From the Department of Molecular Biology, College of Natural
Sciences, Pusan National University, Pusan 609-735, and the
Department of Life Science, Kwangju Institute of Science
and Technology, Kwangju 506-712, Korea
Received for publication, July 10, 2000, and in revised form, September 5, 2000
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ABSTRACT |
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Heat shock induces c-Jun N-terminal kinase (JNK)
activation as well as heat shock protein (HSP) expression through
activation of the heat shock factor (HSF), but its signal pathway is
not clearly understood. Since a small GTPase Rac1 has been suggested to
participate in the cellular response to stresses, we examined whether
Rac1 is involved in the heat shock response. Here we show that moderate
heat shock (39-41 °C) induces membrane translocation of Rac1
and membrane ruffling in a Rac1-dependent manner. In
addition, Rac1N17, a dominant negative mutant of Rac1, significantly
inhibited JNK activation by heat shock. Since Rac1V12 was able to
activate JNK, it is suggested that heat shock may activate JNK via
Rac1. Similar inhibition by Rac1N17 of HSF activation in response to heat shock was observed. However, inhibitory effects of Rac1N17 on heat
shock-induced JNK and HSF activation were reduced as the heat shock
temperature increased. Rac1N17 also inhibited HSF activation by
L-azetidine-2-carboxylic acid, a proline analog, and heavy metals (CdCl2), suggesting that Rac1 may be linked to HSF
activation by denaturation of polypeptides in response to various
proteotoxic stresses. However, Rac1N17 did not prevent phosphorylation
of HSF1 in response to these proteotoxic stresses. Interestingly, a
constitutively active mutant Rac1V12 did not activate the HSF. Therefore, Rac1 activation may be necessary, but not sufficient, for
heat shock-inducible HSF activation and HSP expression, or otherwise a
signal pathway(s) involving Rac1 may be indirectly involved in the HSF
activation. In sum, we suggest that Rac1 may play a critical role(s) in
several aspects of the heat shock response.
Upon exposure to elevated temperatures, cells exhibit a conserved
defense mechanism, the heat shock response (also known as the stress
response). The cellular response to heat shock involves an elevated
expression of highly conserved proteins referred to as the heat shock
proteins (HSPs)1. The HSPs
are known to function as molecular chaperones during protein
folding/assembly and membrane translocation and to prevent aggregation
of misfolded polypeptide chains in cells (for reviews, see Refs. 1 and
2). They are also involved in protection against stress-induced
apoptosis (3). The HSPs can be induced by other proteotoxic stresses
such as L-azetidine-2-carboxylic acid (Azc), a proline
analog, and heavy metals (1). Expression of the HSPs is mediated
through the activation of the heat shock transcription factor (HSF),
which binds to and activates a conserved regulatory site, the heat
shock element (HSE), located in the promoters of heat-inducible HSP
genes (for review, see Refs. 4 and 5). In vertebrates, HSFs 1-4 have
been identified: HSFs 1, 2, and 4 are ubiquitous, whereas HSF3 has been
characterized only in avian species (5). Under nonstressful conditions,
HSF1 exist as a non-DNA-binding form in complex with other regulatory proteins such as HSP90 an HSP70, which are believed to function as
repressors of HSF1 activation in the absence of stress (4-8). It has
been suggested that nonnative polypeptides accumulated by heat shock
and proteotoxic stresses function as a common proximal inducer of HSF1
activation and HSP expression (1, 9, 10). In stressed cells, misfolded
proteins may compete with HSF1 for binding chaperone HSP70, and unbound
HSF1 homotrimerizes, becomes transcriptionally competent and is
hyperphosphorylated (4, 5, 11). Although protein kinases such as
mitogen-activated protein kinase, glycogen synthase kinase 3, and JNK have been demonstrated to down-regulate HSF1 activation in
response to heat shock (12, 13), the signal pathway of heat shock
activation of HSF1 is as yet unclear. Heat shock also activates JNK,
one of mitogen-activated protein kinase family proteins and which is
implicated in a variety of cell regulation such as cell growth and
apoptosis (14-18). A putative JNK phosphatase has been suggested to be
responsible for the heat shock-induced JNK activation (19, 20).
A small GTPase Rac1, a member of Rho GTPase family, has been implicated
in the regulation of various fundamental cellular processes, including
actin cytoskeletal organization, transcriptional activation, and cell
proliferation (for reviews, see Refs. 21-23). Rac1 was originally
found to play essential roles in growth factor-induced membrane
ruffling and cell proliferation (24-28). It also mediates activation
of JNK in response to stresses such as ceramide and Fas (29-31).
Therefore, the signal pathway involving Rac1 is likely involved in the
stress response. Here we show that a distinct signal pathway(s)
involving Rac1 may be implicated in HSF and JNK activation in response
to heat shock. We also demonstrate that Rac1 may be linked to HSF
activation by denaturation of polypeptides by other proteotoxic
stresses such as Azc and heavy metals. However, Rac1N17 did not prevent
phosphorylation of HSF1 in response to these proteotoxic stresses.
Although moderate heat shock induced membrane translocation and
membrane ruffling in a Rac1-dependent manner, a
constitutively active Rac1V12 did not activate the HSF, while it
stimulated the JNK activity. These results suggest that a signal
pathway(s) involving Rac1 may be directly and indirectly linked to heat
shock-induced JNK and HSF activation, respectively.
