プロテインキナーゼを中心とする情報ネットワークの解析

• 研究課題/領域番号: 09273104
• 研究種目: 特定領域研究(A)
• 配分区分: 補助金
• 研究機関: 神戸大学 (1998); 名古屋大学 (1997)
• 研究代表者: 山村 博平 (1998) 神戸大学, 医学部, 教授 (90030882); 日高 弘義 (1997) 名古屋大学, 医学部, 教授 (80100171)
• 研究分担者: 松本 邦弘 名古屋大学, 大学院理学研究科, 教授 (70116375) ; 遠藤 實 埼玉医科大学, 医学部, 教授 (50009990) ; 野澤 義則 岐阜大学, 医学部, 教授 (10021362) ; 中西 真 名古屋市立大学, 医学部, 助教授 (40217774) ; 山村 博平 神戸大学, 医学部, 教授 (90030882)
• 研究期間 (年度): 1998
• 研究課題ステータス: 完了 (1998年度)
• 配分額: 117,000千円 (直接経費: 117,000千円); 1998年度: 45,800千円 (直接経費: 45,800千円); 1997年度: 71,200千円 (直接経費: 71,200千円)
• キーワード: チロシンキナーゼ / Syk / キナーゼhCdsl / ダントロレン / カスバーゼ3 / TRAF6 / TAK1 / TAB2 / 蛋白質リン酸化 / カルシウムシグナリング / 細胞情報伝達 / セリン・スレオニンキナーゼ / 機能探索分子 / 情報ネットワーク / キナーゼカスケード

研究概要

プロテインキナーゼは細胞内情報伝達を行なう最重要な酵素群として働いており、本研究班はこれらのキナーゼの機能を修飾する物質、ターゲットとする新規分子プローブの発見をめざしている。以下に今年度の成果を示す。
B細胞の酸化的及び高浸透圧ストレスによる蛋白質チロシンキナーゼSyk(の活性化機構とその作用の違いについて、2つのSH2部分の変異株を作製して解析した結果、2つのSH2が両ストレスによって、それぞれ異なる作用をしていることを示した。B細胞のLyn、Syk、Btk、PLC-γ2欠損株をもちいてB細胞受容体を活性化したところ、ホスホリパーゼ1)活性にはLynは関与しないが、Syk、Btk、およびPLC-γ2およびPKCが関与することを明らかにした(山村)。G2チェックポイント機構に中心的な役割を演じていると予想される新規のキナーゼhCdslを同定した。このhCdsl遺伝子はG2/M期に特異的に発現しており、精巣あるいは小腸を含む広範囲な組織で発現していた。hCdslキナーゼタンパク質はDNA障害に反応してリン酸化を受けて活性化されることが明らかになった。現在hCdslを特異的に阻害する薬物の開発を行っている(中西)。PC12細胞を1%の低酸素ストレスにさらすと、アポトーシス細胞の出現に先行して、セラミドの増加とスフィンゴミエリンの減少が見られ、細胞内ではMg^<2+>依存型の中性SMaseの活性が上昇していた。また、カスパーゼ3(CPP32)の活性上昇が見られた。C2-セラミドもカスパーゼ3を活性化し、細胞のアポトーシスを誘導した。そしてセラミドの下流でカスパーゼ3が機能している可能性が示唆された。一方、Bcl-2を発現した細胞は、低酸素ストレスおよびC2-セラミドに強い抵抗性を示した。また、Bcl-2は中性SMaseおよびカスパーゼ3に対して抑制的に作用し、アポトーシスを抑制していることが示された(野沢)。ダシトロレンはE-C coupling(興奮収縮連関)における生理的Ca2+放出とCICRの両方を抑制するものの、CICRに対ずる作用はその抑制の程度が条件により大きく変化する。そこでダントロレンの適当な誘導体には、二つの開口様式をもっと明確に区別して認識するものがあると考え、種々のダントロレンの誘導体を作成したところ、興味ある特徴をもつ誘導体(GIF-0082、0090)を見出した。GIF-0082はダントロレンとは異なり生理的Ca2+放出のみに対して抑制効果をもち、CICR機構とのチャネル活性調節メカニズムあるいは開口モードの違いを特異的に認識している可能性を有する(遠藤)。IL-1シグナル伝達経路におけるTAK1の役割について解析を行った結果、TAK1はIL7-1刺激によってTRAF6と複合体を作る。活性型TAK1はAP-1とNF-kB両方を活性化できる。dominant negative型のTAK1はIL-1刺激によるAP-1とNF-κBの活性化を阻害する。TAK1がNIKのリン酸化を誘導し、NIKを介してIKKを活性化する。TAK1の結合蛋白質TAB2が、TAK1とTRAF6をつなぐ役割を果たしている。以上の結果、IL-1刺激によってTAK1はTAB2によってTRAF6複合体にリクルートされ、AP-1とNF-κB活性化につながる2つキナーゼカスケードを活性化すると考えられる(松本)。

研究成果

• [文献書誌] Qin,S.: "Differential regulation of oxidative and osmotic stress-induced Syk activation by both autophosphorylation and SH2 domains." Biochemistry. 37. 5481-5486 (1998)
• [文献書誌] Oishi,I.: "A novel drosophila nuclear protein serine/threonin kinase expressed in the germ line during its establishment." Mech.Dev.71. 49-63 (1998)
• [文献書誌] Yoneya,H.: "Antibodies directed against ZAP-70 cross react with a 66 kDa tyrosine kinase in the rat brain." Biochem.Biophys.Res.Commun.245(1). 140-143 (1998)
• [文献書誌] Rezaul,K.: "lnvolvement of reactive oxygen intermediates in lectin-induced protein-tyrosine phosphorylation of Syk in THP-1 cells." Biochem.Biophys.Res.Commun.246(3). 863-867 (1998)
