1999 Fiscal Year Annual Research Report

細胞内情報伝達機構網の研究

Research Project

Project/Area Number	09NP0601
Research Institution	Kobe University
Principal Investigator	西塚泰美神戸大学, 学長 (10025546)
Co-Investigator(Kenkyū-buntansha)	米澤一仁神戸大学, バイオシグナル研究センター, 教授 (70283900) 斎藤尚亮神戸大学, バイオシグナル研究センター, 教授 (60178499) 吉川潮神戸大学, バイオシグナル研究センター, 教授 (40150354) 深見泰夫神戸大学, 遺伝子実験施設, 助教授 (00156746) 向井秀幸神戸大学, 自然科学研究科, 助教授 (80252758)
Keywords	プロテインキナーゼC / 脂質メッセンジャー / ターゲッティング / 情報伝達機構網 / 構造解析 / 創薬
Research Abstract	1)PKCと高い相同性を示すキナーゼドメインを有するPKBはヒトフォークヘッドドメイン転写因子であるAFXを直接リン酸化し、リン酸化型AFXは細胞質内にとどまり、脱リン酸化により核移行が誘導されることが示された。また、脱リン酸化されたAFXは核内でアポトーシスを誘導することが明らかとなった。従って、PKBはフォークヘッドドメイン転写因子AFXを生理的基質とし、その核移行を抑制することにより細胞生存に貢献していると考えられる。 2)PKC分子種特異的、刺激特異的なターゲッティングに関与するPKC分子内の機能ドメインを決定した。その結果、異なるターゲティングには異なる分子内ドメインが関与することが明らかになった。また、PKCの活性化およびターゲッティングには、phospholipaseCによるジアシルグリセロールの産生以外に、phospholipase A2によって産生された脂肪酸やリゾリン脂質の働きが関与することを見出した。 3)ジアシルグリセロールを代謝する酵素であるジアシルグリセロールキナーゼのターゲッティング機構を解析し、PKCのターゲッティングと時間的、空間的に関連し、機能連関することすることを示唆した。 4)蛋白質リン酸化酵素PKNについて酵素学解析をおこない、PKN分子内の自己抑制領域を同定するとともに、脂肪酸によってその抑制が解除されるメカニズムについて報告した。また、PKNの新規のアイソフォームを同定し、その組織・細胞内分布や、酵素学的な特徴を明らかにした。その他、PKNの結合蛋白質として、新規の巨大蛋白質を単離し、その蛋白質がさまざまな情報伝達因子の足場として機能している可能性を示した。 5)チロシンリン酸化を伴った活性上昇が誘導されるPKCδ分子種の活性調節機構について解析を行い、δ分子種はヒンジ領域および触媒領域内の合計3カ所のチロシン残基がリン酸化を受けることを明らかにした。また、試験管内でのチロシンリン酸化反応によるδ分子種の活性の上昇が示され、細胞内におけるPKCの活性化は細胞刺激の数時間後も持続していることが明かとなり、PKCの活性型への変換は細胞の長期的応答に関与していることが示唆された。 6)PKCファミリーのうち、nPKCδとnPKCεは、mTORkinase(つまり細胞外液のアミノ酸バランスとそれを感知する分子であるPI-kinase関連酵素)によりリン酸化され、活性制御機構を受けることを示した。また、PKC同様AGC kinase familyの一員であるp70S6 kinaseもmTOR kinaseにより活性制御されることを示した。 7)シグナル伝達酵素であるMAPキナーゼの活性調節機構の解析を行い、キナーゼ内のある領域の構造変化が重要であること、また、その結果からデザインされた阻害ペプチドか実際に薬理学的効果を持つことを示した。

Research Products

(43 results)

All Other

All Publications (43 results)

