK1 antibodies, and the kinase activities in the
immunocomplexes were measured by immunocomplex kinase assay using
myelin basic protein as a substrate for ERK2 activity and
GST-c-Jun-(1-79) as a substrate for JNK1 activity. The reaction
mixtures were separated by electrophoresis on SDS-polyacrylamide gel
and subjected to autoradiography. For determination of p38 MAPK
activation, equal amount of protein from control and various times of
PGF2 -treated VSMC were analyzed by Western blotting
using its phosphospecific antibodies. Ang II (100 nM) was
used as a positive control. MBP, myelin basic protein.
|
|
A role for PI3-kinase was implicated both in receptor tyrosine kinase
and GPCR agonist-induced activation of MAPKs (37-39). To find whether
PGF2
activates PI3-kinase in VSMC, growth-arrested cells
were treated with and without 1 µM PGF2
for various times, and the PI3-kinase activity was measured.
PGF2
induced PI3-kinase activity in a
time-dependent manner in VSMC with a peak activity (6-fold)
at 10 min (Fig. 3, upper
panel). The PI3-kinase activity was further confirmed by its
sensitivity to inhibition by wortmannin (Fig. 3, lower
panel) (40, 41). To determine the role of PI3-kinase and
rapamycin-sensitive targets in PGF2
-induced activation
of ERKs, growth-arrested VSMC were treated with and without 1 µM PGF2
in the presence and absence of
different concentrations of wortmannin or 50 ng/ml rapamycin, and ERK2
activity was measured. Wortmannin inhibited the
PGF2
-induced ERK2 activity in a
concentration-dependent manner with maximum effect (>50%)
at 1 µM (Fig.
4A). Rapamycin also partially
(35%) decreased the PGF2
-induced ERK2 activity. On the
other hand, PD 098059 (50 µM), a potent and specific
inhibitor of MEK1 (42), completely blocked the
PGF2
-induced ERK2 activity. To determine whether PI3-kinase and rapamycin-sensitive targets also modulate
PGF2
-induced JNK1 activity, we tested the effect of
wortmannin (1 µM) and rapamycin (50 ng/ml) on the
activation of JNK1 by PGF2
. As shown in Fig.
4B, wortmannin had no effect on PGF2
-induced
JNK1 activation. Rapamycin also did not affect the JNK1 activation by
PGF2
(data not shown).

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 3.
PGF2
activates PI3-kinase in a time-dependent manner in
VSMC. Upper panel, growth-arrested VSMC were treated
with and without 1 µM PGF2 for the
indicated times, and cell lysates were prepared. Equal amount of
protein from control and each treatment was immunoprecipitated with
anti-PI3-kinase antibodies, and the kinase activity in the
immunocomplexes was measured by immunocomplex kinase assay using
phosphatidylinositol as a substrate. Phospholipids were separated by
thin layer chromatography and visualized by autoradiography.
Lower panel, growth-arrested VSMC were treated with and
without 1 µM PGF2 in the presence and
absence of wortmannin (1 µM) for 5 min, and the
PI3-kinase activity was measured as described above. PIP,
phosphatidylinositol 1,4,5-trisphosphate; ORI, origin.
|
|

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of wortmannin, rapamycin, and PD
098059 on PGF2 -induced activation
of MAPKs. Growth-arrested VSMC were treated with and without 1 µM PGF2 in the presence and absence of
wortmannin (100 nM or 1 µM), rapamycin (50 ng/ml), or PD 098059 (50 µM) for 10 min, and the ERK2 and
JNK1 activities were measured as described in Fig. 1. MBP,
myelin basic protein.
|
|
Ribosomal protein S6 kinase, p70S6k, was reported to play
an important role in protein synthesis initiation process (8, 9, 43,
44). We, therefore, wanted to examine the effect of PGF2
on p70S6k. Growth-arrested VSMC were treated with and
without 1 µM PGF2
for various times, and
p70S6k activity was determined. PGF2
activated p70S6k in a time-dependent manner as
determined by the slower mobility of the phosphorylated
p70S6k as compared with its nonphosphorylated form on
SDS-PAGE (Fig. 5, upper
panel). As expected, rapamycin (50 ng/ml), a potent and specific
inhibitor of p70S6k (43, 44), completely blocked the
p70S6k activation by PGF2
. To find the
possible upstream kinases that are involved in
PGF2
-induced p70S6k activation, we next
studied the effect of wortmannin (1 µM) and PD 098059 (50 µM), the potent and specific inhibitors of the PI3-kinase (40, 41) and ERKs (42), respectively, on PGF2
-induced p70S6k activation. Wortmannin completely inhibited the
PGF2
-induced p70S6k activation (Fig. 5,
middle panel). PD 098059 also significantly inhibited the
PGF2
-induced p70S6k activation (Fig. 5,
lower panel).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 5.