Cell Culture, Transfection, Western Blotting, and
Immunoprecipitation--
Rat-2 fibroblast cells were obtained from the
American Type Culture Collection (ATCC, CRL 1764) and grown in
Dulbecco's modified Eagle's medium supplemented with10% (v/v) fetal
bovine serum (Life Technologies, Inc.) and 1% penicillin-streptomycin
(Life Technologies, Inc.) in a 37 °C humidified incubator with 5%
CO2. Rat-2 stable clones expressing a dominant negative
Rac1N17 were prepared as described previously (32). pEXV-Myc and
pEXV-Myc-Rac1N17 plasmids (gifts from Dr. Alan Hall) were expressed as
N-terminally 9E10 epitope-tagged derivatives under SV40 promoter (25).
Rat-2 cells were stably cotransfected with pEXV-Rac1N17 or control
vector plasmid, pEXV, along with the NeoR gene, and clones were
selected and expanded in the presence of G418 (500 µg/ml). For
preparation of constitutively active Rac1V12 cells, full-length wild
type Rac1 cDNA was cloned into a pcDNA3.1 (Invitrogen), and
Gly-to-Val mutagenesis at codon 12 of the Rac1 cDNA was achieved
using a mutagenesis kit (Stratagene). pcDNA3.1-Rac1V12 plasmid was
expressed under CMV promoter. Rat-2 cells were stably transfected with
pcDNA3.1-Rac1V12 or control vector plasmid, pcDNA3.1, and
clones were selected and expanded in the presence of G418. Expression
of mutant Rac1 proteins was determined by Western blotting with
anti-Rac1 antibody (Transduction Laboratories). Metabolic labeling,
SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting,
and immunoprecipitation were carried out as described previously
(32, 33).
Electromobility Shift Assay (EMSA) and Supershift Assay
(EMSSA)--
Cells (1 × 106 cells) were washed three
times with cold phosphate-buffered saline (PBS) and the pellet rapidly
frozen at JNK Activity Assay--
JNK activity was determined as described
in Shin et al. (32). Rat-2 cells were lysed with buffer A
(1% Triton X-100, 25 mM Hepes, 50 mM NaCl, 50 mM NaF, 5 mM EDTA, 10 nM okadaic
acid, 0.1 mM sodium orthovanadate, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin) and the cell extracts incubated with anti-JNK
antibody (Santa Cruz Biotechnology, Inc.) for 1 h and then protein
A-agarose for an additional 1 h. After centrifugation, the
precipitates were incubated in the mixture of 20 mM Hepes,
pH 7.4, 2 mM sodium orthovanadate, 2 mM DDT,
100 mM MgCl2, [32P]ATP, and 4 µl of glutathione S-transferase-c-Jun at 30 °C
for 30 min. To stop the reaction, 4 × Laemmli sample buffer was
added to the precipitates and heated at 100 °C for 5 min. After
centrifugation, the supernatant was analyzed by SDS-PAGE and autoradiography.
Confocal Microscopy--
Rat-2 cells were grown on round
coverslips in multiwell culture plates and exposed to epidermal growth
factor (EGF, 100 ng/ml) and heat shock for 10 min. The cells
were fixed with 3.7% (w/v) formaldehyde in PBS for 30 min on ice. The
fixed cells were then permeabilized by incubating in 0.2% Triton X-100
in PBS for 15 min on ice. For actin staining, the cells were incubated
with 0.165 M NBD-phallacidin for 30 min at room
temperature. The stained cells were washed three times in PBS for 15 min and mounted on slide glasses with gelvitol, which was
prepared by mixing 100 ml of 23% polyvinyl alcohol in PBS with 50 ml
glycerol. For Rac1 staining, the permeabilized cells were blocked with
3% bovine serum albumin containing several drops of horse serum in TBS
for 1 h at room temperature and then incubated with anti-Rac1
antibody (Upstate Biotechnology) overnight at 4 °C. After
washing three times with TBS plus 0.1% Triton X-100 (TBST), the cells
were stained with fluorescein isothiocyanate-conjugated secondary
antibody for 1 h and washed five times in TBST and mounted on
slide glasses with crystal mount. Then the stained cells were
observed under a confocal microscope (LSM510, Carl Zeiss).
Preparation and Characterization of Rac1N17 and Rac1V12
Cells--
To test the possible involvement of Rac1 in the heat shock
response, we prepared and characterized Rat-2 fibroblast cells expressing either a dominant negative Rac1N17 or a constitutively active Rac1V12 (32). Anti-Rac1 antibody was used in Western blotting of
cell extracts to detect simultaneously the amounts of mutant and
endogenous Rac1. Because of the tag, Rac1 mutants had a slower
electrophoretic mobility than endogenous Rac1. The expression levels of
mutant Rac1 proteins were similar to that of endogenous Rac1 (Fig.
1A). Expression of Rac1V12 in
which amino acid 12 was substituted to Val significantly stimulated the
JNK activity, while expression of Rac1N17 in which residue 17 was changed to Asn reduced the JNK activity (Fig. 1A). Similar
results have been reported by several investigators in different cell lines (27, 28). Rac1 has been shown to play a role in mitogen signaling; when injected into quiescent cells, Rac1V12 stimulates cell
cycle progression through G1 and DNA synthesis, while
Rac1N17 blocks serum-induced DNA synthesis (21, 26-28). In agreement with these results, Rac1V12 cells exhibited an increased cell growth,
whereas Rac1N17 cells showed a decreased cell proliferation (Fig.
1B). In addition, upon stimulation of cells with EGF,
Rac1N17 cells did not show any changes in actin filaments, whereas
normal cells exhibited extensive membrane ruffling within 10 min,
followed by stress fiber formation (32).
Activation of Rac1 and Induction of Membrane Ruffling by Heat
Shock--
To test whether Rac1 is activated by heat shock, we
examined its membrane translocation by confocal microscopy using
anti-Rac1 antibody. As shown in Fig.