[Publications] Takaishi,H.: "Regulation of nuclear translocation of Forkhead transcription factor AFX by protein kinase B."Proc.Natl.Acad.Sci.USA. 96. 11836-11841 (1999)
[Publications] Aoto,M.: "A 58-kDa Shc protein is present in Xenopus eggs and is phosphorylated on tyrosine residues upon egg activation."Biochem.Biophys.Res.Commun.. 258. 265-270 (1999)
[Publications] Sato,K.-I.: "Evidence for the involvement of a Src-related tyrosine kinase in Xenopus egg activation."Dev.Biol.. 209. 308-320 (1999)
[Publications] Kiriyama,H.: "Identification of the catalytic subunit of cAMP-dependent protein kinase from the photosynthetic flagellate, Euglena gracilis Z."FEBS Lett.. 450. 95-100 (1999)
[Publications] Fukami,Y.: "Peptide inhibitors of MAPK pathway ; A structure-mimetic peptide corresponding to the conserved IDA (Inter-DFG-APE) region of the kinase domain."Pharmacol.Ther.. 82. 399-407 (1999)
[Publications] Nakazawa.M.: "Biochemical analysis of the interaction between elongation factor 1α and α/β tubulins from a ciliate, Tetrahymena pyriformis."FEBS Lett.. 435. 29-34 (1999)
[Publications] Tokmakov,A.: "Deregulation of MAPK at low pH due to a structural rearrangement of activation segment."Biochim.Biophys.Acta. 1476. 66-74 (1999)
[Publications] Sato K.-I.: "Fertilization signalling and protein-tyrosine kinases."Comp.Biochem.Physiol.. (印刷中).
[Publications] Sato K.-I.: "Regulation of Xenopus oocytes, eggs, and embryos by protein-tyrosine phosphorylation."Curr.Top.Biochem.Res.. (印刷中).
[Publications] Yamazaki,M.: "Identification of the RhoA-binding domain of phospholipase D1."J.Biol.Chem.. 274. 6035-6038 (1999)
[Publications] Takahashi,M.: "Characterization of a novel giant scaffolding protein, CG-NAP, that anchors multiple signaling enzymes to centrosome and the Golgi apparatus."J.Biol.Chem.. 274. 17267-17274 (1999)
[Publications] Yoshinaga,C.: "Mutational Analysis of the Regulatory Mechanism of PKN : The Regulatory Region of PKN Contains an Arachidonic Acid-Sensitive Autoinhibitory Domain."J.Biochem.. 126. 475-484 (1999)
[Publications] Oishi,K.: "Identification and characterization of PKN β, a novel isoform of protein kinase PKN."Biochem.Biophys.Res.Comm.. 261. 808-814 (1999)
[Publications] Shibata H.: "Interaction of PKN with a neuron-specific basic helixloop-helix transcription factor, NDRF/NeuroD2."Mol Brain Res.. 74. 126-134 (1999)
[Publications] Tanaka,M.: "Identification of myosin II as a binding protein to the PH domain of protein kinase B."Biochem.Biophys.Res.Commun.. 255. 169-174 (1999)
[Publications] Fujita,E.: "Akt phosphorylation site found in human caspase-9 is absent in mouse caspase-9."Biochem.Biophys.Res.Commun.. 264. 550-555 (1999)
[Publications] Konishi,H.: "Opposing effects of protein kinase C δ and protein kinase B a on H2O2-induced apoptosis in CHO cells."Biochem.Biophys.Res.Commun.. 264. 840-846 (1999)
[Publications] Bandyopadhyay,G.: "Effects of transiently expressed atypical (ζ λ), conventional (α,β) and novel (δ,ε) protein kinase Cisoforms on insulinstimulated translocation of epitope-tagged GLUT4 glucose transporters in rat adipocytes : specific interchangeable effects of protein kinases C-ζ and C-λ."Biochem.J.. 337. 461-470 (1999)
[Publications] Irie,K.: "Synthesis and phorbol ester-binding studies of the individual cysteine-rich motifs of protein kinase D."Bioorg.Med.Chem.Lett.. 9. 2487-2490 (1999)
[Publications] Tanaka,K.: "Identification of protein kinase B (PKB) as a phosphatidylinositol 3,4,5-trisphosphate binding protein in Dictyostelium discoideum."Biosci.Biotechnol.Biochem.. 63. 368-372 (1999)
[Publications] Standaert,M.L.: "Effects of knockout of the protein kinase C βgene on glucose transport and glucose homeostasis."Endocrinology. 140. 4470-4477 (1999)
