Detecting Phases of Iron

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. 15. 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response in a PBS2 [downstream target of SHO1, SSK2, and SSK22 (13)]–defective yeast strain (K. Irie and K. Matsumoto, unpublished data), which indicates that ASK1 activity observed in TM257-H1 was mediated by the PBS2-HOG1 signaling pathway. 16. J. Rouse et al., Cell 78, 1027 (1994). 17. J. Han, J.-D. Lee, L. Bibbs, R. J. Ulevitch, Science 265, 808 (1994). 18. J. C. Lee et al., Nature 372, 739 (1994). 19. Xenopus MAPK [Y. Gotoh et al., EMBO J. 10, 2661 (1991)] and Xenopus MAPKK (34) were cloned as described. Coding regions for rat SAPKa (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 pSRa456 [Y. Takebe et al., Mol. Cell. Biol. 8, 466 (1988)], yielding pSRa-HA1. The cDNAs encoding MAPK, SAPKa, p38, MAPKK, SEK1, and MKK3 were subcloned into the Bgl II site of pSRa-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 20mM tris-HCl (pH 7.5), 12 mM b-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 mg/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 mMNaCl, 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. 20. 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 mg of each substrate in a final volume of 25 ml of a solution containing 20 mM tris-HCl (pH 7.5), 10 mM MgCl2, and 100 mM [g-32P]ATP (adenosine triphosphate) (0.3 mCi). 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). 21. 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 mg of His-MAPKK, His-SEK1, His-MKK3, or His-MAPKK6 with the immune complex for 15min at 30°C in a final volume of 25 ml of a solution containing 20 mM tris-HCl (pH 7.5), 10 mM MgCl2, and 100 mM ATP and subsequently for 7 min at 25°C with 0.3 mCi of [g-32P]ATP and 3 mg of GST–catalytically inactive MAPK (for MAPKK) or His-tagged catalytically inactive p38 (for SEK1,MKK3, andMAPKK6) in the same solution (final volume, 35 ml). To measure the kinase activity of wild-type p38, we used His-tagged wildtype p38 and ATF2 instead of catalytically inactive p38. Samples were analyzed by SDS-PAGE and image analyzer. 22. Y. Gotoh and E. Nishida, unpublished data. 23. 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. 24. 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). 25. Z. Xia, M. Dickens, J. Raingeaud, R. J. Davis, M. E. Greenberg, Science 270, 1326 (1995). 26. Y.-R. Chen, C. F. Meyer, T.-H. Tan, J. Biol. Chem. 271, 631 (1996). 27. N. L. Johnson et al., ibid., p. 3229. 28. M. Verheij et al., Nature 380, 75 (1996). 29. 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. 30. H. Ichijo and K. Miyazono, unpublished data. 31. J. Raingeaud et al., J. Biol. Chem. 270, 7420 (1995). 32. 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 (4ethoxymethylene-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 b-galactosidase staining) were isolated on phOx-coated magnetic beads with the Capture-Tec kit (Invitrogen), allowed to grow, counted, and treated with TNF-a. Nontransfected Jurkat cells and isolated Jurkat cells that were transfected with pHook1, and control pcDNA3 plasmids were similarly sensitive to TNF-a in the DNA fragmentation assay (30). 33. H. Ichijo et al., J. Biol. Chem. 268, 14505 (1993). 34. H. Kosako, E. Nishida, Y. Gotoh, EMBO J. 12, 787 (1993). 35. 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 3 106 cells were lysed with 200 ml 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 15,000g 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. 36. 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.