PGF2
activates p70S6k in VSMC. Growth-arrested VSMC were
treated with and without 1 µM PGF2 for the
indicated times or for 10 min in the presence and absence of the
indicated inhibitors, and cell lysates were prepared. Equal amount of
protein from control and each treatment was analyzed by immunoblotting
using anti-p70S6k antibodies. Phosphorylated
p70S6k migrates slower than the nonphosphorylated form
on SDS-PAGE.
|
|
A large body of evidence indicates that eIF4E plays a determinant role
in protein synthesis (1, 3, 4, 7). To investigate whether
PGF2
activates eIF4E and, if so, the role of PI3-kinase and ERKs in this phenomenon, we first studied the time course effect of
PGF2
on eIF4E phosphorylation. Growth-arrested and
[32P]orthophosphoric acid-labeled VSMC were treated with
and without 1 µM PGF2
for various times,
and the phosphorylation state of eIF4E was determined by
immunoprecipitation using its specific antibodies followed by SDS-PAGE
(35). PGF2
induced phosphorylation of eIF4E in a
time-dependent manner with a maximum effect (5-fold) at 20 min, which persisted at least for 60 min (Fig.
6, upper panel). To test the
role of PI3-kinase, ERKs and rapamycin-sensitive targets in
PGF2
activation of eIF4E, we then studied the effect of
wortmannin (1 µM), PD 098059 (50 µM), and
rapamycin (50 ng/ml), the PI3-kinase, ERKs, and p70S6k
inhibitors, respectively, on eIF4E phosphorylation induced by PGF2
. All three drugs blocked the
PGF2
-induced eIF4E phosphorylation by 60-70% (Fig. 6,
lower panel). PD 098059 and rapamycin also inhibited the
basal eIF4E phosphorylation.

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 6.
PGF2
induces phosphorylation of eIF4E in VSMC. Growth-arrested and
[32P]orthophosphoric acid-labeled VSMC were treated with
and without 1 µM PGF2 for various times or
for 20 min in the presence and absence of the indicated inhibitors
(wortmannin, 1 µM; PD 098059, 50 µM;
rapamycin, 50 ng/ml), and cell lysates were prepared. Equal amount of
protein from control and each treatment was immunoprecipitated with
anti-eIF4E antibodies, and the immunocomplexes were separated by
electrophoresis on SDS-polyacrylamide gel. The phosphorylated eIF4E was
visualized by autoradiography.
|
|
The activity of eIF4E was reported to be regulated by its binding
proteins 4E-BP1 and 4E-BP2 (17-20). In addition, several studies have
reported that wortmannin-dependent rapamycin-sensitive RAFT1/FRAP/mTOR phosphorylates 4E-BP1 (45-47). Upon phosphorylation, 4E-BP1 dissociates from eIF4E allowing the latter to be phosphorylated and activated. To find whether PGF2
induces
phosphorylation of 4E-BP1, and if so, the role of ERKs, PI3-kinase, and
RAFT1/FRAP/mTOR in this phenomenon, growth-arrested and
[32P]orthophosphoric acid-labeled VSMC were treated with
and without PGF2
(1 µM) in the presence
and absence of wortmannin (1 µM), PD 098059 (50 µM), or rapamycin (50 ng/ml) for 20 min, and the
phosphorylation state of 4E-BP1 was measured by immunoprecipitation using its specific antibodies (36). PGF2
induced 4E-BP1 phosphorylation 4-fold, and this effect was sensitive to inhibition by
all three compounds (Fig. 7).

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 7.
