From the National Creative Research Initiative Center
for Cell Death, Graduate School of Biotechnology, Korea University,
Anam-dong, Sungbuk-ku, Seoul 136-701, South Korea, the ¶ College
of Pharmacy, Seoul National University, Seoul 151-742, South Korea, the
Seoul National University Hospital Clinical Research Institute,
#28 Yongon-dong, Chongno-ku, Seoul 110-799, South Korea, and the
** Laboratory of Cell Signaling, Graduate School, Tokyo Medical and
Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan
Received for publication, June 25, 2000, and in revised form, December 21, 2000
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ABSTRACT |
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Apoptosis signal-regulating kinase 1 (ASK1) is a
mitogen-activated protein kinase kinase kinase that can activate the
c-Jun N-terminal kinase and the p38 signaling pathways. It plays a
critical role in cytokine- and stress-induced apoptosis. To further
characterize the mechanism of the regulation of the ASK1 signal, we
searched for ASK1-interacting proteins employing the yeast two-hybrid
method. The yeast two-hybrid assay indicated that mouse glutathione
S-transferase Mu 1-1 (mGSTM1-1), an enzyme involved in the
metabolism of drugs and xenobiotics, interacted with ASK1. We
subsequently confirmed that mGSTM1-1 physically associated with ASK1
both in vivo and in vitro. The in
vitro binding assay indicated that the C-terminal portion of
mGSTM1-1 and the N-terminal region of ASK1 were crucial for binding one
another. Furthermore, mGSTM1-1 suppressed stress-stimulated ASK1
activity in cultured cells. mGSTM1-1 also blocked ASK1 oligomerization. The ASK1 inhibition by mGSTM1-1 occurred independently of the glutathione-conjugating activity of mGSTM1-1. Moreover, mGSTM1-1 repressed ASK1-dependent apoptotic cell death. Taken
together, our findings suggest that mGSTM1-1 functions as an endogenous inhibitor of ASK1. This highlights a novel function for mGSTM1-1 insofar as mGSTM1-1 may modulate stress-mediated signals by repressing ASK1, and this activity occurs independently of its well-known catalytic activity in intracellular glutathione metabolism.
Apoptosis is an active cellular process that occurs not only
during embryogenesis and metamorphosis but also during post-embryonal life, thus controlling normal development and homeostasis of
multicellular organisms (1-3). Apoptosis is characterized by
morphological changes that include chromatin condensation, membrane
blebbing, and packaging of nuclear fragments into small apoptotic
bodies, which are eliminated through phagocytosis by neighboring cells without eliciting inflammatory reactions (3, 4). Derangement of cells
from the tightly regulated apoptotic process is associated with the
occurrence of many human diseases such as cancer, autoimmune diseases,
and various neurodegenerative disorders (5).
Apoptotic cell death is thought to occur through an orchestrated
sequence of intracellular signaling cascades. In particular, the
mitogen-activated protein kinase
(MAPK)1 signaling pathways
have been shown to be involved in the mechanism for regulation of cell
death and survival (6, 7). The MAPK signaling pathways include three
distinct components of the protein kinase family; MAPKs, MAPK kinases
(MAPKKs), and MAPK kinase kinases (MAPKKKs). When activated, MAPKKKs
phosphorylate and activate MAPKKs, which in turn phosphorylate and
activate MAPKs. The mammalian MAPKs include three subfamilies:
extracellular signal-regulated kinase (ERK), c-Jun N-terminal
kinase/stress-activated protein kinase (JNK/SAPK) and p38 MAPK (6, 7).
The ERK signaling pathway is often stimulated by mitogens, whereas the
JNK/SAPK and the p38 signaling pathways are preferentially stimulated
by pro-inflammatory cytokines such as TNF- Among the MAPKKK family, MEKK1, MEKK2, MEKK3, and Tpl-2/Cot can
stimulate both the ERK and the JNK/SAPK pathways (10). On the other
hand, TAK1 and MTK1/MEKK4 have been shown to activate both the JNK/SAPK
and the p38 pathways. Recently, apoptosis signal-regulating kinase 1 (ASK1) was also identified as an MAPKKK that activates both the
JNK/SAPK and the p38 signaling cascades (11). Overexpression of ASK1
induces apoptotic cell death, and a dominant negative mutant of ASK1
prevents TNF- TNF- Glutathione S-transferases (GSTs) are a family of enzymes
that catalyze the conjugation of reduced GSH to a variety of
electrophiles. GSTs can also function as peroxidases and isomerases
(28). The GSTs possess two binding domains that are critical for their
catalytic activity: a GSH binding site (G-site) and an adjacent
substrate binding site (H-site) (29, 30). In addition to their
catalytic function, GSTs can also serve as nonenzymatic binding
proteins (known as ligandins) interacting with various lipophilic
compounds that include steroid and thyroid hormones (31-33). Some
evidence suggests that GST is involved in cellular defense against a
broad spectrum of toxic agents that may be generated in the environment or within the cell (28). On the basis of their primary structure, the
mammalian cytosolic GSTs have been grouped into five classes, alpha,
mu, pi, sigma, and theta. The most abundant ones are the alpha, mu, and
pi classes (28). All GST isoforms catalyze a similar reaction, but they
share very little amino acid identity, typically no more than 25-30%
(29).