• [文献書誌] Liu,Z-J.: "A critical role for cyclin C in promotion of the hematopoietic cell cycle by cooperation with c-Myc." Molecular and Cellular Biology. 18(6). 3445-3454 (1998)
• [文献書誌] Qin,S.: "A deficiency in Syk enhances ceramide-induced apoptosis in DT40 lymphoma B cells." FEBS Lett.427. 139-143 (1998)
• [文献書誌] Tohyama,Y.: "Outside-in signaling of soluble and solid-phase fibrinogen through integrin α IIB β3 is different and cooperative with cach other in a megakaryoblastic leukemia cell,CMK." Blood. 92(4). 1277-1288 (1998)
• [文献書誌] Liu,Z-J.: "Functional cooperation of cyclin C and c-Myc in mediating homotypic cell,adhesion via very late antigen-4 activation and vascular cell adhesion,molecule-1 induction." Blood. 92(12). 1-13 (1998)
• [文献書誌] Hitomi,T.: "Cross-iinking of the B cell receptor induces activation of phospholipase D through Syk,Btk and phospholipase C-γ2." FEBS Lett.in press (1999)
• [文献書誌] Hashimoto,Y.: "Critical role for the 310 helix region of p57(Kip2) in cyclin-dependent kinase 2 inhibition and growth suppression." J.Biol.Chem.273. 16544-16550
• [文献書誌] Tanikawa,M.: "Potent prostaglandin A1 analogs that suppress tumor cell growth through induction of p21 and reduction of cyclin E." J.Biol.Chem.273. 18522-18527
• [文献書誌] Suzuki,M.: "Rational design of antitumor prostaglandins with high biological stability." J.Med.Chem.41. 3084-3090
• [文献書誌] Menjo,M.: "Critical role for p27Kipl in cell cycle arrest after androgen depletion in mouse mammary carcinoma cells(SC-3)." Oncogene. 17. 2619-2627 (1998)
• [文献書誌] Ishikawa,T.: "Anti-cancer-prostaglandin-induced cell-cycle arrest and its modulation by an inhibitor of the ATP-dependent glutathione S-conjugate export pump(GS-X pump)." Biochem.J.336. 569-576 (1998)
• [文献書誌] Yanagisawa,K.: "Opposite regulation of the expression of cyclin-dependent kinase inhibitors during contact inhibition." J.Biochem.125. 36-40 (1998)
• [文献書誌] Menjo,M.: "Regulation of G1 cyclin-dependent kinases in liver regeneration." J.Gastro.Hepatol.13(suppl.). S100-105 (1998)
• [文献書誌] Nakanishi,K.: "Dual intracellular recording of neocortical neurons in a neuron-glia co-culture system." Brain Res.Protocol. in press.(1998)
• [文献書誌] Sakai,H.: "Molecular cloning of a cDNA encodig a scrine protease homologous to complement Cls precursor from rat C6 glial celfs and its expression during glial differentiation." Gene. 209. 87-94 (1998)
• [文献書誌] Nakamura,M.: "Involvement of tyrosine phosphorylation in inhibition of fMLP-induced PLD activation by N-acetyl-L-cysteine in differentiated HL-60 cells." J.Leuk.Biol.63. 781-789 (1998)
• [文献書誌] Kanoh,H.: "Molecular cloning of a gene encoding phospholipase D from the pathogenic and dimorphic fungus,Candida albicans." Biochim.Biophys.Acta. 1398. 359-364 (1998)
