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INTRODUCTION |
The cellular responses to a huge variety of external stimuli are
mediated by sophisticated arrays of phosphorylation cascades. The
extracellular signal-regulated kinase mitogen-activated protein kinase
(ERK1 MAPK) pathway was one
of the first phosphorylation cascades to be well characterized
following mitogenic stimulation by peptide growth factors (1).
Specifically, the steps following the engagement of specific growth
factor receptor at the plasma membrane include the activation of the
small G-protein Ras, with Ras·GTP then directly interacting with and
regulating the activation of the Ser/Thr kinase, Raf. Raf in turn
phosphorylates the dual specificity kinase MEK (MAPK kinase), which
then phosphorylates and activates the ERK MAPKs. This signaling cascade
of Ras 
Raf MEK ERK MAPKs has been implicated in the
regulation of cellular differentiation, transformation, and
proliferation through activation of intracellular substrates including
transcription factors, such as Elk-1, c-Jun, and ATF2, and other
protein kinases, including p90RSK (2).
Other subfamilies of Ser/Thr kinases, collectively called
stress-activated MAPKs, have been characterized following their preferential activation in response to extracellular stress stimuli such as osmotic shock, UV light, oxidative stress, low pH, or heat
shock (3). These stress-activated protein kinases, which include the
c-Jun NH2-terminal kinase MAPKs (JNK MAPKs) and p38 MAPKs,
act within phosphorylation cascades that show overall similarity in
organization but are distinct from the ERK MAPK cascade (4). Thus the
stress-activated protein kinases are regulated by distinct dual
specificity kinases that are in turn regulated by specific upstream
kinases. These kinase pathways are implicated in signaling apoptosis,
cell differentiation, and transformation (4).
Although specific MAPKs function exclusively within their respective
pathways, it has been recently shown that a single stimulus can
activate two or more MAPK cascades to varying degrees (5-7). The
regulation of a cellular response may therefore be the result of
several signaling cascades working in concert. It is now clear that the
ERK MAPKs can be activated by various stress stimuli including heat
shock, hydrogen peroxide, arsenite, and osmotic shock (8-11). This
dispels the earlier model of ERK MAPK activation exclusively by
mitogenic stimuli (1).
It still remains to be clarified whether all forms of cellular stress
and mitogenic stimuli activate identical signaling events to activate
the ERK MAPKs. The increasing complexity of these signaling events
revealed through recent research suggests that this is not the case.
Although a number of studies have demonstrated that the classical
pathway of Ras Raf MEK mediates ERK MAPK activation in response
to oxidative stress and arsenite (10-12), the ERK MAPK activation
following exposure to pervanadate was not mediated by this series of
events (13). This has led to the suggestion that there may be
MEK-independent routes to ERK MAPK activation when cells are exposed to
different forms of stress (13).
In the current study, we have investigated heat shock-induced
activation of the MAPK cascades in the IL3-dependent,
murine pro-B cell line, BaF3. Surprisingly, we have now found that heat shock stimulated the phosphorylation and activation of the ERK MAPKs.
Furthermore, the "stress-activated" JNK MAPKs but not the p38 MAPKs
were phosphorylated and activated. The robust activation of ERK MAPK
was unexpected. We focused on characterizing the delayed kinetics of
ERK MAPK activation following heat shock. This suggested a novel
stress-activated signaling pathway to ERK MAPKs which, in addition, was
more potent than that elicited following the exposure to the cytokine
IL3. We have demonstrated that heat shock activates ERK MAPKs via a
pathway that involves MEK but is independent of Ras and Raf activation.
We present evidence that suggests that heat shock activated the ERK
MAPKs, not through a sequence of upstream "on" signals but instead
by deactivating a serine/threonine phosphatase-dependent
"off" signal. The resulting activation of ERK MAPKs appears to play
a role in maintaining cell viability following the heat shock insult.
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EXPERIMENTAL PROCEDURES |
Materials--
The monoclonal antibody Ras R02120 was obtained
from Transduction Laboratories, and the monoclonal antibody
anti-phosphotyrosine 4G10 was obtained from Upstate Biotechnology, Inc.
Polyclonal antibodies that recognized ERK1 (C-16) and ERK2 (C-14) were
from Santa Cruz Biotechnology. RPMI 1640 medium, heat-inactivated fetal calf serum, penicillin/streptomycin, glutamine, and phosphate-buffered saline were purchased from Life Technologies, Inc. PD98059,
bisindolylmaleimide, and murine recombinant IL3 were obtained from
Calbiochem, whereas suramin was from ICN Biomedicals Inc. Antibodies
that recognize the total (phosphorylated and non-phosphorylated) forms
of ERK1 (C-16, sc-93), ERK2 (C-14, sc-154), JNK1 (C-17, sc-474), p38
MAPK ((C-20, sc-535), Raf-1, A-Raf, and B-Raf were from Santa Cruz Biotechnology. Phospho-specific p44/42 ERK MAPK
(Thr202/Tyr204, number 9101S), phospho-specific
p38 MAPK (Thr180/Tyr182, number 9211S),
phospho-specific MEK1/2 (Ser217/Ser221, number
9121S), and total MEK (number 9122) were from New England Biolabs. [
32P]ATP was from PerkinElmer Life
Sciences. The Lumi-Light Plus chemiluminescent substrate was from Roche
Molecular Biochemicals. Molecular mass markers and Hyperfilm MP were
from Amersham Pharmacia Biotech. Recombinant Elk-(307-428) and
c-Jun-(1-135) were expressed as glutathione S-transferase
(GST) fusion proteins and purified by glutathione-Sepharose
chromatography for use as substrates for ERK MAPK and JNK MAPK,
respectively. All other reagents were from Sigma.
Cell Culture and Treatment--
All cells were grown at 37 °C
in a controlled atmosphere of 5% CO2, 95% air and 99%
relative humidity. The IL3-dependent BaF3 cell line and
BaF3-derived N6F-20 cell lines were cultured in RPMI 1640 medium
supplemented with 10% (v/v) heat-inactivated fetal calf serum, 10%
(v/v) medium conditioned by WEHI 3BD
cells (as an IL3
source), 2 mM glutamine, 0.01 mM
-mercaptoethanol, and 1000 units/ml penicillin/streptomycin (6,
14).
The conditioned medium containing IL3 was withdrawn for 3 h before
treatment. Cells (2 × 107 cells in 2 ml) were exposed
to the different treatments. For heat shock treatment, BaF3 cells were
sealed in screw cap flasks containing an atmosphere of 5%
CO2, 95% air. These flasks were then immersed completely
in a water bath with a measured temperature of 42 °C. By using this
protocol, the medium within the flask reached 42 °C within 5 min of
immersion. As a control, cells were aliquoted and starved in the same
manner but were immersed in a water bath with a measured temperature of
37 °C.
Some studies required pretreatment of the cells prior to the
experiment. In all cases equal number of cells were treated in an
identical manner either in the presence of an inhibitor or absence of
inhibitors as a control. Isopropyl--thiogalactopyranoside (IPTG) was
added to a final concentration of 5 mM to induce
RasAsn-17 expression in the N6F-20 BaF3 cell line 16 h
prior to and including the 3-h starvation period (15). Suramin, made
fresh daily, was added to a final concentration of 300 µM
during the final hour of the IL3 starvation period, and PD98059 was
added to a final concentration of 30 µM during the final
hour of the starvation period. All subsequent incubations then
proceeded in the presence of these inhibitors.