In the present study, we show that mouse glutathione
S-transferase Mu 1-1 (mGSTM1-1) physically interacts with
ASK1 and, in doing so, functions as a negative regulator of ASK1 inside
cells, repressing ASK1-mediated signals. The ASK1-inhibiting action of mGSTM1-1 appears to be independent of its transferase activity. Thus,
our study uncovered a novel function for mGSTM1-1 insofar as this
enzyme may participate in the regulation of stress-activated signals by
suppressing ASK1 activity.
DNA Constructs--
ASK1 mutants, ASK- Cell Culture and Transfection--
293 human embryonic kidney
cells and HeLa cells were grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum (Life Technologies, Inc.).
For DNA transfection, cells were plated in 100-mm dishes (2.5 × 106 cells/plate), grown overnight, and transfected with
appropriate expression vectors by the calcium phosphate method (36) or
by using LipofectAMINE (Life Technologies, Inc.).
Yeast Two-hybrid Screening--
To search for ASK1-binding
proteins, a yeast two-hybrid screening, was carried out as described in
the manufacturer's protocol (CLONTECH). Briefly, a
full-length ASK1 cDNA was fused in-frame to the LexA DNA-binding
domain in the pLexA bait plasmid. Approximately 2 × 106 clones of a mouse adult brain cDNA library in
pB42AD prey plasmid were screened using a EGY48 yeast strain that had
been transformed with p8op-LacZ. Plasmid DNAs of positive clones were
recovered after transformation into Escherichia coli KC8
cells, and the cDNA inserts were sequenced.
Coimmunoprecipitation--
To test the physical interactions of
ectopically expressed proteins, 293 cells were cotransfected with
plasmids expressing either ASK1-FLAG or ASK1-
To test interactions between endogenous proteins, mouse liver tissue
was minced with scissors and homogenized in buffer A with an IKA Ultra
Turrax homogenizer (IKA Labotechnik). The homogenate was
subjected to centrifugation at 12,000 × g at 4 °C
for 20 min. The soluble fraction was precleared with rabbit preimmune
IgG and protein G-Sepharose and then subjected to immunoprecipitation with rabbit preimmune IgG, rabbit anti-mGSTM1-1 antibody (Calbiochem), or rabbit anti-ASK1 antibody (Santa Cruz Biotechnology). The
immunopellets were analyzed by immunoblot probed with rabbit anti-ASK1 antibody.
Immunocomplex Kinase Assays--
Cells were lysed in buffer A
and subjected to microcentrifugation at 12,000 × g.
The solubilized fraction was then subjected to immunoprecipitation with
the appropriate antibodies, and the immunopellets were assayed for the
indicated protein kinases as described previously (34, 35).
Phosphorylated substrates were visualized and quantified after
SDS-polyacrylamide gel electrophoresis using a Fuji BAS 2500 phosphorescence imager. GST-SEK1(K129R), GST-c-Jun-(1-135),
GST-ATF2-(1-109), or myelin basic protein was used as a substrate for
ASK1, SAPK/JNK, p38, or ERK2, respectively.
In Vitro Binding Assays--
mGSTM1-1 was bacterially expressed
and purified with glutathione-agarose beads. Hexahistidine (His)-tagged
mGSTM1-1, mGSTM1- Luciferase Reporter Assay for c-Jun-dependent
Transcription--
The transcription stimulating activity of c-Jun was
assayed with the PathDetect luciferase reporter kit (Stratagene).
