プロテインキナ-ゼCの特異的分子種発現細胞系の構築及びその選択的抑制剤の開発研究

1989 Fiscal Year Annual Research Report

• Project/Area Number: 63870016
• Research Institution: Kobe University
• Principal Investigator: 荻田 浩司 神戸大学, 医学部, 助手 (60204103)
• Co-Investigator(Kenkyū-buntansha): 深見 泰夫 神戸大学, 遺伝子実験施設, 助教授 (00156746) ; 橘 秀樹 神戸大学, 理学部, 助教授 (70126118) ; 岸本 明 神戸大学, 医学部, 助教授 (60127363)
• Keywords: Protein Kinase C / 血小板 / LTP(長期増強) / T cell / synaptosome / DiC_8 / TPA / 細胞増殖

Research Abstract

代謝内分泌、神経機能のみならず細胞の分化、増殖、発癌機構などprotein kinase C(PKC)が制御する細胞反応は広汎な領域に及ぶ。PKCの分子多様性の存在が確立された現在、これら多彩な生理機能を担う分子種をそれぞれ同定し、PKC分子種の生理意義を明らかにする事が緊急の課題となった。
本年度はPKC分子種特異機能解明を目的とし、以下の成果を得た。
1.放出反応-血小板
海馬より調製したsynaptosomeはα、β、γの3種のPKCを有するが、TPAを前処理する事によりβ、γを選択的にdown regulationさせ、γのみを有するsynaptosome調製に成功した。これらsynaptosomeを用い、PKC分子種によるノルアドレナリン(NA)放出の増強効果を比較検討し、次の結果を得た。NA放出のTPAによる増強効果はβ、α活性有意な海馬及びγ活性のみ有するsynaptosomeにおいても有意差は認められず、γPKCのNA放出反応への関与はないと結論した。
2.神経特異機能-LTP(長期増強)
PKC分子種特異的活性調節剤検索の結果、lipoxin Aは脳、神経系でのみ発現されるγPKCを特異的に活性化する事を見いだした。また、β、aPKCを活性化し得ない低能度のアラキドン酸によりCa^<2+>を必要とせず活性化し得た。一方、記憶のモデルとして知られる海馬長期増強確立には、PKCの活性化と共にアラキドン酸及びその代謝物の関与が示唆されている。したがって、γ分子種の長期増強確立のメカニズム解明を目指す生化学的基盤は確立し得たと考える。
3.細胞分化、増殖-T cell
抗原刺激により惹起されるT cellの活性化はTPAとCa^<2+>ionophoreの協奏作用により再現可能である。本研究はIL-2R発現、細胞増殖能、PKC translocationを指標にT cell活性化におけるPKCの意義解明を目的とし、以下の結論を得た。短期間の膜透過性のDiacylglycerol(DiC_8)は、TPAにより起こるIL-2R発現、細胞増殖とも起こし得ず、traslocationも一時的であったが頻回により再現し得た。従って、細胞増殖は細胞内Diacylglycelerolの一過性上昇で惹起されず、長期的なPKC活性化、即ち、持続的なDG産生を必要とする事を示唆し得た。しかしながら、T cellに存在するβI、βII、a分子種はすべてtranslocationに関し、同様の挙動を示し、細胞増殖シグナルに関与するPKC分子種の同定は今後の課題である。