[Publications] Yamamoto,T.: "Involvement of EF hand motifs in the Ca2+-dependent binding of the pleckstrin homology domain to phosphoinositides."Eur.J.Biochem.. 265. 481-490 (1999)
[Publications] Ogino,C.: "Purification, characterization, and sequence determination of phospholipase D secreted by Streptoverticillium cinnamoneum."J.Biochem.. 125. 263-269 (1999)
[Publications] Konishi,H.: "Activation of protein kinase B induced by H2O2 and heat shock through distinct mechanisms dependent and independent of phosphatidylinositol 3-kinase."J.Biochem.. 126. 1136-1143 (1999)
[Publications] Fujimoto,K.: "Identification of protein kinase C phosphorylation sites involved in phorbol ester-induced desensitization of the histamine H1 receptor."Mol.Pharmacol.. 55. 735-742 (1999)
[Publications] Kitamura,T.: "Insulin-induced phosphorylation and activation of cyclic nucleotide phosphodiesterase 3B by the serine-threonine kinase Akt."Mol.Cell.Biol.. 19. 6286-6296 (1999)
[Publications] Takahashi,T.: "Activation of Akt/protein kinase B after stimulation with angiotensin II in vascular smooth muscle cells."Am.J.Physiol.. 276. H1927-H1934 (1999)
[Publications] Nakai,M.: "Amyloid beta Protein (25-35) Phosphorylates MARCKS through Tyrosine Kinase-Activated Protein Kinase C Signaling Pathway in Microglia."J.Neurochem.. 72. 1179-1186 (1999)
[Publications] Murakami,N.: "An association study of a polymorphism of non-erythroid α-spectrin gene with schizophrenia."American Journal of Medical Genetics.. 88. 291-293 (1999)
[Publications] Kuroda,S.: "Biochemical characterization and expression analysis of neural thrombospondin-1-like protein, NELL1 and NELL2."Biochem.Biophys.Res.Commun.. 265. 79-86 (1999)
[Publications] Ren,Y.: "Immunocytochemical localization of serotonin and serotonin transporter (SET) in taste buds of rat."Mol.Brain Res.. 74. 221-224 (1999)
[Publications] Shimada,F.: "The neuroprotective agent MS-153 stimulates glutamate uptake."Eur.J.Pharmacol.. 386. 263-270 (1999)
[Publications] Sumioka,K.: "Induction of 55 kDa PKN cleavage product by ischemia/reperfusion model in the rat retina."Invest.Opthal.Vis.Sci.. 41. 29-35 (2000)
[Publications] Sakai,N.: "Involvement of the actin cytoskeleton in the regulation of serotonin transporter (SET) activity : possible mechanism underlying SET regulation by protein kinase C."Neurochem.Int.. 36. 567-579 (2000)
[Publications] Oyasu,M.: "Immunocytochemical localization of a neuron-specific thrombospondin-1-like protein, NELL2 : Light and electron microscopic studies in the rat brain."Mol.Brain Res.. 76. 151-160 (2000)
[Publications] Shirai,Y.: "Phospholipase A2 and its products are involved in the purinergic receptor-mediated translocation of protein kinase C in CHO-K1 cells."J.Cell Sci.. (印刷中).
[Publications] Akiyama,H.: "Blood-brain barrier formation of grafted human umbilical vein endothelial cells in athymic mouse brain."Brain Res.. (印刷中).
[Publications] Ueyama,T.: "cDNA cloning of an alternative splicing variant of protein kinase Cδ (PKCδIII), a new truncated form of PKC, in rats."Biochem.Biophys.Res.Commun.. (印刷中).
[Publications] Parekh,D.: "mTOR controls one of two kinase oathways acting upon nPKCδ and nPKCε."J.Biol.Chem.. 274. 34758-34764 (1999)
[Publications] Isotani,S.: "Immunopurified mammalian target of rapamycin phosphorylates and activates p70 S6 kinase α in vitro."J.Biol.Chem.. 274. 34493-34498 (1999)
[Publications] Nanahoshi,M.: "Alpha4 protein as a common regulator of type 2A-related serine/threonine protein phosphatases."FEBS Lett.. 446. 108-112 (1999)
[Publications] Shigemitsu,K.: "Structural requirement of leucine for activation of p70 S6 kinase."FEBS Lett.. 447. 303-306 (1999)
[Publications] Shigemitsu,K.: "Regulation of translational effectors by amino acid and mammalian target of rapamycin signaling pathways."J.Biol.Chem.. 274. 1058-1065 (1999)