Typically, 293 cells were transiently transfected with pFR-Luc,
pFA2-c-Jun, pcDNA3-mGSTM1-1, pcDNA3-ASK1, and
pcDNA3- Apoptotic Cell Death--
Cultured cells were transfected with
pEGFP (CLONTECH) and plasmids expressing the
indicated proteins. At 48 h after transfection, the cells were
washed twice with phosphate-buffered saline, fixed with 0.25%
glutaraldehyde, permeabilized with 0.1% Triton X-100, and stained with
DAPI. The DAPI-stained nuclei in GFP-positive cells were examined for
apoptotic morphology by fluorescence microscopy. The percentage of
GFP-expressing cells that were apoptotic was determined from three
independent dishes.
mGSTM1-1 Physically Interacts with ASK1--
To better understand
the molecular mechanism for the regulation of the ASK1 activity, we
decided to identify proteins that can physically interact with ASK1
using the yeast two-hybrid screening method with a mouse adult brain
cDNA library. Eight strongly positive clones were identified after
the second round of screening using leucine/tryptophan/histidine/uracil-deficient medium and X-gal plates.
Five of the positive clones were identified as thioredoxin, as reported
previously (37). Two other positive clones were identified as a gene
encoding a mouse class mu GST, mGSTM1-1. In subsequent experiments, we
examined which portion of the ASK1 protein was responsible for the
binding to mGSTM1-1 in the yeast two-hybrid system (Fig.
1, A and B).
Several bait plasmids that encoded a full-length, an N-terminally
deleted, or a C-terminally deleted ASK1 fused to the LexA DNA-binding
domain were constructed. ASK1-NT, ASK1-
To identify which region of mGSTM1-1 is crucial for binding to ASK1, we
carried out an in vitro binding assay using His-tagged mGSTM1-1, mGSTM1- mGSTM1-1 Inhibits the Enzymatic Activity of ASK1--
Because our
data indicated that mGSTM1-1 directly interacted with ASK1, we decided
to examine whether mGSTM1-1 could modulate ASK1 activity. The enzymatic
activity of ectopically expressed ASK1 was stimulated by exposure of
the transfected cells to UV radiation or H2O2
(Fig. 3, A and B).
Interestingly, mGSTM1-1 repressed both the UV- and the
H2O2-stimulated activity of ASK1. Because it
had been recently reported that Daxx activates ASK1 (12), the effect of
mGSTM1-1 on Daxx-stimulated ASK1 activity was examined in the following
experiments. Cotransfection of 293 cells with plasmids expressing ASK1
and Daxx-(498-740), an active mutant of Daxx (12), resulted in ASK1
activation (Fig. 3C). The Daxx-(498-740)-stimulated ASK1
activity was suppressed by mGSTM1-1. We also examined whether mGSTM1-1
could suppress ASK1 activity in vitro (Fig.
4A). In the in
vitro kinase assay, ASK1 activity was inhibited by mGSTM1-1, but
not by other GST isoforms such as GST pi and GST alpha. In the separate
in vitro kinase assays, mGSTM1-1 did not inhibit activities
of other protein kinases, including JNK1, p38, and ERK2 (Fig.
4B). Purified recombinant mGSTM1-1 inhibited ASK1 in a
noncompetitive inhibition mode, and the inhibitor constant
(Ki) for ASK1 by GSTµ was 7.3 × 10
Next, we tested whether the GSH-conjugating catalytic activity of
mGSTM1-1 is critical for the ASK1 inhibition. It has been shown that a
conserved N-terminal tyrosine residue in the GST isoforms is critical
for the catalytic activity of the various GSTs (38, 39). We, therefore,
constructed a mutant form of mGSTM1-1, mGSTM1-1(Y6F), in which this
tyrosine residue was replaced by a phenylalanine residue in the 6th
amino acid position. mGSTM1-1(Y6F) was a catalytically inactive form of
mGSTM1-1 by GST enzyme assay using 1-chloro-2,4-dinitrobenzene (data
not shown). Subsequently, the inhibitory effects of wild-type mGSTM1-1
and mGSTM1-1(Y6F) on ASK1 activation were evaluated by cotransfection
studies. Our data demonstrate that mGSTM1-1(Y6F) was as effective as
mGSTM1-1 in inhibiting H2O2-stimulated ASK1
activity (Fig. 5A). We also tested whether an intracellular level of GSH could modulate the inhibitory function of mGSTM1-1 on ASK1 activity by treating the cells
with L-buthionine-S,R-sulfoximine
(BSO), an agent that depletes intracellular GSH by inhibiting GSH
biosynthesis (40-43). The treatment with BSO did not abolish the
inhibitory action of mGSTM1-1 on the
H2O2-stimulated ASK1 activity (Fig.
