p38 Mitogen-activated Protein Kinase Activation Is Required
for Fibroblast Growth Factor-2-stimulated Cell Proliferation but
Not Differentiation*
Pamela
Maher
From the Department of Cell Biology, Scripps Research Institute,
La Jolla, California 92037
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ABSTRACT |
Basic fibroblast growth factor (FGF-2) is a
member of a family of polypeptides that have roles in a wide range of
biological processes. To determine why different cell types show
distinct responses to treatment with FGF-2, the array of FGF receptors present on the surface of a cell which differentiates in response to
FGF-2 (PC12 cells) was compared with that present on the surface of a
cell that proliferates in response to FGF-2 (Swiss 3T3 fibroblasts). Both cell types express exclusively FGFR1, suggesting that there are
cell type-specific FGFR1 signaling pathways. Since mitogen-activated protein kinases function as mediators of cellular responses to a
variety of stimuli, the roles of these proteins in FGF-mediated responses were examined. FGF-2 activates extracellular signal-regulated kinases with similar kinetics in both fibroblasts and PC12 cells, and a
specific inhibitor of extracellular signal-regulated kinase activation
blocks differentiation but has little effect on proliferation. In
contrast, while p38 mitogen-activated protein kinase is activated weakly and transiently in PC12 cells treated with FGF-2, a much stronger and sustained activation of this kinase is seen in
FGF-2-treated fibroblasts. Furthermore, specific inhibitors of this
kinase block proliferation but have no effect on differentiation. This
effect on proliferation is specific for FGF-2 since the same
concentrations of inhibitors have little or no effect on proliferation
induced by serum.
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INTRODUCTION |
Basic and acidic fibroblast growth factors (FGF-2 and
FGF-1)1 are the prototypes
for a large family of multifunctional growth factors, which have been
identified in a wide variety of tissues (for reviews see Refs. 1-4).
Although FGFs were first characterized on the basis of their ability to
stimulate cell proliferation, it is now known that FGFs can modulate a
number of other cellular functions, including promotion or inhibition
of differentiation, survival, protease synthesis and secretion, and
chemotaxis. However, the pathways that mediate these cell type-specific
effects of FGFs are not well understood.
The FGFs interact with two classes of FGF receptors: high affinity
receptors, which bind FGFs with picomolar affinity and are thought to
mediate the cellular responses to FGF; and low affinity receptors,
which bind FGFs with nanomolar affinity and are characterized by the
presence of heparan sulfate moieties. The family of high affinity FGF
receptors contains four closely related members (for reviews, see Refs.
4-7), which all possess intrinsic tyrosine kinase activity. In
addition, a number of alternately spliced mRNAs corresponding to
multiple isoforms of FGF receptor-1 (FGFR1), FGF receptor-2 (FGFR2),
and FGF receptor-3 (FGFR3) (8-11) have been identified. These isoforms
include receptors lacking Ig-like domain I (two Ig-like domain isoform)
and/or alternative sequences for the second half of Ig-like domain III
(receptor isoforms a, b, and c). In a recent study, the interaction of
nine members of the FGF family with the four FGF receptors was examined using the induction of mitogenesis as an end point (12). This study
demonstrated that each of the FGF receptors is capable of binding and
responding to more than one type of FGF. Furthermore, both the two
Ig-like domain isoform and the three Ig-like domain isoform (with
Ig-like domain 1) of FGF receptors 1 and 3 showed similar responses to
all of the FGFs tested, indicating that the first Ig-like domain is
unlikely to play a role in regulating intracellular signaling by the
receptors. In addition, cells transfected with either FGFR1b or FGFR1c
showed a strong mitogenic response to treatment with FGF-2, suggesting
that the third Ig-like domain is also unlikely to play a role in
regulating signaling through FGFR1.
The pathways involved in FGF receptor signal transduction have not yet
been fully elucidated. Since the FGF receptors are tyrosine kinases, an
early event in the cellular response to receptor activation is
substrate binding and tyrosine phosphorylation. Indeed,
autophosphorylation of tyrosines 653 and 654 in the tyrosine kinase
domain of FGFR1 is required for activation of the kinase activity of
the receptor, as well as subsequent substrate phosphorylation and
stimulation of proliferation or differentiation (13). However, many of
the proteins known to be phosphorylated on tyrosine by other receptor
tyrosine kinases do not appear to be phosphorylated in response to the
treatment of cells with FGFs (for examples, see Refs. 7 and 13-15)
suggesting that FGF receptors have a distinct set of substrates.
Furthermore, porcine aortic endothelial cells transfected with
cDNAs encoding either the PDGF receptor or FGFR1 show very
different patterns of protein tyrosine phosphorylation in response to
PDGF or FGF-2, respectively, although both growth factors generate a
mitogenic response (16). Members of the Raf-Ras-MAP kinase pathway have
been implicated in FGFR signaling (17-21), based on the use of
gain-of-function or dominant-negative constructs. However, since this
pathway is activated in cells that show different responses to FGF
treatment (22-26), it is unclear whether it plays a role in mediating
the cell type-specific effects of FGFs. Although it was suggested (27,
28) that the MAP kinase pathway can mediate distinct cellular responses
depending on whether the activation of the pathway is transient or
sustained, the kinetics of kinase activation with respect to distinct
responses to FGF have not been examined.
Two cell lines that show distinct responses to FGF treatment
(proliferation versus differentiation) have been used to
examine the role of receptor-specific versus cell
type-specific signaling pathways in mediating the two different
outcomes. The results presented here demonstrate a previously
unrecognized role for the p38 MAP kinase pathway in FGF-stimulated cell proliferation.
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EXPERIMENTAL PROCEDURES |
Materials--
PD98059 was obtained from Biomol and solubilized
in Me2SO. SB202190, SB203580, and Ro318220 were obtained
from Calbiochem and solubilized in Me2SO. U0126 was
obtained from Promega and solubilized in Me2SO. Recombinant
human FGF-2 was prepared as described (29). PDGF was purchased from R & D Systems.
Cell Culture--
Swiss 3T3 cells were obtained from the
American Type Culture Collection (Rockville, MD) and grown in
Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Inc.)
supplemented with 10% calf serum (Hyclone) and antibiotics. To examine
the effects of FGF-2 or other growth factors on the Swiss 3T3 cells,
they were grown to confluence and then made quiescent by washing with
serum-free DMEM, followed by incubation for 2 days in DMEM with 0.5%
calf serum. PC12 cells were obtained from D. Schubert (Salk Institute) and maintained in DMEM supplemented with 10% fetal calf serum (Hyclone), 5% horse serum (Hyclone), and antibiotics. To examine the
effects of FGF-2 on the PC12 cells, the culture medium was removed and
replaced by the chemically defined N2 medium (Life Technologies, Inc.).
T-47D cells were obtained from the American Type Culture Collection and
grown in DMEM supplemented with 10% fetal calf serum and antibiotics.
