From the National Creative Research Initiatives
Center for ARS Network, Sung Kyun Kwan University, Suwon, Kyunggido
440-746, Korea and the § National Creative Research
Initiatives, Center for Cell Death, Graduate School of Biotechnology,
Korea University, Seoul 136-701, Korea
Received for publication, July 13, 2000, and in revised form, October 26, 2000
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ABSTRACT |
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Glutamine has been known to be an apoptosis
suppressor, since it blocks apoptosis induced by heat shock,
irradiation, and c-Myc overexpression. Here, we demonstrated that HeLa
cells were susceptible to Fas-mediated apoptosis under the condition of
glutamine deprivation. Fas ligation activated apoptosis
signal-regulating kinase 1 (ASK1) and c-Jun N-terminal kinase (JNK;
also known as stress-activated protein kinase (SAPK)) in Gln-deprived
cells but not in normal cells, suggesting that Gln might be involved in
the activity control of ASK1 and JNK/SAPK. As one of the possible mechanisms for the suppressive effect of Gln on ASK1, we investigated the molecular interaction between human glutaminyl-tRNA synthetase (QRS) and ASK1 and found the Gln-dependent association of
the two molecules. While their association was enhanced by the
elevation of Gln concentration, they were dissociated by Fas ligation
within 5 min. The association involved the catalytic domains of the two enzymes. The ASK1 activity was inhibited by the interaction with QRS as
determined by in vitro kinase and transcription assays. Finally, we have shown that QRS inhibited the cell death induced by
ASK1, and this antiapoptotic function of QRS was weakened by the
deprivation of Gln. Thus, the antiapoptotic interaction of QRS with
ASK1 is controlled positively by the cellular concentration of Gln and
negatively by Fas ligation. The results of this work provide one
possible explanation for the working mechanism of the antiapoptotic
activity of Gln and suggest a novel function of mammalian ARSs.
Apoptosis is a genetically regulated process that is essential for
correct morphogenesis during embryogenesis and the maintenance of
tissue homeostasis (1, 2). Since dysregulation of apoptosis has been
implicated in autoimmune disease and atherosclerosis as well as
neurodegenerative disorders and cancer (3-5), tumor suppressor and
proapoptotic genes should tightly regulate apoptosis (6). Apoptosis is
induced by cytokines such as Fas ligand and tumor necrosis factor
(TNF),1 growth factor
withdrawal, ischemia, and amino acid deprivation (5, 7, 8).
Glutamine is a nonessential amino acid, but it is heavily utilized as a
major metabolic fuel as well as a precursor for nucleotide synthesis in
fibroblasts, lymphocytes, and macrophages (9). Since Gln deprivation
induces apoptosis in intestinal epithelial cells, whereas methionine
deprivation does not (8), Gln might play an important role in
protecting cells from apoptosis induced by different stimuli. For
example, Gln supplementation reduces apoptosis induced by heat shock,
irradiation, and c-Myc overexpression (10-12). Furthermore, Gln
stimulates intestinal cell proliferation and activates
mitogen-activated protein kinases such as p42/p44 MAPK and JNK/SAPK
(13, 14), suggesting that Gln could regulate signal transduction
pathways for cellular proliferation and apoptosis. However, the
molecular mechanism of Gln in suppressing apoptosis and stimulating
cellular proliferation remains to be explained.
Aminoacyl-tRNA synthetases (ARSs) catalyze aminoacylation of their
cognate tRNAs and thus play an essential role in protein synthesis.
ARSs have been found in cytoskeleton- or endoplasmic reticulum-associated structures or in cytoplasm, but they are also
found in the nucleus and even in the nucleolus (15-17), suggesting that ARSs have various noncanonical functions in addition to tRNA aminoacylation from eukaryotes. In particular, methionyl-tRNA synthetase is involved in rRNA biogenesis at its localization site in
the nucleolus (17).
