From the Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan 30043, Republic of China
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ABSTRACT |
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We have previously shown that treatment with
okadaic acid (OA) followed by heat shock (HS) (termed OA HS
treatment) leads to rapid transactivation of the 78-kDa
glucose-regulated protein gene (grp78) in 9L rat brain
tumor cells. A cAMP-responsive element-like (CRE-like, TGACGTGA)
promoter sequence and a protein kinase A signaling pathway are involved
in this induction, and activation of both CRE binding protein (CREB)
and activating transcription factor-2 (ATF-2) is required in the above
process. Herein, we report that transactivation of grp78,
as well as phosphorylation/activation of ATF-2, can be completely
annihilated by SB203580, a highly specific inhibitor of p38
mitogen-activated protein kinase (p38MAPK). Activation of
p38MAPK by OA
HS is also substantiated by its own
phosphorylation as well as the phosphorylation and activation of MAPK
activating protein kinase-2 in cells subjected to this treatment. The
involvement of p38MAPK in the activation of ATF-2, which
leads to the transactivation of rat grp78, is confirmed by
electrophoretic mobility shift assay using a probe containing the
CRE-like sequence as well as by transient transfection assays with a
plasmid containing a 710-base pair stretch of the grp78
promoter. Together with our previous studies, these results led us to
conclude that phosphorylation/activation of CREB upon OA
HS
treatment is mediated by cAMP-dependent protein kinase,
whereas that of ATF-2 is mediated by p38MAPK. The
transcription factors may bind to each other to form heterodimers that
in turn transactivate grp78 by binding to the CRE-like
element. This suggests that distinct signaling pathways converge on
CREB-ATF-2, where each subunit is individually activated by a specific
class of protein kinases. This may allow modulation of
grp78 transactivation by diverse external stimuli.
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INTRODUCTION |
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The 78-kDa glucose-regulated protein (GRP78)1 is a calcium-binding molecular chaperone expressed in the endoplasmic reticulum of eucaryotic cells (1, 2). GRP78 is constitutively expressed, and its expression is enhanced up to 20-fold in cells under a variety of stressful conditions that deplete glucose or intracisternal calcium or otherwise disrupt glycoprotein trafficking (3-8). The above changes lead ultimately to accumulation of underprocessed or misfolded proteins in the endoplasmic reticulum and result in the induction of GRP78 (9). This protein is also induced by ethanol via a mechanistic pathway different from that of the "classical inducers" (10). By contrast, GRP78 is usually not induced by heat shock (HS) alone (11, 12).
The mammalian grp78 promoter, lacking any heat shock element, is regulated by a complex interplay of several cis-elements and protein factors binding to these sites (6, 11, 13-17). It has been shown that the highly conserved 36-base pair region within the mammalian grp78 promoter consists of CCAAT-like sequences flanked by GC-rich motifs that are important for basal and enhanced expression of this gene. Using this region as a probe for electrophoretic mobility shift assay (EMSA), at least two specific protein-DNA complexes are detected in the nuclear extracts of HeLa cells (14, 16). Moreover, transactivation of grp78 by several chemical stressors is found to correlate with changes in the activities of protein kinases and phosphatases (18-20). It should also be noted that the induction of grp78 expression by the classical inducers is slow and requires several hours of sustained treatment with lag phases of 3 h to more than 24 h.
We have demonstrated that okadaic acid (OA), a potent inhibitor of
protein phosphatases 1 and 2A, enhances the expression of GRP78 (8) as
well as potentiates the expression of GRP78 in heat-shocked 9L rat
brain tumor (RBT) cells (12). When followed by heat shock at 45 °C
for 15 min, GRP78 is induced within 60 min treatment with a low dose
(200 nM) of OA compared with prolonged treatment of several
hours with classical inducers (12). To understand the signal
transduction mechanism involved in the rapid induction of GRP78, we
determined the functionality of the cis-acting CRE-like
element of rat grp78 promoter in cells treated sequentially with OA and heat shock (OA HS, treatment of 200 nM OA
followed by heat shock at 45 °C for 15 min). We found that
OA
HS-induced GRP78 expression is regulated by binding of protein
factors in nuclear extract to the CRE-like element. The transcription
factors that interact with the grp78 CRE-like element have
been identified as activating factor-2-cAMP responsive element binding
protein (ATF-2-CREB) heterodimers and CREB homodimers, by antibody
interference and supershift assays (17).