Stimulation with Growth Factors--
Quiescent Swiss 3T3 cells
or PC12 cells in N2 medium were treated with the growth factors and
other agents as described in the figure legends and, after the
indicated time periods, the cells were solubilized in SDS-sample buffer
containing 0.1 mM Na3VO4 and 1 mM PMSF, boiled for 5 min, and either analyzed immediately or stored frozen at
70 °C.
SDS-PAGE and Immunoblotting--
Proteins were separated on 10%
SDS-polyacrylamide gels and transferred to nitrocellulose. Transfers
were blocked for 2 h at room temperature with 5% nonfat milk in
TBS, 0.1% Tween 20 and then incubated overnight at 4 °C in the
primary antibody diluted in 5% bovine serum albumin in TBS, 0.05%
Tween 20. The primary antibodies used were: phosphospecific
cAMP-responsive element-binding protein (CREB) antibody (1/1000),
phosphospecific ATF-2 antibody (1/1000), phosphospecific p38 MAPK
antibody (9211; 1/1000), phosphospecific MAPK antibody (9101, 1/1000)
and ATF-2 antibody (1/500) from New England Biolabs; p38 MAP kinase
antibody (sc-728, 1/1000) and CREB-1 antibody (sc-186; 1/1000) from
Santa Cruz Biotechnology; pan ERK antibody (1/5000) from Transduction
Laboratories. The transfers were rinsed with TBS, 0.05% Tween 20 and
incubated for 1 h at room temperature in horseradish
peroxidase-conjugated goat anti-rabbit or goat anti-mouse (Bio-Rad)
diluted 1/5000 in 5% nonfat milk in TBS, 0.1% Tween 20. The
immunoblots were developed with the Super Signal reagent (Pierce).
Measurement of the Stimulation of DNA Synthesis--
Cells were
grown in 96-well dishes (1 × 104 cells/well) for 2 days in DMEM containing 10% calf serum, at which time they were confluent. After washing with serum-free DMEM, they were incubated for
another 2 days in DMEM with 0.5% calf serum. The rate of DNA synthesis
was measured 24 h after the addition of growth factors and other
agents to the cells by the addition of 0.2 µCi/well [methyl-3H]thymidine (6.7 Ci/mol, ICN),
followed by incubation for 5 h. The cultures were then processed
for scintillation counting as described (30).
Receptor Cross-linking and
Immunoprecipitation--
125I-FGF-2 was prepared as
described (31), using lactoperoxidase. The free iodine was removed by
passage over a heparin-Sepharose column. The specific activity was
4-6 × 105 cpm/ng. For cross-linking, cells were
incubated with 106 cpm/ml 125I-FGF-2 in DMEM
containing 20 mM HEPES and 0.2% gelatin for 2 h at
4 °C with shaking. Cells were rinsed twice with PBS and incubated with 0.15 mM disuccinimidyl suberate (Pierce) in PBS for 15 min at room temperature with shaking. The cross-linking was stopped by
the addition of 200 mM ethanolamine and the cells
solubilized in Triton X-100 buffer (1% Triton X-100, 50 mM
HEPES, pH 7.5, 50 mM NaCl, 5 mM EDTA, 1 mM Na3VO4, 1 mM PMSF).
FGFR were immunoprecipitated overnight at 4 °C with rabbit
antibodies against the C-terminal domains of the four different FGFR
(Santa Cruz Biotechnology; sc-121, sc-122, sc-123, and sc-124). The
immunoprecipitates were collected on protein A-Sepharose; washed twice
with 0.1% Triton X-100 in 20 mM HEPES, pH 7.5, 150 mM NaCl; washed once with PBS; and solubilized in
SDS-sample buffer. The samples were separated on 7.5%
SDS-polyacrylamide gels, and the gels were dried overnight and autoradiographed.
MAPKAP Kinase-2 Assay--
Cells in 60-mm dishes were
solubilized in 500 µl of 1% Triton X-100 in 50 mM Tris,
pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM
Na3VO4, 0.1% 2-mercaptoethanol, 5 mM sodium pyrophosphate, 10 mM
-glycerophosphate, 50 mM NaF, 1 mM PMSF, 1 µg/ml aprotinin, and 1 µg/ml leupeptin. MAPKAP kinase-2 in the
supernatants was collected with sheep anti-rabbit MAPKAP kinase-2 (2 µg/immunoprecipitate; Upstate Biotechnology, Inc.) preabsorbed to
protein G-Sepharose. The immunoprecipitates were washed once with
solubilization buffer containing 500 mM NaCl, once with
solubilization buffer, and once with kinase assay buffer and
resuspended in 30 µl of kinase assay buffer (20 mM MOPS,
pH 7.2, 25 mM
-glycerophosphate, 5 mM EGTA,
1 mM Na3VO4, and 1 mM
dithiothreitol) containing 25 mM MgCl2, 150 µM ATP, 10 µCi/assay [
-32P]ATP (ICN),
and 62.5 µM MAPKAP kinase-2 substrate peptide (Upstate Biotechnology). Following incubation at 30 °C for 30 min, the protein G-Sepharose beads were pelleted and the supernatants
transferred to P-81 phosphocellulose paper disks. The disks were washed
three times with 0.85% phosphoric acid, once with H2O, and
counted in a liquid scintillation counter.
Stress-activated Protein Kinase (SAPK)/c-Jun N-terminal Kinase
(JNK) Assay--
Cells were treated with FGF-2 (25 ng/ml) for 10 min
and solubilized with 500 µl of 1% Triton X-100 in 20 mM
Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4,
2.5 mM sodium pyrophosphate, 1 mM
-glycerophosphate, 1 mM PMSF, and 1 µg/ml leupeptin.
Preliminary experiments using an antibody to the activated form of
SAPK/JNK indicated that this time point represented the peak of the
very low level of SAPK/JNK activation induced by FGF-2 in the Swiss 3T3
cells. GST-c-Jun (1-89) bound to glutathione-Sepharose was used to
"pull-down" total SAPK/JNKs from the FGF-treated cell extracts, and
kinase activity was assayed as described (32) in the absence or
presence of 5 µM SB202190 or SB203580 except that c-Jun
phosphorylation was detected with an antibody specific for the
phosphorylated form of the protein (New England Biolabs; 9810; 1/1000)
in conjunction with SDS-PAGE and immunoblotting.
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RESULTS |
To identify the signaling pathways involved in different responses
to FGF-2, two cell lines were chosen for study which show distinct
responses to FGF-2 treatment. When treated with FGF-2, Swiss 3T3
fibroblasts undergo a mitogenic response that can be monitored by
[3H]thymidine incorporation. In contrast, PC12 cells
differentiate and this can be monitored visually by the extension of
neurites. They do not show a mitogenic response as defined by
[3H]thymidine incorporation. To determine if these
distinct cellular responses are mediated at the level of the FGF
receptor, the receptors present at the surface of each cell type and
capable of binding FGF-2 were characterized using
125I-FGF-2 and chemical cross-linking, followed by
immunoprecipitation with antibodies specific to each of the four
different FGF receptors. Fig.
