Negative Regulation of MEKK1-induced Signaling by Glutathione S-Transferase Mu

Abstract

Mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1 (MEKK1) is an important component in the stress-activated protein kinase pathway. Glutathione S-transferase Mu 1-1 (GST M1-1) has now been shown to inhibit the stimulation of MEKK1 activity induced by cellular stresses such as UV and hydrogen peroxide. GST M1-1 inhibited MEKK1 activation in a manner independent of its glutathione-conjugating catalytic activity. In vitro binding and kinase assays revealed that GST M1-1 directly bound MEKK1 and inhibited its kinase activity. Co-immunoprecipitation analysis showed a physical association between endogenous GST M1-1 and endogenous MEKK1 in L929 cells. Overexpressed GST M1-1 interfered with the binding of MEKK1 to SEK1 in transfected HEK293 cells. Furthermore, GST M1-1 suppressed MEKK1-mediated apoptosis. Taken together, our results suggest that GST M1-1 functions as a negative regulator of MEKK1. Mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1 (MEKK1) is an important component in the stress-activated protein kinase pathway. Glutathione S-transferase Mu 1-1 (GST M1-1) has now been shown to inhibit the stimulation of MEKK1 activity induced by cellular stresses such as UV and hydrogen peroxide. GST M1-1 inhibited MEKK1 activation in a manner independent of its glutathione-conjugating catalytic activity. In vitro binding and kinase assays revealed that GST M1-1 directly bound MEKK1 and inhibited its kinase activity. Co-immunoprecipitation analysis showed a physical association between endogenous GST M1-1 and endogenous MEKK1 in L929 cells. Overexpressed GST M1-1 interfered with the binding of MEKK1 to SEK1 in transfected HEK293 cells. Furthermore, GST M1-1 suppressed MEKK1-mediated apoptosis. Taken together, our results suggest that GST M1-1 functions as a negative regulator of MEKK1. Mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1 (MEKK1), 1The abbreviations used are: MEKK1, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1; MEKK1-s, [35S]methionine-labeled MEKK1; MAP3K, mitogen-activated protein kinase kinase kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; SAPK, stress-activated protein kinase; HA, hemagglutinin; GST, glutathione S-transferase; ASK1, apoptosis signal-regulating kinase 1; GFP, green fluorescent protein; SEK, stress-activated protein kinase/extracellular signal-regulated kinase kinase.1The abbreviations used are: MEKK1, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1; MEKK1-s, [35S]methionine-labeled MEKK1; MAP3K, mitogen-activated protein kinase kinase kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; SAPK, stress-activated protein kinase; HA, hemagglutinin; GST, glutathione S-transferase; ASK1, apoptosis signal-regulating kinase 1; GFP, green fluorescent protein; SEK, stress-activated protein kinase/extracellular signal-regulated kinase kinase. a 196-kDa serine/threonine kinase, is one of the first identified members of the mitogen-activated protein kinase kinase kinases (MAP3Ks) (1Lange-Carter C.A. 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Those proteins include α-actin, 14-3-3, p21-activated kinase, nuclear factor κB-inducing kinase, and small guanine nucleotide-binding proteins such as Ras, Rac, and Cdc42 (3Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3564) Google Scholar, 17Fanger G.R. Widmann C. Porter A.C. Sather S. Johnson G.L. Vaillancourt R.R. J. Biol. Chem. 1998; 273: 3476-3483Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 18Russell M. Lange-Carter C.A. Johnson G.L. J. Biol. Chem. 1995; 270: 11757-11760Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 19Fanger G.R. Johnson N.L. Johnson G.L. EMBO J. 1997; 16: 4961-4972Crossref PubMed Scopus (253) Google Scholar, 20Christerson L.B. Vanderbilt C.A. Cobb M.H. Cell Motil. Cytoskeleton. 1999; 43: 186-198Crossref PubMed Scopus (89) Google Scholar, 21Kaga S. Ragg S. Rogers K.A. Ochi A. J. Immunol. 