Glutathione S-Transferase Mu Modulates the Stress-activated Signals by Suppressing Apoptosis Signal-regulating Kinase 1*

Ssang-Goo ChoDagger §, Yong Hee LeeDagger §, Hee-Sae ParkDagger , Kanghyun RyooDagger , Keon Wook Kang, Jihyun ParkDagger , Soo-Jung EomDagger , Myung Jin KimDagger , Tong-Shin ChangDagger , Soo-Yeon ChoiDagger , Jaekyung ShimDagger , Youngho KimDagger , Mi-Sook DongDagger , Min-Jae Lee||, Sang Geon Kim, Hidenori Ichijo**, and Eui-Ju ChoiDagger DaggerDagger

From the Dagger  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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-alpha and interleukin-1beta 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).

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-alpha and Fas-induced apoptosis (11, 12).

TNF-alpha 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-alpha 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-alpha -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.

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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

DNA Constructs-- ASK1 mutants, ASK-Delta N, ASK1-Delta 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-Delta N-(85-218) and mGSTM1-Delta 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 Delta 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).

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-Delta 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 beta -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.

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-Delta N, or mGSTM1-Delta 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.

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-beta -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 beta -galactosidase activity in the same cells.

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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-Delta C, or ASK1-Delta 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-Delta C, but not ASK1-Delta 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-Delta C, or ASK1-NT, but not with ASK1-Delta 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-Delta 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.


View larger version (23K):
[in this window]
[in a new window]
 
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-Delta 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.

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-Delta N, and mGSTM1-Delta C (Fig. 2A). mGSTM1-Delta N and mGSTM1-Delta 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-Delta N but scarcely with mGSTM1-Delta C. It is noteworthy that mGSTM1-Delta 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-Delta C was able to bind to GSH-affinity resin with a high efficiency (data not shown). Furthermore, the binding of mGSTM1-Delta C or mGSTM1-1 to the GSH-agarose beads was reduced in the presence of free GSH (Fig. 2B). Thus, mGSTM1-Delta 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.


View larger version (34K):
[in this window]
[in a new window]
 
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-Delta N, and mGSTM1-Delta 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-Delta N, or GSTµ-Delta 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-Delta 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.

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 × 10-9 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.


View larger version (28K):
[in this window]
[in a new window]
 
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.


View larger version (26K):
[in this window]
[in a new window]
 
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).

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).


View larger version (32K):
[in this window]
[in a new window]
 
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.

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.


View larger version (55K):
[in this window]
[in a new window]
 
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.

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 SAPKbeta activation. In comparison, mGSTM1-1 did not affect SAPKbeta activity stimulated by overexpression of Delta 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.


View larger version (20K):
[in this window]
[in a new window]
 
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, Delta MEKK1, HA-SAPKbeta , 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 SAPKbeta 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-beta -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 beta -galactosidase activity measured.

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-alpha or by the Fas-Daxx pathway (11, 12). We, therefore, tested the effect of mGSTM1-1 on TNF-alpha - or Daxx-induced apoptotic cell death (Fig. 8). Exposure of HeLa cells to TNF-alpha markedly enhanced apoptotic cell death (Fig. 8A), whereas the TNF-alpha -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-alpha -induced apoptosis. mGSTM1-1 alleviated the TNF-alpha -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.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 8.   mGSTM1-1 suppresses TNF-alpha - 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-alpha (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

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.

    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.

    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.