1A shows that this approach is
capable of detecting all four FGF receptors when they are present on a
single cell type, T-47D. Fig. 1B shows the results obtained
with Swiss 3T3 cells and PC12 cells and demonstrates that both cell
lines express only cell surface FGFR1. The two FGFR1 bands seen in the
Swiss 3T3 cells correspond to the two and three Ig-like domain isoforms
of the receptor (data not shown), whereas the single FGFR1 band seen in
the PC12 cells corresponds to the three Ig-like domain isoform of the
receptor. These results suggest that the distinct cellular responses
exhibited by the two cell lines following treatment with FGF-2 are
mediated by different intracellular signaling pathways which are
activated by the same cell surface FGF receptor.

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Fig. 1.
Identification of cell surface FGF
receptors. 125I-FGF-2 was bound to the cells at
4 °C. The ligand was cross-linked with disuccinimidyl suberate and
the ligand receptor complexes were solubilized with Triton X-100 buffer
and immunoprecipitated with antibodies specific to the C-terminal
domains of the four FGF receptors and protein A-Sepharose. The
immunoprecipitates were analyzed by SDS-PAGE and autoradiography.
R1, 2, 3, and 4,
immunoprecipitates with antibodies specific to FGF receptors 1, 2, 3, and 4. A, T47-D cells; B, Swiss 3T3 cells and
PC12 cells. Molecular masses (in kDa) are indicated at right
in B. Data from a single experiment are shown. Similar
results were obtained in three independent experiments.
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As an initial approach to identifying the signaling pathways
responsible for the distinct cellular responses to FGF-2 treatment, the
roles of different members of the MAP kinase family were examined. In
mammalian cells at least three distinct members of the MAP kinase
family are expressed: ERKs (also known as MAPKs), SAPK (also known as
JNK), and p38 MAPK. ERKs are activated by growth factors and are
involved in both cell proliferation and differentiation, whereas
SAPK/JNKs and p38 MAPK are thought to be activated primarily in
response to proinflammatory cytokines and environmental stress. They
are also implicated in inflammatory responses, cell cycle arrest, DNA
repair, and programmed cell death (for reviews, see Refs. 33-35).
However, a number of recent studies have demonstrated a role for p38
MAPK in other cellular processes, including cardiac hypertrophy (36,
37), ischemic preconditioning (38), hemopoietic proliferation (39), T
cell proliferation (40), and neuronal differentiation (41).
As mentioned above, ERKs are implicated in the promotion of both cell
proliferation and differentiation. It has been suggested that the
specific cellular response to ERK activation is dependent upon whether
that activation is transient or sustained (27). Many of the
observations supporting this hypothesis derive from studies with PC12
cells where epidermal growth factor, which failed to promote
differentiation, only activated ERKs for a brief time period whereas
growth factors such as nerve growth factor and FGF-2, which induce
differentiation, promoted long term activation of ERKs (28). To
determine if similar differences in the time course of ERK activation
are seen between Swiss 3T3 cells and PC12 cells treated with FGF-2, ERK
activation was monitored at various time points up to 8 h after
stimulation with FGF-2 using an antibody specific for the dual
phosphorylated, and therefore active, form of the kinase in conjunction
with immunoblotting. As shown in Fig. 2,
sustained ERK activation following addition of FGF-2 was seen in both
cell lines. Similar results were obtained with an
immunoprecipitation/in vitro kinase activity assay and with
an in vitro kinase activity assay on partially purified ERKs (42) (data not shown).

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Fig. 2.
Time course of ERK activation in response to
FGF-2. Swiss 3T3 cells or PC12 cells were untreated
(ct) or treated with 25 ng/ml FGF-2 for 10 min to 8 h,
cell extracts were prepared, and equal amounts of protein were analyzed
by SDS-PAGE and immunoblotting with antibodies specific for the
activated forms of ERKs 1 and 2 (phospho-Thr202/Tyr204) and total ERKs. Data
from a single experiment are shown. Similar results were obtained in
three independent experiments.
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To determine if ERK activity was necessary for either FGF-2 stimulated
differentiation or proliferation, the specific MEK1 inhibitor, PD98059,
was used (43). Treatment of PC12 cells with PD98059 brought about a
significant reduction in FGF-2-induced ERK activation, which decreased
to control levels at 100 µM PD98059 (Fig.
3A). This same concentration
of PD98059 inhibited PC12 cell differentiation by ~90% (Fig.
3B), suggesting that ERK activation is required for the
induction of differentiation by FGF-2. In contrast, the same
concentrations of PD98059 had only a slight effect on ERK activation in
Swiss 3T3 cells (Fig. 3A). Recently, a second MEK1 inhibitor
(U0126), which has a 100-fold higher affinity for MEK1 than does
PD98059, was described (44). In contrast to PD98059, U0126 effectively
blocked ERK activation in Swiss 3T3 cells treated with FGF-2, PDGF, or
10% serum (Fig. 4). Although 10 µM U0126 reduced PDGF-stimulated Swiss 3T3 proliferation
by ~80%, this same concentration of inhibitor had little or no
effect on cell proliferation induced by either FGF-2 or serum (Fig.
3C). These results suggest that ERK activation is not
required for the induction of fibroblast proliferation by FGF-2.

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Fig. 3.
Effect of PD98059 on ERK activation and the
cellular response to FGF-2. A, cells were untreated
(ct) or treated with 25 ng/ml FGF-2 for 10 min in the
absence or presence of the indicated concentrations of PD98059, cell
extracts were prepared, and equal amounts of protein were analyzed by
SDS-PAGE and immunoblotting with antibodies specific for activated ERKs
and total ERKs. Data from a single experiment are shown. Similar
results were obtained in three independent experiments. B,
PC12 cells in N2 medium were treated with 25 ng/ml FGF-2 in the absence
or presence of the indicated concentrations of PD98059 for 48 h at
which time the cells were scored for the presence of neurites.
Ct, control. For each treatment, 100 cells in each of three
separate fields were counted. Cells were scored positive if one or more
neurites >1 cell body diameter in length were observed. The results
presented are the average ± S.D. of three independent
experiments. C, quiescent Swiss 3T3 fibroblasts were treated
for 24 h with 5 ng/ml FGF-2, 5 ng/ml PDGF, or 10% serum in the
absence or presence of the indicated concentrations of U0126 and then
labeled with [3H]thymidine and radioactive label
incorporation analyzed by liquid scintillation counting. The results
are presented as the percentage of the cell proliferation seen with the
growth factor alone and are the average of three independent
experiments with each treatment run in triplicate.