Upon cellular exposure to apoptosis condition, mammalian tyrosyl-tRNA
synthetase is secreted and split into two fragments with distinct
cytokine activities by leukocyte elastase and extracellular protease
(18, 19). The split cytokines contain an endothelial monocyte-activating polypeptide II-like domain and an
interleukin-8-like domain. Thus, tyrosyl-tRNA synthetase has a potent
apoptosis-inducing activity after proteolytic cleavage, since their
split polypepetides stimulate the production of TNF and tissue
factor from target cells and have leukocyte chemotaxis activity to
scavenge apoptotic corpses. The precursor of endothelial
monocyte-activating polypeptide II is associated with the N-terminal
noncatalytic extension of arginyl-tRNA synthetase and enhances
aminoacylation activity (20). Like human tyrosyl-tRNA synthetase, the
cytokine domain of this precursor is released upon apoptosis and exerts
its proapoptotic function (21, 22).
Here, we studied a novel regulatory role of human glutaminyl-tRNA
synthetase (QRS) in inhibiting apoptosis signal-regulating kinase 1 (ASK1). We first searched for the effect of Gln in suppressing Fas-mediated apoptosis and reducing the activation of ASK1 as well as
JNK/SAPK. We also observed that QRS inhibits ASK1 kinase activity and
apoptosis by binding to ASK1. The molecular interaction of QRS and ASK1
was dependent on glutamine concentration, suggesting that glutamine
could repress Fas-mediated apoptosis and ASK1 activation via QRS.
Materials--
QRS (native N-terminal 236 amino acids) was
overexpressed as a His-tagged protein using Escherichia coli
and then purified using nickel affinity chromatography following the
manufacturer's protocol (Invitrogen). Rabbit polyclonal antibody was
then raised against these proteins as described previously (20). The
IgG from antiserum was purified by protein A affinity chromatography according to the manufacturer's protocol (Bio-Rad). Anti-hemagglutinin (HA), -ASK1, and -Myc antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-JNK/SAPK, p38, phospho-p38, p42/p44 MAPK, and phospho-p42/p44 MAPK antibodies were from New England
Biolabs. Anti-Fas antibody for apoptosis induction was obtained from
Upstate Biotechnology, Inc. (Lake Placid, NY). The caspase-3
colorimetric assay kit was obtained from Promega.
The cDNAs encoding the full-length, N-terminal 236 amino acids
(QRS-N) and C-terminal 539 amino acids (QRS-C) of human QRS were
generated using polymerase chain reaction from pM191 (Dr. Shiba, Cancer
Institute, Tokyo, Japan) and ligated into pcDNA3 (Invitrogen)
containing Myc and FLAG tags using the EcoRI and NotI sites. The cDNAs encoding the full-length,
N-terminal 649 amino acids (ASK1-N) and C-terminal 725 amino acids
(ASK1-C) of human ASK1 were cloned into pcDNA3 with an HA epitope
tag at the 3'-end (kind gift of Dr. Hidenori Ichijo, Tokyo Medical and
Dental University).
Cell Cultures, DNA Transfection, and
Immunoprecipitation--
Human embryonic kidney 293 and HeLa cells
were grown in Dulbecco's modified Eagle's medium supplemented with
10% heat-inactivated fetal bovine serum and 50 µg/ml penicillin and
streptomycin in a 5% CO2 incubator. 100-mm dishes of 293 cells were transfected with pcDNA3-HA-ASK1 and pcDNA3-Myc-QRS
using Geneporter (Gene Therapy Systems) according to the
manufacturer's protocol. Twenty-four hours after transfection, cells
were lysed with 20 mM Tris-HCl (pH 7.5) buffer containing
12 mM ASK1 Kinase Assay and Reporter Gene Assay--
The kinase
activity of ASK1 was determined using the immunoprecipitated ASK1
prepared as described above. The precipitated ASK1 was washed three
times with 20 mM Tris-HCl (pH 7.5) buffer containing 150 mM NaCl, 5 mM EGTA, 2 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 1%
Triton X-100 and once with a reaction buffer of 20 mM
Tris-HCl (pH 7.5) and 20 mM MgCl2. The reaction
was carried out in the reaction buffer in the presence of 0.5 µCi of
[
For the serum response element-Luc reporter gene assay, 293 cells grown
in 24-well plates were cotransfected with the indicated plasmids with
50 ng of serum response element-Luc reporter construct. After an
incubation of 24 h, cells were lysed in 150 µl of reporter lysis
buffer (Promega), and 20 µl of the lysate was assayed in a
Luminometer (MicrolumatPlus; EG&G) with a luciferase assay system (Promega).
