MEK inhibition augments Raf activity, but has variable effects
on mitogenesis, in vascular smooth muscle cells
Robert H.
Weiss1,2,3,
Elizabeth
A.
Maga1, and
Al
Ramirez1
1 Division of Nephrology,
Department of Internal Medicine, and
2 Cell and Developmental Biology
Graduate Group, University of California, Davis 95616; and
3 Department of Veterans Affairs
Northern California Health Care System, Pleasant Hill, California 94523
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ABSTRACT |
Proteins
comprising the mitogen-activated protein (MAP) kinase signaling cascade
are activated by a variety of growth factors, but the precise role of
this series of kinase reactions, especially Raf kinase and MAP kinase
kinase (MEK), in vascular smooth muscle (VSM) cell
mitogenesis is not known. In this study, a specific and selective
inhibitor of MEK, PD-98059, was used to examine the role of MEK in DNA
synthesis and Raf-1 activity in VSM cells stimulated with serum as well
as with growth factors encompassing both tyrosine kinase and G
protein-coupled receptor classes. Although significant increases in DNA
synthesis are seen after stimulation of VSM cells with either 10%
serum, platelet-derived growth factor (PDGF)-BB, or
-thrombin,
preincubation of the cells with 50 µM PD-98059 for 1 h inhibits
stimulation by PDGF and thrombin, but not by serum. There is a
dose-dependent inhibition of the mitogenic effect by PD-98059 in all
cases; these results are not affected when PD-98059 is added at times
ranging from 4 h before to 2 h after growth factor addition (times at
which PD-98059 exerts its inhibitory effect). In the presence of
PD-98059, stimulated MAP kinase activity is attenuated when growth
factors are added between 10 min and 4 h, times which correspond to
both early and sustained phases of MAP kinase activity. In addition,
Raf-1 activity is markedly increased by incubation of the cells with
PD-98059, despite attenuation of hyperphosphorylation of this kinase.
Thus growth factors coupled to both tyrosine kinase and G protein
receptors require components of the MAP kinase cascade (MEK
and/or MAP kinase) for VSM cell mitogenesis, whereas serum is
capable of stimulatory effects in the absence of active MEK and MAP
kinase. Furthermore, there exists a functional feedback stimulatory
effect of inhibited MEK on its upstream activator Raf-1 in the case of
serum as well as growth factors coupled to tyrosine kinase and G
protein receptors.
mitogen-activated protein kinase;
-thrombin; platelet-derived
growth factor-BB; PD-98059
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INTRODUCTION |
GROWTH FACTORS TRANSMIT their signals from the cell
surface to the nucleus by utilizing a series of sequentially activated kinase cascades, which are integrated into a complex network known as
the mitogen-activated protein (MAP)/extracellular signal-regulated kinase pathways (reviewed in Ref. 16). Although elements of this
cascade have been shown to be involved in the regulation of the
proliferative response of cells to both tyrosine kinase and G
protein-coupled receptor growth factors, the actual sequence of
molecular events that occurs to transmit the mitogenic response from
the cell surface receptor to the nucleus is not completely understood.
Upon activation of many different cell types by a variety of growth
factor receptors, the MAP kinase pathway becomes activated initially by
the formation of Ras-GTP. The ensuing recruitment of Raf to the plasma
membrane by Ras causes its activation, by unknown factors, and
transmission of the signal through the cascade reactions then occurs
(11, 23). Raf-1 phosphorylates and activates MAP kinase kinase (MEK),
which in turn phosphorylates and activates MAP kinase in a biphasic
fashion (17, 18). Subsequently, active MAP kinase is translocated to
the nucleus where it phosphorylates transcription factors, such as
Elk-1, which leads to transcription of
c-fos (9, 25). Although the elements
of the MAP kinase pathways function to propagate their signals from the
cell membrane to the nucleus, there is evidence that feedback loops
also occur (22, 31).
Although it had been inferred that hyperphosphorylation of Raf was
associated with its activation, we (2) and others (8, 24) recently
showed that hyperphosphorylation of Raf is not required for its
catalytic activity. Other investigators have demonstrated that
hyperphosphorylation of Raf-1 is associated with a decrease in its
affinity with the cell membrane and inactivation of this kinase, at
least in cells stimulated with serum (28), whereas still others have
shown that Raf hyperphosphorylation is the consequence of events
occurring downstream of MAP kinase (22). However, although the
proliferative response of the receptor tyrosine kinases has been
thought to be conferred by the Ras/Raf/MAP kinase pathway, there is
also evidence of Ras-independent signaling by the platelet-derived
growth factor (PDGF) receptor (4).
The recent discovery of PD-98059 has allowed detailed investigation of
the means by which the mitogenic signal is transmitted through Raf to
its downstream effectors. PD-98059 has been shown to be a specific
inhibitor of MEK, acting by preventing phosphorylation and thus
activation of both MEK-1 and MEK-2 (1, 7, 15, 20). Because activation
of the MAP kinase pathway is associated with many mitogenic growth
factors, we asked what effect MEK inhibition has on DNA synthesis in
vascular smooth muscle (VSM) cells stimulated by serum as well as by
growth factors coupled to tyrosine kinase or G protein-coupled
receptors. We now show that, although PDGF-BB and
-thrombin-mediated
mitogenesis is significantly inhibited by PD-98059, this inhibitor
allowed 10% serum to remain stimulatory. Furthermore, in the case of
cells stimulated by PDGF-BB,
-thrombin, or 10% serum, PD-98059
caused inhibition of MAP kinase but stimulation of Raf-1 catalytic
activity, despite loss of Raf-1 hyperphosphorylation.
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MATERIALS AND METHODS |
Materials.
Cultures of A10 embryonic thoracic aorta, smooth muscle, DB1X rat cells
were obtained from the American Type Culture Collection.
