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Activation of Apoptosis Signal-regulating Kinase 1 by Reactive Oxygen Species through Dephosphorylation at Serine 967 and 14-3-3 Dissociation

2004; Elsevier BV; Volume: 279; Issue: 11 Linguagem: Inglês

10.1074/jbc.m311129200

ISSN

1083-351X

Autores

Erinn Hoag Goldman, Lei Chen, Haian Fu,

Tópico(s)

Heat shock proteins research

Resumo

Oxidative stress has been indicated in a variety of pathological processes such as atherosclerosis, diabetes, and neurodegenerative diseases. Understanding how intracellular signaling pathways respond to oxidative insults such as hydrogen peroxide (H2O2) would have significant therapeutic implications. Recent genetic studies have placed apoptosis signal-regulating kinase 1 (ASK1) in a pivotal position in transmitting H2O2-initiated signals. How ASK1 is activated by H2O2, though, remains a subject of intense investigation. Here we report a mechanism by which H2O2 induces ASK1 activation through dynamic control of its phosphorylation at serine 967. We found that treatment of COS7 cells with H2O2 triggers dephosphorylation of Ser-967 through an okadaic acid-sensitive phosphatase, resulting in dissociation of the ASK1·14-3-3 complex with concomitant increase of ASK1 catalytic activity and ASK1-mediated activation of JNK and p38 pathways. Oxidative stress has been indicated in a variety of pathological processes such as atherosclerosis, diabetes, and neurodegenerative diseases. Understanding how intracellular signaling pathways respond to oxidative insults such as hydrogen peroxide (H2O2) would have significant therapeutic implications. Recent genetic studies have placed apoptosis signal-regulating kinase 1 (ASK1) in a pivotal position in transmitting H2O2-initiated signals. How ASK1 is activated by H2O2, though, remains a subject of intense investigation. Here we report a mechanism by which H2O2 induces ASK1 activation through dynamic control of its phosphorylation at serine 967. We found that treatment of COS7 cells with H2O2 triggers dephosphorylation of Ser-967 through an okadaic acid-sensitive phosphatase, resulting in dissociation of the ASK1·14-3-3 complex with concomitant increase of ASK1 catalytic activity and ASK1-mediated activation of JNK and p38 pathways. Reactive oxygen species (ROS) 1The abbreviations used are: ROS, reactive oxygen species; ASK1, apoptosis signal-regulating kinase 1; HA, hemagglutinin; H2O2, hydrogen peroxide; JNK, c-Jun NH2-terminal kinase; MKK, mitogen-activated protein kinase kinase; PMSF, phenylmethylsulfonyl fluoride; PP, protein phosphatase; WT, wild type.1The abbreviations used are: ROS, reactive oxygen species; ASK1, apoptosis signal-regulating kinase 1; HA, hemagglutinin; H2O2, hydrogen peroxide; JNK, c-Jun NH2-terminal kinase; MKK, mitogen-activated protein kinase kinase; PMSF, phenylmethylsulfonyl fluoride; PP, protein phosphatase; WT, wild type. produced through a variety of cellular processes or derived from exogenous sources play important roles in the regulation of normal physiology, including cell proliferation, survival, senescence, and apoptotic cell death (1Martindale J. Holbrook N. J. Cell. Physiol. 2002; 192: 1-15Crossref PubMed Scopus (1923) Google Scholar). However, unchecked and excessive production of ROS may result in severe damage to cellular components including DNA, proteins, and lipids, causing oxidative stress or injury. Oxidative stress has been implicated in a variety of human diseases including atherosclerosis, diabetes, arthritis, cancer, and neurodegenerative disorders (1Martindale J. Holbrook N. J. Cell. Physiol. 2002; 192: 1-15Crossref PubMed Scopus (1923) Google Scholar). Thus, understanding how cellular signaling pathways respond to oxidative insults such as hydrogen peroxide (H2O2) may lead to novel strategies for therapeutic interventions. Accumulating evidence suggests that apoptosis signal-regulating kinase 1 (ASK1) plays a pivotal role in mediating the H2O2-induced stress response. ASK1 is a multifunctional serine/threonine protein kinase involved in the regulation of diverse physiological processes, including cell differentiation and apoptosis (2Takeda K. Matsuzawa A. Nishitoh H. Ichijo H. Cell Struct. Funct. 