• [文献書誌] Leidich,S.D.: "Cloning,and disruption of the Candida albicans phospholipase B gene,CaPLBl." J.Biol.Chem.273. 26078-26086 (1998)
• [文献書誌] Wang,S.: "Molecular cloning and cell cycle-dependent expression of a novel NIMA-related protein kinase(TpNrK) in Tetrahymena cells." Biochem.J.334. 197-203 (1998)
• [文献書誌] Nagata,K.: "Expression of GTP-binding proteins and protein kinase C isozymes in platelet-like Particles derived from megakaryoblastic leukemia cells(MEG-Ol)" Platelets. 9. 291-296 (1998)
• [文献書誌] Banno,Y.: "Evidence for the presence of multiple forms of sphingosine kinase in human platelets." 335. 301-304 (1998)
• [文献書誌] Mizutani,T.: "Changes in the expression of protein kinase C(PKC),phospholipases C(PLC) and D(PLD) isofoms in spleen brain and kidney of the aged rat : RT-PCR and Western blot analysis.Mech." Ageing Develop.105. 151-172 (1998)
• [文献書誌] Hayakawa,K.: "Decreased phospholipase Dactivities in ceramide-induced apoptosis of RBL-2H3 mast cells." Biomed.Res.19. 159-170 (1998)
• [文献書誌] Okamoto,H.: "Studies on blood compatibility of terpolymers composed of methylmethacrylate methoxypolyethylene-glycol methacrylate and dimethylsiloxane methacrylate." J.Biomater.Sci.Polymer Edn.9. 943-959 (1998)
• [文献書誌] Ito,Y.: "Possible involvement of mitogen-activated protein kinase in phospholipase D activation induced by H2O2.but not by carbachol,in rat pheochromocytoma PC12 cells." J.Neurochem.71. 2278-2285 (1998)
• [文献書誌] Nomura,T.: "Identification of GTP cyclohydrolase I activity in the ciliated protozoa,Tetrahymena pyriformis." Comp.Biochem.Physiol.B120. 753-760 (1998)
• [文献書誌] Kasai,T.: "Increased activity of oleate-dependent type phospholipase D during actinomycin D-induced apoptosis in Jurkat T cells." J.Immunol.161. 6469-6474 (1998)
• [文献書誌] Komazaki,S.: "Morphological abnormalities of adrenal gland and hypertrophy of liver in mutant mice lacking ryanodine receptors." Cell Tissue Res.294. 467-473 (1998)
• [文献書誌] Ikemoto,T.: "Effect of calmodulin on Ca2+-induced Ca2+ release of skelctal muscle from mutant mice expressing either ryanodine receptor type 1 or type 3." Pflugers Archiv. 437. 43-48 (1998)
• [文献書誌] Kameya,S.: "α-1 syntrophin gene disruption results altered distribution of nNOS in skeletal muscle." J.Biol.Chem.274. 2193-2200 (1999)
• [文献書誌] Kurokawa,M.: "The oncoprotein Evi-1 represses TGF-β signalling by inhibiting Smad3." Nature. 394. 92-96 (1998)
• [文献書誌] Shimomura,T.: "Functional and physical interaction between Rad24 and Rfc5 in the yeast checkpoint pathways." Mol.Cell.Biol.18. 5485-5491 (1998)
• [文献書誌] Kondo,T.: "Role of a complex containing Radl7,Mec3 and Ddc1 in the yeast DNA damage checkpoint pathway." Mol.Cell.Biol.19. 1136-1143 (1999)
• [文献書誌] Yamaguchi,K.: "XIAP.a cellular memeber of the inhibitor of apoptosis protein family,links the receptors to TAB1-TAK1 in the BMP signaling pathway." EMBOJ.18. 179-187 (1999)