-Thrombin
was kindly supplied to us by Dr. J. Fenton (Albany, NY). PD-98059,
Elk-1 fusion protein, LumiGLO (Phototope-HRP Western Detection System),
phospho-MAP kinase, and phospo-Elk-1 antibodies were obtained from New
England Biolabs (Beverly, MA). Human recombinant PDGF-BB, sheep
polyclonal anti-human c-Raf kinase COOH terminal, inactive glutathione
S-transferase (GST)-MAP kinase kinase 1 (MEK-1), and inactive GST-p42 MAP kinase (MAPK) were obtained from UBI (Lake
Placid, NY). Sheep polyclonal anti-human c-Raf kinase COOH terminal was
obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Goat
anti-rabbit horseradish peroxidase (HRP)-linked IgG was obtained from
Bio-Rad (Richmond, CA). Components for the enhanced chemiluminescence
(ECL) system and
[3H]thymidine were
obtained from Amersham (Arlington Heights, IL). All other reagents were
from Sigma (St. Louis, MO).
Cell culture.
A10 cells were maintained in DMEM with 10% fetal bovine serum,
penicillin (50 U/ml), and streptomycin (50 U/ml) in a humidified atmosphere of 5% CO2-95% air at
37°C. Culture medium was changed every other day until the cells
were confluent. The cells were growth-arrested by placing them in
quiescence medium containing DMEM, 20 mM HEPES (pH 7.4), 5 mg/ml
transferrin, 0.5 mg/ml BSA, 50 U/ml penicillin, and 50 U/ml
streptomycin. Quiescence medium was changed daily for 1-2 days
before each experiment. Cells were harvested with 5 ml trypsin and
subcultured at a 1:10 dilution weekly.
DNA synthesis.
Cultured A10 cells were used at passages 17-26. Before each
experiment, the cells were harvested, plated onto 24-well plates, grown
to confluence, and then serum starved for 24 h. For dose-response studies, 10, 20, 30, 40, 50, or 60 µM of the MEK-1/2 inhibitor PD-98059 was added 1 h before growth factor addition. For time course
studies, 50 µM PD-98059 was added 4, 2, and 1 h before, at the same
time as, and 1 and 2 h after growth factor addition. [3H]thymidine
incorporation was assessed as previously described (30).
MAP kinase activity assay.
A10 cells were grown to confluence in 6-cm dishes and serum starved for
48 h. Cells were treated with 50 µM PD-98059 for 1 h. Control cells
were treated with the same quantity of DMSO. The cells were stimulated
for 10 min with either 10% serum, PDGF-BB (30 ng/ml), or
-thrombin
(1 U/ml). The plates were washed one time with ice-cold PBS, and 0.5 ml
lysis buffer [20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM
EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM
-glycerolphosphate, 1 mM
Na3VO4,
1 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride
(PMSF)] was added to each plate. The plates were incubated on ice
for 5 min, and then the cells were scraped off the plate and
transferred to an Eppendorf tube. The lysate was passed through a
25-gauge needle four times and then centrifuged at 14,000 rpm for 10 min at 4°C. Active MAP kinase was immunoprecipitated from each
lysate by placing normalized amounts of lysate (as determined
spectrophotometrically) in a tube with phospho-MAP kinase antibody in a
1:50 dilution lysate-antibody and incubated overnight at 4°C with
gentle shaking. Protein A-Sepharose beads (50 µl) were added to each
tube and were placed for 1 h at 4°C with shaking. Samples were
centrifuged at 14,000 rpm for 30 s, and the pellet was washed twice
with 500 µl lysis buffer and twice with 500 µl kinase buffer
[25 mM Tris, pH 7.5, 5 mM
-glycerolphosphate, 2 mM
dithiothreitol (DTT), 0.1 mM
Na3VO4,
and 10 mM MgCl2]. The
immunoprecipitate was resuspended in 50 µl kinase buffer containing
100 µM ATP and 1 µg Elk-1 fusion protein, a MAP kinase substrate.
Samples were incubated at 30°C for 30 min, and the reaction was
terminated by adding 25 µl 3× SDS sample buffer. Samples were
boiled for 5 min, vortexed lightly, and centrifuged at 14,000 rpm for 2 min. Thirty microliters of each sample were electrophoresed on a 12.5%
SDS-PAGE gel, and the proteins were electrophoretically transferred to
a nitrocellulose membrane. MAP kinase activity was determined by
Western blotting with an antibody specific for phosphorylated Elk-1.
Membranes were blocked for 1 h with 5% nonfat dry milk-Tris-buffered
saline (TBS)-0.1% Tween at room temperature. Membranes
were incubated with a 1:1,000 dilution of phospho-Elk-1 antibody in 5%
BSA-TBS-0.05% Tween overnight at 4°C with gentle shaking. The
membranes were washed three times for 5 min with TBS-0.1% Tween and
incubated for 1 h at room temperature with a 1:2,000 dilution of
goat-anti rabbit HRP in 5% nonfat dry milk-TBS-0.1% Tween. After
three washes of 5 min with TBS-0.1% Tween, the membranes were placed
in 10 ml of 1× LumiGLO for 1 min, exposed to film for various
times, and developed.
Western blotting.
A10 cells were grown to confluence on 6-cm culture dishes and serum
deprived for 48 h. Cells were treated with 50 µM PD-98059 or DMSO for
1 h. After stimulation for 10 min with either 10% serum, PDGF-BB (30 ng/ml), or
-thrombin (1 U/ml), the cells were washed three times
with ice-cold PBS and lysed in place by the addition of ice-cold RIPA-A
buffer [50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM
sodium orthovanadate, 1 mM sodium fluoride, 1 mM PMSF (freshly
prepared), 1% Nonidet P-40, and 1 µg/ml aprotinin]. After
10-min incubation at 4°C, the lysate was scraped off with a rubber
spatula and centrifuged at 14,000 rpm at 4°C for 10 min. The
supernatant was saved, and the protein concentration was determined spectrophotometrically at 595-nm absorbance with the Bio-Rad protein concentration reagent. Aliquots containing identical amounts of protein
for each lysate were heated with SDS-PAGE gel loading buffer in a
boiling water bath for 5 min, loaded onto a 7.5% polyacrylamide gel
with 5% stacking gel, and electrophoresed at 100 V for 2 h. Molecular
weight standards were electrophoresed on the same gel. Subsequently,
the gel was electrophoretically transferred onto a nitrocellulose
filter in transfer buffer (193 mM glycine, 25 mM Tris, and 20%
methanol, pH 8.3). The nitrocellulose filters were blocked for 1 h with
5% nonfat dry milk in TBS-T (20 mM Tris, 137 mM NaCl, pH 7.5, 0.1%
Tween 20), washed in TBS-T, and incubated at room temperature for 1 h
in a 1:1,000 dilution of Raf-1 antibody in TBS-T. The filter was washed
again in TBS-T and incubated for 1 h with a 1:15,000 dilution of goat
anti-rabbit HRP-coupled IgG in TBS-T. The final washes were with TBS-T
for 15 min and 4 × 5 min. The ECL system was used for detection.