2A, exposure of Rat-2 cells to
moderate heat shock (40 °C) induced membrane translocation of Rac1,
similar to that in cells treated with EGF. Similar results were
obtained in cells exposed to either 39 or 41 °C (data not shown).
Since Rac1 mediates growth factor-induced membrane ruffling and
lamellipodia (22, 23, 25), we examined alterations in the actin
structure in response to heat shock (39-44 °C) by confocal
microscopy using NBD-phallacidin, a probe specific for F-actin. As
exposed to heat shock for 10 min, membrane ruffling was observed (Fig.
2B). Heat shock-induced ruffling pattern was different
depending on the heat shock temperature. Mild heat shock
(39-41 °C)-induced ruffling was similar to that observed in growth
factor-treated cells (22, 23, 25), whereas severe heat shock
(43-44 °C)-induced ruffling was similar to that in cells treated
with phorbol 12-myristate 13-acetate, a protein kinase C activator
(34). Rac1N17 inhibited membrane ruffling in response to mild, but not
to severe, heat shock (Fig. 2B). In addition, protein kinase
C inhibitors, H7 and staurosporine, did not exert inhibitory effects on
severe heat shock-induced ruffling, indicating that protein kinase C is
not likely involved in the severe heat shock-induced actin reorganization (data not shown). These results suggest that mild heat
shock-induced actin ruffling may occur via a Rac1 signal pathway, while
severe heat shock-induced actin remolding is likely regulated by a
Rac1- and protein kinase C-independent mechanism.
Inhibition by Rac1N17 of Heat Shock-induced JNK Activation--
It
was examined whether Rac1 participates in heat shock-induced JNK
activation. Heat shock activated JNK in time- and
temperature-dependent manners (Fig.
3) (18). Rac1N17 completely inhibited
moderate (39-41 °C) heat shock-induced JNK activation, but
it partially inhibited severe (43 °C) heat shock-induced JNK
activation. Thus, a distinct signal pathway involving Rac1 may mediate
JNK activation by mild heat shock, whereas a Rac1-independent pathway
as well as Rac1 signal pathway may be responsible for JNK activation by severe heat shock.
Suppression of Heat Shock-induced HSP Expression and HSF1
Activation by Rac1N17--
We also investigated whether Rac1 is
involved in heat shock-induced HSF activation. Heat shock highly
enhanced the synthesis of HSP70 and HSP90 as described previously (1),
and Rac1N17 significantly prevented the heat shock-induced HSP
expression, while unaffecting synthesis of other proteins (Fig.
4). As shown in Fig.
5A and demonstrated by others
(4-6), HSF1 was involved in the heat shock-induced HSP expression.
Rac1N17 significantly inhibited heat shock-induced HSF1 activation
(Fig. 5B), without affecting the level of HSF1 (Fig.
5C). However, as the heat shock temperature was elevated or
heat shock was imposed for prolonged period, an inhibitory effect of
Rac1N17 was diminished (Figs. 4 and 5B). These results
suggest that Rac1 may be required in heat shock-induced HSP expression
and HSF1 activation, although the Rac1-independent mechanism also
participates in severe heat shock response.
Inhibition by Rac1N17 of HSF Activation in Response to
L-Azetidine-2-Carboxylic Acid and Heavy Metals--
It has
been suggested that mild heat shock induces denaturation of only
nascent polypeptides, while severe heat shock causes misfolding of both
cellular proteins and nascent polypeptides (35, 36). Therefore, it is
possible that Rac1 may be linked to HSF1 activation by denaturation of
nascent polypeptides. First, we tried to confirm whether HSF1
activation in response to mild heat shock response is linked with the
regulation of protein synthesis. As demonstrated by others (35, 36),
preincubation with cycloheximide, an inhibitor of protein synthesis,
significantly blocked HSF1 activation in response to moderate heat
shock of 39-41 °C, but not to severe heat shock of 43-45 °C
(Fig. 6A). We also examined the effects of cycloheximide on HSF1 activation in response to Azc,
which causes denaturation of only nascent polypeptides. As shown in
Fig. 6A, Azc-induced HSF1 activation was significantly inhibited by cycloheximide. With other's previous results (35, 36),
these results support that mild heat shock only affects the proteins
being newly synthesized, whereas severe heat shock causes misfolding of
nascent polypeptides as well as proteins preexisting in cells.
It was then examined whether Rac1 is involved in HSF activation by
denaturation of nascent polypeptides. Azc-induced HSF1 activation was
prevented by Rac1N17 (Fig. 6B). However, Rac1N17 also
prevented HSF (possibly HSF1) activation by CdCl2 (Fig.
6B), which is thought to cause denaturation of both nascent
polypeptides and proteins preexisting in cells. Furthermore, Rac1N17
also prevented HSF1 activation in response to heat shock of 43 °C,
at which cycloheximide did not exert an inhibitory effect on HSF1
activation (Fig. 5B, lower panel). Taking these
results together, we suggest that Rac1 may be linked to activation of
HSF1 by denaturation of cellular polypeptides in response to a variety
of proteotoxic stresses, including heat shock, amino acid analogs, and
heavy metals.
Heat Shock-induced HSF1 Phosphorylation Is Not Prevented by
Rac1N17--
HSF1 has been known to be hyperphosphorylated upon
exposure to proteotoxic stresses such as heat shock and heavy metals
(4, 5, 11). Therefore, the effects of Rac1N17 on heat shock-induced HSF
phosphorylation were also examined. As shown in Fig.
7, HSF1 was hyperphosphorylated in cells
exposed to severe heat shock (43-45 °C) and cadmium chloride, while
its phosphorylation slightly increased in cells treated with mild heat
shock (39-41 °C) and Azc. Rac1N17 did not prevent HSF1
phosphorylation in response to these proteotoxic stresses, indicating
that HSF1 phosphorylation is not likely linked to the Rac1 signal
pathway.
