Activation of the p38 Signaling Pathway by Heat Shock Involves the Dissociation of Glutathione S-Transferase Mu from Ask1
2002; Elsevier BV; Volume: 277; Issue: 34 Linguagem: Inglês
10.1074/jbc.m203642200
ISSN1083-351X
AutoresSonia Dorion, Herman Lambert, Jacques Landry,
Tópico(s)Genomics, phytochemicals, and oxidative stress
ResumoDespite the importance of the stress-activated protein kinase pathways in cell death and survival, it is unclear how stressful stimuli lead to their activation. In the case of heat shock, the existence of a specific mechanism of activation has been evidenced, but the molecular nature of this pathway is undefined. Here, we found that Ask1 (apoptosis signal-regulating kinase 1), an upstream activator of the stress-activated protein kinase p38 during exposure to oxidative stress and other stressful stimuli, was also activated by heat shock. Ask1 activity was required for p38 activation since overexpression of a kinase dead mutant of Ask1, Ask1(K709M), inhibited heat shock-induced p38 activation. The activation of Ask1 by oxidative stress involves the oxidation of thioredoxin, an endogenous inhibitor of Ask1. A different activation mechanism takes place during heat shock. In contrast to p38 induction by H2O2, induction by heat shock was not antagonized by pretreatment with the antioxidantN-acetyl-l-cysteine or by overexpressing thioredoxin and was not accompanied by the dissociation of thioredoxin from Ask1. Instead, heat shock caused the dissociation of glutathioneS-transferase Mu1-1 (GSTM1-1) from Ask1 and overexpression of GSTM1-1-inhibited induction of p38 by heat shock. We concluded that because of an alternative regulation by the two distinct repressors thioredoxin and GSTM1-1, Ask1 constitutes the converging point of the heat shock and oxidative stress-sensing pathways that lead to p38 activation. Despite the importance of the stress-activated protein kinase pathways in cell death and survival, it is unclear how stressful stimuli lead to their activation. In the case of heat shock, the existence of a specific mechanism of activation has been evidenced, but the molecular nature of this pathway is undefined. Here, we found that Ask1 (apoptosis signal-regulating kinase 1), an upstream activator of the stress-activated protein kinase p38 during exposure to oxidative stress and other stressful stimuli, was also activated by heat shock. Ask1 activity was required for p38 activation since overexpression of a kinase dead mutant of Ask1, Ask1(K709M), inhibited heat shock-induced p38 activation. The activation of Ask1 by oxidative stress involves the oxidation of thioredoxin, an endogenous inhibitor of Ask1. A different activation mechanism takes place during heat shock. In contrast to p38 induction by H2O2, induction by heat shock was not antagonized by pretreatment with the antioxidantN-acetyl-l-cysteine or by overexpressing thioredoxin and was not accompanied by the dissociation of thioredoxin from Ask1. Instead, heat shock caused the dissociation of glutathioneS-transferase Mu1-1 (GSTM1-1) from Ask1 and overexpression of GSTM1-1-inhibited induction of p38 by heat shock. We concluded that because of an alternative regulation by the two distinct repressors thioredoxin and GSTM1-1, Ask1 constitutes the converging point of the heat shock and oxidative stress-sensing pathways that lead to p38 activation. Jun N-terminal kinase apoptosis signal-regulating kinase 1 glutathione S-transferase GST Mu1-1 myelin basic protein N-acetyl-l-cysteine tumor necrosis factor TNF receptor-associated factor 2 thioredoxin hemagglutinin mitogen-activated protein Heat shock affects all proteins and structures but nevertheless produces a highly specific stress response aimed at protecting the cells and re-establishing homeostasis. In addition to the well characterized transcriptional activation of the genes coding for heat shock proteins (1Morimoto R.I. Jurivich D.A. Kroeger P.E. Mathur S.K. Murphy S.P. Nakai A. Sarge K. Abravaya K. Sistonen L.T. Morimoto R. Tissières A. Georgopoulos C. The Biology of Heat Shock Proteins and Molecular Chaperones. 