PGF2
induces phosphorylation of 4E-BP1 in VSMC. Growth-arrested and
[32P]orthophosphoric acid-labeled VSMC were treated with
and without 1 µM PGF2 in the presence and
absence of the indicated inhibitors (wortmannin, 1 µM; PD
098059, 50 µM; rapamycin, 50 ng/ml) for 20 min, and cell
lysates were prepared. Equal amount of protein from control and each
treatment was immunoprecipitated with anti-4E-BP1 antibodies, and the
immunocomplexes were separated by electrophoresis on SDS-polyacrylamide
gel. The phosphorylated 4E-BP1 was visualized by autoradiography.
|
|
To relate the above signaling events to PGF2
-induced
protein synthesis, we next studied the effect of wortmannin (1 µM), PD 098059 (50 µM), and rapamycin (50 ng/ml) on PGF2
-induced [35S]methionine
incorporation. As shown in Fig.
8A, PD 098059 significantly inhibited the PGF2
-induced protein synthesis. In
contrast, wortmannin and rapamycin, while inhibiting the basal protein
synthesis, had no significant effect on PGF2
-induced
protein synthesis. To attest the role of ERKs in
PGF2
-induced protein synthesis further, we have tested
the effect of cAMP. Previous studies from other laboratories have
reported that cAMP inhibits the "ERK MAPK" pathway at the Raf-1
level in several cell types including arterial smooth muscle cells (48,
49). As shown in Fig. 8B, cAMP (1 mM) completely
inhibited the PGF2
-induced protein synthesis in VSMC. To
find whether the inhibition of PGF2
-induced protein synthesis by cAMP correlates with decreased ERKs activities, we further
determined the effect of cAMP on PGF2
activation of
ERK2. As shown in Fig. 9, cAMP also
inhibited the PGF2
-induced ERK2 activation. In order to
find the role of the above mechanisms of PGF2
-induced
global protein synthesis in the modulation of a specific protein, whose
expression may be induced by this eicosanoid, we first studied its
effect on the expression of bFGF-2. Immunoblotting analysis of equal
amount of protein from VSMC treated with and without 1 µM
PGF2
for various times showed a
time-dependent increase in the induction of expression of
bFGF-2 by PGF2
(Fig. 10,
upper panel). Maximal increase (6-7-fold) in bFGF-2
expression in response to PGF2
occurred at 8 h,
which then persisted at least for 16 h after the initiation of the
treatment. To study the possible mechanisms underlying
PGF2
-induced bFGF-2 expression, we determined the effect
of wortmannin (1 µM), PD 098059 (50 µM), and rapamycin (50 ng/ml) on PGF2
-induced bFGF-2
expression. Whereas wortmannin inhibited the
PGF2
-induced bFGF-2 expression only partially (30%), PD
098059 completely blocked the effect. Rapamycin had no significant
effect on PGF2
-induced bFGF-2 expression (Fig. 10,
lower panel).

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 8.
PGF2 -induced global protein
synthesis requires ERKs activities. Growth-arrested VSMC were
treated with and without 1 µM PGF2 for
24 h in the presence and absence of the indicated inhibitors
(wortmannin, 1 µM; PD 098059, 50 µM;
rapamycin, 50 ng/ml; cAMP, 1 mM), and protein synthesis was
measured by pulse-labeling the cells for the last 12 h of the 24-h
incubation period with 1 µCi/ml [35S]methionine. *,
p < 0.01 versus control; **,
p < 0.01 versus PGF2
alone.
|
|

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 9.
Effect of cAMP on
PGF2 -induced activation of
ERK2. Growth-arrested VSMC were treated with and without 1 µM PGF2 in the presence and absence of 1 mM cAMP for 10 min, and the ERK2 activity was measured as
described in the legend to Fig. 1. MBP, myelin basic
protein.
|
|

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 10.