1999 Fiscal Year Annual Research Report

細胞内情報伝達機構網の研究

Principal Investigator

西塚 泰美 神戸大学, 学長 (10025546)

Research Products

[Publications] Takaishi,H.: "Regulation of nuclear translocation of Forkhead transcription factor AFX by protein kinase B."Proc.Natl.Acad.Sci.USA. 96. 11836-11841 (1999)

[Publications] Aoto,M.: "A 58-kDa Shc protein is present in Xenopus eggs and is phosphorylated on tyrosine residues upon egg activation."Biochem.Biophys.Res.Commun.. 258. 265-270 (1999)

[Publications] Sato,K.-I.: "Evidence for the involvement of a Src-related tyrosine kinase in Xenopus egg activation."Dev.Biol.. 209. 308-320 (1999)

[Publications] Kiriyama,H.: "Identification of the catalytic subunit of cAMP-dependent protein kinase from the photosynthetic flagellate, Euglena gracilis Z."FEBS Lett.. 450. 95-100 (1999)

[Publications] Fukami,Y.: "Peptide inhibitors of MAPK pathway ; A structure-mimetic peptide corresponding to the conserved IDA (Inter-DFG-APE) region of the kinase domain."Pharmacol.Ther.. 82. 399-407 (1999)

[Publications] Nakazawa.M.: "Biochemical analysis of the interaction between elongation factor 1α and α/β tubulins from a ciliate, Tetrahymena pyriformis."FEBS Lett.. 435. 29-34 (1999)

[Publications] Tokmakov,A.: "Deregulation of MAPK at low pH due to a structural rearrangement of activation segment."Biochim.Biophys.Acta. 1476. 66-74 (1999)

[Publications] Sato K.-I.: "Fertilization signalling and protein-tyrosine kinases."Comp.Biochem.Physiol.. (印刷中).

[Publications] Sato K.-I.: "Regulation of Xenopus oocytes, eggs, and embryos by protein-tyrosine phosphorylation."Curr.Top.Biochem.Res.. (印刷中).

[Publications] Yamazaki,M.: "Identification of the RhoA-binding domain of phospholipase D1."J.Biol.Chem.. 274. 6035-6038 (1999)

[Publications] Takahashi,M.: "Characterization of a novel giant scaffolding protein, CG-NAP, that anchors multiple signaling enzymes to centrosome and the Golgi apparatus."J.Biol.Chem.. 274. 17267-17274 (1999)

[Publications] Yoshinaga,C.: "Mutational Analysis of the Regulatory Mechanism of PKN : The Regulatory Region of PKN Contains an Arachidonic Acid-Sensitive Autoinhibitory Domain."J.Biochem.. 126. 475-484 (1999)

[Publications] Oishi,K.: "Identification and characterization of PKN β, a novel isoform of protein kinase PKN."Biochem.Biophys.Res.Comm.. 261. 808-814 (1999)

[Publications] Shibata H.: "Interaction of PKN with a neuron-specific basic helixloop-helix transcription factor, NDRF/NeuroD2."Mol Brain Res.. 74. 126-134 (1999)

[Publications] Tanaka,M.: "Identification of myosin II as a binding protein to the PH domain of protein kinase B."Biochem.Biophys.Res.Commun.. 255. 169-174 (1999)

[Publications] Fujita,E.: "Akt phosphorylation site found in human caspase-9 is absent in mouse caspase-9."Biochem.Biophys.Res.Commun.. 264. 550-555 (1999)

[Publications] Konishi,H.: "Opposing effects of protein kinase C δ and protein kinase B a on H2O2-induced apoptosis in CHO cells."Biochem.Biophys.Res.Commun.. 264. 840-846 (1999)

[Publications] Irie,K.: "Synthesis and phorbol ester-binding studies of the individual cysteine-rich motifs of protein kinase D."Bioorg.Med.Chem.Lett.. 9. 2487-2490 (1999)

[Publications] Tanaka,K.: "Identification of protein kinase B (PKB) as a phosphatidylinositol 3,4,5-trisphosphate binding protein in Dictyostelium discoideum."Biosci.Biotechnol.Biochem.. 63. 368-372 (1999)

[Publications] Standaert,M.L.: "Effects of knockout of the protein kinase C βgene on glucose transport and glucose homeostasis."Endocrinology. 140. 4470-4477 (1999)

[Publications] Yamamoto,T.: "Involvement of EF hand motifs in the Ca2+-dependent binding of the pleckstrin homology domain to phosphoinositides."Eur.J.Biochem.. 265. 481-490 (1999)