The stability of suramin during incubation at 42 °C was also
assessed. For these studies, suramin was preincubated at 42 °C in
starvation medium at a final concentration of 300 µM. As
a control, suramin was preincubated at 37 °C in starvation medium. After 2 h, the media was re-equilibrated to 37 °C and then used in further studies. Briefly, starved cells were removed from their starvation medium by centrifugation and then resuspended in
suramin-containing medium for 1 h. To test the efficacy of
suramin, cells were then exposed to 20 ng/ml IL3 (10 min).
Preparation of Cell Lysates--
Following treatment, cells were
washed with cold phosphate-buffered saline and then lysed in ice-cold
Buffer A (20 mM HEPES, pH 7.7, 20 mM
-glycerophosphate, 2.5 mM MgCl2, 0.1 mM EDTA, 100 mM NaCl supplemented with 0.05%
(v/v) Triton X-100, 500 µM dithiothreitol, 100 µM Na3VO4, 20 µg/ml leupeptin,
100 µg/ml phenylmethanesulfonyl fluoride). Lysates were centrifuged
(10,000 × g, 4 °C, 10 min), and the supernatants
were retained for further analysis.
Characterization of ERK MAPK and JNK MAPK Activities--
ERK
MAPK activities were assayed by pulldown kinase assays using
GST-Elk-(307-428) substrate (16). Briefly, cell lysate (100 µg) was
incubated with the GST fusion protein (20 µg) for 2 h at
4 °C. The complex was captured on 20 µl of glutathione-Sepharose (1:1 (v/v) slurry in Buffer A) and, after extensively washing in
ice-cold Buffer A, was incubated with reaction buffer (20 mM HEPES, pH 7.6, 20 mM MgCl2, 20 mM -glycerophosphate supplemented with 500 µM dithiothreitol, 100 µM
Na3VO4, 20 µM ATP) supplemented with [32P]ATP (1 µCi per assay) for 25 min at
30 °C. The phosphorylated protein was resolved by SDS-PAGE, detected
by autoradiography, and incorporated 32P quantitated by
Cerenkov counting of the Coomassie-stained GST-Elk-(307-428) band.
JNK MAPK activities were also assayed by a similar pulldown kinase
assay procedure in which GST-c-Jun-(1-135) replaced the GST-Elk
substrate (17).
Associated Ras Interaction Assay--
Activation of Ras
(Ras·GTP) was assayed using the Ras binding domain of Raf in the
associated Ras interaction assay (ARIA) (18). Treated cells were lysed
in Buffer B (25 mM HEPES, pH 7.5, 150 mM NaCl,
1% (v/v) Nonidet P-40, 0.25% (w/v) sodium deoxycholate, 10% (v/v)
glycerol, 25 mM NaF, 10 mM MgCl2, 1 mM EDTA, 1 mM Na3VO4, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). Cell lysates (1 mg) were incubated with GST-Ras binding domain of Raf (50 µg) for 120 min
at 4 °C and then captured on 20 µl of glutathione-Sepharose (1:1
(v/v) slurry in Buffer B) for an additional 60 min. The pellets were
then recovered and washed with Buffer B (3 × 200 µl). SDS-PAGE sample buffer (20 µl) was added and then Ras was detected by immunoblotting.
Raf Activity Assay--
Activation of Raf was assayed by a
linked MEK/ERK kinase assay utilizing MBP as substrate (19). Treated
BaF3 cells were washed in phosphate-buffered saline and then lysed in
Buffer C (10 mM Tris-HCl, pH 7.4, 50 mM NaCl, 5 mM EDTA, 50 mM NaF freshly supplemented with
1% (v/v) Triton X-100, 0.1% (w/v) BSA, 20 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 2 mM
Na3VO4). The prepared cell lysates were then
immunoprecipitated with antibodies (1 µg) for the three isoforms of
Raf (4 °C, 1 h). The antibodies were then captured on protein
A-Sepharose (40 µl of a 1:1 (v/v) slurry in Buffer C) for a further
2 h. The pellets were washed extensively in Buffer C and
resuspended in Buffer D (50 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, 0.5 mM Na3VO4,
and 0.1% (v/v) -mercaptoethanol). The kinase reaction was initiated
by incubating pellets for 30 min at 30 °C with MEK/ERK buffer (30 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, 0.1% (v/v)
-mercaptoethanol, 0.3% (v/v) Brij-35, 6.5 µg/ml GST-MEK, 100 µg/ml GST-ERK2, 10 mM MgCl2, and 0.2 mM ATP). The reaction was terminated by adding 10 µl of
supernatant to 40 µl of ice-cold Buffer C supplemented with 1 mg/ml
BSA. MBP phosphorylation was initiated with 40 µl of MBP buffer (50 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, 25 µM MBP, 12.5 mM MgCl, 0.125 mM
ATP) supplemented with 2.25 µCi of [32P]ATP per
assay. The kinase reaction was allowed to proceed for 30 min at
30 °C and then the MBP phosphorylation reaction was terminated by
spotting onto P81 paper. The paper was washed extensively in 75 mM phosphoric acid, and incorporated radioactivity was
quantitated by Cerenkov counting. An equivalent volume of Buffer C
replaced the antibody to serve as a control for each assay.
Immunoblotting--
Proteins in cell lysates were resolved by
SDS-PAGE and then transferred to polyvinylidene difluoride membranes.
For MAPK or Ras detection, membranes were blocked with 5% (w/v) milk
powder in TBST (10 mM Tris, pH 7.5, 150 mM
NaCl, 0.1% (v/v) Tween 20) and then blotted with primary antibodies
diluted (1/250 to 1/1000, depending on the antibody and determined
experimentally) in the block solution. Horseradish peroxidase-linked
secondary antibodies were diluted 1/20000 in 1% (w/v) non-fat milk
powder in TBST. The conditions of immunoblotting were altered for
phosphotyrosine with the membranes blocked with 1% (w/v) BSA in TBST
followed by incubation with the 4G10 primary antibody diluted 1/2000 in TBST and then the secondary antibody diluted 1/20,000 in 0.2% (w/v)
BSA in TBST. In all cases, final detection was with Lumi-Light Plus
chemiluminescent substrates according to the manufacturer's instructions.
Cell Viability--
Cell numbers and viability were determined
by the trypan blue exclusion method. A small aliquot of cells (50-200
µl) was diluted with 1× phosphate-buffered saline to a volume of 500 µl. An equal volume of 0.4% (w/v) trypan blue was then added, and
the mixture was allowed to stand at room temperature (5 min). Counting
chambers of a hemocytometer were then filled, and the live (unstained) and dead (blue-stained) cells were counted when viewed under a phase
contrast microscope.
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RESULTS |
Heat Shock Stimulation of MAPK Phosphorylation--
We were
interested in understanding the regulation of the ERK MAPK pathway by
diverse extracellular stimuli including cytokines and stress stimuli.
We used the IL3-dependent proB cell line, BaF3, to
investigate activation of MAPKs by a range of different stimuli
including heat shock. First, we treated IL3-deprived BaF3 cells with
either the cytokine IL3 (20 ng/ml, 10 min), oxidative stress (0.5 mM hydrogen peroxide, 10 min), the phorbol ester TPA (1 µM, 5 min), osmotic shock (0.5 M sorbitol, 10 min), or heat shock (42 °C, 60 or 120 min), and we determined the
phosphorylation of three major subfamilies of MAPKs by immunoblotting
with antibodies specific for the phosphorylation of each of these three
subfamilies. As shown in Fig.