2003; 28: 23-29Crossref PubMed Scopus (198) Google Scholar). It was originally discovered as a mitogen-activated protein kinase kinase kinase with proapoptotic activity (3Wang X.S. Diener K. Jannuzzi D. Trollinger D. Tan T.H. Lichenstein H. Zukowski M. Yao Z. J. Biol. Chem. 1996; 271: 31607-31611Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 4Tobiume K. Inage T. Takeda K. Enomoto S. Miyazono K. Ichijo H. Biochem. Biophys. Res. Commun. 1997; 239: 905-910Crossref PubMed Scopus (67) Google Scholar, 5Ichijo 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 (2006) Google Scholar). The kinase activity of ASK1 is activated by many stress signals and proinflammatory cytokines, including H2O2, tumor necrosis factor-α, endoplasmic reticulum stress, serum withdrawal, and chemotherapeutic agents (5Ichijo 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 (2006) Google Scholar, 6Gotoh Y. Cooper J.A. J. Biol. Chem. 1998; 273: 17477-17482Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 7Saitoh 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 (2065) Google Scholar, 8Nishitoh H. Matsuzawa A. Tobiume K. Saegusa K. Takeda K. Inoue K. Hori S. Kakizuka A. Ichijo H. Genes Dev. 2002; 16: 1345-1355Crossref PubMed Scopus (1131) Google Scholar, 9Chen Z. Seimiya H. Naito M. Mashima T. Kizaki A. Dan S. Imaizumi M. Ichijo H. Miyazono K. Tsuruo T. Oncogene. 1999; 18: 173-180Crossref PubMed Scopus (168) Google Scholar, 10Wang T.H. Wang H.S. Ichijo H. Giannakakou P. Foster J.S. Fojo T. Wimalasena J. J. Biol. Chem. 1998; 273: 4928-4936Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). ASK1 stimulation in turn activates both the MKK4/MKK7-JNK pathway and the MKK3/MKK6-p38 kinase pathway, leading to stress responses or apoptosis (3Wang X.S. Diener K. Jannuzzi D. Trollinger D. Tan T.H. Lichenstein H. Zukowski M. Yao Z. J. Biol. Chem. 1996; 271: 31607-31611Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 5Ichijo 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 (2006) Google Scholar). In support of its role in apoptotic signaling, overexpression of ASK1 or a constitutively active fragment, ASK1-KC, can trigger apoptotic cell death in several cell types via a mitochondria-dependent caspase 3 pathway (5Ichijo 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 (2006) Google Scholar, 11Hatai T. Matsuzawa A. Inoshita S. Mochida Y. Kuroda T. Sakamaki K. Kuida K. Yonehara S. Ichijo H. Takeda K. J. Biol. Chem. 2000; 275: 26576-26581Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). Furthermore, the expression of a catalytically inactive mutant of ASK1 exhibits a dominant negative effect, inhibiting apoptosis induced by stress signals such as tumor necrosis factor-α and H2O2 (5Ichijo 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 (2006) Google Scholar, 6Gotoh Y. Cooper J.A. J. Biol. Chem. 1998; 273: 17477-17482Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 7Saitoh 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 (2065) Google Scholar). Significantly, it has been established that ASK1 plays a critical role in mediating oxidative stress signaling (6Gotoh Y. Cooper J.A. J. Biol. Chem. 1998; 273: 17477-17482Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 7Saitoh 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 (2065) Google Scholar, 12Tobiume K. Matsuzawa A. Takahashi T. Nishitoh H. Morita K. Takeda K. Minowa O. Miyazono K. Noda T. Ichijo H. EMBO Rep. 2001; 2: 222-228Crossref PubMed Scopus (1003) Google Scholar). Mouse embryonic fibroblast cells from ASK1-/- mice are resistant to oxidant- and tumor necrosis factor-α-induced apoptosis and fail to maintain sustained levels of JNK and p38 kinase activity upon treatment with H2O2 or tumor necrosis factor-α (12Tobiume K. Matsuzawa A. Takahashi T. Nishitoh H. Morita K. Takeda K. Minowa O. Miyazono K. Noda T. Ichijo H. EMBO Rep. 2001; 2: 222-228Crossref PubMed Scopus (1003) Google Scholar). These lines of evidence suggest