In eucaryotic cells, transcriptional regulation upon stimulation of the
cAMP-dependent protein kinase (PKA) signaling pathway is
mediated by a family of cAMP-responsive nuclear factors including CREB
and ATF-2. These factors contain the basic domain/leucine zipper motifs
and bind as homo- or heterodimers to CRE or CRE-like elements (21). The
binding of CREB to CRE is insufficient to induce transcription.
Activation of transcription requires the phosphorylation of CREB at
Ser-133 (21-23). The transcription factor ATF-2 can not only
efficiently form DNA binding homodimers but also dimerize efficiently
with numerous members of the ATF family and the Jun/Fos family
(24-26). Stimulation of ATF-2-dependent transactivation
requires the phosphorylation of ATF-2 at Thr-69 and Thr-71, presumably
by the stress-activated protein kinase/Jun-N-terminal protein kinase
(27, 28). More recently, it has been demonstrated that ATF-2 is
efficiently phosphorylated by p38 mitogen-activated protein kinase
(MAPK) and that enhancement of ATF-2-dependent gene
expression by p38MAPK (one of the p38MAPK
isoforms) is approximately 20-fold higher than that of other MAPKs
tested (29).
p38MAPK belongs to the MAPK family which is activated through phosphorylation of specific tyrosine and threonine residues in response to mitogens or stress. Distinct MAPK cascades can be activated independently and simultaneously (30). p38MAPK is a potent activator of MAPK-activated protein kinase-2 (MAPKAPK-2), which phosphorylates the small heat shock protein (HSP27) (31-33). Besides MAPKAPK-2, activated p38MAPK phosphorylates transcription factors, including ATF-2, Max, CREB-homologue/growth arrest DNA damage 153 (CHDP/GADD153), important in the regulation cell growth and apoptosis (34). Recently, a group of pyridinyl imidazole compounds have been identified as highly specific inhibitors of p38MAPK (35-37). The inhibitory effect of these compounds toward p38MAPK is attributed to binding of the drug to or near the ATP binding pocket of the kinase (38). A related compound, SB203580, inhibits p38MAPK with an IC50 of 0.6 µM and exhibits no effect even at 100 µM on the activities of 12 other protein kinases tested, including extracellular-regulated protein kinase-2 and stress-activated protein kinase/Jun-N-terminal protein kinase (36). In the past 2 years, SB203580 has been extensively employed to explore the specific roles of p38MAPK in cellular responses in a variety of experimental systems (36, 39-47). For example, this signaling pathway has been demonstrated to be responsible in a variety of processes including platelet aggregation in response to collagen or thromboxane analogue (42), IL-6 synthesis in response to tumor necrosis factor (43), T cell proliferation in response to IL-2 and IL-7 (39), activation of polymorphonuclear leukocytes by chemotactic peptides (45), and phosphorylation of HSP27 upon stress (40, 41).
Herein we report that grp78 transcription induced by
OA HS in 9L RBT cells is
p38MAPK-dependent by using the highly specific
p38MAPK inhibitor SB203580. The OA
HS-induced ATF-2
phosphorylation is regulated by p38MAPK activity. This
activation is blocked by prior incubation with SB203580. These results
point to a novel p38MAPK signaling pathway leading to the
rapid transactivation of grp78 in 9L RBT cells. Moreover,
the synergistic action of PKA as well as p38MAPK on
regulation of rat grp78 are discussed.
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EXPERIMENTAL PROCEDURES |
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Materials--
OA was purchased from Life Technologies, Inc.
Cultureware was obtained from Corning (Corning, NY), and culture medium
components were purchased from Life Technologies, Inc.
[35S]Methionine (specific activity >800 Ci/mmol),
[-32P]dCTP (3,000 Ci/mmol), [
-32P]ATP
(5,000 Ci/mmol), and enhanced chemiluminescence (ECL) Western blotting
detection kit were from Amersham Corp. (Buckinghamshire, UK).