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Fig. 4.
Effect of U0126 on ERK activation induced by
different growth factors. Quiescent Swiss 3T3 cells were untreated
(ct) or treated with 25 ng/ml FGF-2, 25 ng/ml PDGF, or 10%
serum for 5 min in the absence or presence of 10 µM
U0126, cell extracts were prepared, and equal amounts of protein were
analyzed by SDS-PAGE and immunoblotting with antibodies specific for
activated ERKs and total ERKs. Data from a single experiment are shown.
Similar results were obtained in two independent experiments.
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Since p38 MAPK was found to be activated by FGF-2 in several different
cell types (45, 46), its activation by FGF-2 in both the PC12 cells and
Swiss 3T3 cells was tested using an antibody specific for the dual
phosphorylated, and thereby active, form of the kinase. In contrast to
the results with ERKs, quite different time courses of p38 MAPK
activation were seen in PC12 cells as compared with Swiss 3T3 cells
(Fig. 5). Whereas in the FGF-2-treated PC12 cells, only a brief induction of p38 MAPK activation relative to
control levels was observed, in Swiss 3T3 cells, FGF-2 induced a much
more prolonged activation of the same enzyme (Fig. 5). As noted above,
p38 MAPK activation has been associated primarily with the cellular
response to stress rather than proliferation. To determine how the
activation of p38 MAPK by FGF-2 compared with that of stress-inducing
agents, cells were treated with two classical stress-inducing agents
and extracts probed with the phosphospecific p38 MAPK antibody. As
shown in Fig. 6A, the level of
p38 MAPK activation by FGF-2 in Swiss 3T3 cells was very similar to
that seen with either anisomycin or sorbitol, indicating that, in Swiss
3T3 cells, FGF-2 is a strong activator of this MAPK family member.

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Fig. 5.
Time course of p38 MAPK activation in
response to treatment with FGF-2. A, cells were
untreated (ct) or treated with 25 ng/ml FGF-2 for 2.5 min to
8 h, cell extracts were prepared and equal amounts of protein were
analyzed by SDS-PAGE and immunoblotting with antibodies specific for
the activated form (phospho-Thr180/Tyr182) of
p38 MAPK and total p38 MAPK. Data from a single experiment are shown.
Similar results were obtained in four independent experiments.
B, the relative intensities of the anti-phospho-p38 MAPK
bands shown in A were determined by densitometry and plotted
relative to untreated cells (time 0) in order to demonstrate more
clearly the differences in the time courses of activation between the
two cell lines.
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Fig. 6.
A, comparison of p38 MAPK activation by
FGF-2 and stress-inducing agents. Cells were untreated (ct)
or treated with 25 ng/ml FGF-2 for 5 min (FGF), 10 µg/ml
anisomycin for 30 min (anis), or 300 mM sorbitol
for 30 min (sorb), cell extracts were prepared, and equal
amounts of protein analyzed by SDS-PAGE and immunoblotting with
antibodies specific for activated p38 MAPK and total p38 MAPK. Data
from a single experiment are shown. Similar results were obtained in
two independent experiments. B, effect of p38 MAPK
inhibitors on SAPK/JNK activity. Cells were treated with 25 ng/ml FGF-2
for 10 min and then solubilized in Triton X-100 buffer. SAPK/JNKs were
isolated from the cell extract with GST-c-Jun bound to
glutathione-Sepharose and the precipitates assayed for kinase activity
in the absence (ct) or presence of 5 µM
SB202190 (202), 5 µM SB203580
(203), or vehicle (Me2SO (DMSO)).
Phosphorylated c-Jun was detected by SDS-PAGE electrophoresis and
immunoblotting with an antibody specific to the phosphorylated form of
the protein. Data from a single experiment are shown. Similar results
were obtained in two independent experiments.
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To further compare the induction of p38 MAPK activity by FGF-2 in Swiss
3T3 cells with that in PC12 cells, the activation of MAPKAP kinase-2 by
FGF-2 was examined. MAPKAP kinase-2 is a specific and direct substrate
of p38 MAP kinase in vitro and in vivo;
therefore, its activation can serve as a measure of p38 MAP kinase
activity (46, 47). Furthermore, by using MAPKAP kinase-2 activity as a
measure of p38 MAP kinase activity, it is possible to assess the
effects of several specific, reversible p38 MAP kinase inhibitors on
the activity of p38 MAP kinase in FGF-2-treated cells. As shown in Fig.
7, FGF-2 induced a 20-30-fold activation
of MAPKAP kinase-2 activity in Swiss 3T3 cells, whereas the same
concentration of FGF-2 only brought about a 2-3-fold increase in
MAPKAP kinase-2 activity in PC12 cells. These results are consistent
with the data on p38 MAP kinase activation obtained using the antibody
to the phosphorylated form of the enzyme (Fig. 5). Fig. 7 also shows
that low concentrations of the specific p38 MAP kinase inhibitors,
SB202190 and SB203580, reduce MAPKAP kinase-2 activity to near control
levels in FGF-2-treated cells, indicating that these inhibitors can be
used to assess the role of p38 MAP kinase in FGF-induced proliferation
and differentiation. These inhibitors are highly specific for p38
kinase both in vitro and in vivo and, even at
concentrations as high as 100 µM, were found to have no
effect on the activity of many other protein kinases, including other
MAP kinase family members (35, 39, 47, 48). Although inhibition of
SAPK/JNK activity by SB203580 at concentrations above 1 µM was reported recently (32, 49), JNK2 isoforms were
much more susceptible to inhibition than JNK1 isoforms. No inhibition
of SAPK/JNK activity was observed in the Swiss 3T3 cells treated with
FGF-2 in the presence of either SB202190 or SB203580 (Fig.
6B). In addition, neither inhibitor had any effect on ERK
activation (data not shown).

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Fig. 7.
Analysis of p38 MAPK activity using an assay
for the activation of MAPKAP kinase-2, a specific and direct substrate
of p38 MAPK. Cells were untreated (Ct) or treated with
25 ng/ml FGF-2 for 5 min in the absence or presence of the indicated
concentrations of the specific p38 MAPK inhibitors SB202190 or SB203580
and then solubilized in Triton X-100 buffer. MAPKAP kinase-2 was
immunoprecipitated from the extracts and kinase activity assayed using
a peptide substrate and liquid scintillation counting. The results
represent the average of four independent experiments.
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To assess the role of p38 MAPK in the cellular response to FGF-2, the
effects of the two p38 MAPK inhibitors described above on both Swiss
3T3 proliferation and PC12 cell differentiation were tested. Both
inhibitors were very effective at blocking Swiss 3T3 cell
proliferation, as measured by [3H]thymidine
incorporation, with maximal inhibition occurring at 5 µM
(Fig. 8A). These results
correlate well with the results for the inhibition of MAPKAP kinase-2
activation by the two compounds (Fig. 7). In contrast, 5 µM SB202190 or SB203580 had very little effect on cell
proliferation induced by the addition of 10% serum to quiescent cells
(Fig. 8A). Neither p38 MAPK inhibitor had any effect on PC12
differentiation induced by FGF-2 (Fig. 8B).