that ASK1 is a key player in apoptotic signaling, and in particular, ASK1 may function as a pivotal mediator of H2O2-induced stress response. However, the molecular mechanism by which ASK1 transmits H2O2 signals remains a subject of intense investigation. Because of its important role in cell death signaling, the activity of ASK1 is tightly regulated by multiple mechanisms, including phosphorylation, oligomerization, and protein-protein interactions. Phosphorylation of ASK1 at Ser-845 appears to be required for activity because dephosphorylation at this site by PP5 leads to ASK1 inactivation (13Morita K. Saitoh M. Tobiume K. Matsuura H. Enomoto S. Nishitoh H. Ichijo H. EMBO J. 2001; 20: 6028-6036Crossref PubMed Scopus (246) Google Scholar). Phosphorylation at Ser-83 by Akt/protein kinase B, on the other hand, attenuates ASK1 activity (14Kim A.H. Khursigara G. Sun X. Franke T.F. Chao M.V. Mol. Cell. Biol. 2001; 21: 893-901Crossref PubMed Scopus (618) Google Scholar). It has also been demonstrated that intramolecular interaction, probably between the NH2-terminal and COOH-terminal domains of ASK1, may be required to maintain ASK1 in its inactive state (15Chang H.Y. Nishitoh H. Yang X. Ichijo H. Baltimore D. Science. 1998; 281: 1860-1863Crossref PubMed Scopus (531) Google Scholar), whereas oligomerization of its COOH-terminal domains is correlated with ASK1 activation (6Gotoh Y. Cooper J.A. J. Biol. Chem. 1998; 273: 17477-17482Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 13Morita K. Saitoh M. Tobiume K. Matsuura H. Enomoto S. Nishitoh H. Ichijo H. EMBO J. 2001; 20: 6028-6036Crossref PubMed Scopus (246) Google Scholar). The most commonly observed means of ASK1 regulation, however, is through protein-protein interactions. Numerous proteins have been shown to bind ASK1 to exert their regulatory function. For example, binding of TRAF2 (16Nishitoh H. Saitoh M. Mochida Y. Takeda K. Nakano H. Rothe M. Miyazono K. Ichijo H. Mol. Cell. 1998; 2: 389-395Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar) or Daxx (15Chang H.Y. Nishitoh H. Yang X. Ichijo H. Baltimore D. Science. 1998; 281: 1860-1863Crossref PubMed Scopus (531) Google Scholar) promotes ASK1 function, whereas the kinase and proapoptotic activities of ASK1 are inhibited by many other associated proteins, including reduced thioredoxin (7Saitoh 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 (2065) Google Scholar), glutaredoxin (17Song J.J. Rhee J.G. Suntharalingam M. Walsh S.A. Spitz D.R. Lee Y.J. J. Biol. Chem. 2002; 277: 46566-46575Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar), Cdc25A (18Zou X. Tsutsui T. Ray D. Blomquist J.F. Ichijo H. Ucker D.S. Kiyokawa H. Mol. Cell. Biol. 2001; 21: 4818-4828Crossref PubMed Scopus (87) Google Scholar), Hsp 72 (19Park H.S. Cho S.G. Kim C.K. Hwang H.S. Noh K.T. Kim M.S. Huh S.H. Kim M.J. Ryoo K. Kim E.K. Kang W.J. Lee J.S. Seo J.S. Ko Y.G. Kim S. Choi E.J. Mol. Cell. Biol. 2002; 22: 7721-7730Crossref PubMed Scopus (139) Google Scholar), ASK1-interacting protein 1 (20Zhang R. He X. Liu W. Lu M. Hsieh J. Min W. J. Clin. Invest. 2003; 111: 1933-1943Crossref PubMed Scopus (128) Google Scholar), and 14-3-3 proteins (21Zhang L. Chen J. Fu H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8511-8515Crossref PubMed Scopus (313) Google Scholar). It seems probable that ASK1 is suppressed from promoting apoptosis under normal survival or proliferative conditions by specific phosphorylation and regulated protein-protein interactions but becomes proapoptotic when suppression is relieved. One mechanism that mediates the H2O2 effect appears to involve redox-regulated dissociation of ASK1 from bound thioredoxin, leading to ASK1 oligomerization and activation (6Gotoh Y. Cooper J.A. J. Biol. Chem. 1998; 273: 17477-17482Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 7Saitoh 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 (2065) Google Scholar, 22Tobiume K. Saitoh M. Ichijo H. J. Cell. Physiol. 