Polyclonal antibody against phosphorylated CREB (specific for
Ser(P)-133-CREB) was purchased from Upstate Biotechnology Inc. (Lake
Placid, NY). Antibodies for GRP78, p38MAPK, phosphorylated
p38MAPK, CREB, and ATF-2 were purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). Chemicals for electrophoresis
were from Bio-Rad. Other chemicals were purchased from Merck
(Darmstadt, Germany) or Sigma. SB203580 was a kind gift from SmithKline
Beecham Pharmaceuticals (King of Prussia, PA).
Cell Culture-- 9L RBT cells, derived from rat gliosarcoma (48), were a gift of Dr. M. L. Rosenblum (University of California at San Francisco) and were maintained in Eagle's minimum essential medium supplemented with 10% fetal calf serum, 100 units/ml penicillin G, and 100 µg/ml streptomycin. Prior to each experiment, stock cells were plated in 25-cm2 flasks or 6-well plates at a density of 4-6 × 104 cells per cm2. Exponentially growing cells at 80-90% confluency were used.
Drug and Heat Treatment--
For drug treatment, OA and SB203580
stock solutions were diluted with culture medium to the desired
concentration before adding to the cells, and the treatment was
performed at 37 °C. For heat shock, cells in flasks or plates were
sealed with Parafilm and submerged in a water bath pre-set at 45 ± 0.1 °C for 15 min. To study the combined effects of OA and heat
shock (OA HS treatment), cells were preincubated with 200 nM OA for 1 h and then heated at 45 °C for 15 min
in the presence of the drug. Alternatively, the cells were treated
according to various protocols as described below and in the figure
legends.
Metabolic Labeling and SDS-PAGE-- Alterations of protein synthesis and phosphorylation were revealed by [35S]methionine and [32P]orthophosphate labeling, respectively. In vivo 32P labeling was performed with 1 mCi of [32P]orthophosphate in 1 ml of labeling medium (phosphate-free DMEM containing 10% FCS) for 1 h prior to various treatments in the presence of the isotope. Synthesis of GRP78 protein in the treated cells was revealed by [35S]methionine labeling. After treatments, cells were washed twice and incubated with fresh medium at 37 °C for 8 h before labeling with 20 µCi of [35S]methionine in 1 ml of medium for 1 h. After labeling, cells were washed with PBS, lysed in sample buffer (49), and subjected to electrophoresis as described previously (8, 50). The gels were fixed, dried, and processed for autoradiography.
Immunoblot Analysis-- For immunoblot analysis, cell lysates were resolved using a mini-gel apparatus (Hoefer, San Francisco, CA). After electrophoresis, the proteins were electro-transferred onto a nitrocellulose membrane (Hybond-C Super, Amersham Corp.) and probed with antibodies against p38MAPK, phosphorylated p38MAPK, GRP78, CREB, phosphorylated CREB, and ATF-2, separately. The immune complexes were visualized using enhanced chemiluminescence (ECL) according to the manufacturer's protocol (Amersham Corp.).
RNA Isolation and Northern Blotting--
Total RNA was isolated
from 9L cells according to Chomczynski and Sacchi (51). After washing
with PBS, cells (5 × 106) were trypsinized, collected
by centrifugation, and lysed in 1.5 ml of RNA extraction buffer (0.5%
sarcosyl, 4 M guanidine thiocyanate, 0.1 M
-mercaptoethanol, and 25 mM sodium citrate). Total RNA
was extracted by sequential addition of one-tenth volume of 2 M sodium acetate, pH 4.0, an equal volume of
water-saturated phenol, and two-tenths volume of chloroform/isoamyl
alcohol (24:1) followed by precipitation of the aqueous phase with an
equal volume of isopropyl alcohol. The RNA pellets were then
re-dissolved in 0.3 ml of extraction buffer (without
-mercaptoethanol) and precipitated again with isopropyl alcohol. The
pellets were washed with 70% ethanol and dissolved in diethyl
pyrocarbonate-treated water. RNA concentrations were determined
spectrophotometrically at 260 nm. An equal amount of total RNA isolated
from different treatment conditions was blotted onto the nylon membrane
and fixed by using an ultraviolet (UV) cross-linker (Stratagene).