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Fig. 8.
Effects of the p38 MAPK inhibitors on the
cellular responses to FGF-2. A, quiescent Swiss 3T3
fibroblasts were treated for 24 h with 5 ng/ml FGF-2 or 10% serum
in the absence or presence of the indicated concentrations of SB202190
or SB203580 and then labeled with [3H]thymidine and
radioactive label incorporation analyzed by liquid scintillation
counting. The results are presented as the percentage of the cell
proliferation seen with the growth factor alone and are the average of
five independent experiments with each treatment run in triplicate.
, FGF/SB202190; , FGF/SB203580; , serum/SB202190; ,
serum/SB203580. B, PC12 cells were treated with 25 ng/ml
FGF-2 in the absence or presence of the indicated concentrations of
SB202190 or SB203580 for 24 h, at which time the cells were scored
for the presence of neurites. Ct, control. For each
treatment, 100 cells in each of three separate fields were counted.
Cells were scored positive if one or more neurites >1 cell body
diameter in length were observed. The results presented are the
average ± S.D. of three independent experiments.
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The requirement for p38 MAP kinase activity for FGF-2-stimulated cell
proliferation suggests that one or more downstream substrates of this
kinase are essential for the proliferative response. As mentioned
above, one of these substrates is MAPKAP kinase-2, which is activated
by FGF-2 and whose activation is blocked by the p38 MAP kinase
inhibitors. MAPKAP kinase-2 phosphorylates HSP27, which, in turn, can
modulate actin filament dynamics (46). However, how this effect on
microfilaments could contribute to the proliferative response remains
to be determined. p38 MAPK also activates a number of other proteins,
including several kinases and transcription factors (45, 50-56). To
determine whether any of these latter substrates were activated by
FGF-2 in a p38 MAPK-dependent manner in the Swiss 3T3
cells, a combination of inhibitors and phosphospecific antibodies was
used. As shown in Fig. 9, FGF-2
stimulated the phosphorylation of CREB, ATF-1, and ATF-2 at sites
essential for the activation of transcriptional activity. However, the
phosphorylation of none of these transcription factors was inhibited by
the p38 MAPK inhibitors (Fig. 9) alone. The phosphorylation of both
CREB and ATF-1 was, however, inhibited by a combination of the p38 MAP
kinase inhibitors and the MEK1 inhibitor, PD98059 (Fig. 9). Similar
results were reported for nerve growth factor activation of CREB in
PC12 cells (55, 57) and FGF-2 activation of CREB in SK-N-MC cells (55).
In contrast, the phosphorylation of ATF-2 was only slightly inhibited
by the combination of inhibitors (Fig. 9). However, since the p38 MAPK
inhibitors alone have no effect on the activation of CREB, ATF-1 or
ATF-2, but do block FGF-stimulated cell proliferation, it is unlikely
that the activation of any of these transcription factors is required
for the proliferative response to FGF-2.

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[in this window]
[in a new window]
|
Fig. 9.
Analysis of p38 MAPK substrate
activation. Quiescent 3T3 cells were untreated or treated with 25 ng/ml FGF-2 for 5 min in the absence or presence of 5 µM
SB202190, 10 µM SB203580, 100 µM PD98059,
or a combination of the drugs as indicated in the figure. Cell extracts
were prepared and equal amounts of protein analyzed by SDS-PAGE and
immunoblotting with antibodies specific for phosphorylated CREB and
total CREB or phosphorylated ATF-2 and total ATF-2. Data from a single
experiment are shown. Similar results were obtained in three
independent experiments.
|
|
The finding that a combination of inhibitors was required to inhibit
CREB phosphorylation suggested that CREB phosphorylation in response to
FGF-2 treatment of Swiss 3T3 cells may result from the activation of
mitogen and stress-activated protein kinase-1 (MSK-1), a novel protein
kinase recently shown to be activated by both ERKs and p38 kinase in a
variety of different cell lines (55). To test this idea, Ro318220, a
protein kinase inhibitor that can inhibit the activity of MSK-1 (55),
was used. Treatment of cells with Ro318220 blocked the phosphorylation
of CREB by FGF-2 (Fig. 10), as reported
previously (55), but had little or no effect on the phosphorylation of
ATF-2 (Fig. 10). These data indicate that, while FGF-induced
phosphorylation of CREB is likely to be mediated by MSK-1, the
phosphorylation of ATF-2 must be mediated by other, as yet undefined,
kinases.

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 10.
Effect of Ro318220 on p38 MAPK substrate
activation. Quiescent Swiss 3T3 cells were untreated
(ct) or treated with 25 ng/ml FGF-2 for 5 min in the absence
or presence of 1, 5, or 10 µM Ro318220. Cell extracts
were prepared and equal amounts of protein analyzed by SDS-PAGE and
immunoblotting with antibodies specific for phosphorylated CREB and
total CREB or phosphorylated ATF-2 and total ATF-2. Data from a single
experiment are shown. Similar results were obtained in three
independent experiments.
|
|
 |
DISCUSSION |
The mechanisms whereby a single growth factor can induce very
distinct responses in different types of cells is still not clear.
While it is possible that growth factors which interact with multiple
members of a receptor family can have distinct effects on cells
depending on the specific receptor family member expressed by a given
type of cell, it is likely that there are other mechanisms regulating
cell type-specific responses. The results presented here demonstrate
that, in the case of FGF-2, cell type-specific signaling pathways can
mediate the distinct cellular responses to this growth factor. Although
Swiss 3T3 cells and PC12 cells show very different responses to
treatment with FGF-2, both cell lines express only FGFR1 on their cell
surfaces. An earlier study found that a different clone of PC12 cells
expressed FGFR3 and FGFR4 along with FGFR1 but that FGFR1 was the only
receptor required for FGF-2 to induce differentiation (58), consistent
with the data presented here. The failure to detect FGFR3 and FGFR4 in the PC12 cells used in the present study is not due to an inability to
detect these receptors by the method used to analyze receptor expression (Fig. 1A). Thus, the absence of FGFR3 and FGFR4
from the PC12 cells used in the present study is probably due to
variations among the different clones of this cell line.