PGF2
induces bFGF-2 expression in an ERK-dependent manner in
VSMC. Growth-arrested VSMC were treated with and without 1 µM PGF2 for the indicated times or for
8 h in the presence and absence of the indicated inhibitors
(wortmannin, 1 µM; PD 098059, 50 µM;
rapamycin, 50 ng/ml), and cell lysates were prepared. Equal amount of
protein from control and each treatment was analyzed by immunoblotting
using anti-bFGF-2 antibodies.
|
|
 |
DISCUSSION |
The novel findings of the present study are as follows: 1)
PGF2
, an eicosanoid and a G protein-coupled receptor
agonist, activates several early response serine/threonine kinases such as MAPKs, PI3-kinase, and p70S6k and induces eIF4E and
4E-BP1 phosphorylation, global protein synthesis, and bFGF-2 expression
in growth-arrested VSMC; 2) PGF2
-induced global protein
synthesis and bFGF-2 expression to a major extent require activation of
ERKs but not PI3-kinase or p70S6k; 3) PGF2
activates ERK2 in a manner that is dependent and independent of
PI3-kinase; and 4) although multiple signaling pathways seem to be
involved in PGF2
-induced eIF4E phosphorylation, a
correlation was observed only between ERK-dependent eIF4E
phosphorylation and global protein synthesis by PGF2
.
Duckworth and Cantley (37) have reported that both
PI3-kinase-dependent and -independent mechanisms play a
role in the activation of ERKs in Swiss 3T3 cells in response to
platelet-derived growth factor, a receptor tyrosine kinase agonist. In
addition, Seva et al. (39) have shown that gastrin, a GPCR
agonist, activates ERKs via two signal transduction pathways in AR4-2J
cells, of which one is sensitive to inhibition by PI3-kinase
inhibitors. Our findings suggest at least two redundant pathways for
activation of ERKs in VSMC by PGF2
, of which one is
sensitive to PI3-kinase inhibitors. Numerous studies have reported a
requirement for PI3-kinase activity in p70S6k activation by
various stimulants in several cell types (10-12, 40). The present
finding that wortmannin, a potent inhibitor of PI3-kinase, blocks the
PGF2
-induced p70S6k activation is consistent
with the above reports. Since wortmannin blocked the
PGF2
-induced p70S6k activation completely
and ERK2 activation partially, it is likely that PI3-kinase lies
upstream to and signals several kinase cascades leading to gene
expression. Although wortmannin inhibited the PGF2
-induced ERK2 activity by 50%, it had no
significant effect on PGF2
-induced global protein
synthesis, which was sensitive to inhibition by PD 098059, a MEK1
inhibitor, suggesting that PI3-kinase-independent ERK2 activation is
more important than PI3-kinase-dependent ERK2 activation in
the induction of global protein synthesis by PGF2
. This
view can be further supported by the finding that rapamycin, although
it inhibits ERK2 activity partially, fails to block
PGF2
-induced global protein synthesis. These findings
further suggest complexity in the coupling of ERKs to various signaling
kinase cascades targeting modulation of different effector molecules
(25, 37, 38).
The present findings suggest that ERKs play an important role in the
phosphorylation of eIF4E and 4E-BP1, the regulators of protein
synthesis, by PGF2
. Our results also indicate a role for
PI3-kinase-dependent rapamycin-sensitive mechanisms in
PGF2
-induced phosphorylation of eIF4E and 4E-BP1 as
these events were inhibited by wortmannin and rapamycin. Indeed,
insulin-induced eIF4E and PHAS-1 (4E-BP1) phosphorylation was also
reported to be inhibited by wortmannin and rapamycin in myeloid
progenitor cells (50). However, the PI3-kinase-dependent
rapamycin-sensitive eIF4E and 4E-BP1 phosphorylation may also be
mediated by ERKs as the PI3-kinase inhibitor, wortmannin, and the
RAFT1/FRAP/mTOR inhibitor, rapamycin, significantly reduced ERKs
activities induced by PGF2
. ERKs may play a role
downstream to mTOR in the signaling pathway of PI3-kinase-dependent rapamycin-sensitive eIF4E and 4E-BP1
phosphorylation. Based on the sensitivity of eIF4E and 4E-BP1
phosphorylation to inhibition by various kinase inhibitors, it is
tempting to speculate a role for multiple signal transduction pathways
in the modulation of activity of these important regulators of protein
synthesis. Furthermore, different groups of MAPKs may be involved in
the activation of eIF4E by different stimulants (51). To cite an example in support of this notion, Morley and McKendrick (52) have
demonstrated that serum-induced eIF4E phosphorylation was blocked by
the ERK MAPK pathway inhibitor PD 098059, whereas anisomycin-induced eIF4E phosphorylation was attenuated by the p38 MAPK inhibitor SB
203580. It is noteworthy that while PD 098059, wortmannin, and
rapamycin decreased PGF2
-induced phosphorylation of
eIF4E and 4E-BP1 by more than 60%, only PD 098059 significantly
inhibited the protein synthesis stimulated by PGF2
.