DNA Fragmentation Assay--
To determine the degradation of
chromosomal DNA into nucleosome-sized fragments, a 500-µl aliquot of
the lysis buffer (100 mM Tris-HCl, pH 8.5, 5 mM
EDTA, 0.2 M NaCl, 0.2% SDS, and 0.2 mg/ml proteinase K)
was added to the cell pellet (2 × 105 cells) and
incubated at 37 °C overnight. DNA was obtained by ethanol
precipitation, separated in a 1.8% agarose gel, and visualized under
UV light.
Measurement of Caspase-3 Activity--
Caspase-3 activity was
measured according to the manufacturer's protocol. HeLa cells in
100-mm dishes were lysed with 300 µl of chilled cell lysis buffer.
After microcentrifugation (14,000 × g, 20 min,
4 °C), 60 µg of total protein from the clear supernatant was mixed
with 32 µl of caspase assay buffer, 2 µl of Me2SO, 10 µl of 100 mM dithiothreitol, and 2 µl of 10 mM Asp-Glu-Val-Asp-p-nitroanilide. After
incubation at 37 °C for 4 h, samples were read at 405 nm.
Apoptosis Assay--
293 cells were transiently transfected with
0.5 µg of pcDNA3-HA-ASK1-C and 1 µg each of
pcDNA3-Myc-QRS-F, -N, and -C along with 0.5 µg of
pcDNA3-EGFP. Total amounts of the transfected DNA were adjusted to
be the same with pcDNA3. The cells were washed with PBS twice
20 h after transfection, and then normal or Gln-free medium was
added. The transfected cells were fixed with 3% paraformaldehyde with
4,6-diamidino-2-phenylindole 48 h after transfection, and then the
cell death was determined by counting the apoptotic nuclei using
fluorescence microscopy.
Glutamine Deprivation Sensitizes HeLa Cells to Fas-mediated
Apoptosis--
Since c-Myc-dependent apoptosis is reduced
by Gln supplementation (12) and is dependent on Fas signaling (24, 25),
it is tempting to conjecture that Gln itself suppresses Fas-mediated apoptosis. To address this issue, we incubated HeLa cells in Gln-free medium overnight and administrated anti-Fas antibody for inducing apoptosis after supplementing Gln (0 and 4 mM,
respectively). Following the treatment with anti-Fas for 8 h, cell
morphology was observed under an inverted microscope. Blebbing
morphology appeared in cells in the absence of Gln but not in the
presence of Gln (Fig. 1A).
Since the blebbing morphology was not observed in cells untreated with
anti-Fas antibody, Gln deprivation makes cells susceptible to apoptosis
by Fas ligation. The same results were obtained with respect to
intranucleosomal DNA laddering. Following treatment with anti-Fas
antibody for 24 h, DNA fragmentation appeared in cells incubated
in Gln-free medium (Fig. 1B). However, there was very little
DNA fragmentation in cells untreated with anti-Fas or supplemented with
Gln.
To make these data more convincing, the extent of apoptosis activation
after anti-Fas treatment was determined by monitoring caspase-3. Cells
incubated in Gln-free medium showed higher caspase-3 activity than
those supplemented with Gln, and only the cells in Gln-free medium were
sensitive to Fas-induced apoptosis (Fig. 1C). Apoptosis of
HeLa cells in Gln-free medium was increased by the anti-Fas antibody
treatment in a dose-dependent manner, whereas the cells in
the normal medium were not sensitive to Fas ligation (Fig.
1D). Based on these data, we conclude that Gln suppresses
apoptosis induced by Fas ligation.