Since members of the MAPK family have been implicated in both cell
proliferation and differentiation, it was likely that these kinases
were also involved in FGF-2 signaling. Indeed, a number of studies
suggest that MAP kinases play a role in one or more cellular responses
to FGF-2 (20, 24, 25, 59-61). Furthermore, one hypothesis as to how
growth factors induce neuronal differentiation is based on the idea
that prolonged ERK activation is required (28). However, the kinetics
of FGF-2-stimulated ERK activation in PC12 cells and Swiss 3T3 cells
are indistinguishable (Fig. 2), suggesting that other signaling
pathways are necessary for the distinct cellular responses to this
growth factor. Nevertheless, ERK activity does appear to be required
for FGF-2-induced differentiation since an inhibitor of ERK activation
blocks neurite outgrowth by 90%. This result is consistent with
previous studies on FGF-2 signaling in PC12 cells (60, 62), but not
with a recent study on FGF-2 signaling in a conditionally immortalized
rat hippocampal cell line (63). However, this difference may be a
reflection of the fact that these two cell lines are models for
different types of neurons, which, therefore, may show distinct, cell
type-specific responses to FGF-2 treatment.
In contrast, a role for ERK activity in FGF-2-induced cell
proliferation could not be demonstrated. Surprisingly, another member
of the MAP kinase family, p38 MAPK, was found to be specifically required for the proliferative response to FGF-2. p38 MAPK was strongly
activated by FGF-2 in Swiss 3T3 cells for an extended time period,
whereas only a weak activation for a much shorter time period was
observed in the PC12 cells. Furthermore, two specific inhibitors of p38
MAPK blocked FGF-2-induced cell proliferation at concentrations that
had no effect on either serum-stimulated proliferation or FGF-induced
neurite outgrowth in PC12 cells. Although several previous studies
reported activation of p38 MAPK following treatment with FGF-2 (45,
46), this activation was not associated with a specific cellular
response. Thus, the studies described here are the first demonstration
that this member of the MAPK family plays an important role in
FGF-induced cell proliferation.
p38 MAPK was first identified several years ago in
lipopolysaccharide-stimulated mouse macrophages (64) and was shown to be the target of a series of anti-inflammatory pyridinyl imidazole compounds (48). Four different isoforms (
,
,
,
) of this kinase, which can be distinguished on the basis of inhibitor and substrate specificity, have now been characterized (65, 66). Only p38
MAPK
and
are inhibited by the pyridinyl imidazole SB203580 and
MAPKAP kinase 2 is only phosphorylated by p38 MAPK
(65, 66). Thus,
while the specific isoforms(s) of p38 MAPK which is present in the
Swiss 3T3 cells and required for FGF-induced cell proliferation has not
been characterized, the data indicate that it must be p38MAPK
. While
it is possible that the failure to detect an effect of the p38 MAPK
inhibitors on FGF-stimulated neurite outgrowth in PC12 cells could be
due to the presence of p38 MAPK isoforms which are activated by FGF-2
but not affected by these compounds, the finding that the kinetics and
degree of p38 MAPK activation differ substantially between the Swiss
3T3 cells and the PC12 cells suggest that this is unlikely. As
mentioned earlier, p38 MAPK activity has generally been associated with the cellular response to stress. However, a few reports have suggested positive roles for this kinase in promoting cell hypertrophy (36, 37),
ischemic preconditioning (38), and hemopoietic (39) and T cell (40)
proliferation or differentiation (41). The results presented here
provide the first demonstration that this kinase can also play a
critical role in FGF-stimulated cell proliferation.
Although the signaling pathways downstream of p38 MAPK that are
required for FGF-stimulated cell proliferation have not been characterized, the data presented here indicate that several known substrates and signaling pathways downstream of p38 MAPK are not required for this response. These include ATF-1, ATF-2, and CREB. In
contrast, the data suggest that the kinase MAPKAP kinase-2 could play
an important role in the proliferative response to FGF-2 but not PC12
cell differentiation. Several other substrates of p38 MAPK including
the transcription factors MEF2C (53) and CHOP (56) and the kinases
MAPKAP kinase-3 (50), Mnk1 (51, 52), and PRAK (54) have been recently
identified. Whether any of these are required for FGF-induced cell
proliferation remains to be determined. However, Mnk1 can phosphorylate
eukaryotic initiation factor-4E (eIF4E) (52). eIF4E phosphorylation is
induced by treatment of quiescent cells with serum (67) and increased
levels of eIF4E phosphorylation have been directly correlated with
enhanced rates of translation in a variety of cells (for review, see
Ref. 68). Thus, activation of Mnk1 via p38 MAPK may be required for FGF-stimulated cell proliferation. Although activation of PRAK was not
detected in FGF-2-treated HeLa cells (54), HeLa cells were reported to
lack high affinity FGF receptors (69).
In summary, the results presented here demonstrate a critical role for
p38 MAPK in FGF-induced cell proliferation while at the same time
ruling out a role in FGF-stimulated differentiation. Although much
remains to be determined about the activation of this pathway and its
specific consequences within the cell, these data provide convincing
evidence for cell type-specific signaling pathways activated by the
same FGF receptor.
 |
ACKNOWLEDGEMENT |
I thank Dr. David Schubert for helpful
discussions and critical reading of the manuscript.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant GM54604.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.
 |
ABBREVIATIONS |
The abbreviations used are:
FGF, fibroblast
growth factor;
FGFR, fibroblast growth factor receptor;
MAP, mitogen-activated protein;
MAPK, mitogen-activated protein kinase;
SAPK, stress-activated protein kinase;
ERK, extracellular
signal-regulated kinase;
ATF-2, activating transcription factor 2;
CREB, cAMP-responsive element-binding protein;
PAGE, polyacrylamide gel
electrophoresis, PBS, phosphate-buffered saline;
TBS, Tris-buffered
saline;
PMSF, phenylmethylsulfonyl fluoride;
DMEM, Dulbecco's modified
minimal essential medium;
PDGF, platelet-derived growth factor;
GST, glutathione S-transferase;
MAPKAP kinase-2, mitogen-activated protein kinase activated protein kinase-2;
MOPS, 4-morpholinepropanesulfonic acid;
eIF4E, eukaryotic initiation
factor-4E.
 |
REFERENCES |
-
Baird, A.,
and Bohlen, P.
(1990)
in
Peptide Growth Factors and their Receptors (Sporn, M. B., and Roberts, A. B., eds), pp. 369-418, Springer Verlag, Berlin
-
Burgess, W. H.,
and Maciag, T.
(1989)
Annu. Rev. Biochem.
58,
575-606[CrossRef][Medline]
[Order article via Infotrieve]
-
Wagner, J. A.
(1991)
Curr. Top. Microbiol. Immunol.
165,
95-118[Medline]
[Order article via Infotrieve]
-
Fernig, D. G.,
and Gallagher, J. T.
(1994)
Prog. Growth Factor Res.
5,
353-377[Medline]
[Order article via Infotrieve]
-
Partanen, J.,
Vainikka, S.,
and Alitalo, K.
(1993)
Phil. Trans. R. Soc. Lond. B
340,
297-303[Medline]
[Order article via Infotrieve]
-
Givol, D.,
and Yayon, A.