Raf activity assay.
Raf activity was determined by an in vitro kinase cascade reaction.
Briefly, immunoprecipitated active Raf will activate an inactive MEK,
which can then activate an inactive MAP kinase. Any Raf-activated MAP
kinase then phosphorylates the MAP kinase substrate Elk-1. The amount
of Raf activity is related to the amount of phosphorylated Elk-1 by a
phospho-specific Elk-1 antibody.
A10 cells were grown to confluence in 6-cm dishes and serum starved for
48 h. Cells were treated with 50 µM PD-98059 for 1 h. Control cells
were treated with DMSO. The cells were then stimulated for 10 min with
either serum, PDGF-BB (30 ng/ml), or
-thrombin (1 U/ml). All dishes
were washed three times with ice-cold PBS and placed in 500 µl
buffer A (50 mM Tris, pH 7.5, 1 mM
EDTA, 1 mM EGTA, 0.1%
-mercaptoethanol, 1% Triton X-100, 50 mM
sodium fluoride, 5 mM sodium pyrophosphate, 10 mM sodium
glycerophosphate, 0.5 mM
Na3VO4,
0.1 mM PMSF, 1 µg/ml aprotinin, and 1 µg/ml leupeptin) on ice for
10 min. Cells were scraped into an Eppendorf tube, passed through a
25-gauge needle four times, and centrifuged at 14,000 rpm for 10 min at
4°C. Lysates were normalized for protein amount (as determined
spectrophotometrically) and immunoprecipitated overnight with 2 µg
sheep polyclonal anti-human c-Raf kinase COOH terminal at 4°C with
shaking. Protein G agarose beads (100 µl) that had been washed with
PBS were added to each tube and incubated for 1 h at 4°C. The
immunoprecipitates were washed one time with buffer
A plus 0.5 M NaCl followed by one wash with
buffer A. Samples were resuspended in
70 µl assay dilution buffer (20 mM MOPS, pH 7.2, 25 mM
-glycerolphosphate, 5 mM EGTA, 1 mM
Na3VO4,
and 1 mM DTT), and 10 µl of a 75 mM
MgCl2/500 µM ATP mix were added to each tube. Components of the c-Raf kinase cascade were added as
follows: 0.4 µg of inactive GST-MAP kinase kinase 1 (MEK-1) and 1.0 µg of inactive GST-p42 MAP kinase. Samples were incubated at 30°C
for 30 min. Four microliters of this mixture were then added to a new
tube containing 46 µl kinase buffer plus 100 µM ATP and 1 µg
Elk-1 fusion protein. Samples were incubated for 30 min at 30°C.
Detection of Elk-1 phosphorylation was carried out as described above
for the MAP kinase assay.
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RESULTS |
PD-98059 inhibits PDGF-BB- and
-thrombin-, but not
serum-induced, mitogenesis.
To determine the effect of PD-98059 on mitogenesis induced by both
tyrosine kinase and G protein-coupled receptors in VSM cells, we first
examined the effect of these growth factors, as well as serum, on VSM
cell mitogenesis. In the absence of PD-98059, stimulation of the cells
by serum, PDGF-BB, and
-thrombin results in a significant increase
(6.75-, 6.08-, and 2.01-fold control, respectively) in
[3H]thymidine
incorporation (Fig. 1). The relatively
small increase in
[3H]thymidine
incorporation after
-thrombin stimulation is consistent with
previous reports in these cells (19). In subsequent experiments, cells
were preincubated with 50 µM of the MEK inhibitor PD-98059 for 1 h
before stimulation with growth factor or 10% serum; preincubation with
PD-98059 under these conditions causes significant inhibition of MAP
kinase activity (see below). After 18 h of growth factor incubation,
[3H]thymidine was
added for 6 h before TCA precipitation. In all cells except those
treated with serum, DNA synthesis was significantly decreased by
preincubation with PD-98059 (Fig. 2),
suggesting that serum, but not PDGF-BB or
-thrombin, utilizes a
pathway for mitogenesis that is independent of MEK.

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Fig. 1.
Vascular smooth muscle (VSM) cell mitogenesis induced by serum and
growth factors. Confluent, serum-starved VSM cells were stimulated with
10% serum, -thrombin (1 U/ml), or platelet-derived growth factor
(PDGF)-BB (30 ng/ml) for 24 h.
[3H]thymidine (1 µCi/well) was added for the last 6 h of incubation, and
TCA-precipitable radioactivity was assessed by liquid scintillation.
Data are expressed as means ± SE of 8-14 wells per data point.
* P < 0.05 compared with
control.
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Fig. 2.
Effect of PD-98059 on VSM cell mitogenesis. Confluent, serum-starved
VSM cells were incubated with PD-98059 (50 µM; hatched bars) for 1 h
before addition of 10% serum, -thrombin (1 U/ml), or
PDGF-BB (30 ng/ml) for an additional 24 h. Control cells (solid bars)
were incubated with same volume of DMSO solvent.
[3H]thymidine (1 µCi/well) was added for last 6 h of incubation, and TCA-precipitable
radioactivity was assessed by liquid scintillation. Data are expressed
as means ± SE of 3 wells per data point and are representative of 3 separate experiments. * P < 0.05 (+)PD-98059 compared with ( )PD-98059.
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To assess the growth response of VSM cells to varying concentrations of
PD-98059, we next performed dose-response experiments using these
growth factors with the MEK inhibitor. For clarity of comparison among
the different experiments,
[3H]thymidine
incorporation is graphed as percent change relative to cells that were
appropriately stimulated but were not preincubated with PD-98059. The
effect of incubation of the cells with 10% serum (squares in Fig.