Glutamine Deprivation Sensitizes HeLa Cells to SAPK and ASK1
Activation by Fas Ligation--
To explore the molecular mechanism
for suppressing Fas-mediated apoptosis by Gln, we investigated
the activation/phosphorylation of p42/p44 MAPK, JNK/SAPK, p38 MAPK, and
ASK1 after Fas ligation in cells incubated with or without Gln. To
measure the activation status of endogenous JNK/SAPK and ASK1, we
performed an in vitro kinase assay using polyclonal
antibodies that specifically recognize the phosphorylated active forms
of these enzymes. The phosphorylation of p42/p44 MAPK was shown in 10 min after anti-Fas antibody treatment both in cells grown with and
without Gln (Fig. 2, top
row). However, p38 MAPK was not phosphorylated after Fas
ligation in either treatment of the cells (data not shown). Next, we
analyzed JNK/SAPK activation by an immunocomplex kinase assay. The
results showed that stimulation of the Gln-starved HeLa cells with
anti-Fas antibody increased the JNK/SAPK activity starting 10 min after
stimulation of Fas up to 60 min (Fig. 2, middle
row). In contrast, there was no JNK/SAPK activation by Fas
ligation in cells incubated in Gln-containing medium.
Since JNK/SAPK is activated by ASK1 after Fas ligation, we next
investigated the activation of ASK1 after Fas ligation. ASK1 kinase
assay was determined by immunoprecipitation with rabbit polyclonal
anti-ASK1 antibody and reaction with MBP and
[ QRS Is Associated with ASK1--
Gln might suppress Fas-mediated
apoptosis by inhibiting ASK1 or JNK/SAPK that is involved in the
apoptosis pathway. Although various metabolic enzymes could control the
steady state level of cellular free Gln, we were interested in QRS that
ligates Gln to its cognate tRNA. Since human QRS is one of the enzymes
utilizing free Gln, we thought that it could be a good candidate to
explain Gln effect on ASK1 and JNK/SAPK inhibition. To address the
issue, we determined the molecular interaction of QRS with ASK1 in 293 cells. HA epitope-tagged ASK1 was coexpressed with Myc-QRS in 293 cells
and immunoprecipitated with anti-HA antibody. The immune complexes were
subjected to immunoblotting with anti-QRS antibody. As shown in Fig.
3A, QRS was found to
associated with ASK1 in a dose-dependent manner.
Interaction of QRS with ASK1 was not detected by immunoprecipitation
with mock IgG control antibody. However, we failed to detect QRS
coprecipitated with ASK-1 at its endogenous level. Normally, most of
QRS is associated with other ARSs to form a macromolecular protein
complex (26), and thus only a small portion of QRS would be available
for the interaction with ASK1. Also, the steady state level of ASK1 is
low and associated with various cellular signaling molecules (23,
27-32). We suspect that these may be the reason for the difficulty to
detect the association of endogenous QRS and ASK1 by
coimmunoprecipitation.
To map the interaction regions of QRS and ASK1, various deletion
mutants were generated (Fig. 3B). ASK-F, -N, or -C was
precipitated with anti-HA antibody, and coimmunoprecipitated QRS
was detected by immunoblotting with anti-QRS antibody. QRS was
precipitated with ASK1-F or -C but not with ASK1-N, indicating that
ASK1-C is responsible for the interaction (Fig. 3C). Then
Myc-tagged QRS derivatives (Fig. 3B) were transiently
expressed with HA-ASK1 and immunoprecipitated with anti-HA antibody.
The precipitated complex was immunoblotted with anti-Myc antibody.
QRS-F and QRS-C were precipitated with ASK1, whereas QRS-N was not
(Fig. 3D). Thus, the catalytic domains of QRS and ASK-1 are
involved in their association.
QRS Represses ASK1 Activity--
Since QRS interacted with ASK1 as
shown above, we then tested the effect of QRS on ASK1 activity. HA-ASK1
was co-overexpressed with different Myc-QRS derivatives and
immunoprecipitated with anti-HA antibody. The precipitated complexes
were subjected to a kinase assay using myelin basic protein as a
substrate of ASK1. ASK1 was strongly repressed by QRS-F and QRS-C but
not by QRS-N (Fig. 4A),
consistent with the interaction result in Fig. 3D.
We confirmed the QRS-mediated repression of ASK1 by determining the
ability of ASK1 to stimulate the serum response factor (SRF). JNK/SAPK
phosphorylated by ASK1 activates the SRF, which then induces
transcription from the promoter containing the serum response element
(33). Using this principle, the effect of QRS binding to ASK1 was
tested by the induction of luciferase, the expression of which is under
the control of SRF (34). Expression of ASK1 alone in 293 cells
increased the expression of luciferase about 5-fold. However,
coexpression of ASK1 with QRS-F or QRS-C abolished the induction of
SRF-luciferase (Fig. 4B).