5B).
mGSTM1-1 Blocks ASK1 Oligomerization--
A recent study by Gotoh
and Cooper (44) demonstrated that homo-oligomerization of ASK1 is an
important mechanism for ASK1 activation. We, therefore, examined
whether mGSTM1-1 could modulate the oligomerization of ASK1 to suppress
ASK1 activity. 293 cells were cotransfected with ASK1-FLAG and ASK1-HA
constructs in the presence or absence of mGSTM1-1 construct.
Coimmunoprecipitation study demonstrated that ASK1-HA was present in
ASK1-FLAG immunoprecipitates (Fig. 6).
These data indicate that ASK1 oligomerization occurred in the cells
overexpressing both ASK1-FLAG and ASK1-HA. Coexpression of mGSTM1-1
with ASK1-FLAG and ASK1-HA resulted in disruption of the ASK1
oligomerization (Fig. 6). Thus, our data suggest that the inhibition of
ASK1 oligomerization may be at least one mechanism by which mGSTM1-1
suppresses ASK1 activation.
mGSTM1-1 Suppresses ASK1-mediated Activation of the JNK/SAPK
Signaling Cascade--
One of the major downstream signals of ASK1 is
the activation of JNK/SAPK, which in turn results in the stimulation of
the transcriptional activity of c-Jun (7). We tested whether the ASK1
inhibition by mGSTM1-1 could result in a decrease in the activities of the downstream signals. Overexpression of ASK1 by itself
was sufficient to stimulate JNK/SAPK activity in transfected cells even
without any further treatment (Fig.
7A). Our data show that
mGSTM1-1 mitigated the ASK1-induced SAPK mGSTM1-1 Represses ASK1-dependent Apoptotic Cell
Death--
We examined whether mGSTM1-1 would affect
ASK1-dependent apoptotic cell death. ASK1 was initially
discovered as a MAPKKK that could activate apoptosis initiated by
TNF- In our present study, we demonstrate that mGSTM1-1 directly
interacts with the N-terminal portion of ASK1 both in vivo
and in vitro and that this interaction results in
suppression of ASK1 activity as well as ASK1-dependent
apoptotic cell death.
The detoxification reaction catalyzed by GST may be one of the most
important survival tools that living organisms have developed. GST
conjugates reduced glutathione to a variety of electrophilic xenobiotics or products of oxidative stress (28). It is thus involved
in the protection of cells against chemical stress. GST is also able to
nonenzymatically bind, both covalently and noncovalently, various other
chemical compounds, which include steroid and thyroid hormones, bile
acids, bilirubin, heme, and fatty acids, all of which are not
substrates for its enzymatic activity (28). Considering that
intracellular concentrations of GST in many tissues are at micromolar
levels, GST could constitute an intracellular binding pool for
lipophilic ligands. This type of "ligandin" function could serve
the purpose of preventing cytotoxic ligands from interacting with their
intracellular targets (33). It is also noteworthy that one class of
GST, GST pi, can associate with and inhibit JNK (45), thus possibly
keeping basal JNK activity low in nonstressed cells. Our data in this
study indicate that mGSTM1-1 similarly interacts with ASK1 and that
this interaction causes the suppression of the enzymatic activity of
ASK1, which is one of the upstream kinases of JNK. It is tempting to
propose a function for GSTs as endogenous negative regulators of the
JNK signaling pathway through multiple mechanisms: GST mu acts on ASK1,
and GST pi acts on JNK.
It has been well documented that the expression of GSTs can be induced
in many organisms by exposure to a variety of stresses, including
oxidative stress and that c-Jun is one of the transcription factors
involved in this induction (28). Oxidative stress can also activate
ASK1 and other components in the JNK signaling pathway leading to c-Jun
activation (6, 7, 37). Thus, it is possible that oxidative stress and
other stress factors that activate ASK1 can induce mGSTM1-1 expression
and that the expressed mGSTM1-1 protein can suppress the
stress-activated ASK1 activity. This type of regulatory loop may be an
integral part of the defense mechanism by which mGSTM1-1 protects cells
from a variety of stresses, including oxidative stress. On the basis of
our data, therefore, we propose a new function for mGSTM1-1: mGSTM1-1
modulates stress-activated signals by suppressing ASK1 in a way that is
independent of its transferase activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and interleukin-1
and cellular stresses, including UV light, H2O2,
and osmotic shock, and withdrawal of growth factors. Many lines of
evidence demonstrate that the JNK/SAPK signaling pathway plays a role
in apoptotic cell death induced by a variety of stresses (6-9).
and Fas-induced apoptosis (11, 12).