3A) is
consistently stimulatory when compared with control cells that were not
stimulated (triangles in Fig. 3A) at
concentrations from 10 to 50 µM PD-98059. However, incubation of
these cells with PD-98059 abolished the stimulatory effect of both
PDGF-BB (circles in Fig. 3B) and
-thrombin (diamonds in Fig. 3B)
at these same concentrations. This is consistent with the data above
(Fig. 2) showing inhibition of growth by PDGF-BB or
-thrombin, but
not by 10% serum, in cells incubated with PD-98059.

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Fig. 3.
Dose-dependent inhibition of VSM cell mitogenesis with PD-98059.
Confluent, serum-starved VSM cells were incubated with PD-98059 at
concentrations indicated for 1 h before addition of 10% serum
(A) or -thrombin (1 U/ml) or
PDGF-BB (30 ng/ml) (B) for an
additional 24 h.
[3H]thymidine (1 µCi/well) was added for last 6 h of incubation, and TCA-precipitable
radioactivity was assessed by liquid scintillation. Control cells were
incubated with PD-98059 at concentrations indicated but were not
stimulated. Data are expressed as percent change compared with cells
that did not receive PD-98059 and are means ± SE of 3 wells per
data point. * P < 0.05 compared with control cells that were incubated with same
concentrations of PD-98059 but not stimulated. Experiment shown is
pooled from 3 experiments.
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To explore the dependence of activation of MEK on phase of the cell
cycle at which it is inhibited, we next examined the effect of time of
addition of the inhibitor relative to growth factor on synchronized,
serum-starved cells. As above,
[3H]thymidine is
graphed as percent change relative to cells that were appropriately
stimulated but were not preincubated with PD-98059. Serum is
stimulatory in VSM cells exposed to 50 µM PD-98059 (squares in Fig.
4A) as
compared with control cells that were not stimulated (triangles in Fig.
4A), regardless of whether the MEK
inhibitor is added at times ranging from 4 h before to 2 h after serum
addition, although this stimulation only reached statistical
significance when PD-98059 was added before serum. Mitogenesis induced
by both PDGF-BB (circles in Fig. 4B)
and
-thrombin (diamonds in Fig. 4B) is completely inhibited by
PD-98059 at these same times.

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Fig. 4.
Time of PD-98059 addition does not affect growth trend. Confluent,
serum-starved VSM cells were incubated with PD-98059 (50 µM) at
indicated time relative to 10% serum
(A) or -thrombin (1 U/ml) or
PDGF-BB (30 ng/ml) (B) addition. For
example, 2 represents an experiment in which PD-98059 was added
2 h before growth factor addition. Cells were incubated for 24 h after
growth factor addition.
[3H]thymidine (1 µCi/well) was added for last 6 h of incubation, and TCA-precipitable
radioactivity was assessed by liquid scintillation. Control cells were
incubated with PD-98059 at concentrations indicated but were not
stimulated. Data are expressed as percent change compared with control
cells that did not receive PD-98059 and are means ± SE of 3 wells
per data point. * P < 0.05 compared with control cells that were incubated with same
concentrations of PD-98059 but not stimulated. Experiment shown is
pooled from 3 experiments.
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PD-98059 decreases MAP kinase activity.
When activated by MEK, MAP kinase translocates to the nucleus to
stimulate such transcriptional activators as Elk-1 (9). Because MEK is
positioned in the phosphorylation cascade such that it activates MAP
kinase, we next examined several time courses of catalytic activity of
this downstream molecule as measured by phosphorylation of Elk-1 by
immunoprecipitated MAP kinase. In initial experiments, VSM cells were
treated with 50 µM PD-98059 for 1 h and then stimulated with either
10% serum, PDGF-BB, or
-thrombin. In all cases, the presence of the
MEK inhibitor attenuates MAP kinase activity as seen by decreased
phosphorylation of the MAP kinase-specific Elk-1 substrate (Fig.
5). To determine whether PD-98059 is
inhibitory toward MEK (as measured by MAP kinase activity) at times
that would be required for early events in the cell cycle to take
place, we examined MAP kinase activity in cells that had been incubated
with PD-98059 for various times before stimulation with PDGF-BB and
serum. MAP kinase activity was inhibited by PD-98059 when the cells
were incubated with PD-98059 for from 1 to 2 h before serum (Fig.
6A) or
PDGF (Fig. 6B) stimulation, but by 4 h, the inhibition was much less pronounced or absent. It is significant that the maximal inhibition of MAP kinase occurred when PD-98059 was
added 1 h before serum stimulation (Fig.
6A); under these conditions, serum
was maximally stimulatory (
1 in Fig.
4A), further supporting the
independence of serum-stimulated growth from MAP kinase activation.

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Fig. 5.
Mitogen-activated protein (MAP) kinase activity is decreased by
PD-98059. Confluent, serum-starved VSM cells were incubated, where
indicated with an asterisk, with PD-98059 (50 µM) for 1 h.
Subsequently, cells were either unstimulated (C) or stimulated with
10% serum (S), -thrombin (T; 1 U/ml), or PDGF-BB (P; 30 ng/ml) for
10 min and lysed. Lysate was immunoprecipitated with MAP kinase
antibody, and assessment of phosphorylation of Elk-1 by MAP kinase was
determined as described in MATERIALS AND
METHODS. Experiment shown is representative of 3 experiments.
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Fig. 6.
MAP kinase activity is inhibited by PD-98059 for up to 4 h. Confluent,
serum-starved VSM cells were incubated, where indicated with an
asterisk, with PD-98059 (50 µM) for 1, 2, or 4 h before addition of
10% serum (A) or PDGF-BB (30 ng/ml)
(B) for 10 min. Cells not exposed to
inhibitor were incubated in the same volume of DMSO. Control cells (C)
were not stimulated and were lysed at 1 h. Lysates were
immunoprecipitated with MAP kinase antibody, and assessment of
phosphorylation of Elk-1 by MAP kinase was determined as described in
MATERIALS AND METHODS.