2002; 191: 95-104Crossref PubMed Scopus (297) Google Scholar). The inhibitory effect of bound thioredoxin on ASK1, however, may be redox-independent, as shown in an endothelial cell system (23Liu Y. Min W. Circ. Res. 2002; 90: 1259-1266Crossref PubMed Scopus (304) Google Scholar). Among ASK1 regulatory events, the interaction of ASK1 with 14-3-3 proteins is particularly intriguing because this complex formation is controlled by phosphorylation at Ser-967 (21Zhang L. Chen J. Fu H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8511-8515Crossref PubMed Scopus (313) Google Scholar). 14-3-3 belongs to a family of phosphoserine/phosphothreonine-binding molecules that recognize the motif Arg-Ser-X-pSer/pThr-X-Pro or its derivatives (pSer/pThr represents phosphorylated Ser or Thr, and X denotes any amino acid) (24Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1185) Google Scholar, 25Aitken A. Trends Biochem. Sci. 1995; 20: 95-97Abstract Full Text PDF PubMed Scopus (264) Google Scholar, 26Fu H. Subramanian R.R. Masters S.C. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 617-647Crossref PubMed Scopus (1320) Google Scholar, 27Yaffe M.B. FEBS Lett. 2002; 513: 53-57Crossref PubMed Scopus (551) Google Scholar). They can bind to many signaling molecules through specific pSer/pThr motifs, including the proapoptotic protein Bad (28Zha J. Harada H. Yang E. Jockel J. Korsmeyer S. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2246) Google Scholar) and transcription factor FKLR1 (29Brunet A. Bonni A. Zigmond M. Lin M. Juo P. Hu L. Anderson M. Arden K. Blenis J. Greenberg M. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5381) Google Scholar). In fact, the control of 14-3-3/Bad binding by Akt/protein kinase B represents the first molecular link by which a major survival signaling pathway is coupled to the death machinery (30Datta S. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4920) Google Scholar). Functionally, the 14-3-3·Bad complex formation results in inhibition of Bad-mediated apoptosis (28Zha J. Harada H. Yang E. Jockel J. Korsmeyer S. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2246) Google Scholar, 31Datta S.R. Katsov A. Hu L. Petros A. Fesik S.W. Yaffe M.B. Greenberg M.E. Mol. Cell. 2000; 6: 41-51Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar, 32Masters S.C. Yang H. Datta S.R. Greenberg M.E. Fu H. Mol. Pharmacol. 2001; 60: 1325-1331Crossref PubMed Scopus (137) Google Scholar). Through such phosphorylation-dependent protein-protein interactions, 14-3-3 plays an important role in the regulation of many cellular processes including cell proliferation and survival signaling. We have demonstrated previously that 14-3-3 binding to ASK1 through phosphorylated Ser-967 effectively suppresses ASK1-induced apoptosis (21Zhang L. Chen J. Fu H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8511-8515Crossref PubMed Scopus (313) Google Scholar). However, how upstream signals control Ser-967 phosphorylation and ASK1·14-3-3 association remains elusive. Stress signals may utilize the sensitive kinase and phosphatase signaling network to control ASK1 function, achieving a balanced biological outcome such as cell suicide or survival. In this study, we have examined the dynamic regulation of ASK1 by H2O2 and uncovered a critical link between H2O2 signaling and the phosphorylation status of Ser-967. Our findings define a novel mechanism whereby H2O2 triggers the activity of an okadaic acid-sensitive phosphatase upstream of ASK1, resulting in dephosphorylation of Ser-967 and subsequent activation of ASK1-mediated stress pathways. Plasmids, Cell Culture, and DNA Transfection—Expression vectors for ASK1 and its mutants have been described (5Ichijo 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 (2006) Google Scholar, 21Zhang L. Chen J. Fu H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8511-8515Crossref PubMed Scopus (313) Google Scholar). COS7 cells were cultured in Dulbecco's modified Eagle's medium with fetal bovine serum (10%; Mediatech, Washington, D. C.). Transfection was performed with FuGENE 6 (Roche Applied Science) according to the manufacturer's instructions. Reagents—Hydrogen peroxide, N-acetyl-l-cysteine, catalase, okadaic