1998; 160: 4182-4189PubMed Google Scholar, 22Su Y.C. Han J. Xu S. Cobb M. Skolnik E.Y. EMBO J. 1997; 16: 1279-1290Crossref PubMed Scopus (212) Google Scholar, 23Xu S. Cobb M.H. J. Biol. Chem. 1997; 272: 32056-32060Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). The carboxyl-terminal region of MEKK1 contains a catalytic domain that binds and phosphorylates SEK1 (24Xia Y. Wu Z. Su B. Murray B. Karin M. Genes Dev. 1998; 12: 3369-3381Crossref PubMed Scopus (174) Google Scholar). Glutathione S-transferases (GSTs) are a group of detoxification proteins that catalyze the conjugation of reduced GSH to various electrophilic molecules. GSTs also function as nonenzymatic binding ligands that interact with a variety of lipophilic compounds including steroid hormones (25Hayes J.D. Pulford D.J. Crit. Rev. Biochem. Mol. Biol. 1995; 30: 445-600Crossref PubMed Scopus (3214) Google Scholar). The mammalian cytosolic GSTs are classified into five classes based on their primary structure: Alpha; Mu; Pi; Sigma; and Theta (25Hayes J.D. Pulford D.J. Crit. Rev. Biochem. Mol. Biol. 1995; 30: 445-600Crossref PubMed Scopus (3214) Google Scholar). The Alpha, Mu, and Pi are the major ones among these classes. There are many lines of evidence that GSTs have a protective role against a variety of cytotoxic agents (26Yang Y. Cheng J.Z. Singhal S.S. Saini M. Pandya U. Awasthi S. Awasthi Y.C. J. Biol. Chem. 2001; 276: 19220-19230Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 27Cheng J.Z. Singhal S.S. Saini M. Singhal J. Piper J.T. Van Kuijk F.J. Zimniak P. Awasthi Y.C. Awasthi S. Arch. Biochem. Biophys. 1999; 372: 29-36Crossref PubMed Scopus (109) Google Scholar, 28Cho S.G. Lee Y.H. Park H.S. Ryoo K. Kang K.W. Park J. Eom S.J. Kim M.J. Chang T.S. Choi S.Y. Shim J. Kim Y. Dong M.S. Lee M.J. Kim S.G. Ichijo H. Choi E.J. J. Biol. Chem. 2001; 276: 12749-12755Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). The Pi and Alpha classes of GST have been shown to suppress JNK (26Yang Y. Cheng J.Z. Singhal S.S. Saini M. Pandya U. Awasthi S. Awasthi Y.C. J. Biol. Chem. 2001; 276: 19220-19230Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 29Adler 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. Ronai Z. EMBO J. 1999; 18: 1321-1334Crossref PubMed Scopus (948) Google Scholar). We previously reported that GST M1-1 functions as an endogenous inhibitor of apoptosis signal-regulating kinase 1 (ASK1), a MAP3K of the JNK/SAPK and p38 mitogen-activated protein kinase pathways, thereby suppressing ASK1-mediated cell death (28Cho S.G. Lee Y.H. Park H.S. Ryoo K. Kang K.W. Park J. Eom S.J. Kim M.J. Chang T.S. Choi S.Y. Shim J. Kim Y. Dong M.S. Lee M.J. Kim S.G. Ichijo H. Choi E.J. J. Biol. Chem. 2001; 276: 12749-12755Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). To further understand the mechanism underlying the modulation of the JNK/SAPK signaling pathway by GST M1-1, we have investigated a possible effect of GST M1-1 on the components of this pathway and report in this study that GST M1-1 physically associates with MEKK1 and inhibits its kinase activity. GST M1-1, by binding to MEKK1, interferes with an interaction between MEKK1 and its substrate SEK1. Thus, our findings suggest that MEKK1 is a new target of GST M1-1. Cell Culture and DNA Transfection—Human embryonic kidney 293 (HEK293) cells and mouse fibrosarcoma L929 cells were cultured at 37 °C in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Hyclone). For DNA transfection, HEK293 cells were plated with a density of 3 × 106 cells/100-mm culture dish, incubated overnight, and then transfected with the indicated expression vectors by calcium phosphate method (30Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4798) Google Scholar) or with the use of LipofectAMINE (Invitrogen). Plasmid Construction—cDNA encoding JNK1 was amplified by the polymerase chain reaction and subcloned into p3×FLAG-CMV-10 (Sigma). cDNAs for GST-SEK1(K129R), GST-JNK1(K55R), and GST-c-Jun-(1–79) and mouse cDNAs encoding GST M1-1, GST M1-1-FLAG, HA-GST M1-1, and GST M1-1(Y6F) were described previously (31Park J. Kim I. Oh Y.J. Lee K. Han P.L. Choi E.J. J. Biol. Chem. 1997; 272: 16725-16728Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 32Shim J. Lee H. Park J. Kim H. Choi E.J. Nature. 