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The activation of MAP by a variety of growth factors has been shown to
be biphasic in nature, with a rapid phase ending by 30 min and a
sustained phase beginning at ~30 min and lasting up to 4 h (5, 10,
17). We next asked whether PD-98059 is inhibitory during both phases of
activation. Serum-deprived VSM cells were incubated with PDGF-BB
concomitantly with PD-98059 for from 10 min to 24 h before lysis and
determination of MAP kinase activity. Although significant inhibition
of MAP kinase activity was seen with 10 min and 1 h of PDGF and
PD-98059 incubation, times of 4 h and later did not show consistent
inhibition by PD-98059 (Fig. 7). These data
demonstrate that PD-98059 inhibits both the early phase (10 min after
stimulation) and at least part of the sustained phase (1 h after
stimulation) of MAP kinase activation and are consistent with the data
in Fig. 6 showing that there is minimal effect of PD-98059 (at least
toward MAP kinase inhibition) by 4 h of incubation in the media.

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Fig. 7.
MAP kinase is inhibited by PD-98059 when added with growth factor at
times <4 h. Confluent, serum-starved VSM cells were incubated
concurrently with PDGF-BB (30 ng/ml) and, where indicated with an
asterisk, PD-98059 (50 µM). Cells not exposed to inhibitor were
incubated with the same volume of DMSO. After 10 min or 1, 4, 8, or 24 h, cells were lysed. Control cells (C) were not stimulated and were
lysed at 1 h. Lysates were immunoprecipitated with MAP kinase antibody,
and assessment of phosphorylation of Elk-1 by MAP kinase was determined
as described in MATERIALS AND
METHODS.
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PD-98059 inhibits hyperphosphorylation but increases catalytic
activity of Raf-1.
Although Raf lies upstream of MEK in the MAP kinase signaling cascade,
there are reports that the activity of this kinase is affected by more
distal signaling molecules such as MAP kinase (27, 28, 31) or molecules
downstream of MAP kinase (22). Raf activity had traditionally been
assessed either by examination of its phosphorylation state, based on
its migration on an SDS gel, or by determination of its catalytic
activity, but recent data from several laboratories, including our own,
have indicated that these two assays are not equivalent (1, 2, 8).
Hyperphosphorylation is indicated by the shift of the band to a higher
molecular weight as opposed to nonstimulated cells; a slower migrating
band indicates phosphorylation of Raf-1. The state of phosphorylation
of Raf-1 was examined by Western blotting of growth factor-exposed cell lysates that had been pretreated with 50 µM PD-98059. In the absence of PD-98059, the characteristic hyperphosphorylation of Raf was seen
when cells were treated with serum, PDGF-BB, or
-thrombin (Fig.
8). In the presence of PD-98059, the
mobility of Raf-1 is faster (i.e., the band migrates further down the
gel at a lower molecular weight) than without PD-98059 in the case of
serum,
-thrombin, and PDGF, indicating that the hyperphosphorylation of Raf-1 in all three cases is reduced by the MEK inhibitor.

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Fig. 8.
Hyperphosphorylation of Raf is inhibited by PD-98059. Confluent,
serum-starved VSM cells were incubated, where indicated with an
asterisk, with PD-98059 (50 µM) for 1 h. Subsequently, cells were
either unstimulated (C) or stimulated with 10% serum (S), -thrombin
(T; 1 U/ml), or PDGF-BB (P; 30 ng/ml) for 10 min, lysed, and Western
blotted with Raf-1 antibody. Arrowheads indicate control
phosphorylation state of Raf-1. Multiple control lysates were run on
same gel as indicators of mobility of basal unstimulated Raf-1.
Experiment shown is representative of 3 experiments.
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We next examined the catalytic activity of Raf-1 in VSM cells treated
with the MEK inhibitor. Raf-1 was immunoprecipitated from stimulated
cells and subjected to a cascade reaction ultimately leading to
phosphorylation of Elk-1 (see MATERIALS AND
METHODS). Elk-1 phosphorylation was assessed by
Western blotting against a phospho-Elk-1 antibody. In the absence of
PD-98059, Raf activity of cells stimulated with 10% serum,
-thrombin, or PDGF-BB was unchanged from that of control cells after
10 min of growth factor stimulation (Fig.
9A). In
the presence of PD-98059, Raf-1 activity was greatly increased over
that of control cells for 10% serum,
-thrombin, or PDGF-BB,
indicating that, despite abolition of hyperphosphorylation by PD-98059,
the catalytic activity of Raf-1 was increased by the MEK inhibitor. To
control for quantity of immunoprecipitated Raf-1 in the Raf activity
assay, we electrophoresed an aliquot of the immunoprecipitated lysate
used in the assay and performed Western blotting with Raf-1 antibody.
All of the lanes show an equivalent quantity of Raf-1 (Fig.
9B), despite significant changes in
catalytic activity (Fig. 9A).

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|
Fig. 9.
Raf catalytic activity is increased by PD-98059. Confluent,
serum-starved VSM cells were incubated, where indicated with an
asterisk, with PD-98059 (50 µM) for 1 h. Subsequently, cells were
either unstimulated (C) or stimulated with 10% serum (S), -thrombin
(T; 1 U/ml), or PDGF-BB (P; 30 ng/ml) for 10 min and lysed.
A: lysate was immunoprecipitated with
Raf-1 antibody, and assessment of phosphorylation of Elk-1 in an in
vitro cascade reaction was determined as described in
MATERIALS AND METHODS.
B: above immunoprecipitated lysate was
Western blotted with Raf-1 antibody. Experiments shown are
representative of 3 separate experiments.
|
|
 |
DISCUSSION |
The role of the MAP kinase cascade in mitogenic signaling is an area of
intense interest, since growth factors are involved in a variety of
disease processes ranging from atherosclerosis to cancer to kidney
disease. Although mitogenic signals from the tyrosine kinase-coupled
growth factors have been clearly linked, through Grb2, Sos, and Ras, to
the MAP kinase cascade, the mechanism by which the G protein-coupled
receptors activate Ras and the distal MAP kinase cascade effectors is
not known. There is, however, clear evidence that this cascade is
activated by G protein-coupled receptors such as
-thrombin (2) and
angiotensin II (2, 3).
The MEK inhibitor PD-98059 is a highly specific inhibitor of activation
of MEK-1 and MEK-2 and thus of activation of MAP kinase (1, 7, 15, 20).