Template for the GRP78 hybridization probe was a polymerase chain
reaction product from isolated 9L genomic DNA. Polymerase chain
reaction primers for producing the GRP78 probe were
5
-TCGTGGCTCCTCCTG-3
(forward) and 5
-CAACCACCATGCCTA-3
according to
the exon 1 of rat grp78 gene. Rat glyceraldehyde-3-phosphate
dehydrogenase oligonucleotide probe was purchased from
CLONTECH (Palo Alto, CA). GRP78 and
glyceraldehyde-3-phosphate dehydrogenase oligonucleotide probes were
labeled with [
-32P]dCTP by Rediprime DNA labeling
system (Amersham Corp.). Following prehybridization, hybridization, and
autoradiography, membranes were stripped off probes by boiling in
0.1 × SSC (15 mM NaCl, 1.5 mM sodium
citrate, pH 7.0) containing 0.01% SDS for 20 min and then rehybridized
with other probes. Autoradiograms were quantified by densitometric
scanning in two-dimensional mode (Molecular Dynamics).
Construction of Plasmids and Transient Transfection
Assays--
The construction of pGRP78-BGL plasmid was derived from
ligation of a rat grp78 promoter fragment with the
pgal-Promoter reporter vector (CLONTECH). The
p
gal-Promoter vector contains the SV40 early promoter inserted
upstream of the lacZ gene. A 0.7-kilobase pair
BglII/KpnI fragment derived from rat
grp78 promoter containing the CRE-like element (17) was
inserted into the BglII/KpnI vector to generate
pGRP78-BGL. A negative control that lacked eucaryotic promoter and
enhancer sequences (p
gal-Basic) was also included.
Nuclear Extract Preparation--
Nuclear extracts were prepared
as described by Roy et al. (52) with some modifications
(17). In brief, approximately 5 × 108 cells were
trypsinized, collected by centrifugation at 1,000 × g
for 8 min at 4 °C, washed with PBS, and centrifuged. Cells were
suspended in 4 ml of nuclear extraction buffer I (250 mM sucrose, 15 mM Tris-HCl, pH 7.9, 140 mM NaCl, 2 mM EDTA, 0.5 mM EGTA, 0.15 mM
spermidine, 1 mM dithiothreitol, 0.4 mM
phenylmethylsulfonyl fluoride, 25 mM KCl, and 2 mM MgCl2) and homogenized by a Dounce grinder
with 3 strokes. Nonidet P-40 was then added to a final concentration of
0.5%, and the mixture was incubated on ice for about 5 min. Following
another round of homogenization (6 more strokes), nuclei and cell
debris were collected by centrifugation at 1,000 × g
for 8 min as described previously. Nuclei were washed with 5 ml of
buffer I and centrifuged as above. The nuclei were then lysed by
incubating the sample on ice for 5 min in 1 packed cell volume of
nuclear extraction buffer II (buffer I supplemented with 350 mM KCl), followed by a 25-stroke homogenization. The homogenate was transferred to 1.5-ml microcentrifuge tubes and centrifuged at 18,000 × g for 90 min. The supernatant
was dialyzed against dialysis buffer (20 mM Hepes, 100 mM KCl, 0.1 mM EDTA, 0.2 mM
phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, 20% glycerol) for 90 min at 4 °C and then stored frozen at 70 °C until use.