(1992)
FASEB J.
6,
3362-3369[Abstract/Free Full Text]
-
Johnson, D. E.,
and Williams, L. T.
(1993)
Adv. Cancer Res.
60,
1-41[Medline]
[Order article via Infotrieve]
-
Eisemann, A.,
Ahn, J. A.,
Graziani, G.,
Tronick, S. R.,
and Ron, D.
(1991)
Oncogene
6,
1195-1202[Medline]
[Order article via Infotrieve]
-
Hou, J.,
Kan, M.,
McKeehan, K.,
McBride, G.,
Adams, P.,
and McKeehan, W. L.
(1991)
Science
251,
665-668[Medline]
[Order article via Infotrieve]
-
Johnson, D. E.,
Chen, H.,
Werner, S.,
and Williams, L. T.
(1991)
Mol. Cell. Biol.
11,
4627-4634[Medline]
[Order article via Infotrieve]
-
Chellaiah, A. T.,
McEwen, D. G.,
Werner, S.,
Xu, J.,
and Ornitz, D. M.
(1994)
J. Biol. Chem.
269,
11620-11627[Abstract/Free Full Text]
-
Ornitz, D. M.,
Xu, J.,
Colvin, J. S.,
McEwen, D. G.,
MacArthur, C. A.,
Coulier, F.,
Gao, G.,
and Goldfarb, M.
(1996)
J. Biol. Chem.
271,
15292-15297[Abstract/Free Full Text]
-
Mohammadi, M.,
Dikic, I.,
Sorokin, A.,
Burgess, W. H.,
Jaye, M.,
and Schlessinger, J.
(1996)
Mol. Cell. Biol.
16,
977-989[Abstract]
-
Cantley, L. C.,
Auger, K. R.,
Carpenter, C.,
Duckworth, B.,
Graziani, A.,
Kapeller, R.,
and Soltoff, S.
(1991)
Cell
64,
281-302[Medline]
[Order article via Infotrieve]
-
Schlessinger, J.,
and Ullrich, A.
(1992)
Neuron
9,
383-391[Medline]
[Order article via Infotrieve]
-
Wennstrom, S.,
Landgren, E.,
Blume-Jensen, P.,
and Claesson-Welsh, L.
(1992)
J. Biol. Chem.
267,
13749-13756[Abstract/Free Full Text]
-
Amaya, E.,
Musci, T. J.,
and Kirscher, M. W.
(1991)
Cell
66,
257-270[Medline]
[Order article via Infotrieve]
-
MacNicol, A. M.,
Muslin, A. J.,
and Williams, L. T.
(1993)
Cell
66,
571-583
-
Tang, T. L.,
Freeman, R. M.,
O'Reilly, A. M.,
Neel, B. G.,
and Sokol, S. Y.
(1995)
Cell
80,
473-483[Medline]
[Order article via Infotrieve]
-
Umbhause, M.,
Marshall, C. J.,
Mason, C. S.,
and Smith, J. C.
(1995)
Nature
376,
58-62[CrossRef][Medline]
[Order article via Infotrieve]
-
Whitman, M.,
and Melton, D. A.
(1992)
Nature
357,
252-254[CrossRef][Medline]
[Order article via Infotrieve]
-
Kremer, N. E.,
D'Arcangelo, G.,
Thomas, S. M.,
DeMarco, M.,
Brugge, J. S.,
and Halegoua, S.
(1991)
J. Cell Biol.
115,
809-819[Abstract]
-
Spivak-Kroizman, T.,
Mohammadi, M.,
Hu, P.,
Jaye, M.,
Schlessinger, J.,
and Lax, I.
(1994)
J. Biol. Chem.
269,
14419-14423[Abstract/Free Full Text]
-
Shaoul, E.,
Reich-Slotky, R.,
Berman, B.,
and Ron, D.
(1995)
Oncogene
10,
1553-1561[Medline]
[Order article via Infotrieve]
-
Wang, J.-K.,
Gao, G.,
and Goldfarb, M.
(1994)
Mol. Cell. Biol.
14,
181-188[Abstract]
-
Vainikka, S.,
Joukov, V.,
Weenstrom, S.,
Bergman, M.,
Pelicci, P. G.,
and Alitalo, K.
(1994)
J. Biol. Chem.
269,
18320-18326[Abstract/Free Full Text]
-
Cowley, S.,
Paterson, H.,
Kemp, P.,
and Marshall, C. J.
(1994)
Cell
77,
841-852[Medline]
[Order article via Infotrieve]
-
Traverse, S.,
Seedorf, K.,
Paterson, H.,
Marshall, C. J.,
Cohen, P.,
and Ullrich, A.
(1994)
Curr. Biol.
4,
694-701[Medline]
[Order article via Infotrieve]
-
Lappi, D. A.,
Ying, W.,
Barthelemy, I.,
Martineau, D.,
Prieto, I.,
Benatti, L.,
Soria, M.,
and Baird, A.
(1994)
J. Biol. Chem.
269,
12552-12558[Abstract/Free Full Text]
-
Pasquale, E. B.,
Maher, P. A.,
and Singer, S. J.
(1988)
J. Cell. Physiol.
137,
146-156[Medline]
[Order article via Infotrieve]
-
Schubert, D.,
Ling, N.,
and Baird, A.
(1987)
J. Cell Biol.
104,
635-643[Abstract]
-
Clerk, Z.,
and Sugden, P.
(1998)
FEBS Lett.
426,
93-96[CrossRef][Medline]
[Order article via Infotrieve]
-
Robinson, M.,
and Cobb, M.
(1997)
Curr. Opin. Cell Biol.
9,
180-186[CrossRef][Medline]
[Order article via Infotrieve]
-
Kyriakis, J. M.,
and Avruch, J.
(1996)
Bioessays
18,
567-577[Medline]
[Order article via Infotrieve]
-
Cohen, P.
(1997)
Trends Cell Biol.
7,
353-361[CrossRef]
-
Wang, Y.,
Huang, S.,
Sah, V.,
Ross, J.,
Brown, J.,
Han, J.,
and Chien, K.
(1998)
J. Biol. Chem.
273,
2161-2168[Abstract/Free Full Text]
-
Zechner, D.,
Thuerauf, D.,
Hanford, D.,
McDonough, P.,
and Glembotski, C.
(1997)
J. Cell Biol.
139,
115-127[Abstract/Free Full Text]
-
Nagarkatti, D. S.,
and Sha'afi, R. I.
(1998)
J. Mol. Cell. Cardiol.
30,
1651-1664[CrossRef][Medline]
[Order article via Infotrieve]
-
Foltz, I. N.,
Lee, J. C.,
Young, P. R.,
and Schrader, J. W.
(1997)
J. Biol. Chem.
272,
3296-3301[Abstract/Free Full Text]
-
Crawley, J. B.,
Rawlinson, L.,
Lali, F. V.,
Page, T. H.,
Saklatvala, J.,
and Foxwell, B. M. J.