PD-98059 binds to inactive forms of MEK and prevents its activation by
upstream components such as c-Raf (1). Inhibition of MEK by PD-98059 is
not due to competition for ATP or substrate (MAP kinase) binding (7,
15) and is most likely due to an allosteric effect (7).
Because activation of components of the MAP kinase cascade results from
stimulation of cells by a variety of growth factors, it has been
assumed that transmission of the signal along this pathway is essential
for the induction of mitogenesis by these growth factors. In support of
this assumption, inhibition of MEK with PD-98059 has been associated
with a decrease not only in PDGF-stimulated
[3H]thymidine
incorporation in 3T3 cells (7), but also with a block in nerve growth
factor (NGF)-induced differentiation in PC-12 cells (20). We now show
for the first time that attenuation of MEK activity has a
dose-dependent inhibitory effect on DNA synthesis in VSM cells
stimulated by both tyrosine kinase and G protein-coupled receptors as
well as by 10% serum. However, although serum retains a stimulatory
effect at all concentrations of PD-98059 tested, stimulation of these
cells by PDGF-BB and
-thrombin is significantly inhibited at these
same concentrations. PD-98059 inhibits MEK for up to 4 h; the time at
which PD-98059 is added relative to growth factor has no effect on the
overall trend of growth, consistent with data in Swiss 3T3 cells (1) and showing that it is not significant whether MEK is inhibited "early" or "late" in the cell cycle (13, 29) for it to
demonstrate the effects observed. Control cells exhibited an attenuated
growth response to PD-98059, as has been previously reported (21).
We have shown that serum does not require MEK or MAP kinase for its
mitogenic effects. That there exist pathways alternative to the
Ras/Raf/MEK/MAPK cascade leading from receptor tyrosine kinases to the
proliferative machinery has been suggested by other investigators.
Using dominant inhibitory constructs in NIH/3T3 cells, Barone and
Courtneidge (4) showed the existence of a signaling pathway from the
PDGF receptor to the early oncogenes, which is dependent on Src but
which bypasses Ras. Others have shown that dominant negative Ras does
not abolish MAP kinase activation due to NGF (32). These authors
further showed that tyrosine phosphorylation of phospholipase C-
is
not affected by the inhibitory Ras construct, an experiment which
suggests another alternative pathway of receptor tyrosine kinase
activation of mitogenic machinery. Although cellular differentiation is
often associated with cessation of growth, it can also be induced by
prolonged activation of the MAP kinase pathway in neuronal cells by NGF
(6). However, in this mode of MAP kinase activation, there is evidence
that Raf may utilize signaling pathways independent of MEK and MAP
kinase (14), consistent with our finding that Raf activity is increased (by PD-98059) despite inhibition of MAP kinase activity.
There is also evidence of MEK-dependent, but MAP kinase-independent,
activity in response to some growth factors. The finding that insulin
stimulates SOS phosphorylation through MEK (leading to dissociation of
the Grb2-SOS complex), despite complete inhibition of MAP kinase (12),
suggests that there also exist alternative signaling pathways between
MEK and MAP kinase. Conversely, there exist MEK-independent pathways
for MAP kinase activation. One such pathway, mediating the prolonged
phase of MAP kinase activation, has been demonstrated in Swiss 3T3
cells in response to PDGF and was thought to be mediated through
phosphatidylinositol 3-kinase or protein kinase C (10).
Our finding, that PD-98059 inhibits both the early and at least part of
the sustained phase (5, 10, 17) of MAP kinase activation, is consistent
with a previously published report that sustained MAP kinase activation
is linked to the mitogenic potential of MAP kinase when activated by
serum growth factors (18). Whether MAP kinase "recovers" from
inhibition by 4 h or PD-98059 is degraded in the media is not known;
the latter explanation is unlikely given that others have shown
activity of PD-98059 in culture media for up to 16 h (1).
The differences observed among the growth stimulators tested is likely
due to the fact that serum contains other cytokines and growth factors
that act on DNA synthesis through pathways independent of the MAP
kinase cascade. The precise pathway by which serum stimulates growth in
these studies, independent of MEK and MAP kinase, was not determined.
On the other hand, it has been shown that strong activators of growth,
such as epidermal growth factor in 3T3 cells, may still retain enough
residual MEK activity so as to be able to activate the MAP kinase
cascade despite MEK inhibition by PD-98059 (1). Our finding that MEK is
not inhibited after prolonged times (after 4 h) of PD-98059 incubation may be significant, since it is possible that serum could utilize this
pathway whereas the specific growth factors examined may require
earlier MEK activation (18).
The likely existence of feedback loops in the MAP kinase cascade has
already been suggested. This possibility has been strengthened recently
by the findings of Wartmann and Davis (27), who showed that Raf
hyperphosphorylation occurred after serum stimulation despite a
reduction of Raf catalytic activity. These authors later showed that
inhibition of MEK prevented Raf hyperphosphorylation while restoring
levels of membrane-bound Raf (28). Data from another laboratory were
consistent with the possibility that MAP kinase feeds back to
phosphorylate the T-669 site of the epidermal growth factor receptor
(31). In light of these findings, we examined the effect of MEK
inhibition on activation of MAP kinase cascade component activation in
VSM cells activated by a variety of growth factors. That PD-98059
decreased MAP kinase activity was expected since this molecule is the
substrate for activated MEK, but our finding that Raf activity was
increased in the case of all three growth-stimulatory conditions
despite loss of Raf hyperphosphorylation is consistent with previous
work in other cells (1, 27, 28) and extends those results to several classes of growth factor receptor. Whether this increase in Raf activity with PD-98059 is due to a feedback stimulatory effect of
poorly activated MAP kinase on Raf, or whether it is due to an
accumulation of activated Raf which cannot be phosphatased before
activation of its downstream effector, remains to be determined. Indeed, while the growth of serum-stimulated cells is not greatly inhibited by PD-98059, the MAP kinase activity is reduced by 62% (by
laser densitometry measurement of the protein band on the gel),
demonstrating that MEK is being strongly inhibited by PD-98059 and,
furthermore, that the growth seen with serum is due to activation of
other than the MAP kinase pathway.
