Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways

Science

Volumn 275, Issue 5296, 1997, Pages 90-94

  Ichijo, Hidenori ^a   Nishida, Eisuke ^b   Irie, Kenji ^c   Ten Dijke, Peter ^d   Saitoh, Masao ^a   Moriguchi, Tetsuo ^b   Takagi, Minoru ^e   Matsumoto, Kunihiro ^c   Miyazono, Kohei ^a   Gotoh, Yukiko ^b  

^a CANCER INSTITUTE   (Japan)

^b KYOTO UNIVERSITY   (Japan)

^c NAGOYA UNIVERSITY   (Japan)

^d LUDWIG INSTITUTE FOR CANCER RESEARCH   (Sweden)

^e TOKYO MEDICAL AND DENTAL UNIVERSITY   (Japan)

Author keywords

[No Author keywords available]

Indexed keywords

MITOGEN ACTIVATED PROTEIN KINASE; TUMOR NECROSIS FACTOR ALPHA;

AMINO ACID SEQUENCE; APOPTOSIS; ARTICLE; ENZYME ACTIVATION; ETIOLOGY; HUMAN; NONHUMAN; POLYMERASE CHAIN REACTION; PRIORITY JOURNAL; PROTEIN EXPRESSION; SIGNAL TRANSDUCTION; STRESS;

AMINO ACID SEQUENCE; ANIMALS; APOPTOSIS; CA(2+)-CALMODULIN DEPENDENT PROTEIN KINASE; CELL DIVISION; CELL LINE; CELL SURVIVAL; COS CELLS; ENZYME ACTIVATION; JNK MITOGEN-ACTIVATED PROTEIN KINASES; MAP KINASE KINASE 6; MAP KINASE KINASE KINASES; MITOGEN-ACTIVATED PROTEIN KINASE KINASES; MITOGEN-ACTIVATED PROTEIN KINASES; MOLECULAR SEQUENCE DATA; P38 MITOGEN-ACTIVATED PROTEIN KINASES; PHOSPHORYLATION; POLYMERASE CHAIN REACTION; PROTEIN-SERINE-THREONINE KINASES; PROTEIN-TYROSINE KINASES; RECOMBINANT PROTEINS; SACCHAROMYCES CEREVISIAE; SIGNAL TRANSDUCTION; TRANSFECTION; TUMOR NECROSIS FACTOR-ALPHA;

EID: 14444281569     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.275.5296.90     Document Type: Article