Electrophoretic Mobility Shift Assays (EMSA)--
CRE-like
element binding activities of the nuclear extracts were determined by
EMSA using double-stranded oligonucleotide as probes. The CRE-like
oligonucleotide probe was prepared by annealing
5-GCGTACCAGTGACGTGAGTTGCGGAGG-3
with its complementary strand,
followed by end labeling with T4 polynucleotide kinase. Each gel shift
reaction was carried out in a 20-µl volume in the presence of binding
buffer (15 mM Hepes, pH 7.9, 100 mM KCl, 3 mM MgCl2, 1 mM EDTA, 0.5 mM dithiothreitol, 10% glycerol, and 2 µg of
poly(dI-dC)). Nuclear extract was added to the binding buffer, and the
samples were incubated on ice for 15 min. After incubation, the DNA
probe (2 × 104 cpm for each reaction) was added and
incubated at room temperature for 20 min. The reaction mixtures were
loaded onto a 5% (40:1) polyacrylamide gel which had been pre-run for
30 min in 0.5 × TBE buffer (50 mM Tris, 50 mM boric acid, 1 mM EDTA, pH 7.0) at 150 V. 4%
nondenaturing PAGE was run at 150 V for 1.5 h at 4 °C. After
electrophoresis, the gels were dried and exposed to x-ray film.
In Vivo Labeling and Immunoprecipitation--
For in
vivo metabolic labeling, 9L cells were incubated with 1 mCi of
[32P]orthophosphate in 1 ml of phosphate-free DMEM, 10%
fetal bovine serum for 1 h before OA HS treatment. After
treatment, cells were rapidly chilled on ice, washed twice with
ice-cold PBS, and lysed in lysis buffer (20 mM Hepes, pH
7.9, 5 mM EDTA, 10 mM EGTA, 5 mM
NaF, 0.1 µg/ml microcystin-LR, 10% glycerol, 1 mM
dithiothreitol, 0.4 M KCl, 0.4% Nonidet, P-40 and protease
inhibitors as follows: 5 µg/ml leupeptin, 5 µg/ml aprotinin, 5 µg/ml pepstatin, 1 mM benzamidine, 50 µg/ml
phenylmethylsulfonyl fluoride) for 10 min on ice. Insoluble material
was removed by centrifugation (10,000 × g, 20 min,
4 °C). The protein concentration of the cell lysate was determined
by the Bradford assay (Pierce). An equal amount of cell lysate was
incubated with 6 µg of anti-MAPKAPK-2 or anti-ATF-2 antibody for
2 h at 4 °C. Immune complexes were precipitated with protein
G-Sepharose (Pharmacia Biotech Inc.), washed twice with lysis buffer,
and mixed with equal volume of 2 × sample buffer (49). The
phosphorylation of the MAPKAPK-2 or ATF-2 was examined after 10%
SDS-PAGE followed by autoradiography and densitometric analysis
(Molecular Dynamics).
Assays for MAPKAPK-2 Activity--
The MAPKAPK-2 activity in
cell-free lysate prepared from 9L cells was analyzed. After OA HS
treatment and recovery, cell lysate was prepared by sonication in lysis
buffer and then clarified by centrifugation. Protein concentration of
the cell lysate was determined by Bradford assay (Pierce). An equal
amount of cellular proteins was incubated with 6 µg of anti-MAPKAPK-2
or anti-CREB antibody for 2 h at 4 °C. Immunocomplexes were
precipitated with protein G-Sepharose and washed twice with lysis
buffer and once with assay buffer (20 mM MOPS, pH 7.2, 25 mM
-glycerol phosphate, 5 mM EGTA, 1 mM Na3VO4, 1 mM
dithiothreitol). For the kinase assay, 5 µg of substrate peptide
(KKLNRTLSVA) or 1 µg of immunoprecipitated CREB, 0.1 mM
ATP, 15 mM MgCl2, and 10 µCi/ml
[
-32P]ATP were incubated in a total volume of 100 µl, and the reaction was allowed to proceed at 30 °C for 10 min.
The labeled proteins were analyzed by autoradiography and densitometric
analysis.
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RESULTS |
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SB203580 Abolishes OA HS-induced GRP78 Expression and grp78
Promoter Activity--
To evaluate the possible role of
p38MAPK in the rapid induction of GRP78 by OA
HS, we
tested the effect of SB203580, a highly specific inhibitor of
p38MAPK, on OA
HS-induced GRP78 production and
grp78 gene expression in 9L cells by metabolic labeling with
[35S]methionine and Western blotting. We found that, in
the presence of SB205380, GRP78 synthesis and its accumulation were
almost completely diminished in OA
HS-treated cells (Fig.