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Phosphorylation of HSP27 activates a protective function, which may result from the known phosphorylation-modulated function of the protein at the level of the actin microfilaments (6Lavoie J.N. Lambert H. Hickey E. Weber L.A. Landry J. Mol. Cell. Biol. 1995; 15: 505-516Crossref PubMed Scopus (572) Google Scholar, 7Guay J. Lambert H. Gingras-Breton G. Lavoie J.N. Huot J. Landry J. J. Cell Sci. 1997; 110: 357-368Crossref PubMed Google Scholar, 8Landry J. Huot J. Biochem. Soc. Symp. 1999; 64: 79-89PubMed Google Scholar) or from other described protective activities, either as a chaperone (9Jakob U. Gaestel M. Engel K. Buchner J. J. Biol. Chem. 1993; 268: 1517-1520Abstract Full Text PDF PubMed Google Scholar, 10Ehrnsperger M. Graber S. Gaestel M. Buchner J. EMBO J. 1997; 16: 221-229Crossref PubMed Scopus (638) Google Scholar, 11Lee G.J. Roseman A.M. Saibil H.R. Vierling E. 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After a mild heat shock, cells becomes refractory to reinduction of p38 activity by a second heat shock but remained fully responsive to reinduction by other stresses, cytokines, or growth factors (15Dorion S. Berube J. Huot J. Landry J. J. Biol. Chem. 1999; 274: 37591-37597Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). The specificity of this desensitization reinforces the existence of a highly specific heat shock-sensing pathway upstream of p38. Despite its importance for cell survival, the signaling components and the molecular mechanism leading to heat shock-induced p38 activation are unknown. Little is known about the mechanisms of activation of the stress-sensitive pathways. In the case of UV light and hyperosmotic shock, activation of the stress-activated protein kinase JNK1 is triggered by an activation of the receptors for epidermal growth factor, tumor necrosis factor (TNF) α, and interleukin-1 (16Rosette C. Karin M. Science. 1996; 274: 1194-1197Crossref PubMed Scopus (947) Google Scholar). Alterations of receptor conformation by energy absorption or physical perturbation of the cell surface are thought to be the initial triggering events causing the clustering and internalization of these receptors and the subsequent subversion of signaling pathways normally used by growth factors or cytokines (16Rosette C. Karin M. Science. 1996; 274: 1194-1197Crossref PubMed Scopus (947) Google Scholar). In the case of oxidative stress, the sensing mechanism seems to act at the level of Ask1 (apoptosis signal-regulating kinase-1). Ask1 is a MAP kinase kinase kinase that can activate the MAP kinase kinases 3 and 6 leading to the activation of p38, or the MAP kinase kinases 4 and 7 leading to the activation of JNK (17Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (2059) Google Scholar). The redox regulatory protein thioredoxin (Trx) acts as the oxidative stress sensor for this cascade (18Saitoh M. Nishitoh H. Fujii M. Takeda K. Tobiume K. Sawada Y. Kawabata M. Miyazono K. Ichijo H. EMBO J. 1998; 17: 2596-2606Crossref PubMed Scopus (2112) Google Scholar). Under normal conditions, Trx in the reduced state binds to and inhibits Ask1. Upon oxidative stress, oxidation of Trx triggers its dissociation from Ask1, allowing the activation of Ask1 and the subsequent activation of downstream kinases. Here we show that Ask1 is also activated during heat shock and that this activation is responsible for p38 activation. However, heat shock activation of Ask1 does not proceed by a redox-dependent mechanism as shown for oxidative stress. Instead, a new mechanism of Ask1 activation is described involving the heat shock-induced dissociation from Ask1 of a recently identified inhibitor of Ask1, glutathione S-transferase Mu1-1 (GSTM1-1) (19Cho 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 (350) Google Scholar). It is concluded that the alternative regulation of Ask1 by the redox-sensitive repressor Trx or the heat-sensitive repressor GSTM1-1 defines the converging point of the heat shock and oxidative stress-sensing pathway leading to p38 activation. [γ-32P]ATP (3000 Ci/mmol) was purchased from PerkinElmer Life Sciences. H2O2, N-acetyl-l-cysteine (NAC), and myelin basic protein (MBP) were from Sigma. Protein A-Sepharose was from AmershamBiosciences. Chemicals for electrophoresis were obtained from Bio-Rad and Fisher. HA.11 is a mouse monoclonal antibody recognizing the YPYDVPDYA peptide sequence from human influenza hemagglutinin protein (Covance Research Products, Philadelphia, PA). 9E10 is a mouse monoclonal antibody recognizing the EQKLISEEDL peptide sequence from the human c-Myc protein. 