is a pro-inflammatory cytokine whose signals are mediated by
two cell surface receptors, TNF receptor-1 (p55) and TNF receptor-2
(p75) (13). As TNF-
binds to its receptors, receptor aggregation and
the recruitment of cytoplasmic signaling proteins to TNF receptors are
induced (14-19). One of the proteins recruited to TNF receptors is TNF
receptor-associated factor 2 (TRAF2). TRAF2 can interact directly with
TNF receptor-2 (18) while it is recruited to TNF receptor-1 through TNF
receptor-1-associated death domain protein (14-16). Recently, it is
reported that TRAF2 and other TRAF proteins interact with and activate
ASK1 (20, 21). ASK1 is thus a downstream target of TRAF2 in the
TNF-
-dependent intracellular signaling cascade.
Fas/Apo-1/CD95 is a cell surface protein that belongs to the TNF
receptor superfamily (22). Fas can activate at least two distinct
signaling pathways, each of which can lead to apoptotic cell death. In
one of the pathways Fas interacts with Fas-associated death
domain, which in turn recruits caspase-8 and activates the
caspase cascade, resulting in apoptosis (23-26). In another pathway
Fas interacts with the Fas-associated protein Daxx, which can bind and
activate ASK1 (12). The Daxx-induced ASK1 activation leads to
apoptotic cell death through the activation of the JNK/SAPK
signaling pathway (12, 27). Thus, ASK1 is associated with the mechanism
for apoptotic cell death. However, the molecular mechanism by which ASK1 activity is regulated in the cells is not understood completely.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
N, ASK1-
C, and
ASK1-NT were generated by polymerase chain reaction and cloned into
pLexA (CLONTECH) and pcDNA3 (Invitrogen). Mouse
mGSTM1 cDNA was obtained from a mouse adult brain LexA cDNA
library (CLONTECH). ASK1(K709R) and mGSTM1-1(Y6F) were constructed with the QuikChange site-directed mutagenesis kit
(Stratagene). mGSTM1-1 deletion mutants mGSTM1-
N-(85-218) and
mGSTM1-
C-(1-84) were made by polymerase chain reaction and cloned into pET-28a (Novagen). The construction of GST-SEK(K129R), GST-c-Jun-(1-135), and
MEKK1 has been described previously (34, 35). Daxx-(498-740), an active mutant of Daxx, was a generous gift
from Dr. S. H. Kim (Sungkyunkwan University, Korea).
N-FLAG along with
HA-tagged mGSTM1-1 using LipofectAMINE. After 30 h of
transfection, cells were solubilized in buffer A that contained 20 mM Tris-HCl, pH 7.4, 12 mM
-glycerophosphate, 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, 1 µg/ml leupeptin, and
1 µg/ml aprotinin. The soluble fraction was subjected to
immunoprecipitation with anti-HA monoclonal antibody (Roche Molecular
Biochemicals). The resultant immunopellets underwent SDS-polyacrylamide
gel electrophoresis and immunoblot analysis with anti-FLAG monoclonal
antibody (Sigma Chemical Co.) using an enhanced chemiluminescence
detection system (Amersham Pharmacia Biotech). To examine the effect of
mGSTM1-1 on the oligomerization of ASK1, 293 cells were transfected
with expression vectors producing ASK1-FLAG and ASK1-HA along with
mGSTM1-1. After 30 h of transfection, the cell lysates were
subjected to immunoprecipitation with mouse anti-FLAG antibody. Then,
the immunopellets were analyzed by immunoblot using mouse anti-HA antibody.
N, or mGSTM1-
C was bacterially produced and
purified with Ni2+-nitrilotriacetic acid agarose beads.
ASK1 and its mutant counterparts were in vitro translated in
the presence of [35S]methionine using the TnT
reticulocyte lysate system (Promega). The 35S-labeled
proteins were incubated at 4 °C for 3 h with mGSTM1-1 or its
mutant proteins immobilized onto the beads in binding buffer containing
50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1 mM dithiothreitol, 0.1% Nonidet
P-40, and 5 mg/ml bovine serum albumin. The beads were harvested and
washed three times with washing buffer (50 mM Hepes, pH
7.5, 150 mM NaCl, 1 mM EDTA, 1 mM
dithiothreitol, and 0.1% Tween 20). The 35S-labeled
proteins were then eluted from the beads and analyzed by
SDS-polyacrylamide gel electrophoresis and autoradiography.