Rac1V12 Does Not Activate HSP Expression and HSF
Activation--
Finally, we examined whether heat shock-induced Rac1
activation is sufficient for HSP synthesis and HSF activation. In
constitutively active Rac1V12 cells, however, no significant induction
of HSP synthesis and HSF activation was observed (Fig.
8). These results suggest that heat
shock-induced Rac1 activation may be necessary, but not sufficient, for
heat shock-induced HSP expression and HSF activation, or otherwise a
signal pathway(s) involving Rac1 may be indirectly linked to the
HSF activation.
A small GTPase Rac1, one of the Rho family GTPases, has been
implicated in a variety of cell regulation, including actin remodeling, gene transcription, and cell proliferation (21-28). In fact, a constitutively active mutant Rac1V12 stimulated cell proliferation, whereas a dominant negative mutant Rac1N17 repressed cell growth (Fig.
1B). Rac1 has also been shown to participate in the cellular response to stresses (16, 17, 30, 31). For example, upon exposure to
ceramide and Fas, cells induce apoptosis through activating the
Rac1-JNK signal pathway (29). In this study, we examined whether Rac1
is implicated in the heat shock response. We show that mild heat shock
(39-41 °C) induced membrane translocation and possibly activation
of Rac1 (Fig. 2A). In addition, mild heat shock
(39-42 °C) also induced membrane ruffling in a
Rac1-dependent manner (Fig. 2B), similar to that
observed in growth factor-treated cells (22, 23, 25). These results
suggest that mild heat shock can activate Rac1. It has been
demonstrated that PI 3-kinase and PAK, signal molecules that are
involved in the Rac1 signal pathway, are activated by heat shock (37,
38), further supporting heat shock activation of Rac1. However, it is
not clear how heat shock activates Rac1. Heat shock is known to
increase membrane fluidity and to alter the composition of cell
membrane (39-41), which may in turn lead to the activation of various
membrane proteins, including receptor tyrosine kinases. In fact, heat
shock was shown to activate EGF receptor tyrosine kinase in NIH-3T3
cells (37). These results allow us to speculate that multiple receptor
tyrosine kinases, including EGF receptor, can be activated by heat
shock, as shown by other stresses such as UV irradiation and osmotic shock (42), and their activation may result in the activation of Rac1.
Rac1N17 significantly prevented JNK activation in response to heat
shock, although the inhibitory effect of Rac1N17 was reduced as the
heat shock temperature increased (Fig. 3A). Since heat shock
activates Rac1 (Fig. 2A), and a dominant positive mutant Rac1V12 induces the activation of JNK (Fig. 1 A), mild heat
shock-induced JNK activation is likely regulated via Rac1 (Fig.
9). A number of signal pathways have been
reported that mediate JNK activation in response to external stimuli.
It has been well demonstrated by several investigators that JNK
activation by growth factors, inflammatory cytokines, and stressful
agents such as ceramide is mediated through Rac1 (21, 26-28). In
contrast, protein-damaging stresses such as sodium arsenite, oxidative
stress, and ethanol have been shown to induce JNK activation primarily
through inhibiting a putative JNK phosphatase (19, 20, 43). For
example, the reactive oxygen species (ROS) is able to directly inhibit
JNK phosphatase by oxidizing the SH group of the protein (19, 20). Similar to other proteotoxic stresses, heat shock has been suggested to
activate JNK through inhibiting a JNK phosphatase (19, 20). Since heat
shock can produce ROS (44), and the heat shock response such as HSP
expression is prevented by pretreatment of anti-oxidants (45), ROS may
serve as a mediator of the heat shock response. Furthermore, unlike
other Rho family GTPases, Rac1 is able to produce ROS through
activating NADPH oxidase (21, 46). Therefore, we examined in this study
whether ROS is involved in heat shock-induced JNK activation. However,
pretreatment of antioxidants such as ascorbic acid, butylated
hydroxytoluene, and n-propylgallate did not prevent JNK
activation by heat shock (data not shown). Thus, although Rac1 could
produce ROS through activating NADPH oxidase (46), heat shock-induced
JNK activation is likely regulated by the Rac1 signal pathway not
involving ROS and NADPH oxidase. PAK, one of Rac1 downstream effectors
and which is upstream of JNK, has been reported to be activated by heat
shock (38). Therefore, the Rac1-PAK signal pathway may be responsible
for heat shock-induced JNK activation. Alternatively, MKK7, which can
be activated by stresses and is upstream of JNK (47), may be
responsible for the heat shock-induced JNK activation (Fig. 9).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C. After thawing the pellet on ice, it was suspended
in buffer A (10 mM Tris-HCl, pH 7.9, 10 mM KCl,
1.5 mM MgCl2, 0.5 mM DTT). The
pellet was resuspended in modified buffer A (10 mM
Tris-HCl, pH 7.9, 10 mM KCl, 1.5 mM
MgCl2, 0.5 mM DTT, 0.1% Nonidet P-40). The
nuclei were collected by centrifugation at 5,000 rpm for 10 min at
0 °C. The nuclei were gently resuspended in buffer C (20 mM Tris-HCl, pH 7.9, 0.2 mM EDTA, 1.5 mM MgCl2, 0.5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 25% glycerol, 420 mM NaCl) and incubated for 15 min at 4 °C. The lysates