1A, IL3, TPA, and heat shock
stimulated the phosphorylation of the ERK MAPKs. This stimulation by
heat shock exceeded that of the other stress stimuli and was more akin
to stimulation by the mitogenic factor, IL3, or the phorbol ester, TPA.
This suggests that heat shock, unlike these other stress stimuli, can
potently activate the ERK MAPK pathway in BaF3 cells.
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Fig. 1.
Phosphorylation of MAPKs in BaF3 cells.
BaF3 cells (2 × 107) were cytokine-starved for 3 h and then treated with IL3 (20 ng/ml, 10 min), hydrogen peroxide
(H2O2, 0.5 mM, 10 min), phorbol
ester (TPA 1 µM, 5 min), osmotic shock (sorbitol,
SRB, 0.5 M, 10 min), heat shock (42 °C, 60 or
120 min), or left untreated as a control (Con). Lysates were
analyzed by Western blotting with antibodies for either ERK MAPK
(A), p38 MAPK (B), or JNK MAPK (C).
The upper panel in each case shows results obtained with
phospho-specific antibodies, and the lower panel shows that
the total expression of each MAPK did not change significantly during
the course of the experiment and therefore provides a control for
protein loading. The molecular mass markers (kDa) are shown to
the right of the panel. These experiments were repeated
twice with comparable results.
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In contrast, the two stress stimuli of hydrogen peroxide and sorbitol
stimulated phosphorylation of p38 MAPK, whereas heat shock could not
effectively increase the phosphorylation of these MAPKs at the times
tested (Fig. 1B). In addition, the phosphorylation of the
JNK MAPKs was potently stimulated by sorbitol or heat shock and
moderately stimulated by hydrogen peroxide (Fig. 1C). Upon longer exposures of these immunoblots, phosphorylation of JNK MAPKs
could be observed following IL3
exposure2 as previously
reported (20). To confirm the activation of JNK MAPKs by heat shock and
IL3, we also measured JNK MAPK activity using a c-Jun pulldown assay
(17). Whereas JNK MAPK activation was rapid and transient as described
previously (14), the kinetics of JNK MAPK activation by heat shock
showed a peak of activation following heat shock for 60 min and a
decline at the 120-min time point.2 This is in agreement
with the kinetics of heat shock-stimulated JNK MAPK phosphorylation
shown in Fig. 1C.
These combined results confirm that each MAPK subfamily shows distinct
differences in phosphorylation in response to extracellular stimuli.
Our surprising result was that elevated temperature was a potent
stimulus for ERK MAPKs in addition to the JNK MAPKs that have been
traditionally classed as stress-activated protein kinases. With
the unusual kinetics of robust ERK MAPK activation observed following
the heat shock stimulus, we then focused on how the ERK MAPK pathway
could be regulated by this stress. Furthermore, the availability of a
specific chemical inhibitor of the ERK MAPK pathway, PD98059 (21),
would allow us to evaluate the cellular consequences of ERK MAPK
activation under these circumstances.
Heat Shock Stimulates a Strong, Sustained but Delayed Activation of
ERK MAPK--
We were interested in further evaluating the events
leading to phosphorylation of the ERK MAPKs following the heat shock
stimulus. ERK MAPK activity in soluble extracts was assayed in a
pulldown kinase assay using GST-Elk-(307-428) as a substrate (16). As shown in Fig. 2, negligible ERK MAPK
activity was observed during the first 10-30 min exposure to 42 °C
heat shock. However, activation of ERK MAPKs accompanying heat shock
for 60 and 120 min was far greater than that achieved with a 10-min
exposure to the cytokine stimulus, IL3. Cerenkov counting quantitated
the extent of 32P incorporation, and data from three
independent experiments indicated that 42 °C heat shock for 60 min
activated ERK MAPK 60-fold compared with 32-fold activation by IL3 (20 ng/ml) (Fig. 2A). The ERK MAPK activation by heat shock was
further increased during the 2nd hour of heat shock (Fig. 2,
A and B). This contrasts with previous results of
rapid and transient activation of ERK MAPKs by cytokines such as IL3 or
granulocyte colony-stimulating factor (14). Furthermore it differs from
the JNK MAPK activation we have noted in the heat-shocked BaF3 cells,
which is maximal at 60 min and declining in activity and
phosphorylation by 120 min (Fig. 1C and Footnote 2).
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Fig. 2.
Comparison of ERK MAPK activation by heat
shock and IL3. BaF3 cells (2 × 107) were
cytokine-starved (3 h) before treatment with IL3 (20 ng/ml, 10 min),
heat shock (42 °C for the indicated times), or left untreated as a
control (A). Detergent-soluble lysates were prepared, and
ERK MAPK activity was determined by pulldown kinase assay with
GST-Elk-(307-428) as substrate. A pulldown kinase reaction without
cell lysate was also included as a control for the procedure (no
lysate). Autoradiography indicated the incorporation of
32P from [-32P]ATP into the GST-Elk
substrate (upper panel). Coomassie staining confirmed equal
loading of GST-Elk protein (lower panel). B, time
course of heat shock-stimulated ERK MAPK activity. Cerenkov counting of
phosphorylated GST-Elk was used to quantitate the ERK MAPK activity
(cpm) in heat-shocked or IL3-treated BaF3 cells. Values represent
means ± S.D. for four independent experiments.
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The potent, sustained but delayed activation of ERK MAPKs by heat shock
suggested that the pathway initiated by this cellular stress may differ
from that utilized by the cytokine IL3. We therefore set out to examine
which of the steps involved in the classic ERK MAPK pathway (as
utilized by IL3) were the targets for activation by heat shock.
Starting with the most global changes normally associated with growth
factor and cytokine signaling, we examined the effects of these stimuli
on the total tyrosine phosphorylation profile of cellular proteins.
Immunoblot analysis with the 4G10 phosphotyrosine antibody indicated
that IL3 stimulated the greatest increases in tyrosine phosphorylation
of 35-, 60-, 97-, and 220-kDa proteins (Fig.
3, upper panel). When this was
compared with the tyrosine phosphorylation profile stimulated by heat
shock, it was clear that heat shock from 10 to 120 min did not
stimulate significant, reproducible changes in the tyrosine
phosphorylation profile of BaF3 cells (Fig. 3, upper panel).
Although most work reporting activation of ERK MAPKs in these
hemopoietic cells details the role of cytokines such as IL3 (14, 15,
20, 22), our results suggest that the heat shock-stimulated pathway
differs from that activated by IL3. We confirmed equivalent loading of protein from all cell treatments by reprobing these membranes with
antibodies recognizing total ERK MAPK (Fig. 3, lower panel). This also confirmed that ERK MAPK expression did not change during this
relatively prolonged exposure to 42 °C heat shock.
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Fig. 3.
Effects of IL3, oxidative stress, phorbol
ester, or heat shock on tyrosine phosphorylation. Cytokine-starved
BaF3 cells (2 × 107) were treated with IL3 (20 ng/ml,
10 min), oxidative stress elicited by hydrogen peroxide
(H2O2 0.5 mM, 10 min), the phorbol
ester TPA (1 µM, 5 min), heat shock (42 °C for the
indicated times), or left untreated as a control (Con). A
detergent-soluble extract was prepared (50 µg of protein), and
tyrosine phosphorylation was assessed by immunoblotting with the 4G10
antibody (upper panel). The arrowheads to the
left of this panel indicate the positions of proteins with
the greatest changes in phosphotyrosine following IL3 treatment. The
molecular mass markers (kDa) are shown to the right of the
panel. Constant protein loading and no changes in expression of ERK
MAPKs were confirmed by immunoblotting with the ERK1/2 antibodies
(lower panel).