1996; 381: 804-806Crossref PubMed Scopus (240) Google Scholar). Immune Complex Kinase Assay—Cells were lysed in buffer A that contained 40 mm Tris-HCl, pH 8.0, 120 mm NaCl, 1% Triton X-100, 2 mm EDTA, 2 mm EGTA, 25 mm β-glycerophosphate, 10% glycerol, 25 mm NaF, 1 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 10 μg/ml aprotinin. Cell lysates were subjected to microcentrifugation at 12,000 × g, and the resulting supernatants were subjected to immunoprecipitation with the use of the indicated antibodies. The immunoprecipitates were rinsed with buffer A and then with 20 mm Hepes buffer, pH 7.4, and assayed for the indicated protein kinases as described previously (31Park J. Kim I. Oh Y.J. Lee K. Han P.L. Choi E.J. J. Biol. Chem. 1997; 272: 16725-16728Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 32Shim J. Lee H. Park J. Kim H. Choi E.J. Nature. 1996; 381: 804-806Crossref PubMed Scopus (240) Google Scholar). Phosphorylated substrates were separated by SDS-PAGE and analyzed with a phosphorimaging device (BAS-2500; Fuji). GST-SEK1(K129R), GST-JNK1(K55R), or GST-c-Jun-(1–79) was used as a substrate for MEKK1, SEK1, or JNK1, respectively. Co-immunoprecipitation—Cell lysates were lysed with buffer B that contained 50 mm Tris-HCl, pH 8.0, 120 mm NaCl, 0.5% Nonidet P-40, 1 mm EDTA, 10 mm NaF, 2 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 10 μg/ml aprotinin, and the cell lysates were subjected to microcentrifugation at 12,000 × g at 4 °C. The soluble fraction was subjected to immunoprecipitation with the indicated antibodies. The resulting immunoprecipitates were subjected to SDS-PAGE and immunoblot analysis as described previously (28Cho S.G. Lee Y.H. Park H.S. Ryoo K. Kang K.W. Park J. Eom S.J. Kim M.J. Chang T.S. Choi S.Y. Shim J. Kim Y. Dong M.S. Lee M.J. Kim S.G. Ichijo H. Choi E.J. J. Biol. Chem. 2001; 276: 12749-12755Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). In Vitro Binding Assay—Recombinant hexahistidine-tagged GST M1-1 protein was bacterially expressed and purified using Ni2+-nitrilotriacetic acid beads (Qiagen) as described previously (28Cho S.G. Lee Y.H. Park H.S. Ryoo K. Kang K.W. Park J. Eom S.J. Kim M.J. Chang T.S. Choi S.Y. Shim J. Kim Y. Dong M.S. Lee M.J. Kim S.G. Ichijo H. Choi E.J. J. Biol. Chem. 2001; 276: 12749-12755Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). [35S]Methionine-labeled MEKK1 (MEKK1-s), SEK1, and JNK1 were produced by in vitro transcription and translation using the TnT reticulocyte lysate system (Promega). 35S-Labeled proteins were incubated with GST M1-1 immobilized onto Ni2+-nitrilotriacetic acid beads at 4 °C for 2 h in the binding buffer that contained 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 2 mm EDTA, 1 mm 0.1% Nonidet P-40, 0.1 mm sodium orthovanadate, and 5 mg/ml bovine serum albumin. The beads were rinsed with a washing buffer that contained 50 mm Hepes, pH 7.5, 150 mm NaCl, 1 mm EDTA, and 0.1% Tween 20. 35S-Labeled proteins bound on the beads were separated by SDS-PAGE and analyzed with a Fuji BAS 2500 PhosphorImager. Cell Death Assay—HEK293 cells were transiently transfected with pEGFP (Clontech) and the indicated plasmids. After 48 h of transfection, the cells were unexposed or exposed to the indicated agents. Cells were fixed with 4% paraformaldehyde and stained with 4′,6-diamidino-2-phenolindole. The number of cells with apoptotic nuclei was counted among GFP-expressing cells by fluorescence microscopy. The apoptotic cell death was calculated as the percentage of apoptotic cells over total GFP-positive cells. GST M1-1 Inhibits MEKK1 Activity—To investigate whether GST M1-1 regulates MEKK1, HEK293 cells were transfected with a plasmid vector encoding HA-tagged