(1997)
J. Biol. Chem.
272,
15023-15027[Abstract/Free Full Text]
-
Morooka, T.,
and Nishida, E.
(1998)
J. Biol. Chem.
273,
24285-24288[Abstract/Free Full Text]
-
Ahn, N. G.,
Weiel, J. E.,
Chan, C. P.,
and Krebs, E. G.
(1990)
J. Biol. Chem.
265,
11487-11494[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]
-
Favata, M. F.,
Horiuchi, K. Y.,
Manos, E. J.,
Daulerio, A. J.,
Stradley, D. A.,
Feeser, W. S.,
Van Dyke, D. E.,
Pitts, W. J.,
Earl, R. A.,
Hobbs, F.,
Copeland, R. A.,
Magolda, R. L.,
Scherle, P. A.,
and Trzaskos, J. M.
(1998)
J. Biol. Chem.
273,
18623-18632[Abstract/Free Full Text]
-
Tan, Y.,
Rouse, J.,
Zhang, A.,
Cariati, S.,
Cohen, P.,
and Comb, M.
(1996)
EMBO J.
15,
4629-4624[Abstract]
-
Guay, J.,
Lambert, H.,
Gingras-Breton, G.,
Lavoie, J.,
Huot, J.,
and Landry, J.
(1997)
J. Cell Sci.
110,
357-368[Abstract/Free Full Text]
-
Cuenda, A.,
Rouse, J.,
Doza, Y.,
Meier, R.,
Cohen, P.,
Gallagher, T.,
Young, P.,
and Lee, J.
(1995)
FEBS Lett.
364,
229-233[CrossRef][Medline]
[Order article via Infotrieve]
-
Lee, J.,
Laydon, J.,
McDonnell, P.,
Gallagher, T.,
Kumar, S.,
Gree, D.,
McNulty, D.,
Blumenthal, M.,
Heys, J.,
Landvatter, S.,
Strickler, J.,
McLaughlin, M.,
Siemens, I.,
Fisher, S.,
Livi, G.,
White, J.,
Adams, J.,
and Young, P.
(1994)
Nature
372,
739-746[CrossRef][Medline]
[Order article via Infotrieve]
-
Whitmarsh, A. J.,
Yang, S.-H.,
Su, M. S.-S.,
Sharrocks, A. D.,
and Davis, R. J.
(1997)
Mol. Cell. Biol.
17,
2360-2371[Abstract]
-
McLaughlin, M.,
Kumar, S.,
McDonnell, P. C.,
Van Horn, S.,
Lee, J. C.,
Livi, G. P.,
and Young, P. R.
(1996)
J. Biol. Chem.
271,
8488-8492[Abstract/Free Full Text]
-
Fukunaga, R.,
and Hunter, T.
(1997)
EMBO J.
16,
1921-1933[Abstract/Free Full Text]
-
Waskiewicz, A.,
Flynn, A.,
Proud, C.,
and Cooper, J.
(1997)
EMBO J.
16,
1909-1920[Abstract/Free Full Text]
-
Han, J.,
Jiang, Y.,
Li, A.,
Kravchenko, V.,
and Ulevitch, R.
(1997)
Nature
386,
296-299[CrossRef][Medline]
[Order article via Infotrieve]
-
New, L.,
Jiang, Y.,
Zhao, M.,
Liu, K.,
Zhu, W.,
Flood, L.,
Kata, Y.,
Graham, C. N. P.,
and Han, J.
(1998)
EMBO J.
17,
3372-3384[Abstract/Free Full Text]
-
Deak, M.,
Clifton, A. D.,
Lucocq, J. M.,
and Alessi, D. R.
(1998)
EMBO J.
17,
4426-4441[Abstract/Free Full Text]
-
Wang, X.,
and Ron, D.
(1996)
Science
272,
1347-1349[Abstract]
-
Xing, J.,
Kornhauser, J.,
Xia, Z.,
Thiele, E.,
and Greenberg, M.
(1998)
Mol. Cell. Biol.
18,
1946-1953[Abstract/Free Full Text]
-
Lin, Y. -Z.,
Xu, J.,
Ornitz, D. M.,
Halegoua, S.,
and Hayman, M.
(1996)
J. Neurosci.
16,
4579-4587[Abstract/Free Full Text]
-
Weyman, C.,
and Wolfman, A.
(1998)
Endocrinology
139,
1794-1800[Abstract/Free Full Text]
-
Lin, H.,
Xu, J.,
Ischenko, I.,
Ornitz, D.,
Halegoua, S.,
and Hayman, M.
(1998)
Mol. Cell. Biol.
18,
3762-3770[Abstract/Free Full Text]
-
Kouhara, H.,
Hadari, Y. R.,
Spivak-Kroizman, T.,
Schilling, J.,
Bar-Sagi, D.,
Lax, I.,
and Schlessinger, J.
(1997)
Cell
89,
693-702[Medline]
[Order article via Infotrieve]
-
Hadar, Y.,
Kouhara, H.,
Lax, I.,
and Schlessinger, J.
(1998)
Mol. Cell. Biol.
18,
3966-3973[Abstract/Free Full Text]
-
Kuo, W.,
Abe, M.,
Rhee, J.,
Eves, E.,
McCarthy, S.,
Yan, M.,
Templeton, D.,
McMahon, M.,
and Rosner, M.
(1996)
Mol. Cell. Biol.
16,
1458-1470[Abstract]
-
Han, J.,
Lee, J.-D.,
Bibbs, L.,
and Ulevitch, R. J.
(1994)
Science
265,
808-811[Medline]
[Order article via Infotrieve]
-
Wang, X.,
Diener, K.,
Manthey, C.,
Wang, S.,
Rosenzweig, B.,
Bray, J.,
Delaney, J.,
Cole, C.,
Chan-Hui, P.,
Mantlo, N.,
Lichenstein, H.,
Zukowski, M.,
and Yao, Z.
(1997)
J. Biol. Chem.
272,
23668-23674[Abstract/Free Full Text]
-
Enslen, H.,
Raingeaud, J.,
and Davis, R.
(1998)
J. Biol. Chem.
273,
1741-1748[Abstract/Free Full Text]
-
Morley, S. J.,
and McKendrick, L.
(1997)
J. Biol. Chem.
272,
17887-17893[Abstract/Free Full Text]
-
Morley, S. J.
(1996)
in
Protein Phosphorylation in Cell Growth Regulation (Clemens, M. J., ed), pp. 197-224, Harwood Academic Publishers, Amsterdam
-
Wiedlocha, A.,
Paines, P.,
Madshus, I.,
Sandvig, K.,
and Olsnes, S.
(1994)
Cell
76,
1039-1051[Medline]
[Order article via Infotrieve]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.