Molecular Interaction between QRS and ASK1 Is Affected by Fas
Ligation and Glutamine--
To address the physiological significance
and control of the interaction between QRS and ASK1, we investigated
whether the interaction of QRS and ASK1 is affected by an apoptotic
signal. Since ASK1 is activated after cellular exposure to
H2O2, TNF, or agonistic anti-Fas antibody, we
added these reagents to 293 cells, and the interaction between QRS and
ASK1 was tested by coimmunoprecipitation. The two molecules were
dissociated as early as 5 min after the treatment with anti-Fas
antibody but were reassociated in 30 min (Fig.
5A). However, the interaction
was not affected by H2O2 or TNF in this
experimental condition (Fig. 5B).
Since Gln suppresses ASK1 and JNK/SAPK activation by Fas ligation, Gln
itself might affect the molecular interaction of QRS to ASK1. To
explore the possibility, 293 cells were coexpressed with Myc-QRS and
HA-ASK1 in the presence of 0, 2, 4, or 20 mM Gln.
Twenty-four hours after transfection, the molecular association of QRS
to ASK1 was determined by immunoprecipitation. To further confirm the
effect of Gln on the interaction of the two proteins, we also checked
whether the addition of Gln to the immunoprecipitation mixture affects
the association of the two molecules. The expression level of QRS and
ASK1 was not affected by Gln concentration in medium (Fig.
6A, lower
panel), whereas the molecular interaction between QRS and
ASK1 was significantly increased when the cells were cultivated in the
presence of Gln (Fig. 6A, upper
panel). Moreover, the addition of 20 mM Gln to
the immunoprecipitation buffer fortified the interaction of QRS and
ASK1 even when the cells were cultivated without Gln. Moreover, the
ASK1 activity was greatly reduced in the presence of 20 mM
Gln (Fig. 6B), consistent with the effect of Gln on the
interaction of QRS and ASK1. Thus, it is clear that Gln positively
controls the suppressive association of QRS with ASK1.
QRS Blocks ASK1-induced Apoptosis--
We finally investigated
whether QRS can inhibit the cell death mediated by ASK1. Since the
N-terminal domain of ASK1 is inhibitory to its own proapoptotic kinase
activity (35), the truncation of this domain generates the
constitutively active mutant, ASK1-C (also called ASK1 Aminoacyl-tRNA synthetases are a family of enzymes essential for
protein synthesis. However, it has been discovered that these enzymes
are actively involved in a broad repertoire of other critical cellular
activities as well as protein synthesis (36). It has been previously
demonstrated that ARSs have idiosyncratic distribution in cytoplasm,
nucleus, and nucleolus (17, 37). For instance, methionyl-tRNA
synthetase is localized in the nucleolus of rapidly proliferating
mammalian cells and is responsible for ribosomal RNA biosynthesis (17).
Seshaiah and Andrew (38) also showed that each ARS is uniquely
expressed in different tissues and developmental stages of
Drosophila. Thus, the differential expression and cellular localization of each ARS imply that each ARS has its noncanonical function in addition to the catalytic activity for tRNA aminoacylation. Indeed, different ARSs play roles in tRNA maturation (proofreading and
nuclear export), cytokine-like activity, mitochondrial RNA splicing,
and transcriptional and translational regulation (36).
Here, we found that human QRS is not only the enzyme for cell
proliferation but also the protein that plays a regulatory role in cell
death through an antagonistic interaction with ASK1, a protein kinase
that plays a critical role in apoptosis. QRS has been classified as a
component of the multi-tRNA synthetase complex (39, 40). However, QRS
not associated with the multi-tRNA synthetase complex has been found
(41) and shown to be catalytically active (26). Thus, QRS bound to ASK1
may be in dynamic equilibrium with that in the multi-tRNA synthetase complex.
