細胞増殖におけるプロテインキナーゼC群の機能の分子生物学的解析

• Project/Area Number: 04253239
• 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): 秋田 朗子 東京都臨床医学総合研究所, 遺伝情報, 研究員 (40124432) ; 長田 真一 横浜市立大学, 医学部, 助手 (00244484) ; 水野 恵子 横浜市立大学, 医学部, 助手 (90221803) ; 平井 秀一 横浜市立大学, 医学部, 助手 (80228759)
• Project Period (FY): 1993
• Project Status: Completed (Fiscal Year 1993)
• Budget Amount: ¥21,000,000 (Direct Cost: ¥21,000,000); Fiscal Year 1993: ¥11,000,000 (Direct Cost: ¥11,000,000); Fiscal Year 1992: ¥10,000,000 (Direct Cost: ¥10,000,000)
• Keywords: プロテインキナーゼC / 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

昨年度来の研究から、我々は線維芽細胞への各PKC分子種の高発現系と転写応答のレポーター遺伝子とを用いて、生きた細胞内でのPKCの活性化状況をモニターする系を始めて開発した。この系を用いて本年度は新たに_aPKCの活性化をもモニター出来る事を確認した。この系を用いて、_cPKC、_nPKC、_aPKCという3種のPKC亜群の各々の分子種について、その活性化機構を解析することが始めて可能となった。そこで本年度はこの点を集中的に解析し、PKCの生理的な重要性の発見以来約10年ぶりともいえる画期的な発見をすることが出来た。それは一言でいうとPKCの活性化経路には、従来から知られていたPLC/DAGを介する経路以外に、PI3キナーゼを介する全く新しい経路が関わっているという点である。チロシンキナーゼ型受容体の代表格と言えるPDGF受容体のシグナル異変体や、PI3キナーゼサブユニットの発現系を用いて、各PKC分子種の活性化に関わる経路を生きた細胞を用いて分子生物学的に始めて解析し、従来のPLCを介した経路が_cPKC、_nPKCの活性化に関わることを確認すると同時に、PI3キナーゼを介する経路がこれらの活性化に関わることを始めて見いだした。更に、_aPKCの活性化にはPLCではなく、PI3キナーゼが関わることを始めて見いだした。