These findings suggest that enhanced phosphorylation of eIF4E and
4E-BP1 alone may not be sufficient for global protein synthesis induced
by PGF2
. From these findings, it is also conceivable
that coordinate regulation of both transcriptional and translational
events may be required for increased protein synthesis by
PGF2
. Indeed, this view further supports the role of
ERKs in PGF2
-induced protein synthesis as these kinases
modulate the activities of both translation regulators such as eIF4E
and its binding proteins 4E-BP1/2 and transcriptional factors such as
AP-1 (7, 53). Additional evidence in support of a role for
transcriptional mechanisms in the induction of global protein synthesis
comes from the recent findings that calcineurin, a
calcium-dependent phosphatase, plays a role in cardiac
hypertrophy (54). Calcineurin modulates the activity of several
transcriptional factors including nuclear factor of activated T cells
(55). Although further studies are required to test whether
calcineurin-nuclear factor of activated T cells plays a role in
PGF2
-induced protein synthesis, the present findings
together with those of Sussman et al. (54) and Molkentin
et al. (55) clearly suggest a role for both transcriptional and translational events in induced global protein synthesis.
PI3-kinase was reportedly shown to play a role in the activation of
p70S6k in a manner that is sensitive to inhibition by
rapamycin (10-12). A similar mechanism appears to be operative in the
activation of p70S6k by PGF2
as this event
is blocked by both wortmannin and rapamycin, the PI3-kinase, and mTOR
inhibitors, respectively. Rapamycin, complexed with its receptor
FKBP12, binds to mTOR (mammalian target of rapamycin) protein and
inhibits its function (56). mTOR plays a role in the activation of
p70S6k (47, 57). In addition, it was reported that mTOR
regulates the activities of p70S6k and 4E-BP1 in a parallel
manner (57). Although our results demonstrate the ability of
PGF2
in the activation of both of these signaling
pathways in VSMC, their role, in particular of p70S6k, in
PGF2
-induced hypertrophy is obscure as the inhibitors of
these pathways failed to significantly reduce the protein synthesis induced by this agonist. However, ang II, a peptide hormone and a G
protein-coupled receptor agonist, was reported to stimulate global
protein synthesis via a mechanism involving p70S6k
activation in VSMC (31). It was also reported that ang II induces 4E-BP1 phosphorylation via a MAPK-independent mechanism (58). In
addition, we have previously shown that ang II induces eIF4E phosphorylation and PKC plays a role in this event (59). Based on these
observations as well as the present findings, it is likely that
different signaling mechanisms are involved in the induction of protein
synthesis by different G protein-coupled receptor agonists in VSMC.
PGF2
activation of the p70S6k pathway may be
linked to the regulation of a subset of genes whose identity has not
yet been addressed.
The present study demonstrates that PGF2
, an arachidonic
acid metabolite and a G protein-coupled receptor agonist, induces protein synthesis via activation of ERKs in VSMC. These findings also
provide the first evidence for the role of
PI3-kinase-dependent and -independent ERK MAPK pathways in
the phosphorylation of eIF4E induced by PGF2
in VSMC. In
addition, these results indicate a major role for
PI3-kinase-independent ERK-mediated eIF4E phosphorylation in
PGF2
-induced global protein synthesis and bFGF-2
expression in VSMC.
 |
ACKNOWLEDGEMENT |
We sincerely thank Rachel Stella for typing
the manuscript.