-gal, as indicated. After 48 h of transfection,
cells were lysed and subjected to microcentrifugation at 4 °C for 10 min. The soluble fraction was assayed for luciferase activity using a
luciferase assay kit (Promega). The luciferase activity in the
transfected cells was normalized with reference to the
-galactosidase activity in the same cells.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
C, or ASK1-
N encode amino
acids 1-656, 1-936, or 649-1375 of the ASK1 protein, respectively
(Fig. 1A). In the two-hybrid assay, mGSTM1-1 was able to
bind the full-length ASK1, ASK1-NT, and ASK1-
C, but not ASK1-
N
(Fig. 1B). It was further tested whether mGSTM1-1 could
directly interact with ASK1 in an in vitro binding assay
(Fig. 1C). Purified recombinant mGSTM1-1 did indeed
physically associate with in vitro translated
35S-labeled ASK1 (WT, wild-type), ASK1-
C, or ASK1-NT,
but not with ASK1-
N. mGSTM1-1 bound ASK1 with a ratio of 2:1 in an
in vitro cross-linking experiment using disuccinimidyl
suberate (data not shown). To confirm that mGSTM1-1 interacted with
ASK1 in intact mammalian cells, 293 cells were cotransfected with
expression vectors producing FLAG-tagged ASK1 and HA-tagged mGSTM1-1.
The lysed transfected cells were then subjected to immunoprecipitation with anti-HA antibody. Immunoblot analysis of the immunoprecipitated protein complexes demonstrated that mGSTM1-1 was coimmunoprecipitated with full-length ASK1, but not with ASK1-
N (Fig. 1D).
Next, the interaction of the two endogenous proteins ASK1 and mGSTM1-1
in mouse liver tissue was examined. The liver lysate was
immunoprecipitated with either rabbit preimmune IgG or anti-GSTM1-1
antibody, and the immunocomplexes were analyzed with anti-ASK1 antibody
by immunoblot (Fig. 1E). The immunoblot data show that
mGSTM1-1 was physically associated with ASK1 in cells from mouse liver.
Collectively, our data suggest that ASK1 directly interacts with
mGSTM1-1 and that the N-terminal portion of ASK1 is critically involved
in this binding.
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Fig. 1.
mGSTM1-1 interacts with ASK1.
A, schematic diagram of ASK1 and its mutants. The
hatched boxes designate the kinase domain. B,
pLexA bait plasmids encoding the indicated forms of ASK1 were
cotransformed with a prey plasmid, pB42AD, encoding mGSTM1-1 into EGY48
yeast strains. The transformants were streaked onto a selective plate
that contained X-gal. C, in vitro translated
35S-labeled ASK1 or ASK1 mutant proteins were applied to
mGSTM1-1 immobilized onto glutathione-agarose beads. Bound proteins
were eluted and separated by SDS-polyacrylamide gel electrophoresis.
The 35S-labeled proteins were visualized by
autoradiography. The input 35S-labeled proteins
(one-fifteenth) are also shown. D, 293 cells were
cotransfected with plasmids expressing HA- mGSTM1-1, ASK1-FLAG, and
ASK1- N-FLAG as indicated. Cell lysates from the transfected cells
were subjected to immunoprecipitation with anti-HA antibody, and the
immunopellets were analyzed by immunoblot analysis with anti-FLAG
antibody (left). In addition, the cell lysate blots were
also immunoanalyzed with anti-FLAG or anti-HA antibody
(right). E, the soluble fraction of a mouse liver
homogenate was precleared with rabbit preimmune IgG and then
immunoprecipitated with rabbit anti-ASK1, anti-GSTM1-1
polyclonal antibody, or preimmune IgG. The immunocomplexes were
subjected to SDS-PAGE on 8% polyacrylamide gel, blotted, and
immunoanalyzed with rabbit anti-ASK1 polyclonal antibody.
N, and mGSTM1-
C (Fig.
2A). mGSTM1-
N and
mGSTM1-
C produce amino acid residues 85-218 and 1-84 of mGSTM1-1,
respectively. In vitro translated 35S-labeled
ASK1 associated well with mGSTM1-1 and mGSTM1-
N but scarcely with
mGSTM1-
C. It is noteworthy that mGSTM1-
C (amino acid residues
1-84 of mGSTM1-1) includes the amino acid residues necessary for the
GSH binding (28). Indeed, our in vitro binding study shows
that mGSTM1-
C was able to bind to GSH-affinity resin with
a high efficiency (data not shown). Furthermore, the binding of
mGSTM1-
C or mGSTM1-1 to the GSH-agarose beads was reduced in the
presence of free GSH (Fig. 2B). Thus, mGSTM1-
C
appears to be functionally active for the GSH binding, but it binds
little, if any, to ASK1. Taken together, these data suggest that the
N-terminal region (amino acids 1-84) of mGSTM1-1 may not be critical
for an interaction with ASK1.