were centrifuged at 10,000 rpm for 10 min at 4 °C, and the
supernatants were diluted to modified buffer D (20 mM
Tris-HCl, pH 7.9, 50 mM KCl, 20% glycerol, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM
phenylmethylsulfonyl fluoride) and stored at
80 °C. Between
steps the samples were kept on ice. Double-stranded
oligonucleotides containing the HSE consensus sequence
(5'-GATCCTCGAATGTTCGCGAAAAG-3') were labeled using Klenow polymerase (Promega) and [
-32P]dCTP (Amersham
Pharmacia Biotech, 3,000 Ci/mmol, 10 mCi/ml). 20 µg of nuclear
protein was preincubated for 15 min at 0 °C in 17 µl of binding
buffer (20 mM Tris-HCl, pH 7.5, 5% glycerol, 40 mM NaCl, 4 mM MgCl2, 1 mM EDTA, 1 mM DTT, 0.05 mg/ml bovine serum
albumin), 1 µg of poly(dI-dC) and protease inhibitors (mixture solution; Sigma). Thereafter, binding reaction was performed for 40 min
at room temperature with 200 nCi of radioisotope-labeled oligonucleotide in a final volume of 20 µl. HSF·HSE
complexes were resolved by electrophoresis on a 4% acrylamide
(acrylamide/bisacrylamide, 29:1) gel at 30 mA for 1 h. Prior to
sample loading, the gel was run for 30 min at 20 mA. After
electrophoresis, the gel was exposed to an x-ray film for 12-24 h at
80 °C. For EMSSA, nuclear extracts were incubated with anti-HSF1
(SPA-901, StressGen Biotechnologies Corp.) up to 20 min at room
temperature prior to the addition of reaction mixture containing the
radioisotope-labeled HSE and analyzed by electrophoresis in a similar
fashion to EMSA.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Characterization of Rac1N17 and Rac1V12
cells. A, Rat-2 cells expressing wild type Rac1
(WT), a dominant negative Rac1N17, or a dominant positive
mutant Rac1V12 were prepared as described under "Experimental
Procedures." Cellular proteins were analyzed either by Western
blotting with anti-Rac1 (upper panel) or by JNK activity
assay using glutathione S-transferase-c-Jun-(1-79) as a
substrate (lower panel). B, Rat-2 wild type
(open circles), Rac1N17 (filled circle), and
Rac1V12 cells (filled squares) were plated at a density of
5 × 104 cells/ml, and the cell number was counted at
48 and 72 h after plating.
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Fig. 2.
Activation of Rac1 and induction of ruffling
by mild heat shock. A, Rat-2 wild type (WT)
and Rac1N17 cells were serum-starved for 24 h and exposed to 100 µg/ml EGF or heat shock (40 °C) for 10 min. The cells were
stained with anti-Rac1 antibody (Upstate Biotechnology) and fluorescein
isothiocyanate-conjugated secondary antibody and observed under a
confocal microscope (× 630, Carl Zeiss, LSM510). B, Rat-2
wild type (WT) and Rac1N17 cells were serum-starved for
24 h and exposed to 40 or 43 °C for 10 min. Then the cells were
fixed with 3.7% (w/v) formaldehyde. The fixed cells were permeabilized
with 0.2% Triton X-100 and stained with 0.165 M
NBD-phallacidin. The cells were observed with a confocal microscope (× 630, Carl Zeiss, LSM510).
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Fig. 3.
Inhibition by Rac1N17 of heat shock-induced
JNK activation. Rat-2 wild type (WT) and Rac1N17 cells
were exposed to heat shock of 39, 41, and 43 °C for the times
indicated. 200 µg of the protein extracts were immunoprecipitated
with anti-JNK antibody (Santa Cruz Biotechnology, Inc.) and subjected
to JNK activity assay with glutathione
S-transferase-c-Jun-(1-79) as a substrate (A).
Phosphorylation was quantified by densitometric scanning
(B). The kinase activity of wild type (black
bars) and Rac1N17 cells (gray bars) is expressed as the
value relative to that for the untreated wild type cells.
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Fig. 4.
Suppression by Rac1N17 of heat shock-induced
HSP expression. Rat-2 wild type (WT) and Rac1N17 cells
were exposed to either different heat shock temperatures (39, 41, and
43 °C) for 10 and 20 min (A and B) or 42 °C
for the times indicated (C). The cells were labeled with
[35S]methionine for 4 h at 37 °C during recovery
period, and the cell lysates were then analyzed by SDS-PAGE and
fluorography (A) or by immunoprecipitation with monoclonal
anti-HSP72 antibody (SPA-810, StressGen Biotechnologies Corp.)
(B and C).
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Fig. 5.
Inhibition by Rac1N17 of heat shock-induced
HSF1 activation. A, Rat-2 wild type cells were exposed
to 42 °C for 20 min. Twenty µg of nuclear proteins were
preincubated in the absence or presence of anti-HSF1 antibody and
incubated with 32P-labeled HSE consensus sequence
(5'-GATCCTCGAATGTTCGCGAAAAG-3') in a DNA binding buffer. After
separation on a 4% polyacrylamide gel, DNA-protein complexes were
visualized by autoradiography. B, Rat-2 wild type
(WT) and Rac1N17 cells were exposed to either 42 °C for
the times indicated or exposed to various temperatures for 20 min.
Twenty µg of nuclear proteins were then incubated with
32P-labeled HSE and analyzed by EMSA. Binding reaction was
also done with nuclear extracts from wild type cells exposed to
45 °C heat shock in the presence of 100-fold excess of competitor
DNA (×100 HSE). C, wild type (WT) and Rac1N17
Rat cells were cultured at normal growth condition and the cellular
proteins analyzed by SDS-PAGE and Western blotting with anti-HSF1 (Neo
markers).
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Fig. 6.
