細胞接着と細胞増殖におけるC-キナーゼの役割

• Project/Area Number: 06283222
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Research Institution: Yokohama City University
• Principal Investigator: 大野 茂男 横浜市立大学, 医学部, 教授 (10142027)
• Co-Investigator(Kenkyū-buntansha): 鈴木 厚 横浜市立大学, 医学部, 助手 (00264606) ; 水野 恵子 横浜市立大学, 医学部, 助手 (90221803) ; 長田 真一 横浜市立大学, 医学部, 助手 (00244484) ; 平井 秀一 横浜市立大学, 医学部, 講師 (80228759)
• Project Period (FY): 1996
• Project Status: Completed (Fiscal Year 1996)
• Budget Amount: ¥37,000,000 (Direct Cost: ¥37,000,000); Fiscal Year 1996: ¥15,000,000 (Direct Cost: ¥15,000,000); Fiscal Year 1995: ¥11,000,000 (Direct Cost: ¥11,000,000); Fiscal Year 1994: ¥11,000,000 (Direct Cost: ¥11,000,000)
• Keywords: プロテインキナーゼC / S6キナーゼ / ストレス応答 / NFkB / メタロプロティナーゼ / PI3キナーゼ / ホスホリパーゼC / プロテインキナーゼC結合タンパク質 / 血清飢餓 / DNA合成 / PKC関連酵素 / ホルボールエステル / 転写活性化 / ジアシルグリセロール

Research Abstract

PKCは細胞内シグナル経路の調節因子として極めて重要であることが様々な実験から推測されてきたが、未だにその具体的な作用点は不明である。本研究では、従来の研究で集積したPKCに関する独自の材料を用いて、PKCの具体的な作用点を解明することを目的とし、以下の二つの方向からのアプローチを行い、以下の結果を得た。
1)PI3キナーゼ経路の代表的な下流因子、p70S6キナーゼの活性化機構を解析し、これにaPKCλが関わっていることを明らかにした。
2)nPKCδ結合蛋白質として細胞膜貫通型のタンパク質を同定した。これはメタロプロテイナーゼ/ディスインテグリンファミリーの仲間であり、PKCδはこの細胞質ドメインに特異的に結合する。細胞接着とシグナル伝達経路との接点となっている事が予測され、きわめて興味深い。
3)aPKCλ結合タンパク質として、PDZドメインを有する新規タンパク質を見いだした。これは線虫の卵の不等分裂に関わる遺伝子のほ乳類版であった。
4)Rafを介したMAPキナーゼの活性化過程において、Ras以外に必要な因子として、PKCδを同定した。
5)ストレス応答MAPキナーゼ(SAPK/JNK)経路の上流キナーゼとして、MUK、MST/MLK2を同定し、これらがSEKを介してSAPK/JNKを活性化することを示した。SAPK/JNKの上流には、MEKKに加えてMUK/MLKファミリーが存在する。
6)SAPK/JNK上流因子の一つであるMEKKがTNFαによるNFkBの活性化経路に関わることを見出した。