acid, calyculin A, and cyclosporin A were obtained from Sigma. Calyculin and cyclosporin A were diluted in ethanol, and okadaic acid was prepared in methanol. All other reagents were diluted in water. Calf intestinal phosphatase was purchased from New England Biolabs. Generation of Phospho-specific pSer-967 Antibodies—Polyclonal antibodies were produced by immunizing two rabbits with a synthetic phospho-peptide (keyhole limpet hemocyanin-coupled) corresponding to residues around Ser-967 of human ASK1. From each rabbit, antibodies were purified by protein A and a nonphospho-peptide affinity chromatography (antibodies 2 and 4). The flow-through of non-phosphopeptide chromatography, in which the non-phospho activity was depleted, was collected and applied to a phosphopeptide chromatography for final purification (antibodies 1 and 3). Western Blotting—COS7 cells (4 × 105) were lysed in 200 μl of lysis buffer (0.2% Nonidet P-40, 10 mm HEPES, pH 7.4, 150 mm NaCl, 5 mm NaF, 2 mm Na3VO4, 5 mm Na4P2O7, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride (PMSF)). Cell extracts were clarified by centrifugation, prepared in SDS sample buffer, boiled for 5 min, and resolved on SDS-PAGE (12.5%) for Western blotting. The enzyme-linked immunoblotting procedures were performed essentially as described previously (21Zhang L. Chen J. Fu H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8511-8515Crossref PubMed Scopus (313) Google Scholar). Corresponding secondary antibodies were used against each primary antibody: horseradish peroxidase-conjugated goat anti-mouse IgG for monoclonal antibodies and horseradish peroxidase-conjugated goat anti-rabbit IgG for polyclonal antibodies (Santa Cruz Biotechnology). Cross-reacting materials were visualized using the ECL detection reagents (Amersham Biosciences). Pull-down Assays—COS7 cells were transiently transfected with HA-ASK1 and His6-tagged-14-3-3γ as indicated. 40 h after transfection cells were treated with 1 mm H2O2 and 25 mm aminotriazole and lysed in 1% Nonidet P-40 lysis buffer (1% Nonidet P-40, 137 mm NaCl, 1 mm MgCl2, 40 mm Tris-HCl, 1 mm PMSF, 60 mm imidazole, 5 mm Na4P2O7, 5 mm NaF, 2 mm Na3VO4, 10 μg/ml aprotinin, 10 μg/ml leupeptin). His6-tagged 14-3-3γ was immobilized on Ni2+-charged iminodiacetic acid-Sepharose 6B beads (33Zhang L. Wang H. Liu D. Liddington R. Fu H. J. Biol. Chem. 1997; 272: 13717-13724Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). The 14-3-3 complexes were washed four times with lysis buffer and resolved on SDS-PAGE (12.5%) for Western blotting with anti-ASK1 antibody (Santa Cruz Biotechnology) and anti-14-3-3 antibody (Santa Cruz Biotechnology). Immunoprecipitation and Kinase Assay—The kinase activity of ASK1 was determined essentially as described previously (5Ichijo 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 (2006) Google Scholar, 6Gotoh Y. Cooper J.A. J. Biol. Chem. 1998; 273: 17477-17482Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). 40 h after transfection, media were replaced with Dulbecco's modified Eagle's medium without serum for H2O2 treatment. Cells (4 × 105) were lysed with a β-glycerophosphate-containing lysis buffer (20 mm Tris-Cl, pH 7.5, 10 mm β-glycerophosphate, 5 mm EGTA, 5 mm NaF, 1 mm sodium pyrophosphate, 150 mm NaCl, 1% Nonidet P-40, 2 mm dithiothreitol, 1 mm sodium orthovanadate, 10 μg/ml aprotinin, 1 mm PMSF). After clarification by centrifugation, HA-ASK1 or endogenous JNK was immunoprecipitated with anti-ASK1 (Santa Cruz Biotechnology) or anti-JNK (Cell Signaling) and protein G-Sepharose. After washing the immunoprecipitates three times with the lysis buffer and once with a kinase assay buffer solution (20 mm Tris-Cl, pH 7.5, 10 mm MgCl2,2mm EGTA, 1 mm dithiothreitol, 1 mm PMSF), they were subjected to kinase assays. To measure the ability of ASK1 to phosphorylate p38, the immunoprecipitates were incubated with 1 μg of His-tagged MKK6 before adding 2 μg of His-tagged kinase-inactive p38 as described previously (5Ichijo 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 (2006) Google Scholar). For the