Research Products

• [Publications] Ono,Y: "The Structure expression,and properties of additional members of protein kinase C family." J.Biol.Chem.263. 6927-6932 (1988)
• [Publications] Kosaka,Y.: "The heterogerogeneity of protein kinase C in various rat tissues." Biochem.Biophys.Res.Commun.151. 973-981 (1988)
• [Publications] Tsukuda,M.: "Properties of protein kinase C subspecies in human platelets." Biochem.Biophys.Res Commun.155. 1387-1395 (1988)
• [Publications] Asaoka,Y.: "Activation of a brain-specific protein kinase C subspecies in the presence of phosphatidylethanol." FEBS Lett.231. 221-224 (1988)
• [Publications] Ase,K.: "Differential down-regulation of protein kinase C subspecies in KM3 cells." FEBS Lett.236. 396-400 (1988)
• [Publications] Shearman,M.S: "Isolation of protein kinase C subspecies from a preparation of human T-lymphocytes." FEBS Lett.234. 387-391 (1988)
• [Publications] Saito,N.: "Distribution of protein kinase C-like immunoreactive neurons in rat brain." J.Neuroscience. 8. 369-382 (1988)
• [Publications] Hashimoto,T: "Postnatal development of a brain specific subspecies of protein kinase C in rat." J.Neuroscience. 8. 1678-1683 (1988)
• [Publications] Ase,K.: "Distinct cellular expression of βI-and βII-subspecies of protein kinase C in rat cerebellum." J.Neuroscience. 8. 3850-3856 (1988)
• [Publications] Kose,A.: "Electron microscopic localization of type I protein kinase C in rat Purkinje cells." J.Neuroscience. 8. 4262-4268 (1988)
• [Publications] Sekiguchi,K.: "Mode of activation and kinetic properties of three distinct forms of protein kinase C from rat brain." J.Biochem.103. 759-765 (1988)
• [Publications] Shearman,M.S.: "Molecular heterogeneity and differential expression of multiple protein kinase C subspecies in central nervous tissue." Biochem.Soc.Trans.16. 460-463 (1988)
• [Publications] Naor,Z.: "Calcium-independent activation of hypothalamic type 1 protein kinase C by unsaturated fatty acids." Mol. Endocrinol.2. 1043-1048 (1988)
• [Publications] Kikkawa,U.: "Protein kinase C in transmembrane signalling." Adv. Second Messenger Phosphoprotein Res.21. 67-74 (1988)
• [Publications] Shearman,M.S.: "A rapid method for the resolution of protein kinase C subspecies from rat brain tissue." Methods Enzymol.168. 286-290 (1988)
• [Publications] Nishizuka,Y.: "Phospholipid turnover in signal transduction;protein kinase C and calcium ion as two synergetic mediators." CRC Critical Reviews,CRC Press,. (1988)
• [Publications] Kikkawa,U.: "The heterogeneity and differential expression of protein kinase C in nervous tissues." Phil.Trans.Royal Soc.London. 320. 313-324 (1988)
• [Publications] Kikkawa,U.: "The family of protein kinase C;its molecular heterogeneity and differential expression." Cold Spring Harbor Symp.Quant.Biol.53. 97-102 (1988)
• [Publications] Ono,Y.: "Protein kinase C ζ subspecies from rat brain: Its structure,expression and properties." Proc.Natl.Acid.Sci.86. 3099-3103 (1989)
• [Publications] Berry,N.: "Human T cell activation by phorbol esters and diacylglycerol analogues." J.Immunol.143. 1407-1413 (1989)
• [Publications] Kikkawa,U.: "The protein kinase C family:heterogeneity and its implications." Ann.Rev.Biochem.58. 31-44 (1989)
• [Publications] Ogita,K.: "The structure and function of multispecies of protein kinase C." Adv.in Prostaglandin, Thromboxane,Thromboxane,and Leukotriesne Res.19. 49-56 (1989)
• [Publications] Bignon,E: "Modes of inhibition of protein kinase C by triphenylacrylonitrile antiestrogens." Biochem. Biopys.Res Commun.163. 1377-1383 (1989)
• [Publications] Kishimoto,A.: "The protein kinase C family in the brain: heterogenety and its implications." Ann.N.Y.Acad.Sci.568. 181-186 (1989)
• [Publications] Ono,Y.: "Nuleotide sequences of cDNAs for α and γ subspecies of rat brain protein kinase C." Nucleic Acids Research. 16. 5199-5200 (1988)
• [Publications] Tachibana,H.: "Local similarity in the amino acid sequence between the non-catalytic region of Bous sarcoms virus oncogene product p60^<v-src> and intermediate filament proteins." J.Biochem.104. 869-872 (1988)
• [Publications] Kishimoto,A.: "Limites proteolysis of protein kinase C Subspecies by calcium-dependent neutral protease(calpain)." J.Biol.Chem. 264. 4088-4092 (1989)
• [Publications] Sheaman,M.S.: "Selective activation of the γ-subspecies of protein kinase C from bovine cerebellum by arachidonic acid and its lipoxygenase metabolites." FEBS Lett.243. 177-182 (1989)
• [Publications] Hosoda,K.: "Immunocytochemical localization of the βI subspecies of protein kinase C in rat brain." Proc.Natl.Acad.Sci.86. 1393-1397 (1989)
• [Publications] Kikkawa,U.: "Immunocytochemical localization of the βII subspecies of protein kinase C in rat brain." Proc.Natl.Acad.Sci.86. 3409-3413 (1989)
• [Publications] Suda,T.: "The tumor rejection antigen separated from Rous sarcoma virusinduced murine fibrosacrcoma exhibits a molecular weight of approximately 60kD but differs from functional pp60^<src>" Jap.J.Cancer Res.80. 879-886 (1989)
• [Publications] Ito,A: "Immunocytochemical localization of α subspecies of protein kinase C in rat brain." Proc.Natl.Acad.Sci. (1990)
• [Publications] Ogita,K.: "The hethrogeneity of protein kinase C in signal transduction cascade." Adv.Second Messenger and Phosphoprotein Res.,. (1990)
• [Publications] Bignon,E.: "Dual action of hydroxylated diphenylethylenes on protein kinase C." Biochem.Biophys.Res.Commun. (1990)
• [Publications] Yasuda,I.: "A synthetic peptide substrate for selective assay of protein kinase C." Biochem.Biophys.Res.Commun.166. 1220-1227 (1990)
• [Publications] Berry,N.: "Activation of resting human T cells requires prolonges stimulation of protein kinase C." Proc.Natl.Acad Sci.(1990)
• [Publications] Kishimoto,A.: "Preparation and use of protein kinase C subspecies-specific anti-peptide antibody." Methods Enzymol.(1990)
• [Publications] Ogita,K.: "The expression,separation and assay of protein kinase C subsupecies." Methods Enzymol.(1990)