 |
FOOTNOTES |
*
This work was supported in part by a grant-in-aid from the
American Heart Association (to G. N. R.).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: 4.124B Old John Sealy
Hospital, University of Texas Medical Branch, 301 University Blvd.,
Route 0567, Galveston, TX 77555-0567. Tel.: 409-747-1851; Fax:
409-772-1861; E-mail: grao{at}utmb.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
PI3-kinase, phosphatidylinositol 3-kinase;
bFGF-2, basic fibroblast growth
factor-2;
eIF4E, eukaryotic translation initiation factor 4E;
4E-BP1, eIF4E binding protein 1;
ERK2, extracellular signal-regulated kinase 2;
JNK1, Jun N-terminal kinase 1;
MAPKs, mitogen-activated protein
kinases;
PGF2
, prostaglandin
F2
;
VSMC, vascular smooth muscle cells;
GST, glutathione S-transferase;
PAGE, polyacrylamide gel
electrophoresis;
PBS, phosphate-buffered saline;
PMSF, phenylmethylsulfonyl fluoride;
MOPS, 4-morpholinepropanesulfonic acid;
GPCR, G protein-coupled receptor;
ang II, angiotensin II;
mTOR, mammalian target of rapamycin.
 |
REFERENCES |
-
Rhoads, R. E.
(1988)
Trends Biochem. Sci.
13,
52-56[CrossRef][Medline]
[Order article via Infotrieve]
-
Morley, S. J.,
and Traugh, J. A.
(1990)
J. Biol. Chem.
265,
10611-10616[Abstract/Free Full Text]
-
Hershey, J. W. B.
(1991)
Annu. Rev. Biochem.
60,
717-755[CrossRef][Medline]
[Order article via Infotrieve]
-
Redpath, N. T.,
and Proud, C. G.
(1994)
Biochim. Biophys. Acta
1220,
147-162[Medline]
[Order article via Infotrieve]
-
Krieg, J.,
Hofsteenge, J.,
and Thomas, G.
(1988)
J. Biol. Chem.
263,
11473-11477[Abstract/Free Full Text]
-
Sturgill, T. W.,
and Wu, J.
(1991)
Biochim. Biophys. Acta
1092,
350-357[Medline]
[Order article via Infotrieve]
-
Sonenberg, N.
(1996)
in
Translational Control (Hershey, J. W. B., Mathews, M. B., and Sonenberg, N., eds), pp. 245-269, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
-
Ferrari, S.,
and Thomas, G.
(1994)
Crit. Rev. Biochem. Mol. Biol.
29,
385-413[Abstract]
-
Banerjee, P.,
Ahmad, M. F.,
Grove, J. R.,
Kozlosky, C.,
Price, D. J.,
and Avruch, J.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
8550-8554[Abstract]
-
Cheatham, B.,
Vlahos, C. J.,
Cheatham, L.,
Wang, L.,
Blenis, J.,
and Kahn, C. R.
(1994)
Mol. Cell. Biol.
14,
4902-4911[Abstract]
-
Weng, O. P.,
Andrabi, K.,
Klippel, A.,
Kozlosky, M. T.,
Williams, L. T.,
and Avruch, J.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
5744-5748[Abstract]
-
Reif, K.,
Burgering, B. M. T.,
and Cantrell, D. A.
(1997)
J. Biol. Chem.
272,
14426-14433[Abstract/Free Full Text]
-
Haghighat, A.,
and Sonenberg, N.
(1997)
J. Biol. Chem.
272,
21677-21680[Abstract/Free Full Text]
-
Morley, S. J.,
Dever, T. E.,
Etchison, D.,
and Traugh, J. A.
(1991)
J. Biol. Chem.
266,
4669-4672[Abstract/Free Full Text]
-
Whalen, S. G.,
Gingras, A. C.,
Amankwa, L.,
Mader, S.,
Branton, P. E.,
Aebersold, R.,
and Sonenberg, N.
(1996)
J. Biol. Chem.
271,
11831-11837[Abstract/Free Full Text]
-
Waskiewicz, A. J.,
Flynn, A.,
Proud, C. G.,
and Cooper, J. A.
(1997)
EMBO J.
16,
1909-1920[Abstract/Free Full Text]
-
Hu, C.,
Pang, S.,
Kong, X.,
Velleca, M.,
and Lawrence, J. C.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
3730-3734[Abstract]
-
Lin, T. A.,
Kong, X.,
Haystead, T. A.,
Pause, A.,
Belsham, G. J.,
Sonenberg, N.,
and Lawrence, J. C.
(1994)
Science
266,
653-656[Medline]
[Order article via Infotrieve]
-
Pause, A.,
Belcham, G. J.,
Gingras, A. C.,
Donze, O.,
Lin, T. A.,
Lawrence, J. C.,
and Sonenberg, N.