9E10 was prepared from hybridoma cells (American Type Culture Collection). Monoclonal anti-human thioredoxin was obtained from Serotec (Missisauga, ON, Canada). All other antibodies used are polyclonal antibodies raised in rabbit. Anti-p38 recognizes the C-terminal sequence PPLQEEMES of murine p38 (7Guay J. Lambert H. Gingras-Breton G. Lavoie J.N. Huot J. Landry J. J. Cell Sci. 1997; 110: 357-368Crossref PubMed Google Scholar). Antibody against phosphorylated p38 was obtained from New England Biolabs (Beverly, MA). Anti-Ask1 is a novel antibody developed for this study. It was raised in rabbits against the C-terminal sequence of human Ask1 protein (KAIIDFRNKQT) as described before for anti-p38 (7Guay J. Lambert H. Gingras-Breton G. Lavoie J.N. Huot J. Landry J. J. Cell Sci. 1997; 110: 357-368Crossref PubMed Google Scholar). Anti-GSTM1–1 is an affinity-purified polyclonal antibody (20Whelan R.D. Hosking L.K. Townsend A.J. Cowan K.H. Hill B.T. Cancer Commun. 1989; 1: 359-365Crossref PubMed Scopus (60) Google Scholar). Chinese hamster CCL39 and human Hela cells were cultivated in Dulbecco's modified Eagle's medium containing 2.2 g/l NaHCO3 and 4.5 g/l glucose, and supplemented with 5 or 10% fetal bovine serum, respectively. Cultures were maintained at 37 °C in a 5% CO2 humidified atmosphere. Exponentially growing cells (106 cells per 60 × 15-mm culture dish plated the day before the experiment) were used for all treatments. For heat shock treatment, the dishes were sealed with parafilm and immersed into a circulating water bath thermoregulated at 44 ± 0.05 °C for the indicated period of time. All other inducers used were added directly into culture medium, and the cells were maintained at 37 °C for the duration of treatments. NAC was used after adjusting the pH to 7.4 with NaOH. The plasmids pEBGp38, pCMV5-Myc-Trx, pHM6GSTM1-1(Y6F), pcDNA3-HA-Ask1-WT and K709M were used for expression of glutathione S-transferase (GST)-tagged p38, Myc-tagged human thioredoxin, a HA-tagged catalytically inactive form of mouse GSTM1-1, a HA-tagged human Ask1, and a catalytically inactive Ask1 mutant, respectively (17Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (2059) Google Scholar, 19Cho 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 (350) Google Scholar, 21Liu H. Nishitoh H. Ichijo H. Kyriakis J.M. Mol. Cell. Biol. 2000; 20: 2198-2208Crossref PubMed Scopus (452) Google Scholar, 22Zanke B.W. Rubie E.A. Winnett E. Chan J. Randall S. Parsons M. Boudreau K. McInnis M. Yan M. Templeton D.J. Woodgett J.R. J. Biol. Chem. 1996; 271: 29876-29881Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). CCL39 cells were plated 24 h before transfection at a concentration of 1 × 106cells/75 cm2 culture flask. Transfection by calcium phosphate precipitation was done as described before, using 20 μg of plasmid per flask (23Landry J. Chrétien P. Lambert H. Hickey E. Weber L.A. J. Cell Biol. 1989; 109: 7-15Crossref PubMed Scopus (597) Google Scholar). The cells were replated 24 h later and used 48 h after transfection. After treatments, cells were scraped and extracted in lysis buffer containing 20 mm Tris-HCl, pH 7.5, 12 mm β-glycerophosphate, 150 mmNaCl, 5 mm EGTA, 1 mmNa3VO4, 10 mm NaF, 1% Triton X-100, 0.5% deoxycholate, 20 μg/ml aprotinin, 3 mmdithiothreitol, and 1 mm phenylmethylsulfonyl fluoride. The extracts were vortexed and centrifuged at 17,000 × gfor 12 min at 4 °C. The clarified supernatants were frozen on dry ice and stored at −80 °C. The further steps were carried out at 4 °C. To assay Ask1 activity, an equal volume of Ask1 cell lysate normalized for Ask1 protein was incubated with 10 μl of anti-Ask1 antibody for 1 h and harvested with 15 μl of protein A-Sepharose 50% v/v in lysis buffer. After 30 min, the samples were centrifuged for 15 s and washed twice with 300 μl of 20 mmTris-HCl, pH 7.5, containing 250 mm NaCl, 5 mmEGTA, 1% Triton X-100, 2 mm dithiothreitol, and 1 mm phenylmethylsulfonyl fluoride. This was followed by two washes with 20 mm Tris-HCl, pH 7.5, containing 5 mm EGTA, 2 mm dithiothreitol, and 1 mm phenylmethylsulfonyl fluoride. Immunoprecipitate was directly used for the kinase assays. The assay was carried out in 20 μl of kinase buffer (100 μm ATP, 3 μCi of [γ-32P]ATP, 20 mm Tris-HCl, pH 7.5, 20 mm MgCl2, and 6.5 μg of MBP). Ask1 activity was assayed for 12 min at 30 °C and was stopped by the addition of 10 μl of SDS-PAGE loading buffer. Kinase