We now show that the presence of PD-98059 alters the state of
PDGF-induced Raf-1 hyperphosphorylation in VSM cells, consistent with
data from another laboratory using D-609 to stimulate Chinese hamster
ovary cells overexpressing Raf-1 (28). In the absence of
PD-98059, Raf is hyperphosphorylated after serum, PDGF-BB, and
-thrombin stimulation, whereas the magnitude of Raf-1
hyperphosphorylation is diminished after incubating the cells with
PD-98059. Active MEK has been shown to interact with Raf-1 in NIH/3T3
cells and to cause the mobility shift (hyperphosphorylation) of a
COOH-terminal fragment of Raf-1 (8). In that study, cells possessing
active MAP kinase had hyperphosphorylated Raf-1, whereas those with the MAP kinase activity inactivated by PD-98059 had Raf-1 that was not
hyperphosphorylated.
The data in this study are also consistent with our previous report
that angiotensin II causes catalytic activation of Raf-1 despite the
fact that hyperphosphorylation was not observed (2). It is possible
that hyperphosphorylation occurs before activation (28) and that at the
point when our cells were lysed in that study (10 min after
stimulation), angiotensin II-induced hyperphosphorylation had already
occurred. However, because we did observe concurrent hyperphosphorylation and catalytic activation when the cells were stimulated with thrombin and 10% serum (2), it is further possible that different growth stimulants display different kinetics toward these discrete events.
Employing an in vitro cascade reaction with MEK and MAP kinase to
measure specific Raf-1 catalytic activity, we observed an increase in
Raf-1 activity in stimulated cells in the presence of PD-98059. This
could be due to accumulation of Raf activity, since its substrate MEK
is made unavailable. However, this scenario seems unlikely due to the
fact that MEK inhibition also affects the phosphorylation state of
Raf-1. Active MEK has been shown to be capable of causing
hyperphosphorylation of Raf-1, and inactivated MEK abolished the
hyperphosphorylated state of Raf-1, indicating that active MEK is
responsible for keeping Raf-1 in the hyperphosphorylated and inactive
state (8). We believe that these data support the theory of a
negative-feedback model of Raf activity (26, 28, 31). PD-98059 has been
shown to inhibit the hyperphosphorylation of Sos (15), and it has been
suggested that this would prevent the inactivation of Ras, thereby
allowing Raf-1 to remain active (1). The resulting Raf-1 activity due
to the inhibition of MAP kinase activity may also be a continued
negative feedback of the entire pathway, as active Raf-1 may continue
the signal of "no growth" to signaling molecules further
upstream.
 |
ACKNOWLEDGEMENTS |
This work was supported by the Research Service of the United
States Department of Veterans Affairs and by a gift from Dialysis Clinics, Inc.
 |
FOOTNOTES |
Address for reprint requests: R. H. Weiss, Div. of Nephrology, TB 136, Dept. of Internal Medicine, Univ. of California, Davis, CA 95616.
Received 9 December 1997; accepted in final form 25 February 1998.
 |
REFERENCES |
1.
Alessi, D. R.,
A. Cuenda,
P. Cohen,
D. T. Dudley,
and
A. R. Saltiel.
PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo.
J. Biol. Chem.
270:
27489-27494,
1995[Abstract/Free Full Text].
2.
Apostolidis, A.,
and
R. H. Weiss.
Divergence in the G protein-coupled receptor mitogenic signaling cascade at the level of Raf kinase.
Cell. Signal.
6:
439-445,
1997.
3.
Arai, H.,
and
J. A. Escobedo.
Angiotensin II type 1 receptor signals through Raf-1 by a protein kinase C-dependent, Ras-independent mechanism.
Mol. Pharmacol.
50:
522-528,
1996[Abstract].
4.
Barone, M. V.,
and
S. A. Courtneidge.
Myc but not Fos rescue of PDGF signalling block caused by kinase-inactive Src.
Nature
378:
509-512,
1995[Medline].
5.
Cook, S. J.,
J. Beltman,
K. A. Cadwallader,
M. McMahon,
and
F. McCormick.
Regulation of mitogen-activated protein kinase phosphatase-1 expression by extracellular signal-related kinase-dependent and Ca2+-dependent signal pathways in Rat-1 cells.
J. Biol. Chem.
272:
13309-13319,
1997[Abstract/Free Full Text].
6.
Cowley, S.,
H. Paterson,
P. Kemp,
and
C. J. Marshall.
Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells.
Cell
77:
841-852,
1994[Medline].
7.
Dudley, D. T.,
L. Pang,
S. J. Decker,
A. J. Bridges,
and
A. R. Saltiel.
A synthetic inhibitor of the mitogen-activated protein kinase cascade.
Proc. Natl. Acad. Sci. USA
92:
7686-7689,
1995[Abstract].
8.
Ferrier, A. F.,
M. Lee,
W. B. Anderson,
G. Benvenuto,
D. K. Morrison,
D. R. Lowy,
and
J. E. DeClue.
Sequential modification of serines 621 and 624 in the Raf-1 carboxyl terminus produces alterations in its electrophoretic mobility.
J. Biol. Chem.
272:
2136-2142,
1997[Abstract/Free Full Text].
9.
Gille, H.,
M. Kortenjann,
O. Thomae,
C. Moomaw,
C. Slaughter,
M. H. Cobb,
and
P. E. Shaw.
ERK phosphorylation potentiates Elk-1-mediated ternary complex formation and transactivation.
EMBO J.
14:
951-962,
1995[Abstract].
10.
Grammer, T. C.,
and
J. Blenis.
Evidence for MEK-independent pathways regulating the prolonged activation of the ERK-MAP kinases.
Oncogene
14:
1635-1642,
1997[Medline].
11.
Hallberg, B.,
S. I. Rayter,
and
J. Downward.
Interaction of Ras and Raf in intact mammalian cells upon extracellular stimulation.
J. Biol. Chem.
269:
3913-3916,
1994[Abstract/Free Full Text].
12.
Holt, K. H.,
B. G. Kasson,
and
J. E. Pessin.
Insulin stimulation of a MEK-dependent but ERK-independent SOS protein kinase.