Research Products

• [Publications] Fukumoto, S.: "Protein kinase C delta isoform inhibits the proliferation of vascular smooth muscle cells through suppressiong G1 cyclins expression." J.Biol.Chem.272(in press.).
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• [Publications] Hirano, M.: "MEK kinase is involved in TNFα-mediated activation of NFκB and degradation of IκBα." J.Biol.Chem.271. 13234-13238 (1996)
• [Publications] Ueda, E.: "The η isoform of protein kinase C mediates transcriptional activation of the human transglutaminase 1 gene." J.Biol.Chem.271. 9790-9794 (1996)
• [Publications] Akimoto, K.: "EGF and PDGF receptors activate atypical PKCλ through phosphatidylinositol 3-kinase." EMBO J.15. 788-798 (1996)
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• [Publications] Moriya, S.: "PDGF activates protein kinase Cε through redundant and independent signaling pathways involving phosphpholipase Cγ or phosphatidylinositol 3-kinase." Proc Natl.Acad.Sci.U.S.A.93. 151-155 (1996)
• [Publications] Morishira,R.,Nakayama,H.,Isobe,T.,Matsuda,T.,Hashimoto,Y.,Okano,T.,Fukuda,Y.,Mizuno,K.,Ohno,S.,Kozawa,O.,Kato,K.and Asano,T.: "Primary structure of a γ subunit of G protein,γ 10 and its phosphorylation by protein kinase C." J.Biol.Chem.270. 29469-29475 (1995)
• [Publications] Ogawara,M.,Inagaki,M.,Tsujimura,K.,Takai,Y.,Sekimata,Y.,Hye Ha,M.,Imajoh-Ohmi,S.,Hirai,S.,Ohno,S.,Sugiura,H.and Yamauchi,T.: "Differential Targeting of protein kinase C and CaM kinase II signaling to vimentin." J.Cell.Biol.131. 1055-1056 (1995)
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• [Publications] Kinouchi,T.,Sorimachi,H.,Maruyama,K.,Mizuno,K.,Ohno,S.,Ishiura,S.and Suzuki,K.: "Conventional protein kinase C(PKC)-α and novel PKCε,but not -δ,increase the secretion of an N-terminal fragment of Alzheimer's disease amyloid precursor protein from PKC cDNA transfected 3Y1 fibrobrasts." FEBS Lett.364. 203-206 (1995)
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• [Publications] Hirano,M.,Hirai,S-I.,Mizuno,K.,Osada,S.,Hosaka,M.and Ohno,S.: "A protein kinase C isozyme,nPKCε,is involved in the activation of NF-κB by 12-0-tetradecanoylphorbol-13-acetate(TPA)in rat 3Y1 fibroblasts." Biochem.Biophys.Res.Commun.206. 429-436 (1995)
• [Publications] Ohno,S.and Suzuki,K.: "YKR2 PK(S.cerevisiae)." The protein kinase factbook,Academic Press Limited.80-88 (1995)
• [Publications] Hirano,M.,Osada,S.,Aoki,T.,Hirai,S.,Hosaka,M.,Inoue,J.and Ohno,S.: "MEK kinase is involved in TNF α-mediated activation of NFkB and degradation of IkB α." J.Biol.Chem.(in press).
• [Publications] Akimoto,K.,Takahashi,R.,Moriya,S.,Nishioka,N.,Takayanagi,J.,Kimura,K.,Fukui,Y.,Osada,S.,Mizuno,K.,Hirai,S.,Kazlauskas,A.and Ohno,S.: "EGF and PDGF receptors activate atypical PKCλ through phosphatidylinositol 3-kinase." EMBOJ.(in press).
• [Publications] Ueda,E.,Ohno,S.,Kuroki,T.,Livneh,E.,Yamada,K.,Yamanishi,K.,Yasuno,H.: "The η isoform of protein kinase C mediates transcriptional activation of the human transglutaminase 1 gene." J.Biol.Chem.(in press).
• [Publications] Hirai,S.,Izawa,N.,Osada,S.,Spyrou,G.and Ohno,S.: "Activation of the JNK pathway by distantly related protein kinases,MEKK and MUK." Oncogene. 12. 641-650 (1996)
• [Publications] Moriya,S.Kazlauskas,A.,Akimoto,K.,Hirai,S.,Mizuno,K.,Takenawa,T.,Fukui,Y.,Watanabe,Y.,Ozaki,S.and Ohno,S.: "PDGF activates protein kinase C ε through redundant and independent signaling pathways involving phosphpholipase Cγ or phosphatidylinositol 3-kinase." Proc Natl.Acad.Sci.U.S.A.93. 151-155 (1996)
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• [Publications] Hirai,S-I.,Izumi.Y.,Higa,K.,Mizuno,K.,Osada,S.,Kaibuchi,K.,Suzuki,K.and Ohno,S.: "Ras-dependent signal transduction is indispensable but not sufficientfor the activation of AP1/Jun by PKCδ." EMBOJ.13. 2331-2340 (1994)
• [Publications] Akimoto,K.,Mizuno,K.,Osada,S.,Hirai,S-I.,Tanuma,S.,Suzuki.K.and Ohno,S.: "A new member of the third classof the protein kinaseC family,PKCλ,ubiquitously expressed dominantly in an undifferentiated mouse embyonal carcinoma cell line in and also many tissues and cells." J.Biol.Chem.269. 12677-12683 (1994)
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• [Publications] Koyama,H.,Inaba,M.,Nishizawa,Y.,Ohno,S.and Mori,H.: "Protein kinase C is involved in24-hydroxylase gene expression inducedby 1,25(OH)_2D_3 in rat intestinal epithelial cells." J.Cellular Biochem.55. 230-240 (1994)
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• [Publications] Akita,Y.,Ohno,S.,Yajima,Y.,Konno,Y.,Saido,T.C.,Mizuno,K.,Chida,K.,Osada,S.,Kuroki,T.,Kawashima,S.and Suzuki,K.: "Overproduction of a Ca^<2+>-independent protein kinase Cisozyme,nPKCε,increases the secretion of prolactin from thyrotropin-releasing hormone-stimulated rat pituitary CH_4C_1 cells." J.Biol.Chem.269-6. 4653-4660 (1994)