activity was evaluated by measuring incorporation of the radioactivity into MBP after resolution by SDS-PAGE and quantification using a PhosphorImager (Molecular Dynamics). To assay endogenous or transfected GST-tagged p38 activity, cells were lysed directly in SDS-PAGE loading buffer. Proteins were separated by SDS-PAGE on 10% acrylamide gels. Under these conditions, the transfected tagged protein is distinguished from the endogenous protein due to its slower migration. Proteins were then transferred onto nitrocellulose as previously described (6Lavoie J.N. Lambert H. Hickey E. Weber L.A. Landry J. Mol. Cell. Biol. 1995; 15: 505-516Crossref PubMed Scopus (572) Google Scholar). After reacting the membranes with anti-phospho-p38 antibody, proteins were detected using an enhanced chemiluminescence (ECL) detection kit (Pierce). Equal loading of the kinase on different lanes was verified by immunoblotting with anti-p38 antibody. After treatments, cells were scraped and extracted in coimmunoprecipitation buffer containing 20 mm Tris-HCl, pH 7.5, 10% glycerol, 150 mmNaCl, 10 mm EDTA, 1 mmNa3VO4, 1% Triton X-100, 0.5% deoxycholate, 20 μg/ml aprotinin, and 1 mm phenylmethylsulfonyl fluoride. The extracts were vortexed and centrifuged at 17,000 ×g for 12 min at 4 °C. The clarified supernatants were incubated with anti-Trx or anti-GSTM1-1 antibody for 1 h, and the immune complexes were harvested with 10 μl of protein A-Sepharose 50% suspension in coimmunoprecipitation buffer. After 30 min, samples were centrifuged for 15 s and washed at least three times with 300 μl of coimmunoprecipitation buffer. Proteins were separated using SDS-PAGE, transferred onto nitrocellulose, and immunoblotted with the appropriate antibody. Heat shock strongly activates the stress-activated protein kinase p38, but the upstream signaling pathway leading to this activation is not known. The MAP kinase kinase kinase Ask1 has been shown to be an upstream activator of p38 during exposure to several stressful stimuli (17Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (2059) Google Scholar, 19Cho S.G. Lee Y.H. Park H.S. Ryoo K. Kang K.W. Park J. Eom S.J. Kim M.J. 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Ask1 activity was determined using MBP as substrate. Ask1 activity increased in a time-dependent manner upon heating (Fig.1A). To determine whether Ask1 was an obligatory component of the heat shock-induced p38 activation pathway, we examined the effect of overexpressing Ask1(K709M), a catalytically inactive mutant of Ask1, on p38 activation. Cells were transfected with expression vectors encoding HA-tagged Ask1(K709M) and GST-tagged p38 and exposed to heat shock or other known activators of p38. p38 activity was measured in transfected cells using a phosphospecific p38 antibody (Fig. 1B, left panels). Western blot analysis confirmed equivalent levels of expression of GST-tagged p38 and increasing levels of expression of HA-Ask1(K709M) (Fig. 1B, middle and right panels). Expression of Ask1(K709M) inhibited heat shock-induced p38 activation in a dose-dependent manner. The inhibitory effect was specific. In agreement with previous reports (18Saitoh M. Nishitoh H. Fujii M. Takeda K. Tobiume K. Sawada Y. Kawabata M. Miyazono K. Ichijo H. EMBO J. 1998; 17: 2596-2606Crossref PubMed Scopus (2112) Google Scholar, 21Liu H. Nishitoh H. Ichijo H. Kyriakis J.M. Mol. Cell. Biol. 2000; 20: 2198-2208Crossref PubMed Scopus (452) Google Scholar), Ask1(K709M) blocked the activation by H2O2; however, it had no effect on the activation of p38 by hyperosmotic shock (sorbitol) or sodium arsenite. These findings indicate that Ask1 is an essential upstream activator of p38 in response to heat shock. The specificity of the inhibitory effect indicates that Ask1 mediates only a subset of the stimuli that activates p38. We previously reported that following a first heat shock treatment cells become temporarily desensitized to further p38 activation by heat shock but remain fully responsive to p38 activation by cytokines, growth factors, and stresses (15Dorion S. Berube J. Huot J. Landry J. J. Biol. Chem. 1999; 274: 37591-37597Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). This suggests the existence of a specific mechanism for p38 activation by heat shock. We examined whether this homologous heat desensitization process also affected Ask1 activity. Cells were first