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Fig. 2.
The C-terminal region of mGSTM1-1 is
important for the interaction with ASK1. A,
hexahistidine (His)-tagged mGSTM1-1 wild type
(WT), mGSTM1- N, and mGSTM1-
C proteins were immobilized
onto Ni2+-nitrilotriacetic acid agarose beads and subjected
to in vitro binding assay using in vitro
translated 35S-labeled ASK1. The bound
35S-labeled proteins were analyzed by SDS-PAGE on 10%
polyacrylamide gel and autoradiography. The input
35S-labeled proteins (one-fifteenth) are also shown. To
show the amount of mGSTM1-1, mGSTM1-
N, or GSTµ-
C bound on the
beads, a lower part of the polyacrylamide gel was cut out and stained
with Coomassie Brilliant Blue. B, purified His-tagged GSTµ
or mGSTM1-
C (4 µg of protein) was mixed with GSH-agarose beads
(50% slurry in 15 µl) in 20 mM Hepes buffer, pH 7.4, in
the absence or presence of 10 mM GSH. The bound proteins in
the GSH-agarose beads were subjected to SDS-PAGE on 12% polyacrylamide
gel and visualized by Coomassie Brilliant Blue staining.
9 M (data not shown). Taken together, the
data indicate that mGSTM1-1 can function as a specific inhibitor of
ASK1 in vitro and in intact cells.
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Fig. 3.
Effect of ectopic mGSTM1-1 on ASK1 activity
in cells. 293 cells were cotransfected with plasmids expressing
ASK1-FLAG, mGSTM1-1, and Daxx-(498-740), as indicated. Where
indicated, cells were treated with UV light (60 J/m2)
(A) or H2O2 (2 mM, 20 min) (B) after 48 h of transfection. Cell lysates were
subjected to immunoprecipitation with anti-FLAG antibody. The
immunopellets were assayed for ASK1 activity by immunocomplex kinase
assay.
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Fig. 4.
In vitro effects of GST isoforms
on ASK1 activity. A, 293 cells were transfected with
pcDNA3-ASK1-FLAG and irradiated with UV light (60 J/m2)
at 48 h after transfection. The cell lysates then underwent
immunoprecipitation with anti-FLAG antibody. The immunopellets were
assayed for ASK1 activity by immunocomplex kinase assay in the presence
of mouse GSTM1-1, human GSTP1-1, or human GSTA1-1. B, 293 cells were transfected with HA-JNK1, p38-FLAG, or HA-ERK2 construct.
After 48 h of transfection, the cells were exposed either to 60 J/m2 UV light (for JNK1 and p38 activation) or to 100 nM phorbol 12-myristate 13-acetate for 10 min (for ERK2
activation). The cell lysates were subjected to immunoprecipitation
with anti-HA or anti-FLAG antibody, and the immunopellets were assayed
for the indicated kinase activity in the presence of purified
GSTµ protein (4 µg/assay).
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Fig. 5.
mGSTM1-1 inhibits ASK1 activity in a manner
independent of its GSH-conjugating activity. A, a
catalytically inactive form of mGSTM1-1, mGSTM1-1(Y6F), as well as
mGSTM1-1 inhibits ASK1 activity. B, BSO does not prevent
mGSTM1-1 from suppressing ASK1 activity. In A and
B, 293 cells were cotransfected with plasmids expressing
ASK1-FLAG and HA-tagged mGSTM1-1 or mGSTM1-1(Y6F), as indicated. After
48 h of transfection, the cells were exposed to 2 mM
H2O2 for 20 min and then cell lysates were
measured for ASK1 activity by immunocomplex kinase assay. Where
indicated in panel B, cells were pretreated with 0.5 mM BSO for 24 h prior to the
H2O2 treatment.
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Fig. 6.
mGSTM1-1 blocks ASK1 oligomerization.
293 cells were transfected with expression plasmids producing
ASK1-FLAG, ASK1-HA, and mGSTM1-1, as indicated. After 30 h of
transfection, the cell lysates were subjected to immunoprecipitation
with mouse anti-FLAG antibody. The immunoprecipitates were then
analyzed by immunoblot probed with mouse anti-HA antibody. The cell
lysates were also subjected to immunoblotting using anti-FLAG, anti-HA,
or anti-GSTM1-1 antibody to confirm expression of transfected
constructs.
activation. In comparison,
mGSTM1-1 did not affect SAPK
activity stimulated by overexpression
of
MEKK1, a constitutively active form of MEKK1. Overexpressed ASK1
can also induce the transcription stimulating activity of c-Jun (Fig.