• [文献書誌] Tsuji,J.: "The kinase TAKI can activate the NIK-IkB as well as the MAP kinase cascade in the IL-1 signalling pathway." Nature. in press. (1999)
• [文献書誌] Yamada,T.: "p38 mitogen-activated protein kinase can be involved in transforming growth factor β superfamily signal transduction in Drosophila wing morphogenesis." Mol.Cell.Biol.in press. (1999)
• [文献書誌] Nakajima,S.: "Molecular cloning and expression of a stress-responsive MAPK-related kinae from Tetrahymena cells." J.Biol.Chem.in press. (1999)
• [文献書誌] Sada,K.: "Effect of lectins on protein kinase activity. In Lectin Methods and Protocols." Edited by J.M.Rhodes and J.D.Milton.Humana Press Inc., 423-432 (1998)
• [文献書誌] Minami,Y.: "Roles of Syk protein tyrosine kinase in stress-induced signaling in B cells. Redox Regulation of Cell Signaling and its Clinical Applications." Marcel Dekker Inc.NY,USA.(in press), (1998)
• [文献書誌] Yokokura H.: "Isolation and comparison of rat cDNAs encoding Ca^<2+>/calmodulin-dependent protein kinase lisoforms." Biochim.Biophys.Acta. 1338. 8-12 (1997)
• [文献書誌] Iida Y.: "Myosin light chain phosphorylation controls insulin secretion at a proximal step in the secretory cascade." Am.J.Physiol.E782-E789 (1997)
• [文献書誌] Naito Y.: "Isoform-specific activation and structural diversity of CaM kinase I." J.Biol.Chem.272・51. 32704-32708 (1997)
• [文献書誌] Watanabe H.: "An essential role of myosin light-chain kinase in the regulation of agonist-and fluid-flow stimulated Ca^<2+>influx in endothelial cells." FASEB J.(in press). (1998)
• [文献書誌] Sudo T.: "Altemative splicing regions in the N-terminal regulatory doamins of Annexin XI isoorms are involved in Calcyclin (S100A6)binding and Ca^<2+>-independent membrane association." J.Biol.Chem.(in press). (1998)
• [文献書誌] Qin,S.: "Distinctive Functions of Syk of and Lyn in Mediating Osmotic Stress and Ultraviolet C Irradiation-induced Apoptosis in Chicken B cells." J.Biol.Chem.272. 17994-17999 (1997)
• [文献書誌] Sada,K.: "Relocation of Syk protein-tyrosine kinase to the actin filament network and subsequent association with Fak." Eur.J.Biochem.248. 827-833 (1997)
• [文献書誌] Morisaki,H.: "Cell cycle-dependent phosphorylation of p27 cyclin-dependent kinase(Cdk)inhibitor by cyclin E/Cdk2." Biochem.Biophys.Res.Commun.240. 386-390 (1997)
• [文献書誌] Ohguchi,K.: "Increased activity of small GTP-binding protein-dependent phospholipase D during differentiation in human promyelocytic leukemic HL60 cells." J.Biol.Chem.272. 1990-1996 (1997)
• [文献書誌] Yoshimura,Y.: "Changes in the activity and mRNA levels of phospholipase D during ceramide-induced apoptosis in rat glial cells." J.Neurochem.69. 713-720 (1997)
• [文献書誌] Ikemoto,T.: "Functions of the sarcoplasmic reticulum and morphological features of myocytes from mutant mice lacking both ryanodine receptors type 1 and type 3" J.Physiol.501・2. 305-312 (1997)