ASK1 is a MAPK kinase kinase that activates SEK1/MKK4 and MKK3/MKK6,
which in turn activate JNK/SAPK and p38, respectively (42). The kinase
activity of ASK1 is stimulated by a variety of apoptosis stimuli such
as Fas ligation, TNF, reactive oxygen species, and anti-cancer drugs
cisplatin and paclitaxel (27, 30, 35, 42,
43). Since ASK1 overexpression
induces apoptosis, and a kinase-inactive mutant of ASK1
reduces TNF, Fas and Daxx-induced apoptosis and JNK activation (35,
42), ASK1 is thought to be a pivotal kinase in the TNF and Fas
signaling pathway leading to apoptosis. Indeed, ASK1 directly interacts
with TRAFs and Daxx in the TNF- and Fas-mediated signal pathways (28,
35). In addition, it interacts with various negative and positive
modulators such as thioredoxin, p21Cip1/WAF1, and 14-3-3 (31, 32).
Binding of thioredoxin to ASK1 is controlled by its oxidation and
reduction, thereby linking the cellular redox potential with the
apoptotic process (31). We now add QRS as a novel negative modulator of ASK1 that mediates the Fas-induced apoptosis in a manner controlled by
Gln (Figs. 5-7).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glycerolphosphate, 150 mM NaCl, 5 mM EGTA, 10 mM NaF, 1% Triton X-100, 0.5%
deoxycholate, 3 mM dithiothreitol, 1 mM sodium
orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 5 µg/ml aprotinin. ASK1 in the cell lysate was reacted with anti-HA
antibody (5 µg) at 4 °C for 1 h. After the addition of 50 µl of protein A-agarose, the mixture was incubated at 4 °C for an
additional 4 h. The beads were washed four times with 20 mM Tris-HCl (pH 7.5) buffer containing 150 mM
NaCl, 5 mM EGTA, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 1% Triton X-100. The
precipitated proteins were resolved on 10% SDS-PAGE and transferred to
nitrocellulose membranes. The immunoprecipitates were analyzed by
immunoblotting with anti-QRS and -HA antibodies.
-32P]ATP for 10 min at 30 °C using myelin basic
protein (MBP) (40 µg/ml) (Sigma) as an exogenous substrate (23). The
samples were resolved by SDS-polyacrylamide gel electrophoresis and
subjected to autoradiography, and the phosphorylated MBP was quantified by a phosphor image analyzer (Fuji; FLA-3000).
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ABSTRACT
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Fig. 1.
Gln deprivation sensitizes HeLa cells to
Fas-mediated apoptosis. A, HeLa cells were incubated in
Gln-free medium for 12 h, supplemented with 0 or 4 mM
Gln 1 h before the treatment with anti-Fas antibody (100 ng/ml).
Eight hours after Fas ligation, cellular morphology was observed under
an inverted microscope. B, HeLa cells were incubated
in Gln-free medium for 12 h and supplemented with 0, 0.1, 0.2, 0.5, 1, and 2 mM Gln 1 h before the treatment with
anti-Fas antibody (100 ng/ml). DNA laddering was determined according
to "Experimental Procedures" 24 h after anti-Fas antibody
treatment (100 ng/ml). C, HeLa cells were incubated in
Gln-free medium for 12 h and supplemented with 0 or 4 mM Gln. Caspase activity was measured 12 h after
anti-Fas treatment according to "Experimental Procedures."
D, HeLa cells in the normal or Gln-free medium were treated
with the indicated amounts of anti-Fas antibody, and the apoptotic
cells were counted.
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Fig. 2.
Glutamine deprivation sensitizes HeLa cells
to ASK1 and JNK/SAPK activation by Fas ligation. HeLa cells were
incubated in Gln-free medium for 12 h and supplemented with 0 or 4 mM Gln 1 h before the treatment with anti-Fas (100 ng/ml). After 0, 10, 20, 30, and 60 min following anti-Fas treatment,
whole cell lysates were prepared and analyzed by immunoblotting with
antiphospho-MAPK. The kinase assay for SAPK and ASK1 was determined as
described under "Experimental Procedures."
-32P]ATP. As shown in Fig. 2, ASK1 was significantly
activated 10 min after Fas ligation in cells incubated in Gln-free
medium. However, there was, if any, little activation of ASK1 in cells incubated in 4 mM Gln-containing medium. Taken together, we
can conclude that Gln suppresses ASK1 and JNK/SAPK activation by Fas ligation.
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Fig. 3.