Research Products

• [Publications] Hirai,S.,Izumi,Y.,Higa,K.,Mizuno,K.,Osada,S.,Kaibuchi,K.,Suzuki,K.and Ohno,S.: "Ras-dependent signal transduction is indispensable but not sufficient for the activation of AP1/Jun by PKCdelta・" EMBOJ.(in press).
• [Publications] Akimoto,K.,Mizuno,K.,Osada,S.,Hirai,S.,Tanuma,S.,Suzuki,K.and Ohno,S.20GB02:A new member of the third class of the protein kinase C family,PKC lambda,ubiquitously expressed dominantly in an undifferentiated mouse embyonal carcinoma cell line and also in many tissues and cells.: J.Biol.Chem.(in press).
• [Publications] Ohno,S.and Suzuki,K.: "Protein kinase C:Reassessment.Function of the novel members of the protein kinase C family." FESEB J.(in press).
• [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 aCa_+-independent protein kinase C isozyme,_nPKC epsilon,increases the secretion of protein from thyrotropin-releasing hormone-stimulated rat pituitary GII_4C_1 cells." J.Biol.Chem.(in press).
• [Publications] Chida,K.,Sagara,H.,Suzuki,Y.,Murakami,A.,Osada,S.,Ohno,S.,Hirosawa,K.and Kuroki,T.: "The eta isoform of protein kinase C(_nPKC_<eta>)is localized on rough endoplasmic reticulum." Molecular and Cellular Biol.(in press).
• [Publications] Koyama,H.,Inaba,M.,Nishizawa,Y.,Ohno,S.and Morii,H.: "Protein kinase C is involved in 24-hydroxylase gene expression induced by 1,25(OH)_2D_3 in rat intestinal epithelial cells." J.Cellular Biochemistry. (in press).
• [Publications] Suzuki,K,Akita,Y.and Ohno,S.: "Structural and functional diversities of protein kinase C family members." Recent Advances in Molecular and Biochemical Research on proteins.(in press).
• [Publications] Ohno,S.,Akita,Y.,Hata,A.,Osada,S.,Kubo,K.,Konno,Y.,Akimoto,K.,Mizuno,K.,Saido,T.C.,Kuroki,T.and Suzuki,K.: "Structural and functional diversities of a family of signal transducing protein Kinases;protein Kinase C family." Yokohama Med Bull.43. 139-140 (1993)
• [Publications] Ohno,S.: "Diversity and specificity of multiple protein kinase C." Progress in Endocrinology,Parthenon Press.63-66 (1993)
• [Publications] Koizumi,H.,Kohno,Y.,Osada,S.,Ohno,S.,Ohkawara,A.and Kuroki,T.: "Differentiation-associated localization of _nPKC eta,_aC_a^<++>-independent protein kinase C,in normal human skin and skin diseases." J.Investigative Dermatology. 6. 858-863 (1993)
• [Publications] Mischak,H.,Goodnight,J.,Henderson,D.W.,Osada,S.,Ohno,S.and Mushinski,F.: "Unique expression pattern of protein kinase C-theta:high _mRNA levels in normal mouse testes and in T-lymphocytic cells and neoplasms." FEBS Lett.326. 51-55 (1993)
• [Publications] Sublette,E.,Naik,M.U.,Jiang,X.,Osten,P.,Valsamis,H.,Osada,S.,Ohno, S.and Sacktor,T.C.: "Evidence for a new,high-molecular weight isoform of protein kinase C in rat hippocampus." Neuroscience Lett.159. 175-178 (1993)
• [Publications] Mizuno,K.,Saido,T.C.,Ohno,S.,Tamaoki,T.and Suzuki,K.: "Staurosporine-related compounds,K252_a and UCN-01,inhibits both _cPKC and _nPKC." FEBS Lett.330. 114-116 (1993)
• [Publications] Park,H.- Y.,Russakovsky,V.,Ohno,S.and Gilchrest,B.A.: "The beta-Isoform of protein kinase C stimulates human melanogenesis by activating tyrosinase in pigment cells.21GC14:J.Biol.Chem." 268. 11742-11749 (1993)
• [Publications] Hata,A.,Akita,Y.,Suzuki,K.and Ohno,S.: "Functional divergence of protein kinase C(PKC)family members:PKC_<gamma> differs from PKC alpha,betaII,and _nPKC epsilon in its competence to mediate 12-O-tetradecanoyl phorbol 13-acetate(TPA)-responsive transcriptional activation through a TPA-response element." J.Biol.Chem.268. 9122-9129 (1993)
• [Publications] Osada.S.,Hashimoto,Y.,Nomura,S.,Konno,Y.,Chida,K.,Tajima,O.,Kubo,K.,Akimoto,K.,Koizumi,H.,Kitamura,Y.,Suzuki,K,Ohno,S.and Kuroki,T.: "Predominant expression of novel protein kinase C eta,_aC_a^<2+>-independent isoform of protein kinase C in epithelial tissues,in association with epithlial differentiation." Cell Growth & Differentiation. 4. 167-175 (1993)
• [Publications] Wang,F.,Naik,U.P.,Ehrlich,Y.H.,Freyberg,Z.,Osada,S.,Ohno,S.,Kuroki,T.,Suzuki,K.and Kamocki,E.: "A new protein kinase C,_nPKC eta′and PKC theta are expressed in human platelets:involvement of _nPKC_n′and _nPKC theta in signal transduction stimulated by PAF." Biochem.Biophys.Res.Commun.191. 240-246 (1993)
• [Publications] 大野茂男: "Cキナーゼファミリーの活性調節" 細胞増殖の制御,南江堂. 105-114 (1993)
• [Publications] 大野茂男: "細胞増殖と疾患-細胞増殖の細胞内シグナル伝達機構" Mebio,メジカルビュー社. 10,6. 30-38 (1993)
• [Publications] Hata,A.,Akita,Y.,Suzuki,S.and Ohno,S.: "Functional divergence of protein kinase C(PKC)family members;PKCγdiffers from PKC α,βII,and nPKC ε in its competence to mediate 12-O-tetradecanoyl phorbol 13-acetate(TPA)-responsive transcriptional activation a TPA-response slsment." J.Biol.Chem.
• [Publications] Osada,S.,Hashimoto,Y.,Nomura.,S,Konnno.,Y.,Chida,K.,Tajima,O.,Kubo,K.,Akomoto,K.,Koizumo,H.,Kitamura,Y.,Suzuki,K.,Ohno.,S.and Kuroki,T.: "Predominant expression of novel protein kinase C η,aCa2+-independent isoform of protein kinse C in epithelial tissues,in association with epithlial differentiation." Cell Growth&Differentiation.
• [Publications] Kim,H-M.,Hirota,S.,Onore,H.,Hirata,T.,Suzuki,K.,Ohno,S.,Kuroki,T.,Kitamura,Y.and Nomura,S.: "Localization and development expression of a novel protein kinase C δ gene." Developmental Brain Research.
• [Publications] Osada,S.,Mizuno,K.,Saido,T.C.,Suzuki,K.,and Ohno,S.: "A new member of the protein kinase C family,nPKC θ,predominantly expressed in skeletal muscle." Molec.Cell.Biol.12. 3930-3938 (1992)
• [Publications] Maizcls,E.T.,Miller,J.B.,Cutler Jr.,R.E.,Jackiw,V.,Camey,E.M.,Mizuno,K.,Ohno,S.,and Hunzicker-Dunn,M.: "Estrogen modulates Ca2+-independent lipid-stimulated kinase in the rabbit corpus liteum of pseudopregnancy.Identification of lureal estrogen-modulated lipid-stimulated kinase as δ-C-kinase." J.Biol.Chem.267. 17061-17068 (1992)
• [Publications] Gruber,J.R.,Ohno,S.,and Niles,R.M.: "Increased expression of PKC α plays a key role in retinoic acid induced melanoma differentiation." J.Biol.Chem.267. 13356-13360 (1992)
• [Publications] Hata,A.,Ohno,S.,and Suzuki,K.: "Transcriptional activation of the gene for the large subunit of human m-calpain by 12-o-tetradecanoyl-phorbol-13-acetate." FEBS Letters. 304. 241-244 (1992)
• [Publications] Niino,Y.,Ohno,S.and Suzuki,K.: "Positive and negative regulation of the transcription of the human protein Kinase C β gene." J.Biol.Chem.267. 6158-6163 (1992)
• [Publications] Saedo,T.C.,Mizuno,K.,Konno,Y.,Osada,S.,Ohno,S.and Suzuki,K.: "Purification and characterization of protein Kinase C ε(nPKC ε)from rabbit brain." Biochemistry. 31. 482-490 (1992)
• [Publications] 水野 恵子,大野 茂男: "プロテインキナーゼCアイソザイムの同定" 実験医学. 11(3). 163-169 (1993)
• [Publications] Ohno,S.: "Diversity and specificity of multiple protein kinase C." Progress in Endocrinology,Parthenon Press.,
• [Publications] 大野 茂男: "C-キナーゼとシグナル伝達" 腎臓学Key Notes,東京医学社, 2 (1992)