JNK assay, glutathione S-transferase-c-Jun was used as a substrate (5Ichijo 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 (2006) Google Scholar). After resolution on SDS-PAGE (12.5%), phosphorylation of ASK1, c-Jun, or p38 was visualized by a Typhoon PhosphorImager and quantified using ImageQuant software version 5.2 (Molecular Dynamics, Inc.). Phosphorylation of ASK1 at Ser-967 in Vivo—We have shown previously that Ser-967 of ASK1 is harbored in a critical recognition motif for the pSer/pThr-binding protein 14-3-3 (21Zhang L. Chen J. Fu H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8511-8515Crossref PubMed Scopus (313) Google Scholar). Phosphorylation of this residue enables ASK1 to bind 14-3-3, which leads to suppression of ASK1-induced apoptosis. Thus, Ser-967 of ASK1 may serve as an integration point that couples survival signaling pathways to this death-promoting kinase. To understand how Ser-967 of ASK1 is regulated, we developed antibodies that allow us to monitor the phosphorylation status of this critical site in vivo. Four batches of rabbit antisera were raised against keyhole limpet hemocyanin-conjugated Ser-967-containing phosphopeptides from ASK1. Purified antibodies from these sera showed strong cross-reactivity with ASK1/WT expressed in COS7 cells (Fig. 1A). Two appeared to be specific to the phosphorylated Ser-967 (pSer-967) epitope in ASK1 because they were unable to recognize ASK1/S967A, a mutant of ASK1 which is unphosphorylatable at Ser-967. However, it is possible that the inability of the pSer-967 antibodies to recognize ASK1/S967A might be the result of mutation-induced conformational or structural changes of the epitope. To address this concern, we employed an alternative approach to dephosphorylate Ser-967 by treating ASK1/WT with a broad spectrum protein phosphatase, calf intestinal phosphatase. As shown in Fig. 1B, pSer-967 antibodies failed to recognize dephosphorylated ASK1/WT, confirming the specificity of the antibody for phosphorylated Ser-967. To define the utility of the pSer-967 antibody, we tested its sensitivity. A testament to its usefulness for the monitoring of Ser-967 phosphorylation in vivo, the anti-pSer-967 antibody could recognize pSer-967 when lysate with 0.25 μg of total protein was immunoblotted (Fig. 1C). This level of sensitivity is comparable with that of the pan-ASK1 antibody. Furthermore, antibody titration showed that the phospho-antibody can recognize phosphorylated Ser-967 with a 1:20,000 dilution (Fig. 1D). These experiments demonstrate the sensitivity and specificity of the antibody for pSer-967 of ASK1, providing us with a powerful tool for monitoring ASK1 phosphorylation under various physiological conditions. ROS Induce Dephosphorylation at Ser-967—The above tests also demonstrate that Ser-967 is phosphorylated in cells under growth conditions in the presence of serum (Fig. 1), which is consistent with its role in cell survival signaling. It is possible that the death promoting function of ASK1 is suppressed under growth conditions but becomes proapoptotic when suppression is relieved. To test this hypothesis, we sought to determine whether the phosphorylation status of Ser-967 is responsive to stress signals. It has been well established that ASK1 plays an important role in the apoptotic response induced by ROS, in particular H2O2 (6Gotoh Y. Cooper J.A. J. Biol. Chem. 1998; 273: 17477-17482Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 7Saitoh 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 (2065) Google Scholar, 12Tobiume K. Matsuzawa A. Takahashi T. Nishitoh H. Morita K. Takeda K. Minowa O. Miyazono K. Noda T. Ichijo H. EMBO Rep. 2001; 2: 222-228Crossref PubMed Scopus (1003) Google Scholar). It has been shown that cells derived from ASK1 knock-out mice are resistant to H2O2-induced apoptosis (12Tobiume K. Matsuzawa A. Takahashi T. Nishitoh H. Morita K. Takeda K. Minowa O. Miyazono K. Noda T. Ichijo H. EMBO Rep. 2001; 2: 222-228Crossref PubMed Scopus (1003) Google Scholar). However, the mechanism by which ASK1 mediates the ROS effect remains