1, A and B). The
cell viability was unaffected by the dose of SB203580 used (data not
shown), but protein translation was significantly inhibited at higher
concentrations (up to 50 µM) (Fig. 1A). We have previously demonstrated that OA
HS augments GRP78 production primarily by an increase in the transcription rate. To investigate the
level at which SB203580 affects production of GRP78 in
OA
HS-treated 9L cells, we performed a Northern blot analysis of
total cytoplasmic RNA using a 32P-labeled rat
grp78 probe. Cells were pretreated for 1 h with an
increasing concentration of SB203580 (up to 100 µM),
followed by OA
HS treatment. The induction of grp78
mRNA increased markedly at 1 h recovery after OA
HS
treatment and could be completely prevented by SB203580 (Fig.
2, A and B). A 50%
inhibition was observed at about 5 µM concentration of
the drug and almost complete inhibition at 20 µM (Fig.
2B). We also determined whether the transcriptional activity
of rat grp78 promoter was activated by OA
HS treatment
and whether the activation was affected by prior treatment of SB203580.
The 710-base pair grp78 promoter-
-galactosidase reporter
construct (designated as pGRP78-BGL) was, therefore, tested in the
transient transfection assay. pGRP78-BGL showed much higher activity in
cells after 1 h recovery of OA
HS stimulation (69.19 ± 7.46 units/mg protein) compared with either OA or heat shock alone
(8.36 ± 2.17 and 23.47 ± 3.27 units/mg protein,
respectively). The degree of promoter activation induced by OA
HS
was markedly reduced to that of heat shock stimulation in cells
subjected to prior treatment with SB203580 (27.63 ± 5.61 units/mg
protein) (Fig. 3). However, we normally
did not observe enhanced expression of grp78 mRNA in
heat-shocked cells, and the increase of
-galactosidase activity upon
heat shock may be due to the temperature effect on
-galactosidase.
These data clearly demonstrate that the induction of GRP78 by
OA
HS treatment can be completely prevented by SB203580, indicating the involvement of p38MAPK in this process.
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OA HS Activates the p38MAPK Signal Transduction
Pathway in 9L RBT Cells--
To demonstrate that OA
HS activates
the p38MAPK pathway in 9L rat brain tumor cells, we studied
the effect of OA
HS on phosphorylation of p38MAPK in
cells recovering from the treatment. The tyrosine phosphorylation of
p38MAPK was determined by double immunoblotting using a
phospho-p38MAPK antibody and anti-p38MAPK
antibody to assess the changes in phosphorylation and total expression of p38MAPK. The OA
HS treatment resulted in a
7.5-fold increase in p38MAPK phosphorylation on Tyr-182 and
was reached maximum at 30 min of recovery (Fig.
4, A and B),
whereas the amount of p38MAPK remained constant during the
treatment (Fig. 4A). Furthermore, preincubation of the cells
with 20 µM SB203580 had negligible effect on the
phosphorylation of p38MAPK, demonstrating that the
inhibitor does not interfere with the upstream activators of
p38MAPK (Fig. 4). This experiment provides unique evidence
that in 9L cells, OA
HS treatment results in Tyr-182
phosphorylation of p38MAPK.
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p38MAPK Dependence of OA HS-induced ATF-2
Phosphorylation--
We have previously found that, in
OA
HS-treated cells, ATF-2 protein is markedly increased, and it
binds to the CRE-like element of rat grp78 promoter. To test
whether ATF-2 is phosphorylated by p38MAPK in
vivo, we examined the effect of OA
HS in ATF-2
phosphorylation. We measured the phosphorylation level of ATF-2 in
[32P]orthophosphate-labeled 9L cells with or without
pretreatment of 20 µM SB203580. ATF-2 was
immunoprecipitated from 9L cell lysates and subjected to SDS-PAGE. We
found that treatment with OA
HS caused a significant increase in
ATF-2 phosphorylation (Fig. 6), which was
completely abolished by preincubation of the cells with SB203580.
However, prior treatment of SB203580 did not affect the increase in
ATF-2 induced by OA
HS (Fig. 6, lane 3).