(1994)
Nature
371,
762-767[CrossRef][Medline]
[Order article via Infotrieve]
-
Graves, L. M.,
Bornfeldt, K. E.,
Argast, G. M.,
Krebs, E. G.,
Kong, X. M.,
Lin, T. A.,
and Lawrence, J. C.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
7222-7226[Abstract]
-
Ross, R.
(1993)
Nature
362,
801-809[CrossRef][Medline]
[Order article via Infotrieve]
-
Berk, B. C.,
Vekshtein, V.,
Gordon, H. M.,
and Tsuda, T.
(1989)
Hypertension
13,
305-314[Abstract]
-
Shubeita, H. E.,
McDonough, P. M.,
Harris, A. N.,
Knowlton, K. U.,
Glembotski, C. C.,
Brown, J. H.,
and Chien, K. R.
(1990)
J. Biol. Chem.
265,
20555-20562[Abstract/Free Full Text]
-
Lindner, V.,
and Reidy, M. A.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
3739-3743[Abstract]
-
Dorn, G. W., II,
Becker, M. W.,
and Davis, M. G.
(1992)
J. Biol. Chem.
267,
24897-24905[Abstract/Free Full Text]
-
Adams, J. W.,
Migita, D. S., Yu, M. K.,
Young, R.,
Hellickson, M. S.,
Castro-Vargas, F. E.,
Domingo, J. D.,
Lee, P. H.,
Bui, J. S.,
and Henderson, S. A.
(1996)
J. Biol. Chem.
271,
1179-1186[Abstract/Free Full Text]
-
Murphy, T. J.,
Alexander, R. W.,
Griendling, K. K.,
Runge, M. S.,
and Bernstein, K. E.
(1991)
Nature
351,
233-236[CrossRef][Medline]
[Order article via Infotrieve]
-
Simon, M. I.,
Strathmann, M. P.,
and Gautam, N.
(1991)
Science
252,
802-808[Medline]
[Order article via Infotrieve]
-
Fleming, J. W.,
Wisler, P. L.,
and Watanabe, A. M.
(1992)
Circulation
85,
420-433[Abstract]
-
Watanabe, T.,
Waga, I.,
Honda, Z.,
Kurokawa, K.,
and Shimizu, T.
(1995)
J. Biol. Chem.
270,
8984-8990[Abstract/Free Full Text]
-
Giasson, E.,
and Meloche, S.
(1995)
J. Biol. Chem.
270,
5225-5231[Abstract/Free Full Text]
-
Servant, M. J.,
Giasson, E.,
and Meloche, S.
(1996)
J. Biol. Chem.
271,
16047-16052[Abstract/Free Full Text]
-
Rao, G. N.
(1996)
Oncogene
13,
713-719[Medline]
[Order article via Infotrieve]
-
Rao, G. N.,
and Runge, M. S.
(1996)
J. Biol. Chem.
271,
20805-20810[Abstract/Free Full Text]
-
Frederickson, R. M.,
Montine, K. S.,
and Sonenberg, N.
(1991)
Mol. Cell. Biol.
11,
2896-2900[Medline]
[Order article via Infotrieve]
-
Gingras, A. C.,
Svitkin, Y.,
Belsham, G. J.,
Pause, A.,
and Sonenberg, N.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
5578-5583[Abstract/Free Full Text]
-
Duckworth, B. C.,
and Cantley, L. C.
(1997)
J. Biol. Chem.
272,
27665-27670[Abstract/Free Full Text]
-
Lopez-Ilasaca, M.,
Crespo, P.,
Pellici, P. G.,
Gutkind, J. S.,
and Wetzker, R.
(1997)
Science
275,
394-397[Abstract/Free Full Text]
-
Seva, C.,
Kowalski, A.,
Daulhac, L.,
Barthez, C.,
Vaysse, N.,
and Pradayrol, L.
(1997)
Biochem. Biophys. Res. Commun.
238,
202-206[CrossRef][Medline]
[Order article via Infotrieve]
-
Myers, M. G., Jr.,
Grammer, T. C.,
Wang, L. M.,
Sun, X. J.,
Pierce, J. H.,
Blenis, J.,
and White, M. F.
(1994)
J. Biol. Chem.
269,
28783-28789[Abstract/Free Full Text]
-
Tang, X.,
and Downes, C. P.