unclear. It is possible that ROS induces ASK1 activation and cell death through dephosphorylation of Ser-967. To test this model, we monitored the in vivo phosphorylation status of Ser-967 in COS7 cells. Indeed, treatment of cells with a high (5 mm), intermediate (1 mm), or low (0.5 mm) dose of H2O2 resulted in marked dephosphorylation of Ser-967 (Fig. 2, A and C). In support of a role of ROS in inducing Ser-967 dephosphorylation, pretreatment of cells with N-acetyl-l-cysteine, a ROS scavenger, effectively blocked the H2O2 effect (Fig. 2B). To explore whether phosphorylation of Ser-967 is a regulated process responsive to varying conditions in the cell, we tested the effects of H2O2 using a prolonged time course that allowed for the metabolic clearance of ROS. COS7 cells expressing ASK1 were treated with a low (0.5 mm) or intermediate (1 mm) dose of H2O2 for various times. We show that the dephosphorylation induced by ROS is reversible, with marked Ser-967 dephosphorylation after 30 min followed by increased phosphorylation as early as 60 min post-treatment (Fig. 2C). This reversibility suggests that phosphorylation at Ser-967 is a dynamic process, presumably regulated by a kinase(s) and phosphatase(s), and could be a major mode of ASK1 regulation. Ser-967 Dephosphorylation Is Correlated with Dissociation of 14-3-3—We discovered previously that ASK1 contains a recognition motif for 14-3-3, Arg-Ser-Ile-pSer967-Leu-Pro, in its COOH-terminal regulatory domain (21Zhang L. Chen J. Fu H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8511-8515Crossref PubMed Scopus (313) Google Scholar). ASK1 and 14-3-3 interact both in vitro and in vivo. Ser-967 was shown to be a critical component of the 14-3-3 interaction site of ASK1, and phosphorylation of this residue was proposed to be a major mode of regulation of 14-3-3 binding to ASK1 (21Zhang L. Chen J. Fu H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8511-8515Crossref PubMed Scopus (313) Google Scholar). The availability of the pSer-967-specific antibodies allows us to test this hypothesis by directly monitoring Ser-967 phosphorylation and its relationship to 14-3-3 binding. COS7 cells expressing HA-ASK1 and His6-14-3-3γ were treated for various times with 1 mm H2O2 and 25 mm catalase inhibitor aminotriazole. By inhibiting the primary player in H2O2 degradation with aminotriazole, we were able to increase the longevity of H2O2-induced Ser-967 dephosphorylation. The 14-3-3γ protein complex was pulled down with Ni2+-charged Sepharose beads, and the presence of ASK1 was detected by immunoblotting. Significant dephosphorylation of ASK1 at Ser-967 was observed by 90 min after H2O2 treatment (Fig. 3, upper panels). This dephosphorylation was correlated with dissociation of ASK1 from 14-3-3 (Fig. 3, lower panels). Interestingly, coexpression of His6-14-3-3γ with ASK1 appears to delay H2O2-induced Ser-967 dephosphorylation compared with COS7 cells transfected with HA-ASK1 alone (compare Fig. 3 with Fig. 2A). The H2O2 signal apparently controls levels of Ser-967 phosphorylation and 14-3-3 association. Ser-967 Dephosphorylation Is Associated with Increased ASK1 Catalytic Activity—We demonstrated previously that disruption of the ASK1/14-3-3 interaction dramatically accelerated ASK1-induced cell death (21Zhang L. Chen J. Fu H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8511-8515Crossref PubMed Scopus (313) Google Scholar). The mechanism by which this increased cell death occurred, however, remains unknown. Because the catalytic activity of ASK1 is required for its death promoting function, it is possible that removing 14-3-3 from ASK1 may relieve an inhibitory mechanism, leading to ASK1 activation and the subsequent activation of the JNK and p38 kinases. Thus, having established that Ser-967 dephosphorylation is correlated with dissociation of 14-3-3, we examined whether this H2O2-induced dephosphorylation affects the kinase activity of ASK1. COS7 cells were transfected with HA-ASK1/WT or HA-ASK1/K709R, a kinase-inactive mutant

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