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To eliminate further the possibility that IL3-associated signaling
events were involved in the heat shock response, we exposed BaF3 cells
to heat shock in the continued presence of IL3 (i.e. "unstarved" BaF3 cells). In this way, the IL3-responsive pathways would remain desensitized through their continuous exposure to IL3.
Under these conditions, heat shock was still capable of activating the
ERK MAPKs to approximately the same extent.2 We therefore
conclude a pathway independent of IL3 receptor activation mediates the
activation of ERK MAPKs in response to prolonged heat shock.
PD98059 Inhibits ERK MAPK Activation and Phosphorylation--
It
has been widely demonstrated that MEK1 and -2 are the highly conserved
upstream kinases involved in the stimulation of the ERK MAPK cascade by
various mitogenic and stress stimuli (2). The protein kinase inhibitor
PD98059 has been used extensively to implicate MEK1/2 in regulating the
ERK MAPKs (21). As shown in Fig. 4, we
tested the ability of PD98059 to inhibit ERK MAPK activation by IL3 or
heat shock. We chose two concentrations of PD98059 (10 and 30 µM) that lie within the range that maintains the
specificity for MEK1/2 inhibition (21). We found that control, IL3, or
heat shock-stimulated levels of ERK MAPK activity were inhibited by at
least 40% by 10 µM PD98059 (Fig. 4A), and the level of inhibition increased to at least 50% for 30 µM
(Fig. 4B). In both cases, the level of inhibition achieved
by PD98059 was greatest for heat-shocked cells. In all cases, controls
with appropriate volumes of Me2SO were included to account
for the fact that stock solutions of PD98059 had been prepared in
Me2SO (Fig. 4, A-D).
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Fig. 4.
PD98059 attenuates ERK MAPK activation by
either IL3 or heat shock. Cytokine-starved BaF3 cells (2 × 107) were pretreated with PD98059 at a final concentration
of either 10 µM (A) or 30 µM
(B) during the final hour of the 3-h cytokine starvation
period. For control cells, exposure to the appropriate volume of
Me2SO vehicle was included during this final hour. Cells
were then treated with IL3 (20 ng/ml, 10 min), heat shock (42 °C, 60 min), or left untreated as a control. Lysates were analyzed for ERK
MAPK activation using the GST-Elk pulldown kinase assay. The activity
of the cells pretreated with the appropriate concentration of
Me2SO was defined as 100%, and the other activities were
calculated as a percentage of maximum stimulation. The data presented
are means ± S.D. (n = 3 independent
observations). In all treatments, * indicates a significant inhibition
of ERK MAPK activ ity by PD98059 (p < 0.05, one-way analysis of
variance test with Fischer's Least Significant Difference test) when
compared with the controls preincubated in the presence of
Me2SO alone. C, BaF3 cells (2 × 107) were treated exactly as described and then analyzed by
immunoblotting for either phosphorylated ERK1/2 (upper
panel) or total ERK1/2 MAPKs (lower panel). This
experiment was performed twice with comparable results. D,
an independent set of cells, pretreated with PD98059 (30 µM), was stimulated identically as described. Lysates
were analyzed for JNK activation using the GST-Jun pulldown kinase
assay. A sample in which cell lysate was replaced with lysis buffer
only was used as a negative control. Autoradiography of GST-Jun
resolved by SDS-PAGE indicated incorporation of 32P from
[-32P]ATP into GST-Jun. Coomassie staining confirmed
equal loading of GST-Jun protein (lower panel). This
experiment was performed twice with comparable results.
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We confirmed that 30 µM PD98059 abrogated heat shock and
IL3-stimulated phosphorylation of ERK1 and ERK2 (Fig. 4C, upper
panel), while not altering the total levels of expression of the
ERK MAPKs (Fig. 4C, lower panel). In contrast, we confirmed
that the activation of JNK MAPKs by IL3 or heat shock was not inhibited
by 30 µM PD98059 (Fig. 4D, upper panel), and
under these conditions the total levels of expression of JNK MAPKs did
not change (Fig. 4D, lower panel). This is in agreement with
previous studies showing that the activation of JNK MAPKs is not
inhibited by PD98059 treatment (21).
The involvement of MEK was further confirmed by the observation of
increased phosphorylation of MEK by immunoblotting cell extracts
prepared from heat-shocked cells with antibodies recognizing the
phosphorylated, activated form of MEK1 and -2.2 These
results confirm that the involvement of MEK upstream is conserved in
regulating heat shock-stimulated ERK MAPK phosphorylation and activity.
Heat Shock Stimulates a Ras-independent Pathway to Activate ERK
MAPK--
The next step in identifying the signaling route to ERK MAPK
activation by heat shock entailed the identification of the potential candidates for membrane proximal signaling events. The small G-protein, Ras, has been implicated in the regulation of ERK MAPK activity stimulated by various growth factor receptors (23). In addition, recent
studies have demonstrated that Ras-dependent signaling pathways are stimulated by oxidative stress elicited by hydrogen peroxide (12). The role of Ras in heat shock signaling was examined by
investigating the effects of a dominant-negative mutant of Ras,
RasAsn-17 (14, 15, 20). In the BaF3 cell line this has been
facilitated by the stable introduction of an expression plasmid that
permits the inducible expression of RasAsn-17
when cells are cultured overnight in the presence of IPTG (14, 15, 20).
This system has successfully demonstrated that Ras is essential in the
IL3-induced activation of c-Raf, ERK MAPKs, and JNK MAPKs (15, 20) but
that these Ras-dependent pathways may not be essential in
the IL3 stimulation of proliferation (15).
We induced expression of RasAsn-17 in the N6F-20 BaF3 cell
line by overnight incubation with IPTG (14). As demonstrated in Fig. 5A, although this induction of
RasAsn-17 significantly attenuated the ERK MAPK response to
IL3 (ANOVA, p < 0.05), heat shock-stimulated ERK MAPK
activity at 60 or 120 min remained uninhibited. We confirmed that the
levels of RasAsn-17 remained elevated during the heat shock
protocol by immunoblotting cell lysates for expression of total Ras
protein (Fig. 5B, upper panel). Reprobing the immunoblot
with the ERK MAPK 1/2 antibody confirmed that the expression of ERK
MAPKs remained constant and there was equivalent ERK MAPK loading in
all lanes (Fig. 5B, lower panel). Thus, the failure of
RasAsn-17 to inhibit ERK MAPKs under these heat shock
conditions could not be attributed to instability of the
RasAsn-17 protein at 42 °C. We could not further
evaluate the effectiveness of the RasAsn-17 following heat
shock (for example by ensuring that the RasAsn-17 expressed
in the heat-shocked cells could inhibit subsequent IL3 activation of
ERK MAPKs) because the heat shock activation of ERK MAPKs remained
sustained for more than 2 h following heat shock (see Fig.