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31. Xenopus MARK [Y. Gotoh et al., EMBO J. 10, 2661 (1991)] and Xenopus MAPKK (34) were cloned as described. Coding regions for rat SAPKα (4), human p38 [J. Han, B. Richter, Z. Li, V. V. Kravchenko, R. J. Ulevitch, Biochim. Biophys. Acta 1265, 224 (1995)] , mouse SEK1 (5), and human MKK3 (6) were amplified by PCR. An HA tag was introduced into the Bgl II and Eco Rl sites of a mammalian expression vector pSRα456 [Y. Takebe et al, Mol. Cell. Biol. 8, 466 (1988)], yielding pSRα-HA1. The cDNAs encoding MAPK, SAPKα, p38, MAPKK, SEK1, and MKK3 were subcloned into the Bgl II site of pSRα-HA1. ASK1 cDNA was introduced into another mammalian expression vector, pcDNAS (Invitrogen). For transient expression, COS7 cells were transfected with lipofectamine (Life Technologies) according to the manufacturer's instructions. For preparing extracts, cells were lysed in a buffer solution containing 2OmM tris-HCl (pH 7.5), 12 mM β-glycerophosphate, 150 mM NaCl, 5 mM EGTA, 10 mM NaF, 1% Triton X-100, 0.5% deoxycholate, 3 mM dithiothreitol (DTT), 1 mM sodium vanadate, 1 mM phenylmeth-ylsulfonyl fluoride (PMSF), and aprotinin (20 μg/ml). Cell extracts were clarified by centrifugation at 15,000g for 10 min. For immunoprecipitation, the supernatants were incubated with polyclonal antiserum to ASK1 (24) or monoclonal antibody to HA (12CA5) for 1 hour at 4°C. After the addition of protein A-Sepharose (Pharmacia Biotech), the lysates were incubated for an additional 1 hour. The beads were washed twice with a solution containing 500 mM NaCl, 20 mM tris-HCl (pH 7.5), 5 mM EGTA, 1% Triton X-100, 2 mM DTT, and 1 mM PMSF, then twice with a solution containing 150 mM NaCl, 20 mM tris-HCl (pH 7.5), 5 mM EGTA, 2 mM DTT, and 1 mM PMSF and subjected to kinase assays.
32. Xenopus MARK [Y. Gotoh et al., EMBO J. 10, 2661 (1991)] and Xenopus MAPKK (34) were cloned as described. Coding regions for rat SAPKα (4), human p38 [J. Han, B. Richter, Z. Li, V. V. Kravchenko, R. J. Ulevitch, Biochim. Biophys. Acta 1265, 224 (1995)] , mouse SEK1 (5), and human MKK3 (6) were amplified by PCR. An HA tag was introduced into the Bgl II and Eco Rl sites of a mammalian expression vector pSRα456 [Y. Takebe et al, Mol. Cell. Biol. 8, 466 (1988)], yielding pSRα-HA1. The cDNAs encoding MAPK, SAPKα, p38, MAPKK, SEK1, and MKK3 were subcloned into the Bgl II site of pSRα-HA1. ASK1 cDNA was introduced into another mammalian expression vector, pcDNAS (Invitrogen). For transient expression, COS7 cells were transfected with lipofectamine (Life Technologies) according to the manufacturer's instructions. For preparing extracts, cells were lysed in a buffer solution containing 2OmM tris-HCl (pH 7.5), 12 mM β-glycerophosphate, 150 mM NaCl, 5 mM EGTA, 10 mM NaF, 1% Triton X-100, 0.5% deoxycholate, 3 mM dithiothreitol (DTT), 1 mM sodium vanadate, 1 mM phenylmeth-ylsulfonyl fluoride (PMSF), and aprotinin (20 μg/ml). Cell extracts were clarified by centrifugation at 15,000g for 10 min. For immunoprecipitation, the supernatants were incubated with polyclonal antiserum to ASK1 (24) or monoclonal antibody to HA (12CA5) for 1 hour at 4°C. After the addition of protein A-Sepharose (Pharmacia Biotech), the lysates were incubated for an additional 1 hour. The beads were washed twice with a solution containing 500 mM NaCl, 20 mM tris-HCl (pH 7.5), 5 mM EGTA, 1% Triton X-100, 2 mM DTT, and 1 mM PMSF, then twice with a solution containing 150 mM NaCl, 20 mM tris-HCl (pH 7.5), 5 mM EGTA, 2 mM DTT, and 1 mM PMSF and subjected to kinase assays.
33. Xenopus MARK [Y. Gotoh et al., EMBO J. 10, 2661 (1991)] and Xenopus MAPKK (34) were cloned as described. Coding regions for rat SAPKα (4), human p38 [J. Han, B. Richter, Z. Li, V. V. Kravchenko, R. J. Ulevitch, Biochim. Biophys. Acta 1265, 224 (1995)] , mouse SEK1 (5), and human MKK3 (6) were amplified by PCR. An HA tag was introduced into the Bgl II and Eco Rl sites of a mammalian expression vector pSRα456 [Y. Takebe et al, Mol. Cell. Biol. 8, 466 (1988)], yielding pSRα-HA1. The cDNAs encoding MAPK, SAPKα, p38, MAPKK, SEK1, and MKK3 were subcloned into the Bgl II site of pSRα-HA1. ASK1 cDNA was introduced into another mammalian expression vector, pcDNAS (Invitrogen). For transient expression, COS7 cells were transfected with lipofectamine (Life Technologies) according to the manufacturer's instructions. For