7B). We, therefore, examined the action of mGSTM1-1 on the
ASK1-dependent transactivating activity of c-Jun. mGSTM1-1
repressed the ASK1-induced stimulation of c-Jun-mediated luciferase
reporter activity.
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Fig. 7.
mGSTM1-1 suppresses ASK1-mediated JNK/SAPK
activation and transcription stimulating activity of c-Jun.
A, 293 cells were cotransfected with plasmids expressing
ASK1, MEKK1, HA-SAPK
, or mGSTM1-1, as indicated. At 48 h
after transfection, cell lysates were subjected to immunoprecipitation
with anti-HA antibody, and the resultant immunopellets were tested for
SAPK
activity by immunocomplex kinase assay. B, 293 cells
were cotransfected with luciferase reporter plasmid (pFR-Luc),
pFA-cJun, pcDNA3-ASK1, and pcDNA3-mGSTM1 as indicated.
pcDNA3-
-gal was also included in all transfections. After
48 h of transfection, cell lysates were assayed for luciferase
activity. Luciferase activity in each sample was normalized according
to the
-galactosidase activity measured.
or by the Fas-Daxx pathway (11, 12). We, therefore, tested the
effect of mGSTM1-1 on TNF-
- or Daxx-induced apoptotic cell death
(Fig. 8). Exposure of HeLa cells to
TNF-
markedly enhanced apoptotic cell death (Fig. 8A), whereas the
TNF-
-induced apoptosis was reduced by the expression of ASK1(K709R),
a catalytically inactive form of ASK1. This suggests that ASK1 is
associated with the mechanism operating in TNF-
-induced apoptosis.
mGSTM1-1 alleviated the TNF-
-induced apoptosis of transfected HeLa
cells to the same extent as ASK1(K709R) (Fig. 8A).
Overexpression of Daxx-(498-740), an active mutant of Daxx, enhanced
cell death in transfected 293 cells (Fig. 8B), and this cell
death was suppressible by ASK1(K709R). mGSTM1-1 also suppressed the
Daxx-(498-740)-induced cell death.
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Fig. 8.
mGSTM1-1 suppresses
TNF- - and Daxx-induced apoptotic cell
death. HeLa cells (A) or 293 cells (B) were
transiently transfected with plasmids expressing mGSTM1-1, ASK1(K709R),
or Daxx-(498-740), as indicated, along with pEGFP. After 48 h of
transfection, the cells were fixed, permeabilized, and stained with
DAPI. GFP-expressing cells were analyzed for apoptotic nuclei with a
fluorescence microscope. In A, transfected cells were
exposed to TNF-
(5 ng/ml) plus actinomycin D (100 nM)
for 12 h prior to the DAPI staining.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Drs. S. H. Kim, L. I. Zon, D. Baltimore, M. Karin, and J. Woodgett for providing cDNA clones and Dr. G. Hoschek for critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported by the Creative Research Initiatives Program of the Korean Ministry of Science and Technology (to E.-J. C.).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.
§ Both authors contributed equally to this work.
To whom correspondence should be addressed: Graduate School of
Biotechnology, Korea University, Seoul, 136-701, South Korea. Tel.:
82-2-3290-3446; Fax: 82-2-927-9028; E-mail:
ejchoi@mail.korea.ac.kr.
Published, JBC Papers in Press, January 18, 2001, DOI 10.1074/jbc.M005561200
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ABBREVIATIONS |
---|
The abbreviations used are:
MAPK, mitogen-activated protein kinase;
MAPKK, MAPK kinase;
MAPKKK, MAPK
kinase kinase;
ERK, extracellular signal-regulated kinase;
JNK, c-Jun
N-terminal kinase;
SAPK, stress-activated protein kinase;
ASK1, apoptosis signal-regulating kinase 1;
GST, glutathione
S-transferase;
TNF, tumor necrosis factor;
TRAF, TNF
receptor-associated factor;
HA, hemagglutinin, BSO,
L-buthionine-S,R-sulfoximine;
X-gal, 5-bromo-4-chloro-3-indolyl -D-galactopyranoside;
MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase
kinase;
GSH, reduced glutathione;
DAPI, 4',6-diamidino-2-phenyl-indole;
GFP, green fluorescence protein;
WT, wild type;
PAGE, polyacrylamide
gel electrophoresis.
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