QRS interacts with ASK1. A,
plasmids pcDNA-Myc-QRS (0, 3, or 6 µg) and pcDNA-HA-ASK1 (0.5 µg) were cotransfected into 293 cells grown in 100-mm dishes.
Twenty-four hours after transfection, the samples were
immunoprecipitated with anti-HA antibody and immunoblotted with
anti-QRS. B, schematic presentation of Myc-QRS and HA-ASK1
deletion mutants used for coimmunoprecipitation assays. C,
interaction of HA-ASK1-F, -N, and -C with Myc-QRS-F. Each of the ASK1
deletion mutants was immunoprecipitated (IP) with anti-HA
antibody, and the coprecipitation of QRS was determined with anti-Myc
antibody. Whole cell lysate (WCL) of each transfectant
contains a similar amount of QRS. D, interaction of
Myc-QRS-F, -N, and -C with HA-ASK1-F. HA-ASK1 was immunoprecipitated
with anti-HA antibody, and coprecipitation of QRS mutants was
determined with anti-Myc antibody. Each of the cells contains a similar
amount of HA-ASK1.
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Fig. 4.
QRS represses ASK1 activity.
A (left), HA-ASK1 was coexpressed in 293 cells
with Myc-QRS-F, -N, or -C. HA-ASK1 was precipitated with anti-HA
antibody, and the kinase activity of the immunoprecipitated ASK1 was
determined using MBP as an exogenous substrate in the presence of
[ -32P]ATP. The phosphorylated MBP and
autophosphorylated ASK1 were visualized by autoradiography.
EV, empty vector. Right, the amount of
phosphorylation was quantified using a phosphor image analyzer.
B, the ASK1 activity was determined by
JNK/SAPK-dependent activation of the reporter gene,
luciferase. The reporter plasmid in which the expression of luciferase
is under the control of SRF was transfected into 293 cells with the
combination of HA-ASK1 and Myc-QRS. The expression of luciferase was
monitored using luminometer, and three independent experiments were
carried out.
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Fig. 5.
QRS is dissociated from ASK1 by Fas
ligation. 293 cells transfected with pcDNA3-Myc-QRS (1 µg)
and pcDNA3-HA-ASK1 (0.3 µg) were treated with agonistic anti-Fas
antibody (2 µg/ml) (A), TNF (200 ng/ml) (B,
left), and H2O2 (1 mM)
(B, right). The cells were harvested at the
indicated times. The proteins were extracted from the harvested cells,
and HA-ASK1 was immunoprecipitated with anti-HA antibody. The amount of
coimmunoprecipitated QRS was determined by anti-QRS antibody.
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Fig. 6.
Gln fortifies the molecular interaction of
QRS to ASK1. A, 293 cells transfected with Myc-QRS (2 µg) and HA-ASK1 (0.5 µg) in the presence of 0, 2, 4, and 20 mM Gln were subjected to immunoprecipitation with anti-HA
antibody. The precipitated complexes were analyzed by immunoblotting
with anti-Myc antibody. Gln (0 or 20 mM) was added to the
immunoprecipitation (IP) buffer in order to observe the
effect of Gln on the interaction between QRS and ASK1 more clearly.
B, HA-ASK1 was cotransfected with Myc-QRS to 293 cells in
the presence of 0, 2, 4, and 20 mM Gln. Immunoprecipitates
by anti-HA antibody were subjected to in vitro kinase assay
for ASK1 activity according to "Experimental Procedures."
N). Apoptosis
of 293 cells was induced by the transient transfection of ASK1-C to
about 25% in normal medium, and it was further enhanced to 35% when
the cells were cultivated in Gln-free medium (Fig.
7). The ASK1-C-induced cell death was decreased by the coexpression of QRS-F or -C but not with QRS-N in the
normal medium (Fig. 7, left). However, the antiapoptotic activity of QRS-F or -C was less apparent in Gln-free medium (Fig. 7,
right). This result is consistent with the molecular
association of QRS with ASK and its in vitro effect on the
activity of ASK1 shown above.
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Fig. 7.
QRS blocks ASK1-induced apoptosis in
Gln-dependent manner. 293 cells were cultivated in the
normal or Gln-free medium. Apoptosis of 293 cells was induced by
transient transfection of ASK1-C, which is a constitutive active
mutant. The effect of QRS-F, -N, and -C on ASK1-C-induced apoptosis was
monitored by counting apoptotic cells. The values are the average of
two independent experiments.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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Fig. 8.