exposed to a 20-min heat shock at 44 °C and then exposed 7 h later to a second heat shock or to a H2O2 treatment. As previously demonstrated for p38 (15Dorion S. Berube J. Huot J. Landry J. J. Biol. Chem. 1999; 274: 37591-37597Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar) and confirmed here (Fig.2A), a heat shock pretreatment desensitized cells for the activation of Ask1 by a second heat shock but had no effect on H2O2-induced Ask1 activation (Fig. 2B). These results suggested that heat shock might activate Ask1 by a mechanism different from that used by H2O2. Ask1 is known to be responsive to the cellular redox state. We therefore investigated whether a perturbation in the redox state was a necessary event for heat shock activation of the p38 pathway. Cells were treated for 60 min with increasing concentration of the antioxidant NAC prior to exposure to heat shock or H2O2 treatment. Pretreatment with NAC at concentrations higher than 10 mm inhibited H2O2 activation of p38 but had no effect on the stimulation induced by heat shock (Fig.3A). Thus, whereas generation of reactive oxygen metabolites is an essential event upstream of p38 activation in response to H2O2, oxidative stress is not a key triggering element for the induction of p38 in response to heat shock. We next investigated if Trx, an endogenous inhibitor of Ask1, could block p38 activation by heat shock. CCL39 cells were transfected with expression vectors encoding GST-tagged p38 and Myc-tagged Trx and exposed to heat shock or H2O2 treatments. p38 activity was measured using a phosphospecific p38 antibody (Fig.3B, upper panels). Equivalent levels of expression of GST-tagged p38 and increasing levels of expression of Myc-tagged Trx were confirmed by Western blot analysis (Fig.3B, middle and bottom panels). Expression of Trx inhibited H2O2 activation of GST-tagged p38 in a dose-dependent manner but had no effect on heat shock activation of GST-tagged p38. We examined the effect of heat shock treatment on the in vivo interaction between Ask1 and Trx. Hela cells were transfected with an expression vector encoding HA-tagged Ask1-WT or with an empty vector and exposed to heat shock or H2O2 treatment. Endogenous Trx was immunoprecipitated with monoclonal anti-human Trx antibody, and the immunocomplexes were analyzed by immunoblot with anti-HA antibody (Fig.3C, upper panel). Western blot analyses confirmed equivalent levels of expression of HA-tagged Ask1-WT and endogenous Trx (Fig. 3C, middle and bottom panels). An association between Trx and Ask1 could be demonstrated under control conditions. H2O2 treatment, but not heat shock, caused a dissociation of Trx from Ask1. GSTM1-1 was recently identified as another endogenous inhibitor of Ask1 activity (19Cho 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 (350) Google Scholar). We tested if GSTM1-1 could possibly modulate p38 activation by heat shock. CCL39 cells were transfected with expression vectors encoding GST-tagged p38 and HA-tagged GSTM1-1(Y6F) and exposed to heat shock, H2O2, or sorbitol treatment. GST-tagged p38 phosphorylation was measured using a phosphospecific p38 antibody (Fig.4A, left panels). Equivalent levels of expression of GST-tagged p38 and increasing levels of expression of HA-tagged GSTM1-1(Y6F) were confirmed by Western blot analysis (Fig. 4A, middle and right panels). Expression of GSTM1-1 inhibited in a dose-dependent manner heat shock-induced activation and, to a lesser extent, H2O2-induced activation of p38. It had no effect on sorbitol activation of p38. This result was consistent with the fact that GSTM1-1 is an inhibitor of Ask1 and that Ask1 was not involved in the activation of p38 by sorbitol (Fig.1B). We next examined the effect of heat shock treatment on the interaction of Ask1 with GSTM1-1 in vivo. Cells were transfected with expression vector encoding HA-tagged Ask1-WT and exposed to heat shock or H2O2 treatment. Endogenous GSTM1-1 was immunoprecipitated with polyclonal anti-GSTM1-1 antibody, and the immunocomplexes were analyzed by immunoblot using anti-HA antibody (Fig. 4B). Western blot analyses confirmed equivalent levels of expression of HA-tagged Ask1-WT and endogenous GSTM1-1 (Fig. 4B, middle and bottom panels). The immunoblot data revealed an association between GSTM1-1 and Ask1 under control conditions. 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