8C). Therefore, this would interfere with any ability to
detect a comparatively smaller activation of ERK MAPKs by IL3 even
after a 2-h recovery period. Furthermore, during any longer recovery
periods we might anticipate the de novo synthesis of
RasAsn-17 molecules, and this would interfere with
interpretation of these results.
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Fig. 5.
RasAsn-17 induction fails to
attenuate heat shock-stimulated ERK MAPK activity. The N6F-20 BaF3
clone was induced for 16 h in the presence of IPTG (5 mM) to express the dominant-negative mutant of Ras
(RasAsn-17). Following cytokine starvation (3 h), N6F-20
cells (2 × 107) were treated with IL3 (20 ng/ml, 10 min), heat shock (42 °C, 60 or 120 min), or left unstimulated as a
control (Con). An equal number of uninduced cells were
treated identically for comparison. Detergent-soluble lysates were then
prepared. A, ERK MAPK activity was analyzed by GST-Elk
pulldown kinase assay as described under "Experimental Procedures."
Values represent mean ± S.D. for three independent experiments. *
indicates a significant difference to identically treated cells in the
absence of IPTG (ANOVA, p < 0.05). B, to
confirm enhanced levels of Ras protein following the induction of
RasAsn-17, total lysate protein samples were immunoblotted
with the Ras antibody (upper panel). Constant protein
loading and no changes in expression of ERK MAPKs were confirmed by
immunoblotting with the ERK1/2 antibodies (lower panel).
This was repeated with similar results.
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Because recent evidence suggests that RasAsn-17 may not
effectively inhibit all mechanisms of Ras activation (24), we sought
further evidence to confirm or refute the involvement of Ras in the
activation of ERK MAPKs by measuring Ras activation. This ARIA utilizes
the Ras-binding domain of Raf to isolate active Ras (i.e.
Ras·GTP) (18). A time course of IL3 exposure revealed that maximal
Ras activation occurs within 2 min (Fig.
6A). Activation of both Ras and ERK MAPKs by IL3 was therefore rapid and transient (15). However,
when Ras·GTP levels in heat shock-stimulated cells were assessed, we
observed these were not consistently greater than those observed in
control cells (Fig. 6B). We acknowledge that it is possible
that any heat shock elevation of Ras·GTP was transient and missed in
our time course. However, transient Ras activation is most often
associated with transient ERK MAPK activation (15). The inability of
RasAsn-17 to block heat shock-stimulated ERK MAPK activity,
coupled with the finding of negligible increases in Ras·GTP levels
during heat shock, indicates that the pathway utilized by heat shock to
activate ERK MAPK is most likely one that is independent of Ras. In the BaF3 cell line, pathways other than the well characterized Ras/ERK MAPK
pathway must play a role in the response to heat shock.
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Fig. 6.
Heat shock fails to activate Ras.
Cytokine-starved BaF3 cells (2 × 107) were
stimulated with IL3 (20 ng/ml), TPA (1 µM), hydrogen
peroxide (H2O2, 0.5 mM), or heat
shocked (42 °C). Unstimulated cells were used as a control
(Con). A detergent-soluble protein extract was made, and 1 mg of protein was used to isolate activated Ras by ARIA. Activated Ras
(Ras·GTP) was detected by immunoblotting with a Ras antibody.
A, cells stimulated with IL3 or TPA for various times were
used to investigate the kinetics of Ras activation (Ras·GTP) by
mitogenic stimuli. B, in a separate experiment, heat shock
at various time points was used to investigate the activation of Ras
(upper panel). The response stimulated by IL3 (20 ng/ml, 10 min), H2O2 (0.5 mM, 10 min), and
TPA (1 µM, 5 min), in addition to unstimulated cells
(Con), were also included for comparison. Immunoblot
analysis of the total cell lysate (100 µg) for Ras provides a control
for loading (lower panel). These results are representative
of three independent experiments performed with similar findings.
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Heat Shock Activation of ERK MAPKs Is Independent of Raf--
The
activation of ERK MAPKs is not exclusively mediated by a
Ras-dependent pathway in all cell types. It has been
documented that PKC can directly phosphorylate serine residues of Raf
to modulate its kinase activity (25). We therefore sought to
investigate the kinase activity of Raf stimulated by heat shock and
determine its role in regulating the ERK MAPKs. There are three Raf
isoforms as follows: c-Raf (which is also known as Raf-1), A-Raf, and
B-Raf. The activity of each individual Raf isoform in response to heat shock and IL3 was measured by a MEK/ERK-linked Raf kinase assay as
described under "Experimental Procedures." As shown in Fig. 7, a 2-min exposure to IL3 stimulated
substantial Raf activity over control. Treatment of BaF3 cells with the
cytokine stimulated an 18-, 5.5-, and 2.4-fold activation over control
of c-Raf, A-Raf, and B-Raf kinase activity, respectively. The
relatively lower fold activation stimulated by IL3 in B-Raf was due to
a higher degree of constitutive basal activity,2 and this
is consistent with previously published results with B-Raf assays (26).
In contrast, 42 °C heat shock for 15, 30, 45, and 60 min failed to
stimulate comparable activity from any of the three Raf isoforms (Fig.
7, A-C). It could be argued that we may have missed a
transient Raf activation due to our chosen time points, but this is
unlikely when we would expect sustained Raf activity to regulate
sustained heat shock-induced ERK MAPK activity as has been previously
demonstrated (19).
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Fig. 7.
Heat shock fails to activate Raf
isoforms. Cytokine-starved BaF3 cells (2 × 107)
were stimulated with IL3 (20 ng/ml, 2 min), left untreated as a control
(Con), or heat-shocked (42 °C) for the indicated times. A
detergent-soluble protein extract was made and used to isolate c-Raf
(A), A-Raf (B), or B-Raf
(C) by immunoprecipitation with Raf isoform-specific
antibodies. Kinase activity was then subsequently measured by a
MEK/ERK-linked Raf assay as described under "Experimental
Procedures." The Raf activity in control cells was set at 1, and the
stimulated Raf activity expressed as fold activation over untreated
cells (Con).
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An involvement of PKC was also discounted when we found that the
PKC-specific inhibitor, bisindolylmaleimide I (10 µM),
failed to consistently attenuate heat shock-induced ERK MAPK
activity.2 The inter-batch variation observed with
bisindolylmaleimide I prompted investigation using an inactive form of
this compound, bisindolylmaleimide V, as well as chronic exposure to
phorbol esters to down-regulate expression of phorbol ester-sensitive PKC isoforms. Again, the results with bisindolylmaleimide were inconsistent. However, we consistently observed that down-regulation of
PKC with prolonged overnight exposure to TPA (1 µM)
failed to attenuate the ERK response (data not shown). Taken together these results indicate that heat shock-induced MEK and ERK MAPK activity does not involve upstream regulation by the three Raf isoforms
or the phorbol ester-sensitive PKC isoforms.