Mol. Cell. Biol.
16:
577-583,
1996[Abstract].
13.
Huang, C. L.,
and
H. E. Ives.
Growth inhibition by protein kinase C late in mitogenesis.
Nature
29:
849-850,
1987.
14.
Kuo, W. L.,
M. Abe,
J. Rhee,
E. M. Eves,
S. A. McCarthy,
M. Yan,
D. J. Templeton,
M. McMahon,
and
M. R. Rosner.
Raf, but not MEK or ERK, is sufficient for differentiation of hippocampal neuronal cells.
Mol. Cell. Biol.
16:
1458-1470,
1996[Abstract].
15.
Lazar, D. F.,
R. J. Wiese,
M. J. Brady,
C. C. Mastick,
S. B. Waters,
K. Yamauchi,
J. E. Pessin,
P. Cuatrecasas,
and
A. R. Saltiel.
Mitogen-activated protein kinase kinase inhibition does not block the stimulation of glucose utilization by insulin.
J. Biol. Chem.
270:
20801-20807,
1995[Abstract/Free Full Text].
16.
Marshall, C. J.
MAP kinase kinase kinase, MAP kinase kinase and MAP kinase.
Curr. Opin. Genet. Dev.
4:
82-89,
1994[Medline].
17.
Meloche, S.,
G. Pages,
and
J. Pouyssegur.
Functional expression and growth factor activation of an epitope-tagged p44 mitogen-activated protein kinase, p44mapk.
Mol. Biol. Cell
3:
63-71,
1992[Abstract].
18.
Meloche, S.,
K. Seuwen,
G. Pages,
and
J. Pouyssegur.
Biphasic and synergistic activation of p44mapk (ERK1) by growth factors: correlation between late phase activation and mitogenicity.
Mol. Endocrinol.
6:
845-854,
1992[Abstract].
19.
Molloy, C. J.,
J. E. Pawlowski,
D. S. Taylor,
C. E. Turner,
H. Weber,
M. Peluso,
and
S. M. Seiler.
Thrombin receptor activation elicits rapid tyrosine phosphorylation and stimulation of the Raf-1/MAP kinase pathway preceding delayed mitogenesis in cultured rat aortic smooth muscle cells.
J. Clin. Invest.
5:
1173-1183,
1996.
20.
Pang, L.,
T. Sawada,
S. J. Decker,
and
A. R. Saltiel.
Inhibition of MAP kinase kinase blocks the differentiation of PC-12 cells induced by nerve growth factor.
J. Biol. Chem.
270:
13585-13588,
1995[Abstract/Free Full Text].
21.
Pumiglia, K. M.,
and
S. J. Decker.
Cell cycle arrest mediated by the MEK/mitogen-activated protein kinase pathway.
Proc. Natl. Acad. Sci. USA
94:
448-452,
1997[Abstract/Free Full Text].
22.
Shibuya, E. K.,
J. Morris,
U. R. Rapp,
and
J. V. Ruderman.
Activation of the Xenopus oocyte mitogen-activated protein kinase pathway by Mos is independent of Raf.
Cell Growth Differ.
7:
235-241,
1996[Abstract].
23.
Stokoe, D.,
S. G. MacDonald,
K. Cadwallader,
M. Symons,
and
J. F. Hancock.
Activation of raf as a result of recruitment to the plasma membrane.
Science
264:
1463-1467,
1994[Medline].
24.
Takishima, K.,
I. Griswold-Prenner,
T. Ingebritsen,
and
M. R. Rosner.
Epidermal growth factor (EGF) receptor T669 peptide kinase from 3T3-L1 cells is an EGF-stimulated "MAP" kinase.
Proc. Natl. Acad. Sci. USA
88:
2520-2524,
1991[Abstract].
25.
Treisman, R.
Ternary complex factors: growth factor regulated transcriptional activators.
Curr. Opin. Genet. Dev.
4:
96-101,
1994[Medline].
26.
Ueki, K.,
S. Matsuda,
K. Tobe,
Y. Gotoh,
H. Tamemoto,
M. Yachi,
Y. Akanuma,
Y. Yazaki,
E. Nishida,
and
T. Kadowaki.
Feedback regulation of mitogen-activated protein kinase kinase kinase activity of c-Raf-1 by insulin and phorbol ester stimulation.
J. Biol. Chem.
269:
15756-15761,
1994[Abstract/Free Full Text].
27.
Wartmann, M.,
and
R. J. Davis.
The native structure of the activated Raf protein kinase is a membrane-bound multi-subunit complex.
J. Biol. Chem.
269:
6695-6701,
1994[Abstract/Free Full Text].
28.
Wartmann, M.,
P. Hofer,
P. Turowski,
A. R. Saltiel,
and
N. E. Hynes.
Negative modulation of membrane localization of the Raf-1 protein kinase by hyperphosphorylation.
J. Biol. Chem.
272:
3915-3923,
1997[Abstract/Free Full Text].
29.
Weiss, R. H.,
C. L. Huang,
and
H. E. Ives.
Sphingosine reverses growth inhibition caused by activation of protein kinase C in vascular smooth muscle cells.
J. Cell. Physiol.
149:
307-312,
1991[Medline].
30.
Weiss, R. H.,
and
R. Nuccitelli.
Inhibition of tyrosine phosphorylation prevents thrombin-induced mitogenesis, but not intracellular free calcium release, in vascular smooth muscle cells.
J. Biol. Chem.
267:
5608-5613,
1992[Abstract/Free Full Text].
31.
Wood, K. W.,
H. Qi,
G. D'Arcangelo,
R. C. Armstrong,
T. M. Roberts,
and
S. Halegoua.
The cytoplasmic raf oncogene induces a neuronal phenotype in PC12 cells: a potential role for cellular raf kinases in neuronal growth factor signal transduction.
Proc. Natl. Acad. Sci. USA
90:
5016-5020,
1993[Abstract].
32.
Wood, K. W.,
C. Sarnecki,
T. M. Roberts,
and
J. Blenis.
Ras mediates nerve growth factor receptor modulation of three signal-transducing protein kinases: MAP kinase, Raf-1, and RSK.
Cell
68:
1041-1050,
1992[Medline].
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