Implication of Rac1 in HSF activation in
response to proteotoxic stresses. A, Rat-2 wild type
cells were incubated in the presence or absence of 60 µg/ml
cycloheximide (CHX) for 2 h and then exposed to either
heat shock of 39-45 °C for 20 min or 5 mM Azc for
6 h, and the nuclear extracts were analyzed by EMSA. DNA-protein
complexes (arrowhead) were visualized by autoradiography.
B, suppression of L-azetidine-2-carboxylic acid
and heavy metal-induced HSF activation by Rac1N17. Rat-2 wild type
(WT) and Rac1N17 cells were treated with either 5 mM Azc for 6 h or 200 µM
CdCl2 (Cd) for 4 h. The nuclear extracts
were incubated with anti-HSF1 antibody and then analyzed by
electromobility shift assay. Binding reaction was also done with
nuclear extracts from Rat cells exposed to 5 mM Azc in the
presence of 100-fold excess of competitor DNA (×100 HSE).
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Fig. 7.
Rac1N17 does not inhibit heat shock-induced
phosphorylation of HSF1. Wild type (WT) and Rac1N17
cells were exposed to heat shock for 20 min, 5 mM Azc for
6 h, or 200 µM Cd for 4 h. The protein extracts
were analyzed by SDS-PAGE and Western blotting with anti-HSF1 antibody
(Neo markers).
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Fig. 8.
Rac1V12 does not stimulate HSP expression and
HSF activation. A, Rat-2 wild type (WT),
Rac1N17, and Rac1V12 cells were exposed to heat shock of 43 °C for
20 min and labeled with [35S]methionine at 37 °C for
4 h. The cellular proteins were immunoprecipitated with
anti-HSP72, and the immunoprecipitates were then analyzed by SDS-PAGE
and autoradiography. B, Rat-2 wild type (WT) and
Rac1V12 cells were exposed to 5 mM Azc for 6 h and
heat shock of 43 °C for 20 min, and the nuclear extracts were
analyzed by EMSA.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 9.
Roles of the Rac1 signal pathway in the heat
shock response. Each solid line and arrow
represents a step in an activating pathway. The T-shaped
line represents inactivation or inhibition.
We also show that HSF activation in response to heat shock is significantly prevented by Rac1N17, although the inhibitory effect of Rac1N17 on HSF activation is diminished in severe heat shock-treated cells (Figs. 4 and 5). These results suggest that a specific signal pathway involving Rac1 is likely linked to heat shock-induced HSF activation and HSP expression, while a Rac1-independent mechanism is also involved in the severe heat shock response. Rac1N17 also prevented HSF activation by L-azetidine-2-carboxylic acid, a proline analog, and heavy metals (CdCl2) (Fig. 6), indicating that Rac1 may be implicated in HSF activation by denaturation of polypeptides in response to proteotoxic stresses. In agreement with these findings, it has been demonstrated that mechanical stress-induced HSF activation and HSP expression in vascular smooth muscle cells are prevented by Rac1N17 (48). In addition, Ozaki et al. (2000) has recently shown that Rac1N17 prevents HSF1 activation and HSP expression in response to hypoxia/reoxygenation and sodium arsenite (49). They have also shown that ROS is involved in HSF activation in response to hypoxia/reoxygenation and sodium arsenite. However, we could not find any involvement of ROS in heat shock-induced HSF activation, since several different antioxidants such as ascorbic acid, butylated hydroxytoluene, n-propylgallate, diphenyleneiodonium, and pyrrolidine dithiocarbamate did not exert inhibitory effects on heat shock-induced HSF activation and HSP expression (data not shown). Therefore we suggest that although ROS may participate in HSF activation by hypoxia/reoxygenation and sodium arsenite, it is unlikely to be involved in heat shock-induced HSF activation and HSP expression.
Since Rac1N17 prevents HSF activation and HSP expression in response to a variety of stresses, it can be proposed that the Rac1 signal pathway may be directly linked to the activation of HSF. However, we show that a constitutively active Rac1V12 cannot induce HSF activation and HSP expression in the absence of stress (Fig. 8). A similar result was obtained in HepG2 cells transfected with Rac1V12 (49). Therefore, although heat shock activates Rac1, Rac1 activation may not be directly linked to HSF activation and HSP expression. Even though we do not know how Rac1 is involved in the heat shock response, we postulated two possibilities as follows. One is that besides activating Rac1, heat shock may activate other factor(s), which in combination with Rac1 regulate the heat shock-induced HSF activation. Another possibility is that a signal pathway(s) involving Rac1 may be indirectly linked to heat shock-induced HSF activation. Nonnative polypeptide chains appeared to be a common proximal inducer of HSF1 activation and HSP gene expression in the stressed cells (1, 9, 10). Heat shock and proteotoxic stresses trigger the rapid conversion of HSF1 from inert monomer to homotrimer, resulting in nuclear translocation and increased HSE binding affinity and hyperphosphorylation (4, 5, 11). Rac1N17 did not exert inhibitory effects on hyperphosphoryation in response to proteotoxic stresses such as heat shock and cadmium chloride, suggesting that the Rac signal pathway may be not linked to HSF1 phosphorylation (Fig. 7). Therefore, the signal pathway involving Rac1 may play a critical role(s) in the conversion of HSF from an inactive monomer to the transcriptionally competent form, such as trimer formation, nuclear translocation (Fig. 9).
