Induction of Apoptosis by ASK1, a Mammalian MAPKKK That Activates SAPK/JNK and p38 Signaling Pathways
Tóm tắt
Từ khóa
Tài liệu tham khảo
Sánchez I., et al., ibid. 372, 794 (1994).
Lin A., et al., ibid. 268, 286 (1995).
Han J., et al., ibid. 271, 2886 (1996);
ten Dijke P., et al., Oncogene 8, 2879 (1993);
An amplified oligo(dT)-primed λgt11 cDNA library from human erythroleukemia (HEL) cells [M. Poncz et al . Blood 69 219 (1987)] was screened with a 32 P-labeled PCR fragment. Hybridization and purification of positive bacteriophage were performed as described (33). Nucleotide sequencing was done on both strands with a Sequenase DNA sequencing kit (U.S. Biochemical). Among 18 clones obtained the 3 longest clones termed clone 20 clone 27 and clone 72 were entirely sequenced. The sequence of clone 72 started from the middle of the open reading frame and ended by a stretch of polyadenylate. The sequences of clones 20 and 27 covered the 5′ part of ASK1 cDNA and the overlapping parts with clone 72 were identical in sequence. The ASK1 cDNA sequence combining the clone 20 and clone 72 yielded a 4533-base pair (bp) sequence with an ATG codon starting at position 268 followed by a 4175-bp open reading frame encoding 1375 amino acids.
Clone 27 contained a 4-bp deletion at position 805 to 808 with in-frame upstream stop codons resulting in an NH 2 -terminally truncated protein product of ASK1 (Fig. 1A). Although the functional importance of this truncated form of ASK1 is unknown the longer form of ASK1 derived from the overlapping clones 20 and 72 was used for the functional studies throughout this report.
ASK1 cDNA was introduced into a yeast expression plasmid pNV11 [H. Shibuya et al . Nature 357 700 (1992)]. SHO1 is an SH3 domain-containing transmembrane osmosensor that constitutes another signaling pathway leading to hyperosmolarity responses by way of HOG1 activation independently of SSK2 or SSK22 (13). Single or double mutant strains of SHO1 SSK2 or SSK22 are resistant to hyperosmotic medium; however strains with defects in SHO1 SSK2 and SSK22 are unable to grow in hyperosmotic medium.
ASK1 could not restore the osmotic response in a PBS2 [downstream target of SHO1 SSK2 and SSK22 (13)]-defective yeast strain (K. Irie and K. Matsumoto unpublished data) which indicates that ASK1 activity observed in TM257-H1 was mediated by the PBS2-HOG1 signaling pathway.
Xenopus MAPK [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 RI 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 pcDNA3 (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 20 mM 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 phenylmethylsulfonyl fluoride (PMSF) and aprotinin (20 μg/ml). Cell extracts were clarified by centrifugation at 15 000 g 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.
Myelin basic protein was from Sigma. ATF2 was provided by S. J. Baker and T. Curran (St. Jude Children's Research Hospital). Hexahistidine (His)-tagged c-Jun (7) and glutathione-S-transferase (GST)-catalytically inactive (K57D) Xenopus MAPK (34) were prepared as described. MPK2 (16) a Xenopus counterpart of mammalian p38 was used as a substrate for SEK1 and MKK3. SEK1 phosphorylates and activates p38 as well as SAPK at least in vitro (6). His-tagged catalytically inactive (K54R) p38 was prepared as described [T. Moriguchi et al . J. Biol. Chem. 270 12969 (1995)]. To measure the activity to phosphorylate MBP c-Jun ATF2 or catalytically inactive MAPK or p38 we incubated the immune complex for 30 min at 30°C with 3 μg of each substrate in a final volume of 25 μl of a solution containing 20 mM tris-HCl (pH 7.5) 10 mM MgCl 2 and 100 μM [γ- 32 P]ATP (adenosine triphosphate) (0.3 μCi). The reaction was stopped by addition of Laemmli's sample buffer and boiling. After SDS-polyacrylamide gel electrophoresis (PAGE) phosphorylation of these proteins was quantified with an image analyzer (Fujix BAS2000).
His-tagged Xenopus MAPKK and SEK1 (XMEK2) and human MKK3 and MAPKK6 were bacterially expressed and purified as described [Y. Gotoh et al . Oncogene 9 1891 (1994)]. To measure the activity of an immune complex we first incubated 0.2 μg of His-MAPKK His-SEK1 His-MKK3 or His-MAPKK6 with the immune complex for 15 min at 30°C in a final volume of 25 μl of a solution containing 20 mM tris-HCl (pH 7.5) 10 mM MgCl 2 and 100 μM ATP and subsequently for 7 min at 25°C with 0.3 μCi of [γ- 32 P]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.
Gotoh Y. Nishida E. unpublished data.
To avoid the possibility that constitutively expressed ASK1 might induce cell death resulting in a failure to obtain stable transformants we used a metallothionein-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.
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).
Johnson N. L., et al., ibid.3229.
To measure the activity of SAPK we subjected each cell extract to a kinase detection assay within a polyacrylamide gel (in-gel kinase assay) containing c-Jun as a substrate as described (7). To examine the activity of p38 we immunoprecipitated p38 with polyclonal antibody to p38 (C-20 Santa Cruz) as described (19) except for the presence of 0.1% SDS during the immunoprecipitation and the kinase activity was detected with ATF2 as a substrate.
Ichijo H. Miyazono K. unpublished data.
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).
Cytoplasmic small fragmented DNA was isolated as described [K. S. Selins and J. J. Cohen J. Immunol. 139 3199 (1987)] with minor modifications. Briefly 3 × 10 6 cells were lysed with 200 μl of a buffer containing 20 mM tris-HCl (pH 7.5) 10 mM EDTA and 0.5% Triton X-100. Cell extracts were clarified by centrifugation at 15 000 g 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 phenol-chloroform extraction.
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. Inage 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.