Simplified working model explaining the role
of Gln and QRS in ASK1 activity regulation. The ASK1 activity is
repressed by the interactions with thioredoxin or QRS. (For simplicity,
other ASK1 modulators and signaling pathways are not shown.) When
thioredoxin is oxidized by reactive oxygen species (ROS)
induced by TNF, it is dissociated from ASK1. The antagonistic
interaction of QRS with ASK1 is broken by the competition with Daxx
that is activated by Fas ligation. QRS associated with ASK1 may be in
dynamic equilibrium with that associated with the multi-ARS
complex.
Extensive investigations have revealed important effects of exogenous Gln in nutritional rehabilitation, stimulating intestinal villus regrowth after damage from chemotherapy and improving intestinal barrier function. In addition, Gln blocks apoptosis induced by heat shock, irradiation, and c-Myc-overexpression. In Figs. 1 and 2, we show that Gln prevents the activation of stress kinases such as ASK1 and JNK/ASK1 and then apoptosis initiated by Fas ligation, suggesting that Gln is an apoptosis suppressor. The cellular susceptibility to Fas-mediated signaling by Gln deprivation could not be explained by the protein synthesis inhibition, because ASK1 and JNK/SAPK were activated within 10 min after Fas ligation in Gln-free cells but not in normal cells. Therefore, Gln could suppress apoptosis by inhibiting ASK1 and then JNK/SAPK.
Although the similar amount of exogenous QRS was expressed in cells grown in complete and Gln-free medium, the molecular interaction between QRS and ASK1 was fortified and then the ASK1 activity was strongly inhibited in the presence of Gln (Fig. 6), indicating the positive effect of Gln on the interaction of the two molecules. It has been already demonstrated that amino acids themselves can be signaling molecules. For instance, glutamate acts as a messenger in glucose-induced insulin exocytosis because exogenous glutamate directly stimulates insulin exocytosis, independently of mitochondrial function in permeabilized cells (44). Here, we suggest that Gln could be a signaling messenger for blocking ASK1 activation and suppressing apoptosis via binding to QRS.
Some types of apoptosis are suppressed by the activation of
AKT/protein kinase B and MAPK. Activation of AKT/protein
kinase B by extracellular survival factors prevents apoptosis by
inactivating caspase-9, BAD, Forkhead, and IKK that are involved in
the apoptosis pathway (45-51). Since Fas-mediated apoptosis is
prevented by activating MAPK through overexpression of K-Ras and basic
fibroblast growth factor (52) and is accelerated by inhibiting MAPK
with PD 98059 (53), MAPK could be another survival signal inhibiting
apoptosis. In addition to AKT/protein kinase B and MAPK, we
propose that Gln and QRS are intracellular survival factors for
regulating apoptosis. Although further investigations are necessary to
better understand the mechanism and control for the role of QRS in
apoptosis, the results of this work provide the first evidence that one
of the mammalian ARSs has a direct antagonistic interaction with a
kinase that plays a key role in apoptosis.
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ACKNOWLEDGEMENTS |
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We thank Drs. Guy A. Thompson, Jr. and Wongi Seol for critical discussions.
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FOOTNOTES |
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* This work was supported by a grant from the National Creative Research Initiatives of the Ministry of Science and Technology of 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 may be addressed: National Creative Research Initiatives Center for ARS Network, Sung Kyun Kwan University, 300 Chunchundong, Jangangu Suwon, Kyunggido 440-746, Korea. Tel.: 82-31-290-5680; Fax: 82-31-290-5682; E-mail: shkim@yurim.skku.ac.kr.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M006189200
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ABBREVIATIONS |
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The abbreviations used are: TNF, tumor necrosis factor; ARS, aminoacyl-tRNA synthetase; QRS, glutaminyl-tRNA synthetase; ASK1, apoptosis signal-regulating kinase 1; JNK, c-Jun N-terminal kinase; SAPK, stress-activated protein kinase; MAPK, mitogen-activating protein kinase; HA, hemagglutinin; MBP, myelin basic protein.
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