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SB203580 Prevents the Factor Occupancy of the CRE-like Element in
grp78 Promoter Induced by OA HS Treatment--
We further
analyzed whether the factor occupancy of CRE-like element induced by
OA
HS was affected by prior treatment of SB203580. We examined
DNA binding activity in nuclear extracts from 9L cells, subjected to
OA
HS treatment with or without pretreatment with SB203580, by
using CRE-like element-containing oligonucleotide derived from rat
grp78 promoter. The upper band, identified as ATF-2/CREB
heterodimer previously, was absent in SB203580 pretreated nuclear
extract (Fig. 7). Whereas the binding factor in the lower band, determined to be CREB only, was almost unaffected. Taken together, these data confirm the hypothesis that
p38MAPK is the upstream molecule of the transcription
factor ATF-2 and is responsible for the rapid transactivation of
grp78 gene in 9L cells by sequential treatment with OA and
heat shock.
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OA HS-induced MAPKAPK-2 Activity Has Little Effect on the
Phosphorylation Level of CREB and the Kinase Does Not Phosphorylate
CREB in Vitro--
To assay the activation of MAPKAPK-2 in
OA
HS-treated cells and for studying whether the kinase exhibited
activity toward CREB, we immunoprecipitated the enzyme and CREB, the
substrate, from 9L cell extracts. As earlier, OA
HS strongly
activated MAPKAPK-2 within 15 min, and the activation of the kinase was annihilated by preincubation with SB203580 (Fig.
8). Furthermore, activated MAPKAPK-2,
which is strongly activated in OA
HS-treated cells, does not
phosphorylate the CREB protein in vitro (Fig. 8,
bottom panel).
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DISCUSSION |
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Herein we show that the p38MAPK pathway is activated
by OA HS treatment and that this pathway is involved in the
OA
HS-induced rapid transactivation of the grp78 gene
and expression of GRP78 in 9L RBT cells. This has been achieved by
exploiting SB203580, a highly specific inhibitor of p38MAPK
(36). OA
HS increased the phosphorylation of p38MAPK
which, in turn, promoted the activation (phosphorylation) of its
downstream effector, MAPKAPK-2, and the transcription factor, ATF-2.
Both activation of MAPKAPK-2 and phosphorylation of ATF-2 were
prevented by SB203580, confirming the OA
HS-induced activation via p38MAPK signaling pathway.
Thus far three MAPK subgroups have been characterized in mammalian
cells as follows: extracellular-regulated protein kinases, stress-activated protein kinase/Jun-N-terminal protein kinases, and
p38MAPKs. These kinases are at the center of three distinct
but closely related phosphorylation cascades, playing critical roles in
signal transductions (53, 54). p38MAPK was first identified
as the signaling system in response to lipopolysaccharide in human
monocytes, leading to the production of IL-1 and tumor necrosis
factor- (55, 56). Sequence comparison of p38MAPK with
other MAPKs led to identification of a Thr-Gly-Tyr (TGY) dual
phosphorylation motif in p38MAPK distinct form other MAPKs
having Thr-Glu-Tyr (TEY) or Thr-Pro-Tyr (TPY) dual phosphorylation
motifs. This points to the uniqueness of this subgroup of MAPK and
their distinct activation mechanism (57-61). In the present study, we
have demonstrated that p38MAPK provides a signal necessary
for rapid activation of grp78 gene caused by sequential
treatment of a relatively low dose of OA (200 nM for 1 h) and heat shock (45 °C for 15 min) in specific order. We also
found that OA
HS treatment induces phosphorylation of
p38MAPK in 9L cells. Phosphorylation of p38MAPK
is detected within 1 h after treatment, and the process is blocked by pretreatment of cells with a protein tyrosine kinase inhibitor herbimycin A (data not shown). Several upstream kinases, including the
dual specific kinases MAPK kinase-3 (MKK3), MKK4, and MKK6, have been
implicated in the phosphorylation and activation of p38MAPK
(62-64). Herein, we have not identified the upstream effector(s) of
p38MAPK in the transactivation of rat grp78, and
further investigation is warranted.