Role of Growth Factors Receptors in Regulating the Heat
Shock-stimulated ERK MAPK Activity--
Several reports have
implicated growth factors and their receptors as mediators in the
response to extracellular stress. In these models, stress acts to
stimulate release of growth factors, which then act through an
autocrine/paracrine mechanism to activate the traditional Ras Raf
MEK ERK MAPK cascade (11, 12). Although we have already shown
that heat shock does not activate ERK MAPKs through the traditional
cascade involving Ras or Raf, our observation of delayed kinetics of
heat shock-stimulated ERK MAPK activation may suggest the involvement
of an autocrine signaling loop. As the activation of the ERK MAPK
signaling pathway by peptide growth factors and cytokines is mediated
by the hetero- or homo-multimerization of ligand-specific receptors
localized at the plasma membrane, inhibiting these events at the
membrane may effectively abrogate signaling. We tested the involvement
of growth factor receptors in heat shock signaling with the use of
suramin. This analog of heparin sulfate has been implicated as an
inhibitor of a heterogeneous population of growth factor and cytokine
receptors and has been shown to inhibit ERK MAPK activation by serum,
epidermal growth factor, platelet-derived growth factor, tumor
necrosis factor-
, as well as ERK MAPK activation by oxidative
stress elicited by hydrogen peroxide and arsenite (11, 12, 27,
28).
Fig. 8A represents a typical
experiment where pretreatment of cells with 300 µM
suramin abrogated ERK MAPK activity stimulated by IL3. This
concentration of suramin also inhibited ERK MAPK activation following
heat shock for 60 min by 80% (ANOVA, p < 0.05), but
it failed to significantly inhibit ERK MAPK activity stimulated by heat
shock for 120 min (ANOVA, p > 0.05) (Fig.
7B). We tested whether this failure of suramin to inhibit
the 120-min heat shock response could be due to different mechanisms of
signaling at the two time points or be due to problems of stability of
suramin under these incubation conditions. Preheating of 300 µM suramin in starvation medium at 42 °C for 120 min
prevented its ability to subsequently abrogate IL3-stimulated ERK MAPK
activity (ANOVA, p < 0.05) (Fig. 8B). Thus
suramin was not stable for 120 min under these conditions at 42 °C,
and we cannot draw any conclusions on whether the mechanisms involved
in ERK MAPK activation at 60 and 120 min of heat shock differ.
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Fig. 8.
Effect of suramin on heat shock-stimulated
ERK MAPK activity. Cytokine-starved BaF3 cells (2 × 107) were pretreated with suramin (300 µM)
for 60 min prior to treatment with IL3 (20 ng/ml, 10 min), heat shock
(42 °C for 60 min or 120 min, HS60 and HS120, respectively), or left
unstimulated as a control (Con). A set of cells was treated
identically in the absence of suramin for comparison
(Suramin). To test the stability of suramin under these
incubation conditions, suramin, or an equal volume of media, was also
preincubated at 42 °C (IL3 ) in the absence of cells
before testing its effects on IL3-stimulated ERK MAPK activation.
A, ERK MAPK activity was measured by GST-Elk pulldown kinase
assay as described under "Experimental Procedures." Autoradiography
of GST-Elk indicated the incorporation of 32P from
[-32P]ATP into the substrate (upper panel).
Coomassie staining confirmed equal loading of GST-Elk protein
(lower panel). B, the activity of ERK MAPK was
determined by Cerenkov counting of GST-Elk. Results from three
independent experiments were calculated as a percentage of maximum
stimulation (100%) defined by cells treated in absence of suramin. *
indicates a significant difference to identically treated cells in the
absence of inhibitor as determined by analysis of variance with
Fischer's Least Significant Difference test (p < 0.05). C, cytokine-starved BaF3 cells (2 × 107) were treated with heat shock (42 °C) for either 60 or 120 min and then transferred to 37 °C for a further 60 min of
recovery. Detergent-soluble cell lysates were then prepared, and ERK
MAPK activity was measured by pulldown kinase assay as described.
Values represent mean ± S.E. from three independent
experiments.
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We sought further support for a role of released growth factors
mediating the heat shock ERK MAPK response. We tested whether ERK MAPKs
would remain activated following removal from the elevated temperatures. Our results in Fig. 8C demonstrate that heat
shock-stimulated (42 °C, 60-120 min) ERK MAPK activity remains
elevated after a 60-min recovery period at 37 °C. Thus ERK MAPK
activation by the 60-min heat shock followed by 60 min of recovery was
similar to the activation observed following 120 min of heat shock.
This may suggest the presence of extracellular signaling molecules released during the heat shock treatment. Next, we tested the effects
of media conditioned during the heat shock of these cells. However,
this conditioned media failed to activate ERK MAPK activity when
applied to starved BaF3 cells.2 Finally, we
attempted to prevent ERK MAPK activation with the use of a protein
synthesis inhibitor, cycloheximide. We found that the heat shock
activation of ERK MAPKs was not inhibited by preincubation with 10 µM cycloheximide.2 The combined results from
these studies suggest that, although growth factor receptors may be
involved in the heat shock response, they may be directly modulated
during heat shock without requirement of release of autocrine/paracrine factors.
Exposure to the Phosphatase Inhibitor Okadaic Acid Also Stimulates
Delayed ERK MAPK Activation--
An alternative mechanism to account
for a delayed and sustained activation of a protein kinase could
involve stress-induced inhibition of a protein phosphatase. Whereas
inhibition of tyrosine phosphatases by oxidative stress is a well
recognized modulator of MAPK pathways (29, 30), in the situation of
heat-shocked BaF3 cells the lack of substantial changes in tyrosine
phosphorylation together with modulation of the pathway at the level of
MEK suggests that serine/threonine phosphatases may be more likely
candidates for targets. To evaluate this hypothesis we exposed BaF3
cells to 1 µM okadaic acid (31-34) for up to 2 h.
We did not observe activation of ERK MAPKs following the short term
exposure (10 min) to okadaic acid,2 and this is consistent
with a delayed activation of ERK MAPKs we have previously observed upon
okadaic acid exposure of cardiac myocytes (33). At 60 and 120 min of
exposure to okadaic acid, we noted at least 9.5-fold activation of ERK
MAPKs (Fig. 9A) and increased
phosphorylation of MEK (Fig. 9B). Although the level of ERK
MAPK activation following okadaic exposure was not as potent as heat
shock stimulation of these kinases, it was noted that suramin was also
effective at abrogating the effects of both okadaic acid and heat shock
(Fig. 9). Suramin (300 µM) inhibited the ERK activity
stimulated by 120 min of treatment with okadaic acid or 60 min of heat
shock (ANOVA, p < 0.05) (Fig. 9). Although the statistical analysis did not indicate a significant difference for the
effects of suramin during 60 min of okadaic acid treatment, there was a
trend toward lower ERK activation in the presence of suramin (Fig. 9).
These results suggest that the results of experiments using suramin
should be treated with caution because it is currently unlikely that
effects of okadaic acid are mediated by secondary events of growth
factor release and/or growth factor receptor signaling. Instead, we
would suggest that delayed ERK MAPK activation mediated by either heat
shock or okadaic acid is a result of an attenuated control of negative
regulators of the protein kinases MEK and/or ERK MAPKs.
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Fig. 9.
Suramin attenuates okadaic acid-stimulated
ERK MAPK activation and MEK phosphorylation. Cytokine-starved BaF3
cells (2 × 107) were pretreated with suramin (300 µM) for 30 min prior to treatment with okadaic acid (1 µM, 60 or 120 min), heat shock (42 °C, 60 or 120 min),
or left unstimulated as a control (Con). Identically treated
cells were pretreated with an equivalent volume of starve media for
comparison (suramin). A, ERK MAPK activity was measured by
GST-Elk pulldown kinase assay as described under "Experimental
Procedures." The activity of ERK MAPK was determined by Cerenkov
counting of GST-Elk. Results are expressed as fold activation over
control levels and are expressed as means ± S.D.