• [文献書誌] Suzuki,M.: "Decrease in cerebral free magnesium concentration following closed head injury and effects of VA-045 in rat." Gen.Pharmacol.28. 119-121 (1997)
• [文献書誌] Watanabe,Y.: "Characterization of a serum response factor-like protein in Saccharomyces cerevisiae,Rim1,which has transcriptional activity regulated by the Mpk1(Slt2)mitogen-activated protein kinase pathway." Mol.Cell.Biol.17. 2615-2623 (1997)
• [文献書誌] Ichijo,H.: "Incuction of apoptosis by ASK1,a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathway." Science. 275. 90-94 (1997)
• [文献書誌] Shibuya,H.: "Role of TAK1 and TAB1 in BMP signaling in early Xenopus development." EMBO J.(in press). (1998)
• [文献書誌] Omura H,: "Mitogen-activated protein kinase and P70 ribosomal protein S6 kinase are not involved in the insulin-dependent stimulation of cAMP phosphodiesterase kinase in rat adipocytes." Biochimica Biophysica Acta. (in press). (1998)
• [文献書誌] Tokuyama T,: "Expression of human islet amyloid polypeptide/amylin impairs insulin secretion in mouse pancreatic b cells." Metabolism. 46. 1044-1051 (1997)
• [文献書誌] Sakuma H.: "Molecular Cloning and Functional Expression of a cDNA encoding a New member of Mixed Lineage protein kinase from Human Brain" J.Biol.Chem.272・45. 28622-28629 (1997)
• [文献書誌] Terayama K.: "Cloning and functional expression of a novel glucuronyltransferase involved in the biosynthesis of the carbohydrate epitope,HNK-1" Proc.Natl.Acad.Sci.U.S.A.94・12. 6093-6098 (1997)
• [文献書誌] Yamaji T.: "Neutral glycosphingolipids induce cell-cell aggregation of a variety of hematopoietic cell lines." Eur.J.Biochem.247・1. 21-29 (1997)
• [文献書誌] Morio H.: "Renal expression of serum-type mannan-binding protein in rat." Eur.J.Biochem.243・3. 770-774 (1997)
• [文献書誌] Senda,T.: "Three-dimensional structure of an extradiol type catechol ring cleavage dioxygenase BphC derived from Pseudomonas sp.Strain KKS102.-Structural feature pertinent to substrate specificity and reaction mechanism-" Oxygen Homeostasis and its Dynamics(Ishimura ed.). 276-28 (1997)
• [文献書誌] Mitsui,Y.: "Elucidation of the basic three-dimensional structure of Type I interferons and its functional and evolutionary implications." J.Interferon and Cytokine Res.17. 319-326 (1997)
• [文献書誌] Nomura,T.: "Overexpression of α and β isoforms of Ca^<2+>/calmodulin-dependent protein kinase II in neuroblastoma cells-H-7 promotes neurite outgrowth." Brain Res.766. 129-141 (1997)
• [文献書誌] Yoshimura,Y.: "Phosphorylation-dependent reversible association of Ca^<2+>/calmodulin-dependent protein kinase II with the postsynaptic densities." J.Biol.Chem.272. 26354-26359 (1997)
• [文献書誌] Nakao A,: "Identification of Smad7,a TGFβ-inducible antagonist of TGF-β signalling." Nature. 389. 631-635 (1997)