In this study, we show that Rac1 may play a critical role(s) in several
aspects of the heat shock response, i.e. HSP expression and HSF
activation, JNK activation, and actin reorganization. Inhibitory
effects of Rac1N17 on the heat shock response were more prominent in
cells experiencing mild heat shock than those exposed to severe heat
shock. It is difficult to define the terms "mild" and "severe,"
because the effects of heat shock are determined by both heat shock
temperature and exposure time. As temperature increases by 1 °C, the
time required for the same extent of the heat shock response is reduced
by 2-fold (50). Thus, a severe heat shock response can be obtained by
increasing either heat shock temperature or exposure time. Mild heat
shock has been suggested to confer cells with a capacity to survive
against harsh heat shock or other harmful stresses (1), but the
molecular mechanism underlying this event is not still clear. Mild heat
shock can be seen in the regulation of body temperature in mammals,
including human, whose body temperature is finely regulated in a
homeostatic manner. Although a human gets a fever, body temperature
increases only by 1-2 °C. In such a case, fever can be considered
as mild heat shock. Therefore, Rac1-dependent signal
pathway may play an important role(s) in the physiological heat shock
response such as fever. Rac1 has been implicated in a variety of
cellular functions, including growth factor-induced actin
reorganization and cell proliferation, apoptosis, gene expression, and
survival (21-23). Thus, fever may function as an important signal,
which is required for resetting our body conditions, rather than acting as a proteotoxic stress. Further studies on functions of Rac1 in the
mild heat shock response may provide a new insight into the elucidation
of roles of heat shock in organism.
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ACKNOWLEDGEMENTS |
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We thank D. Y. Moon, J. H. Chung, and H. S. Kim for review of this manuscript and K. J. Lee for critical comments.
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FOOTNOTES |
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* This work was supported by Basic Science Research Institute Program BSRI-1998-15-D00248, Ministry of Education and Grant 96-0401-16-01-3 from the Korea Science and Engineering Foundation, Korea.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Molecular Biology, College of Natural Sciences, Pusan National University, Pusan 609-735, Korea. Tel.: 82-51-510-2275; Fax: 82-51-513-9258; E-mail: hspkang@hyowon.cc.pusan.ac.kr.
Published, JBC Papers in Press, October 24, 2000, DOI 10.1074/jbc.M006042200
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ABBREVIATIONS |
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The abbreviations used are: HSP, heat shock protein; HSF, heat shock factor; HSE, heat shock element; JNK, c-Jun N-terminal kinase; PAGE, polyacrylamide gel electrophoresis; ROS, reactive oxygen species; EMSA, electromobility shift assay; EMSSA, electromobility supershift assay; Azc, L-azetidine-2-carboxylic acid; PBS, phosphate-buffered saline; DTT, dithiothreitol; EGF, epidermal growth factor; NBD, 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl; TBS, Tris-buffered saline.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Welch, W. J., Kang, H. S., Beckmann, R. P., and Mizzen, L. A. (1991) Curr. Top. Microbiol. Immunol. 167, 31-55[Medline] [Order article via Infotrieve] |
2. | Herrmann, J. M., and Neupert, W. (2000) Curr. Opin. Microbiol. 3, 210-214[CrossRef][Medline] [Order article via Infotrieve] |
3. | Mosser, D. D., Carone, A. W., Bourget, L., Denis-Larose, C., and Massie, B. (1997) Mol. Cell. Biol. 17, 5317-5327[Abstract] |
4. | Wu, C. (1995) Annu. Rev. Cell Dev. Biol. 11, 441-469[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Morimoto, R. I.
(1998)
Genes Dev.
12,
3788-3796 |
6. | Abravaya, K., Myers, M. P., Murphy, S. P., and Morimoto, R. I. (1992) Genes Dev. 7, 1153-1164 |
7. | Zou, J., Guo, Y., Guettouche, T., Smith, D. F., and Voellmy, R. (1998) Cell 94, 471-480[Medline] [Order article via Infotrieve] |
8. |
Ali, A.,
Bharadwaj, S.,
O'Carroll, R.,
and Ovsenek, N.
(1998)
Mol. Cell. Biol.
18,
4949-4960 |
9. | Ananthan, J., Goldberg, A. L., and Voellmy, R. (1986) Science 232, 522-524 |
10. |
Dubois, M. F.,
Hoanessian, A. G.,
and Bersaude, O.
(1991)
J. Biol. Chem.
266,
9707-9711 |
11. | Baler, R., Dahl, G., and Voellmy, R. (1993) Mol. Cell. Biol. 13, 2486-2496[Abstract] |
12. |
Chu, B.,
Soncin, F.,
Price, B. D.,
Stevenson, M. A.,
and Calderwood, S. K.
(1996)
J. Biol. Chem.
271,
30847-30857 |
13. |
Dai, R.,
Frejtag, W.,
He, B.,
Zhang, Y.,
and Mivechi, N. F.
(2000)
J. Biol. Chem.
275,
18210-18218 |
14. | Kyriakis, J. M., Banerjee, P., Nikolakaki, E., Dai, T., Rubie, E. A., Ahmad, M. F., Avruch, J., and Woodgett, J. R. (1994) Nature 369, 156-160[CrossRef][Medline] [Order article via Infotrieve] |
15. | Kyriakis, J. M., and Avruch, J. (1996) Bioessays 18, 567-577[Medline] [Order article via Infotrieve] |
16. | Xia, Z., Dickens, M., Raingeaud, J., Davis, R. J., and Greenberg, M. E. (1995) Science 270, 1326-1331[Abstract] |
17. | Verheij, M., Bose, R., Lin, X. H., Yao, B., Jarvis, W. D., Grant, S., Birrer, M. J., Szabo, E., Zon, L. I., Kyriakis, J. M., Haimovitz-Friedman, A., Fuks, Z., and Kolesnick, R. N. (1996) Nature 380, 75-79[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Adler, V.,
Schaffer, A.,
Kim, J.,
Dolan, L.,
and Ronai, Z.