preparing extracts, cells were lysed in a buffer solution containing 2OmM tris-HCl (pH 7.5), 12 mM β-glycerophosphate, 150 mM NaCl, 5 mM EGTA, 10 mM NaF, 1% Triton X-100, 0.5% deoxycholate, 3 mM dithiothreitol (DTT), 1 mM sodium vanadate, 1 mM phenylmeth-ylsulfonyl fluoride (PMSF), and aprotinin (20 μg/ml). Cell extracts were clarified by centrifugation at 15,000g for 10 min. For immunoprecipitation, the supernatants were incubated with polyclonal antiserum to ASK1 (24) or monoclonal antibody to HA (12CA5) for 1 hour at 4°C. After the addition of protein A-Sepharose (Pharmacia Biotech), the lysates were incubated for an additional 1 hour. The beads were washed twice with a solution containing 500 mM NaCl, 20 mM tris-HCl (pH 7.5), 5 mM EGTA, 1% Triton X-100, 2 mM DTT, and 1 mM PMSF, then twice with a solution containing 150 mM NaCl, 20 mM tris-HCl (pH 7.5), 5 mM EGTA, 2 mM DTT, and 1 mM PMSF and subjected to kinase assays.
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35. 32P]ATP and 3 μg of GST-catalytically inactive MAPK (for MAPKK) or His-tagged catalytically inactive p38 (for SEK1, MKK3, and MAPKK6) in the same solution (final volume, 35 μl). To measure the kinase activity of wild-type p38, we used His-tagged wild-type p38 and ATF2 instead of catalytically inactive p38. Samples were analyzed by SDS-PAGE and image analyzer.
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37. To avoid the possibility that constitutively expressed ASK1 might induce cell death, resulting in a failure to obtain stable transformants, we used a metal-lothionein-inducible promoter system. ASK1 and ASK1 (K709R) cDNAs were subcloned into pMEP4 vector (Invitrogen) at convenient enzyme cleavage sites. Transfection of cDNAs was done with Transfectam (Promega) according to the manufacturer's instructions, and selection by hygromycin B was done as described [M. Saitoh et al., J. Biol. Chem. 271, 2769 (1996)]. Several independent colonies were cloned, and the expression of ASK1 protein was determined by immunoprecipitation (33) with antiserum to ASK1 (24). Two independent positive clones were used for the assays with essentially the same results.
38. Antiserum to ASK1 was raised against the peptide sequence TEEKGRSTEEGDCESD (amino acids 554 to 669) that was coupled to keyhole limpet hemocyanin by a glutaraldehyde method, mixed with Freund's adjuvant, and used to immunize rabbits as described (33).
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46. The pcDNA3-ASK1(K709R) plasmid was transfected into Jurkat cells by DMRIE-C reagent (Life Technologies) together with pHook-1 plasmid (Invitrogen), which encodes a single-chain antibody fusion protein directed to the hapten phOx (4. ethoxymethylene-2-phenyl-2-oxazolin-5-one) and thereby allows the selective isolation of transfected cells with magnetic beads coated with phOx. ASK1(K709R)-transfected populations of cells (cotransfection efficiency was nearly 100% as determined by β-galactosidase staining) were isolated on phOx-coated magnetic beads with the Capture-Tec kit (Invitrogen), allowed to grow, counted, and treated with TNF-α. Nontransfected Jurkat cells and isolated Jurkat cells that were transfected with pHook-1, and control pcDNA3 plasmids were similarly sensitive to TNF-α in the DNA fragmentation assay (30).
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49. 6 cells were lysed with 200 μl of a buffer containing 20 mM tris-HCI (pH 7.5), 10 mM EDTA, and 0.5% Triton X-100. Cell extracts were clarified by centrifugation at 15,000g for 5 min. The lysates were incubated with proteinase K (0.2 mg/ml) and ribonuclease A (0.1 mg/ml) at 42°C for 1 hour. DNA was then purified by ethanol precipitation after phenolchloroform extraction.
50. We thank S. J. Baker and T. Curran for ATF2; T. Maeda for TM257-H1; M. Poncz for HEL cDNA library; H. Okazaki and T. Sudo (Kirin Brewery, Japan) for oligonucleotides and advice; T. Kitagawa and C.-H. Heldin for valuable comments; A. Hanyu for technical assistance; U. Engström for preparing the synthetic peptide; and K. Saeki, T. lnage, K. Takeda, H. Nishitoh, and K. Tobiume for discussion. Supported by Grants-in-Aid for scientific research from the Ministry of Education, Science, and Culture of Japan. H.I. and K.M. are supported by grants from Mochida Memorial Foundation for Medical and Pharmaceutical Research and Toray Scientific Foundation.