(n = 3 independent observations). In all treatments, *
indicates a significant inhibition of ERK MAPK activity by suramin
(p < 0.05, one-way analysis of variance test with
Fischer's Least Significant Difference test) when compared with
controls. B, MEK phosphorylation was measured by
immunoblotting with a phospho-MEK-specific antibody (upper
panel) as described under "Experimental Procedures."
Immunoblots of identical samples for total MEK protein levels were
included as a control for loading (lower panel).
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Heat Shock-stimulated ERK MAPK Activity Mediates Cell Viability
following Stress Insult--
It has been suggested that the activation
of the ERK MAPK cascade in response to stress is involved in a
concerted defense mechanism to protect the cells (10, 12). The ability
of BaF3 cells to survive heat shock was therefore measured by trypan
blue staining. Attenuation of ERK MAPK activity by the MEK inhibitor PD98059 (30 µM) was used to investigate the role of heat
shock-stimulated ERK MAPK in maintaining cell viability. We measured
cell viability directly after a 1-h exposure to heat shock or 24 h
after this treatment (Fig. 10). There
was no significant difference in the number of viable cells immediately
after heat shock in the presence or absence of the MEK inhibitor
PD98059 (Fig. 10A). From this result, we would suggest that
ERK MAPK activation is not critical for the short term survival in
these cells.
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Fig. 10.
ERK MAPK activity prevents heat
shock-stimulated loss of cell viability. Non-starved BaF3 cells
(2 × 107), pretreated with the vehicle,
Me2SO (0.3% (v/v)), or PD98059 (30 µM) for
60 min were treated with heat shock (42 °C, 60 min) or left
unstimulated at 37 °C. Cell viability was determined immediately
after treatment (A) or after a 24-h period (B).
Data from three independent experiments are presented as mean ± S.D. * denotes a significant difference between vehicle and PD98059
within a particular treatment. ns denotes no significant
difference between vehicle and PD98059 within a particular
treatment.
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After a 24-h period at 37 °C, the number of viable cells under
control conditions had decreased from 98 to 86% (Fig. 10B). In the presence of PD98059, this percentage of viable cells decreased from 95 to 76% (ANOVA p > 0.05). The presence of
PD98059 therefore did not appear to significantly affect survival of
control cells, although this result will require further investigation.
When we examined the effects of heat shock, we observed that the
viability of heat-shocked cells had decreased to 40% after 24 h
of further culture at 37 °C. Attenuation of ERK activity by PD98059
resulted in a further decrease of viability to 26% which was
significantly different (ANOVA p < 0.05) to viability
of heat-shocked cells in the absence of the inhibitor (Fig.
10B). These results indicate that an attenuation of ERK MAPK
activity by PD98059 is accompanied by increased loss of viability in
the BaF3 cell population. This implicates ERK MAPKs in maintaining the
viability of BaF3 cells following stress from heat shock.
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DISCUSSION |
It is now recognized that both the extent and kinetics of ERK MAPK
activation are critical determinants of the cellular response to growth
factors and cytokines (35). Transient ERK MAPK activation is most
commonly associated with cellular proliferation, whereas prolonged ERK
MAPK has been associated with nuclear translocation of these kinases
and cellular differentiation (36). Presumably the translocation of ERK
MAPKs to the nucleus following prolonged activation permits access to
different subsets of transcription factor substrates essential for the
differentiation process (35).
There is now increasing evidence that activation of the ERK MAPKs can
also be stimulated by a variety of stress stimuli. In most cases, ERK
MAPK activation in response to these stresses is transient. For
example, ERK MAPK activation by hydrogen peroxide, arsenite, or osmotic
stress is maximal within 10-20 min and rapidly returns to basal levels
within the 1st hour of exposure (11, 12, 37, 38). Heat shock has been
well documented as a stress that initiates long term changes within
cells. The best characterized of these changes is the stimulation of
heat shock protein synthesis in a protective response to prevent
further cell damage (39, 40). Indeed, heat shock has been proposed as
an experimental method for enhancing heat shock protein synthesis to
protect against further cell insults such as ischemia (39). Few studies
have considered earlier events in the detection of heat shock by cells or whether heat shock also activates cytoplasmic protein kinase signaling cascades. In a number of cell types, heat shock apparently fails to activate ERK MAPKs but does activate the stress-activated MAPKs such as the JNK MAPKs (3, 38). However, these responses are
apparently cell type-dependent, and heat shock prevents
activation of JNK MAPKs and p38 MAPKs in U937 cells but does activate
ERK MAPKs in H56 hepatoma cells, NIH 3T3 fibroblasts, human HeLa cells, human KB carcinoma cells, or during whole animal hyperthermia (41-45).
In all of these examples, the activation of ERK MAPK was not examined
beyond an initial 60 min of heat shock, and so any long term effect on
signaling pathways was not evaluated.
In the present study, we have demonstrated that ERK and JNK MAPKs, but
not p38 MAPKs, are activated by 42 °C heat shock at 60 and 120 min
(Fig. 1). In the heat-shocked BaF3 cells, we observed maximal
phosphorylation and activation of JNK MAPKs at 60 min. Due to the lack
of a specific chemical inhibitor of the JNK MAPKs and their complex
pathways of upstream regulators, the elucidation of the mechanisms
underlying this activation and its subsequent cellular effects would
prove difficult. Furthermore, the activation of JNK MAPKs by heat shock
was first reported when these protein kinases were cloned (3) and has
been subsequently shown in numerous studies (for recent examples see
Refs. 46-48). Therefore, we further examined the activation of ERK
MAPKs by heat shock, concentrating on the mechanism of activation and
the possible downstream events regulated by this signal transduction pathway.
We characterized the kinetics of ERK MAPK activation in the
IL3-dependent proB cell line BaF3 as sustained at maximal
levels over a 2-h period (Fig. 2). We have observed similar results in the IL3-dependent myeloid cell line NFS-60.2
Activation was also delayed (Fig. 2), and this suggested that in these
cells ERK MAPK is activated either by a novel pathway or by the
classical MAPK pathway following the release of growth factors and/or cytokines.
Apart from these important aspects of the long duration and delayed
nature of ERK MAPK activation by heat shock in our system, it was also
apparent that the level of ERK MAPK activation exceeded that stimulated
by the cytokine IL3 (Fig. 2). IL3 is a critical requirement for the
proliferation and survival of these cells. We were therefore interested
in identifying the upstream events stimulated by heat shock that would
potently activate the ERK MAPKs. Specifically we evaluated whether heat
shock stimulated the well characterized Ras Raf MEK ERK
MAPK cascade or whether a more potent alternative pathway to ERK MAPK
activation may be recruited when these cells were stressed by exposure
to elevated temperatures. An alternative pathway could provide a
rational approach to designing alternative ways to stimulate ERK MAPKs in these cells, thus altering cell proliferation and/or survival, independent of the effects of IL3.
MEK was implicated in heat shock signaling in the BaF3 cells when we
showed that the MEK-specific inhibitor, PD98059, inhibited heat
shock-stimulated ERK activity (Fig. 4). The specificity of this
inhibitor has been documented (21), and it is now widely used in
evaluating the functional consequences of ERK MAPK activation. Furthermore, heat shock stimulated phosphorylation of MEK.2
However, the current study also demonstrated that a dominant-negative mutant of Ras (RasAsn-17) failed to attenuate heat shock
stimulation of ERK MAPK activity (Fig. 5). This was confirmed when we
demonstrated that Ras·GTP levels were not elevated during heat shock
(Fig. 6). These data support a model in which a Ras-independent
signaling pathway leads to potent ERK MAPK activation under these
stress conditions.