• [文献書誌] Imamura T,: "Smad6 inhibits signalling by the TGF-β superfamily." Nature. 389. 626-622 (1997)
• [文献書誌] Kobayashi,K.: "p140Sra-1(Specifically Rac1-associated protein) is a novel specific target for Rac1 small GTPase." J.Biol.Chem.273. 291-295 (1998)
• [文献書誌] Fukata,M.: "Regulation of cross-linking of actin filament by IQGAP1,a target for Cdc42." J.Biol.Chem.272. 29579-29583 (1997)
• [文献書誌] Kurosu H.: "Heterodimeric phosphoinositide 3-kinase consisting of p85 and p110β is synergistically activated by the bg subunits of G proteins and phosphotyrosyl peptide." J.Biol.Chem.272. 24252-24256 (1997)
• [文献書誌] Gotoh,N.: "Tyrosine phosphorylation sites at amino acids 239 and 240 of Shc are involved in epidermal growth factor-induced mitogenic signaling that is distinct from Ras/mitogen-activated protein kinase activation." Mol.Cell.Biol.17. 1824-1831 (1997)
• [文献書誌] Sato,K.-I.: "Tyrosine residue 239 and 240 of Shc are phosphatidylinositol 4,5-bisphosphate-dependent phosphorylation sites by c-Src." Biochem.Biophys.Res.Commun.240. 399-404 (1997)
• [文献書誌] Kihara T,: "Nicotinic receptor stimulation protects neurons against beta amyloid toxicity." Ann.Neurol.42. 159-163 (1997)
• [文献書誌] Shimohama S: "Characterization of plospholipase C isoizymes in human brain and changes in Alzheimer's disease." Neuroscience. 82. 999-1007 (1998)
• [文献書誌] Nakai K.: "Regulation of myosin phosphatase through phosphorylation of the myosin-binding subunit in platelet activation." Blood.90. 3936-3942 (1997)
• [文献書誌] Kihira H.,: "Possible involvement of calcineurin in retinoic acid-induced inhibition of leukemic HL-60 cell proliferation." Int.J Oncol.(in press). (1998)
• [文献書誌] Okumura M,: "Increased serum concentration of soluble CD30 in patients with Graves' disease and Hashimoto's thyroiditis." J Clin Endocrinol Metab. 82. 1757-1760 (1997)
• [文献書誌] Hidaka Y: "Increased serum concentration of interleukin-5 in patients with Graves' disease and Hashimoto's thyroiditis." Thyroid. (in press). (1998)
• [文献書誌] Shibata,K.: "Fibronectin secretion from human peritoneal tissue induces Mr92,000 type IV collagenase expression and invasion in ovarian cancer cell lines." Cancer R.57. 5416-5420 (1997)
• [文献書誌] Thant,A.A.: "c-Ras is required for the activation of the matrix metalloproteinases by Concanavalin A in 3Y1 cells." FEBS Lett.406. 28-30 (1997)
• [文献書誌] Yasue,T.: "A critical role of Lyn and Fyn for B cell responses to CD38 ligation and interleukin 5." Proc.Natl.Acad.Sci.USA.94. 10307-10312 (1997)
• [文献書誌] Baba,M.: "Mouse germinal center B cells with the xid mutation retain responsiveness to anti-mouse CD40 antibodies but diminish IL-5 responsiveness." Intl.Immunol.9. 1463-1473 (1997)
• [文献書誌] Ohkusu,K.: "Protein kinase Ca-mediated chronic signal transduction for immunosenescence." J.Immunol. 159. 2082-2084 (1997)