Activation of the p38MAPK pathway leads to the
phosphorylation and activation of MAPKAPK-2 and ATF-2. Currently, three
isoforms of p38MAPK have been identified. Although ATF-2
can be phosphorylated by p38MAPK and
p38MAPK
, in vitro and in vivo
experiments show a strong substrate preference by
p38MAPK
for ATF-2. It has been shown that enhancement of
ATF-2-dependent gene expression by p38MAPK
is approximately 20-fold greater than that of p38MAPK
and other MAPKs tested (29). On the other hand, transcription factor
ATF-2 is phosphorylated in vitro by p38MAPK on
Thr-69 and Thr-71 (62, 65). Phosphorylation of ATF-2 at these sites
increases the transcriptional activation potential (27, 28). In
addition to forming DNA binding homodimers, ATF-2 also efficiently
forms heterodimers with numerous other members of the ATF family as
well as members of the Jun/Fos family (28, 66-72). Such promiscuity in
dimerization makes ATF-2 an important constituent of factor complexes
exhibiting subtle differences in DNA binding specificity and regulatory
potential. Indeed, a number of studies have demonstrated different
binding properties of ATF-2 homodimers, ATF-2/c-Jun heterodimers, and
ATF-2/C/EBP
heterodimers (24-26, 72).
Previously, we have shown that both ATF-2-CREB heterodimer and CREB
homodimer, referred to as the complex I and II in the gel shift assays,
are involved in the transactivation of rat grp78 gene in 9L
RBT cells treated by OA HS. Activation of CREB is apparently
mediated by the PKA pathway since binding activity to EMSA probe
containing the CRE-like sequence is completely abolished by H-89, a
specific inhibitor of PKA (17). Although it has been shown that
MAPKAPK-2 phosphorylates CREB at Ser-133 in vitro (73), we
did not observe an increase in phosphorylation of CREB by MAPKAPK-2 activated by OA
HS. This indicates that CREB is phosphorylated by
PKA but not by the kinases in the p38MAPK pathway. A simple
explanation for these observations is that HS activates CREB via a PKA
signaling pathway, and OA activates ATF-2 via a p38MAPK
signaling pathway. Phosphorylation of CREB by PKA and ATF-2 by p38MAPK activates the transcription factors which bind to
each other to form heterodimers that in turn transactivate the
grp78 by binding to the CRE-like element. Taken together,
our data demonstrate that distinct signaling pathways converge on
CREB-ATF-2, where each subunit is individually activated by a specific
class of protein kinases. This may allow modulation of grp78
transactivation by a diverse external stimuli. It should be noted that
there are other regulatory elements in the promoter of the
grp78 gene; therefore, the functions of these elements as
well as the cooperativity of the corresponding transcription factors,
including the CREB-ATF-2, warrant further investigation. Our system
provides a detailed analysis of the mechanism leading to the rapid
transactivation of rat grp78 gene.
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ACKNOWLEDGEMENT |
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We thank Dr. A. A. Vyas for valuable comments on this manuscript.
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FOOTNOTES |
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* This work was supported in part by Research Grants NSC87-2311-B007-012 and NSC87-2311-B007-001-B12 (to Y.-K. L.) from the Republic of China National Science Council.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 Life Science,
National Tsing Hua University, Hsinchu, Taiwan 30043, Republic of
China. Fax: 886-3-5715934; E-mail: lslyk{at}life.nthu.edu.tw; URL:
http://life.nthu.edu.tw/.
1 The abbreviations used are: GRP78, 78-kDa glucose-regulated protein; OA, okadaic acid; HS, heat shock; HSP, heat shock protein; RBT cells, rat brain tumor cells; CRE, cyclic-AMP responsive element; PAGE, polyacrylamide gel electrophoresis; EMSA, electrophoretic mobility shift assay; ATF-2, activating factor-2; CREB, CRE binding protein; PKA, protein kinase A or cyclic AMP-dependent protein kinase; p38MAPK, p38 mitogen-activated protein kinase; MAPKAPK-2, MAPK activating protein kinase-2; IL, interleukin; ECL, enhanced chemiluminescence; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5(4-pyridyl) imidazole; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; MOPS, 4-morpholinepropanesulfonic acid.
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