It has been previously reported that the Raf/MEK/ERK cascade can be
regulated independently of Ras through PKC directly regulating Raf
through serine phosphorylation (49). Although this Ras-independent, PKC-dependent mechanism has only been infrequently reported
to follow stimulation with a stress factor (50, 51), the role of PKC
was nevertheless investigated. We have subsequently eliminated participation of PKC in heat shock signaling when a PKC-specific chemical inhibitor, bisindolylmaleimide I, and down-regulation of
phorbol ester-sensitive PKC isoforms upon prolonged TPA exposure failed
to attenuate consistently heat shock-stimulated ERK MAPK activity (data
not shown). More surprisingly, additional analysis of Raf activation
revealed that none of the three Raf isoforms (Raf-1, A-Raf, and B-Raf)
were activated during the 1st hour of heat shock (Fig. 7).
We have further established that heat shock-stimulated ERK MAPK
activity does not involve an autocrine release of growth factors or
cytokines. There is some evidence that supports cytokine induction following whole body heat shock (52). Furthermore, there is evidence
that the activation of ERK MAPKs in response to stress may be mediated
by an autocrine release of growth factors or cytokines then acting via
their specific receptors to stimulate the classical Ras-dependent ERK MAPK pathway (11, 12). Most of this
evidence comes from the use of suramin that has been demonstrated to
bind to heparin-binding growth factor receptors such as the epidermal growth factor receptor, fibroblast growth factor receptor, and platelet-derived growth factor receptor (28, 53). To examine whether
growth factor receptors may be involved in our system, we exposed BaF3
cells to suramin before and during the heat shock treatment. We found
that suramin attenuated the heat shock activation of ERK MAPKs (Fig. 8,
A and B). We sought further support for a role of
growth factors, and we noted that heat shock-stimulated ERK MAPK
activity remained sustained following a 60-min recovery period (Fig.
8C). This supported the idea that heat-shocked BaF3 cells
were producing secondary signaling molecules, which would remain to
stimulate the pathway even after the removal of the heat shock stimulus.
When we continued investigation of the involvement of growth factors,
we found that medium conditioned in the presence of heat-shocked cells
failed to stimulate ERK MAPK activity.2 This indicated that
any autocrine growth factors were not released into media. This may be
possible when cell surface-binding proteoglycans, such as heparin
sulfate, bind growth factors and act as growth factor reservoirs to
facilitate ligand receptor interaction at the cell surface (53).
Alternatively, heat shock may stimulate ERK MAPK activity through a
more direct influence on the growth factor receptors. The physical
state of the cell membrane has been recognized to change in response to
stress such as increased temperature, and this may modulate
membrane-associated events such as receptor activation (54). In support
of this general notion, the exposure of cells to ultraviolet light and
osmotic stress perturbs the cell surface and alters the conformation of a number of different receptors leading to their clustering,
activation, and subsequent alterations in MAPK signaling (55).
Similarly, the activation of the platelet-derived growth factor-
receptor following mechanical stress of vascular smooth muscle cells
occurs by perturbation of the receptor without engagement of the
platelet-derived growth factor-binding site of the receptor (56).
However, our failure to observe enhanced tyrosine phosphorylation
following heat shock (Fig. 3) also opens the question of whether a
growth factor-mediated pathway is in operation.
More recently there have been reports of ERK activity being regulated
by mechanisms other than the traditional Raf MEK ERK MAPK
cascade. Meriin et al. (46) described the activation of JNK MAPKs by heat shock through a decreased rate of protein dephosphorylation (46). In this model, the basal activity of the MAPKs
and MEK of non-stimulated cells is controlled by various serine/threonine phosphatases such as PP1 and PP2A. If the phosphatases are inhibited in any way, phosphorylation of their substrates will be
favored. We have shown that okadaic acid, an inhibitor of the
serine/threonine kinases PP1 and PP2A, stimulated ERK activity and MEK
phosphorylation with delayed kinetics which resembled the activation
following exposure to heat shock (Fig. 9). Surprisingly, the
stimulation of MEK and ERK MAPK by okadaic acid was also sensitive to
inhibition by suramin (Fig. 9), presumably revealing another nonspecific action of suramin in addition to its effects on a number of
important intracellular signal transduction regulators including
protein-tyrosine phosphatases, PKC, Cdc2, phosphatidylinositol 3'-kinase (see Ref. 58 and references therein). We propose that heat
shock may activate MAPKs through the novel mechanism of inhibiting serine/threonine phosphatases.
The potent activation of ERK MAPK prompted our investigation into the
physiological role that potent activation of ERK MAPK may play in heat
shock signaling. We should emphasize, however, that we have not
discounted a role for JNK MAPKs in the response to heat shock, but
rather we have chosen to focus on the effects that the
potentially-protective ERK MAPK pathway may play by exploiting the use
of the specific MEK inhibitor PD98059 (21). Heat shock is synonymous
with the universal down-regulation of general transcription and
translation with a concurrent increase in the production of a set of
specific proteins, termed heat shock proteins (HSPs) (40). These
proteins serve protective roles against cellular damage caused by the
stress stimuli (39). Transcription of several HSPs, such as Hsp70, have
been found to be regulated by heat shock transcription factor 1. Although there is some evidence that heat shock transcription factor 1 can be phosphorylated by MAPKs in vitro, the regulation of
HSPs by these kinases remains undefined (59). We have demonstrated a
relationship between ERK MAPK activity and cell viability. Attenuation
of ERK MAPK activity with a MEK-specific inhibitor (PD98059) resulted
in a loss of viability (Fig. 10), suggesting the activation of the ERK
MAPK cascade may be involved in maintaining long term cell survival.
This confirms previous studies demonstrating the requirement for ERK
MAPK for cell survival following exposure to stress stimuli
(e.g. Refs. 12, 57, and 60).
In summary, we propose a model whereby the signaling mechanism
activated by heat shock for 60 min involves a deactivation of
serine/threonine phosphatases that negatively regulate MEK and ERK. A
recent study suggests phosphatase regulation by a chaperone heat shock
protein (46). The precise mechanism of phosphatase regulation of ERK
MAPK signaling will require further investigation.
and
TOP
ABSTRACT
INTRODUCTION
120 min) manner.
These characteristics suggested a novel mechanism of ERK MAPK
activation and became the focus of this study. A MEK-specific
inhibitor, PD98059, inhibited heat shock ERK MAPK activation by >75%.
Surprisingly, a role for Ras in the heat shock response was eliminated
by the failure of a dominant-negative
RasAsn-17 mutant to inhibit ERK MAPK
activation and the failure to observe increases in Ras·GTP. Heat
shock also failed to stimulate activation of A-, B-, and c-Raf.
Instead, a serine/threonine phosphatase inhibitor, okadaic acid,
activated ERK MAPK in a similar manner to heat shock. Furthermore,
pretreatment with suramin, generally recognized as a broad range
inhibitor of growth factor receptors, inhibited both okadaic
acid-stimulated and heat shock-stimulated ERK MAPK activity by >40%.
Inhibiting ERK MAPK activation during heat shock with PD98059 enhanced
losses in cell viability. These results demonstrate Ras- and
Raf-independent ERK MAPK activation maintains cell viability following
heat shock.