MEKK1 alone or together with a plasmid encoding FLAG-tagged GST M1-1. The exposure of the transfected cells to UV light resulted in an increase in MEKK1 activity, and this activation was inhibited by GST M1-1 (Fig. 1A). GST M1-1 also inhibited H2O2-induced stimulation of MEKK1 activity (Fig. 1B). In comparison, GST M1-1 did not affect the UV-induced stimulation of MLK3 (Fig. 1D) or TAK1 (data not shown). MLK3 and TAK1 are other MAP3Ks in the JNK signaling pathway (3Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3564) Google Scholar, 33Schlesinger T.K. Fanger G.R. Yujiri T. Johnson G.L. Front. Biosci. 1998; 3: D1181-D1186Crossref PubMed Google Scholar). Cdc42, a member of the small GTP-binding protein Rho family, has been shown to with and MEKK1 G.R. Johnson N.L. Johnson G.L. EMBO J. 1997; 16: 4961-4972Crossref PubMed Scopus (253) Google Scholar). The of a of Cdc42, resulted in the activation of MEKK1 (Fig. GST M1-1 inhibited the activation of MEKK1. MEKK1 a of GST whether GST M1-1 inhibits MEKK1 by directly on the kinase. In vitro kinase study revealed that MEKK1 activity was inhibited by GST M1-1 protein not by of GST, GST (Fig. In comparison, GST M1-1 did not inhibit SEK1 or JNK1 activity in vitro (Fig. in vitro binding indicated that GST M1-1 directly bound to MEKK1 not to SEK1 or JNK1 (Fig. In in vitro binding GST M1-1 also bound a MEKK1 to the of MEKK1 (data not M1-1 physically associates with hexahistidine-tagged GST M1-1 protein was bacterially expressed and immobilized onto Ni2+-nitrilotriacetic acid MEKK1, SEK1, and JNK1 were and in vitro in the of 35S-Labeled proteins were incubated at 4 °C for 2 h with GST M1-1 immobilized on the The bound proteins were separated by SDS-PAGE and analyzed with a HEK293 cells were transfected for 48 h with expression encoding FLAG-tagged GST M1-1 and HA-tagged MEKK1 or encoding HA-tagged GST M1-1 and FLAG-tagged Cell lysates were subjected to immunoprecipitation with or and the resulting immunoprecipitates were analyzed by with or Cell lysates were also subjected to immunoblot analysis with the indicated antibodies. L929 cell lysates were subjected to immunoprecipitation with or M1-1 The immunoprecipitates were analyzed by with of cell lysates was also We to investigate a physical interaction between GST M1-1 and MEKK1 in cells. HEK293 cells were with expression vectors encoding FLAG-tagged GST M1-1 and HA-tagged MEKK1. analysis using of the immunoprecipitates indicated that GST M1-1 physically with MEKK1 in the cells (Fig. The interaction between MEKK1 and GST M1-1 was not by the exposure of cells to UV light (data not shown). In a GST M1-1 physically with (Fig. Furthermore, GST M1-1 inhibited the kinase activity of in transfected HEK293 cells (data not shown). We an interaction between endogenous GST M1-1 and MEKK1 proteins in L929 fibrosarcoma cells. L929 cells have been shown to of Mu GST proteins J. Biol. Chem. Full Text PDF PubMed Google Scholar). Co-immunoprecipitation that endogenous GST M1-1 physically with endogenous MEKK1 in L929 cells (Fig. Taken together, our results suggest that GST M1-1 physically associates with and inhibits MEKK1. GST M1-1 with an between MEKK1 and that GST M1-1 physically with MEKK1, we investigated whether GST M1-1 the binding of MEKK1 to its substrate SEK1. HEK293 cells were transfected with expression vectors encoding and in the or the of the GST M1-1 A study showed that MEKK1 physically with SEK1 in the transfected cells and that this interaction was inhibited by GST M1-1 (Fig. In comparison, GST M1-1 did not inhibit an interaction between and SEK1. GST M1-1 did not affect the binding of MEKK1 to JNK1 (Fig. MEKK1 has been shown to the by physically with SEK1 and JNK G. Davis R.J. Biochem. Sci. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). GST M1-1 Inhibits MEKK1 of M1-1 is an that a conjugation of GSH to a substrate The in the acid of GST M1-1 is to for the activity J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, P. R. J. R. EMBO J. PubMed Scopus Google Scholar). To investigate whether the of GST M1-1 on MEKK1 the catalytic activity of GST M1-1, HEK293 cells were transfected with encoding and GST M1-1 or GST M1-1(Y6F) and for UV-induced MEKK1 GST M1-1(Y6F) is a of GST M1-1. GST M1-1(Y6F) inhibited the kinase activity of MEKK1 as as GST M1-1 did (Fig. Thus, these results suggest that the activity of GST M1-1 is for the of on MEKK1. GST M1-1 Inhibits MEKK1-mediated has been shown to for JNK activation and cell death induced by microtubule-disrupting agents such as R. J. T. Cheng G. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). we whether GST M1-1 inhibits the stimulation of MEKK1 and JNK1 as as apoptotic cell death (Fig. the kinase of MEKK1 (Fig. and JNK1 (Fig. and these were suppressed in the cells GST M1-1. The exposure of HEK293 cells to also resulted in a increase in apoptotic cell The apoptosis was inhibited by a of MEKK1, not by a of (Fig. suggest that MEKK1 apoptosis. The expression of GST M1-1 suppressed apoptosis. Overexpressed GST M1-1 also inhibited UV-induced apoptosis. GST the conjugation of reduced glutathione to electrophilic or of also binds a variety of compounds that include and (25Hayes J.D. Pulford D.J. Crit. Rev. Biochem. Mol. Biol. 1995; 30: 445-600Crossref PubMed Scopus (3214) Google Scholar). in and detoxification of GSTs to modulate the signaling induced by cellular The Pi GST directly with JNK as a at the thereby a JNK activity 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. Ronai Z. EMBO J. 1999; 18: 1321-1334Crossref PubMed Scopus (948) Google Scholar). GST inhibits the hydrogen of JNK and as as hydrogen apoptosis in cells (26Yang Y. Cheng J.Z. Singhal S.S. Saini M. Pandya U. Awasthi S. Awasthi Y.C. J. Biol. Chem. 2001; 276: 19220-19230Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). We previously showed that GST M1-1, by directly binding ASK1, activation and apoptosis (28Cho S.G. Lee Y.H. Park H.S. Ryoo K. Kang K.W. Park J. Eom S.J. Kim M.J. Chang T.S. Choi S.Y. Shim J. Kim Y. Dong M.S. Lee M.J. Kim S.G. Ichijo H. Choi E.J. J. Biol. Chem. 2001; 276: 12749-12755Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). findings in this study that GST M1-1 physically associates with MEKK1 in vitro and in cells and that GST M1-1 inhibits the MEKK1 activity. In comparison, GST M1-1 not inhibit MLK3 and of are other MAP3Ks of the JNK/SAPK pathway. GST M1-1 to the stress-activated protein kinase pathway by directly MEKK1 and GST M1-1 binds the carboxyl-terminal of MEKK1 that contains the catalytic domain and inhibits the interaction between MEKK1 and SEK1. In comparison, GST M1-1 binds the amino-terminal regulatory domain of and inhibits (28Cho S.G. Lee Y.H. Park H.S. Ryoo K. Kang K.W. Park J. Eom S.J. Kim M.J. Chang T.S. Choi S.Y. Shim J. Kim Y. Dong M.S. Lee M.J. Kim S.G. Ichijo H. Choi E.J. J. Biol. Chem. 2001; 276: 12749-12755Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). Thus, GST M1-1 to inhibit MEKK1 and in studies are for a role of MEKK1 in apoptosis induced by microtubule-disrupting agents, our study that MEKK1 apoptosis. However, is that studies have shown that MEKK1 function as a or an in cell T. Sather S. Fanger G.R. Johnson G.L. Science. 1998; 282: 1911-1914Crossref PubMed Scopus (280) Google Scholar, R. J. T. Cheng G. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). embryonic cells are to and the expression of MEKK1 the transfected embryonic cells apoptosis T. Sather S. Fanger G.R. Johnson G.L. Science. 1998; 282: 1911-1914Crossref PubMed Scopus (280) Google Scholar). In are to apoptosis with cells R. J. T. Cheng G. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). studies are to understand these of MEKK1. Taken together, our findings suggest that MEKK1 is a new target of GST M1-1 and that the of MEKK1 activation is an of the mechanism underlying the negative of signaling by GST M1-1. We G. R. J. L. I. K. J. S. A. and K. for MEKK1, JNK1, SEK1, and TAK1 vector and A. for of the