(1997)
J. Biol. Chem.
272,
14193-14199[Abstract/Free Full Text]
-
Alessi, D. R.,
Cuenda, A.,
Cohen, P.,
Dudley, D. T.,
and Saltiel, A. R.
(1995)
J. Biol. Chem.
270,
27489-27494[Abstract/Free Full Text]
-
Chung, J.,
Kuo, C. J.,
Crabtree, G. R.,
and Blenis, J.
(1992)
Cell
69,
1227-1236[Medline]
[Order article via Infotrieve]
-
Price, D. J.,
Grove, J. R.,
Calvo, V.,
Avruch, J.,
and Bierer, B. E.
(1992)
Science
247,
973-977
-
van Manteuffel, S. R.,
Gingras, A. C.,
Ming, X. F.,
Sonenberg, N.,
and Thomas, G.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
4076-4080[Abstract/Free Full Text]
-
van Manteuffel, S. R.,
Dennis, P. B.,
Pullen, N.,
Gingras, A. C.,
Sonenberg, N.,
and Thomas, G.
(1997)
Mol. Cell. Biol.
17,
5426-5436[Abstract]
-
Burnett, P. E.,
Barrow, R. K.,
Cohen, N. A.,
Snyder, S. H.,
and Sabatini, D. M.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
1432-1437[Abstract/Free Full Text]
-
Graves, L. M.,
Bornfeldt, K. E.,
Rains, E. W.,
Potts, B. C.,
Macdonald, S. G.,
Ross, R.,
and Krebs, E. G.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
10300-10304[Abstract]
-
Hordjik, P. L.,
Verlaan, I.,
Jalink, K.,
van Corven, E. J.,
and Moolenaar, W. H.
(1994)
J. Biol. Chem.
269,
3534-3538[Abstract/Free Full Text]
-
Mendez, R.,
Meyers, M. G.,
White, M. F.,
and Rhoads, R. E.
(1996)
Mol. Cell. Biol.
16,
2857-2864[Abstract]
-
Wang, X.,
Flynn, A.,
Waskiewicz, A. J.,
Webb, B. L. J.,
Vries, R. G.,
Baines, I. A.,
Cooper, J. A.,
and Proud, C. G.
(1998)
J. Biol. Chem.
273,
9373-9377[Abstract/Free Full Text]
-
Morley, S. J.,
and McKendrick, L.
(1997)
J. Biol. Chem.
272,
17887-17893[Abstract/Free Full Text]
-
Karin, M.
(1995)
J. Biol. Chem.
270,
16483-16486[Free Full Text]
-
Sussman, M. A.,
Lim, W. H.,
Gude, N.,
Taigen, T.,
Olson, E. N.,
Robbins, J.,
Colbert, M. C.,
Gualberto, A.,
Wieczorek, D. F.,
and Molkentin, J. D.
(1998)
Science
281,
1690-1693[Abstract/Free Full Text]
-
Molkentin, J. D.,
Lu, J. R.,
Antos, C. L.,
Markham, B.,
Richardson, J.,
Robbins, J.,
Grant, S. R.,
and Olson, E. N.
(1998)
Cell
93,
215-228[Medline]
[Order article via Infotrieve]
-
Brown, E. J.,
Beal, P. A.,
Keith, C. T.,
Chen, J.,
Shin, T. B.,
and Schreiber, S. L.
(1995)
Nature
377,
441-446[CrossRef][Medline]
[Order article via Infotrieve]
-
Hara, K.,
Yonezawa, K.,
Kozlowski, M. T.,
Sugimoto, T.,
Andrabi, K.,
Weng, Q. P.,
Kasuga, M.,
Nishimoto, I.,
and Avruch, J.
(1997)
J. Biol. Chem.
272,
26457-26463[Abstract/Free Full Text]
-
Fleurent, M.,
Gingras, A. C.,
Sonenberg, N.,
and Meloche, S.
(1997)
J. Biol. Chem.
272,
4006-4012[Abstract/Free Full Text]
-
Rao, G. N.,
Griendling, K. K.,
Frederickson, R. M.,
Sonenberg, N.,
and Alexander, R. W.
(1994)
J. Biol. Chem.
269,
7180-7184[Abstract/Free Full Text]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.