(1995)
J. Biol. Chem.
270,
26071-26077 |
19. | Meriin, A. B., Yaglom, J. A., Gabai, V. L., Zon, L., Ganiatsas, S., Mosser, D. D., Zon, L., and Sherman, M. Y. (1999) Mol. Cell. Biol. 19, 2547-2555 |
20. |
Nguyen, A. N.,
and Shiozaki, K.
(1999)
Genes Dev.
13,
1653-1663 |
21. |
Van Aelst, L.,
and Dsouza-Achorey, C.
(1997)
Genes Dev.
11,
2295-2322 |
22. | Tapon, N., and Hall, A. (1997) Curr. Opin. Cell Biol. 9, 86-92[CrossRef][Medline] [Order article via Infotrieve] |
23. |
Hall, A.
(1998)
Science
279,
509-514 |
24. | Ridley, A. J., and Hall, A. (1992) Cell 70, 389-399[Medline] [Order article via Infotrieve] |
25. | Ridley, A. J., Paterson, H. F., Johnston, C. L., Diekmann, D., and Hall, A. (1992) Cell 70, 401-410[Medline] [Order article via Infotrieve] |
26. | Olson, M. F., Ashworth, A., and Hall, A. (1995) Science 269, 1270-1272 |
27. | Lamarche, N., Tapon, N., Stowers, L., Burbelo, P. D., Aspenström, P., Bridges, T., Chant, J., and Hall, A. (1996) Cell 87, 519-529[Medline] [Order article via Infotrieve] |
28. |
Joneson, T.,
McDonough, M.,
Bar-Sagi, D.,
and Van Aelst, L.
(1996)
Science
274,
1374-1376 |
29. |
Brenner, B.,
Koppernhoefer, U.,
Weinstock, C.,
Linderkamp, O.,
Lang, F.,
and Gulbins, E.
(1997)
J. Biol. Chem.
272,
22173-22181 |
30. | Coso, O. A., Chiariello, M., Yu, Y., Teramoto, H., Crespo, P., Xu, N., Miki, T., and Gutkind, J. S. (1995) Cell 81, 1137-1146[Medline] [Order article via Infotrieve] |
31. | Minden, A., Lin, A., Claret, F. X., Abo, A., and Karin, M. (1995) Cell 81, 1147-1157[Medline] [Order article via Infotrieve] |
32. | Shin, E. A., Kim, K. H., Han, S. I., Ha, K. S., Kim, J. H., Kang, K. I., Kim, H. D., and Kang, H. S. (1999) FEBS Lett. 452, 355-359[CrossRef][Medline] [Order article via Infotrieve] |
33. | Kim, H. R., Kang, H. S., and Kim, H. D. (1999) IUBMB Life 48, 429-433[CrossRef][Medline] [Order article via Infotrieve] |
34. | Myat, M. M., Anderson, S., Allen, L. A., and Aderem, A. (1997) Curr. Biol. 7, 611-614[Medline] [Order article via Infotrieve] |
35. | Baler, R., Welch, W. J., and Voellmy, R. (1992) J. Cell Biol. 117, 1151-1159[Abstract] |
36. |
Tanabe, M.,
Nakai, A.,
Kawazoe, Y.,
and Nagata, K.
(1997)
J. Biol. Chem.
272,
15389-15395 |
37. |
Lin, R. Z.,
Hu, Z. W.,
Chin, J. H.,
and Hoffman, B. B.
(1997)
J. Biol. Chem.
272,
31196-31202 |
38. | Maroni, P., Bendinelli, P., Zuccorononno, C., Schiaffonati, L., and Piccoletti, R. (1999) Cell Biol. Int. 24, 145-152[CrossRef] |
39. | Vigh, L., Maresca, B., and Harwood, J. L. (1998) Trends Biochem. Sci. 23, 369-374[CrossRef][Medline] [Order article via Infotrieve] |
40. | Anderson, R. L., and Parker, R. (1982) Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 42, 57-69[Medline] [Order article via Infotrieve] |
41. |
Murakami-Murofushi, K.,
Nishikawa, K.,
Hirakawa, E.,
and Murofushi, H.
(1997)
J. Biol. Chem.
272,
486-489 |
42. |
Rosette, C.,
and Karin, M.
(1996)
Science
274,
1194-1197 |
43. | Kamata, H., and Hirata, H. (1999) Cell Signal. 11, 1-14[CrossRef][Medline] [Order article via Infotrieve] |
44. |
Davison, J. F.,
Whyte, B.,
Bissinger, P. H.,
and Schiestl, R. H.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
5116-5121 |
45. | Gorman, A. M., Heavey, B., Creagh, E., Cotter, T. G., and Samali, A. (1999) FEBS Lett. 445, 98-102[CrossRef][Medline] [Order article via Infotrieve] |
46. |
Mizuno, T.,
Kaibuchi, K.,
Ando, S.,
Musha, T.,
Hiraoka, K.,
Takaishi, K.,
Asada, M.,
Nunoi, H.,
Matsuda, I.,
and Takai, Y.
(1992)
J. Biol. Chem.
267,
10215-10218 |
47. |
Moriguchi, T.,
Toyoshima, F.,
Masuyama, N.,
Hanafusa, H.,
Gotoh, Y.,
and Nishida, E.
(1997)
EMBO J.
16,
7045-7053 |
48. |
Xu, Q.,
Schett, G.,
Li, C.,
Hu, Y.,
and Wick, G.,.
(2000)
Circ. Res.
86,
1122-1128 |
49. |
Ozaki, M.,
Deshpande, S. S.,
Angkeow, P.,
Suzuki, S.,
and Irani, K.,.
(2000)
J. Biol. Chem.
275,
35377-35383 |
50. | Dewey, W. C. (1989) Radiat. Res. 120, 191-204[Medline] [Order article via Infotrieve] |