Dagger Dagger 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

    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 beta -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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Cohen, J. J. (1993) Immunol. Today 14, 126-130[Medline] [Order article via Infotrieve]
2. Raff, M. C. (1992) Nature 356, 397-400[CrossRef][Medline] [Order article via Infotrieve]
3. Wyllie, A. H. (1980) Nature 284, 555-556[Medline] [Order article via Infotrieve]
4. Kerr, J. F., Wyllie, A. H., and Currie, A. R. (1972) Br. J. Cancer 26, 239-257[Medline] [Order article via Infotrieve]
5. Thompson, C. B. (1995) Science 267, 1456-1462[Medline] [Order article via Infotrieve]
6. Minden, A., and Karin, M. (1997) Biochim. Biophys. Acta 1333, F85-F104[CrossRef][Medline] [Order article via Infotrieve]
7. Ip, Y. T., and Davis, R. J. (1998) Curr. Opin. Cell Biol. 10, 205-219[CrossRef][Medline] [Order article via Infotrieve]
8. Xia, Z., Dickens, M., Raingeaud, J., Davis, R. J., and Greenberg, M. E. (1995) Science 270, 1326-1331[Abstract]
9. Verheij, M., Bose, R., Lin, X. H., Yao, B., Jarvis, W. D., Grant, S., Birrer, M. J., Szabo, E., Zon, L. I., Kyriakis, J. M., Haimovitz-Friedman, A., Fuks, Z., and Kolesnick, R. N. (1996) Nature 380, 75-79[CrossRef][Medline] [Order article via Infotrieve]
10. Ichijo, H. (1999) Oncogene 18, 6087-6093[CrossRef][Medline] [Order article via Infotrieve]
11. Ichijo, H., Nishida, E., Irie, K., ten Dijke, P., Saitoh, M., Moriguchi, T., Takagi, M., Matsumoto, K., Miyazono, K., and Gotoh, Y. (1997) Science 275, 90-94[Abstract/Free Full Text]
12. Chang, H. Y., Nishitoh, H., Yang, X., Ichijo, H., and Baltimore, D. (1998) Science 281, 1860-1863[Abstract/Free Full Text]
13. Tartaglia, L. A., and Goeddel, D. V. (1992) Immunol. Today 13, 151-153[CrossRef][Medline] [Order article via Infotrieve]
14. Hsu, H., Huang, J., Shu, H. B., Baichwal, V., and Goeddel, D. V. (1996) Immunity 4, 387-396[Medline] [Order article via Infotrieve]
15. Hsu, H., Shu, H. B., Pan, M. G., and Goeddel, D. V. (1996) Cell 84, 299-308[Medline] [Order article via Infotrieve]
16. Hsu, H., Xiong, J., and Goeddel, D. V. (1995) Cell 81, 495-504[Medline] [Order article via Infotrieve]
17. Rothe, M., Pan, M. G., Henzel, W. J., Ayres, T. M., and Goeddel, D. V. (1995) Cell 83, 1243-1252[Medline] [Order article via Infotrieve]
18. Rothe, M., Wong, S. C., Henzel, W. J., and Goeddel, D. V. (1994) Cell 78, 681-692[Medline] [Order article via Infotrieve]
19. Shu, H. B., Takeuchi, M., and Goeddel, D. V. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13973-13978[Abstract/Free Full Text]
20. Hoeflich, K. P., Yeh, W. C., Yao, Z., Mak, T. W., and Woodgett, J. R. (1999) Oncogene 18, 5814-5820[CrossRef][Medline] [Order article via Infotrieve]
21. Nishitoh, H., Saitoh, M., Mochida, Y., Takeda, K., Nakano, H., Rothe, M., Miyazono, K., and Ichijo, H. (1998) Mol. Cell 2, 389-395[Medline] [Order article via Infotrieve]
22. Ashkenazi, A., and Dixit, V. M. (1998) Science 281, 1305-1308[Abstract/Free Full Text]
23. Abbas, A. K. (1996) Cell 84, 655-657[Medline] [Order article via Infotrieve]
24. Boldin, M. P., Goncharov, T. M., Goltsev, Y. V., and Wallach, D. (1996) Cell 85, 803-815[Medline] [Order article via Infotrieve]
25. Muzio, M., Chinnaiyan, A. M., Kischkel, F. C., O'Rourke, K., Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J. D., Zhang, M., Gentz, R., Mann, M., Krammer, P. H., Peter, M. E., and Dixit, V. M. (1996) Cell 85, 817-827[Medline] [Order article via Infotrieve]
26. Nagata, S. (1997) Cell 88, 355-365[Medline] [Order article via Infotrieve]
27. Yang, X., Khosravi-Far, R., Chang, H. Y., and Baltimore, D. (1997) Cell 89, 1067-1076[Medline] [Order article via Infotrieve]
28. Hayes, J. D., and Pulford, D. J. (1995) Crit. Rev. Biochem. Mol. Biol. 30, 445-600[Abstract]
29. Mannervik, B., and Danielson, U. H. (1988) CRC Crit. Rev. Biochem. 23, 283-337[Medline] [Order article via Infotrieve]
30. Rushmore, T. H., and Pickett, C. B. (1993) J. Biol. Chem. 268, 11475-11478[Free Full Text]
31. Ishigaki, S., Abramovitz, M., and Listowsky, I. (1989) Arch. Biochem. Biophys. 273, 265-272[Medline] [Order article via Infotrieve]
32. Ketley, J. N., Habig, W. H., and Jakoby, W. B. (1975) J. Biol. Chem. 250, 8670-8673[Abstract]
33. Litwack, G., Ketterer, B., and Arias, I. M. (1971) Nature 234, 466-467[Medline] [Order article via Infotrieve]
34. Park, J., Kim, I., Oh, Y. J., Lee, K., Han, P. L., and Choi, E. J. (1997) J. Biol. Chem. 272, 16725-16728[Abstract/Free Full Text]
35. Shim, J., Lee, H., Park, J., Kim, H., and Choi, E. J. (1996) Nature 381, 804-806[CrossRef][Medline] [Order article via Infotrieve]
36. Chen, C., and Okayama, H. (1987) Mol. Cell. Biol. 7, 2745-2752[Medline] [Order article via Infotrieve]
37. Saitoh, M., Nishitoh, H., Fujii, M., Takeda, K., Tobiume, K., Sawada, Y., Kawabata, M., Miyazono, K., and Ichijo, H. (1998) EMBO J. 17, 2596-2606[Abstract/Free Full Text]
38. Lee, H. C., Toung, Y. P., Tu, Y. S., and Tu, C. P. (1995) J. Biol. Chem. 270, 99-109[Abstract/Free Full Text]
39. Reinemer, P., Dirr, H. W., Ladenstein, R., Schaffer, J., Gallay, O., and Huber, R. (1991) EMBO J. 10, 1997-2005[Abstract]
40. Gopalakrishna, R., Chen, Z. H., and Gundimeda, U. (1997) Arch. Biochem. Biophys. 348, 37-48[CrossRef][Medline] [Order article via Infotrieve]
41. Meister, A. (1994) Cancer Res. 54, 1969s-1975s[Medline] [Order article via Infotrieve]
42. Plummer, J. L., Smith, B. R., Sies, H., and Bend, J. R. (1981) Methods Enzymol. 77, 50-59[Medline] [Order article via Infotrieve]
43. Yokomizo, A., Kohno, K., Wada, M., Ono, M., Morrow, C. S., Cowan, K. H., and Kuwano, M. (1995) J. Biol. Chem. 270, 19451-19457[Abstract/Free Full Text]
44. Gotoh, Y., and Cooper, J. A. (1998) J. Biol. Chem. 273, 17477-17482[Abstract/Free Full Text]
45. Adler, V., Yin, Z., Fuchs, S. Y., Benezra, M., Rosario, L., Tew, K. D., Pincus, M. R., Sardana, M., Henderson, C. J., Wolf, C. R., Davis, R. J., and Ronai, Z. (1999) EMBO J. 18, 1321-1334[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.