[Publications] Ogino,C.: "Purification, characterization, and sequence determination of phospholipase D secreted by Streptoverticillium cinnamoneum."J.Biochem.. 125. 263-269 (1999)

[Publications] Konishi,H.: "Activation of protein kinase B induced by H2O2 and heat shock through distinct mechanisms dependent and independent of phosphatidylinositol 3-kinase."J.Biochem.. 126. 1136-1143 (1999)

[Publications] Fujimoto,K.: "Identification of protein kinase C phosphorylation sites involved in phorbol ester-induced desensitization of the histamine H1 receptor."Mol.Pharmacol.. 55. 735-742 (1999)

[Publications] Kitamura,T.: "Insulin-induced phosphorylation and activation of cyclic nucleotide phosphodiesterase 3B by the serine-threonine kinase Akt."Mol.Cell.Biol.. 19. 6286-6296 (1999)

[Publications] Takahashi,T.: "Activation of Akt/protein kinase B after stimulation with angiotensin II in vascular smooth muscle cells."Am.J.Physiol.. 276. H1927-H1934 (1999)

[Publications] Nakai,M.: "Amyloid beta Protein (25-35) Phosphorylates MARCKS through Tyrosine Kinase-Activated Protein Kinase C Signaling Pathway in Microglia."J.Neurochem.. 72. 1179-1186 (1999)

[Publications] Murakami,N.: "An association study of a polymorphism of non-erythroid α-spectrin gene with schizophrenia."American Journal of Medical Genetics.. 88. 291-293 (1999)

[Publications] Kuroda,S.: "Biochemical characterization and expression analysis of neural thrombospondin-1-like protein, NELL1 and NELL2."Biochem.Biophys.Res.Commun.. 265. 79-86 (1999)

[Publications] Ren,Y.: "Immunocytochemical localization of serotonin and serotonin transporter (SET) in taste buds of rat."Mol.Brain Res.. 74. 221-224 (1999)

[Publications] Shimada,F.: "The neuroprotective agent MS-153 stimulates glutamate uptake."Eur.J.Pharmacol.. 386. 263-270 (1999)

[Publications] Sumioka,K.: "Induction of 55 kDa PKN cleavage product by ischemia/reperfusion model in the rat retina."Invest.Opthal.Vis.Sci.. 41. 29-35 (2000)

[Publications] Sakai,N.: "Involvement of the actin cytoskeleton in the regulation of serotonin transporter (SET) activity : possible mechanism underlying SET regulation by protein kinase C."Neurochem.Int.. 36. 567-579 (2000)

[Publications] Oyasu,M.: "Immunocytochemical localization of a neuron-specific thrombospondin-1-like protein, NELL2 : Light and electron microscopic studies in the rat brain."Mol.Brain Res.. 76. 151-160 (2000)

[Publications] Shirai,Y.: "Phospholipase A2 and its products are involved in the purinergic receptor-mediated translocation of protein kinase C in CHO-K1 cells."J.Cell Sci.. (印刷中).

[Publications] Akiyama,H.: "Blood-brain barrier formation of grafted human umbilical vein endothelial cells in athymic mouse brain."Brain Res.. (印刷中).

[Publications] Ueyama,T.: "cDNA cloning of an alternative splicing variant of protein kinase Cδ (PKCδIII), a new truncated form of PKC, in rats."Biochem.Biophys.Res.Commun.. (印刷中).

[Publications] Parekh,D.: "mTOR controls one of two kinase oathways acting upon nPKCδ and nPKCε."J.Biol.Chem.. 274. 34758-34764 (1999)

[Publications] Isotani,S.: "Immunopurified mammalian target of rapamycin phosphorylates and activates p70 S6 kinase α in vitro."J.Biol.Chem.. 274. 34493-34498 (1999)

[Publications] Nanahoshi,M.: "Alpha4 protein as a common regulator of type 2A-related serine/threonine protein phosphatases."FEBS Lett.. 446. 108-112 (1999)

[Publications] Shigemitsu,K.: "Structural requirement of leucine for activation of p70 S6 kinase."FEBS Lett.. 447. 303-306 (1999)

[Publications] Shigemitsu,K.: "Regulation of translational effectors by amino acid and mammalian target of rapamycin signaling pathways."J.Biol.Chem.. 274. 1058-1065 (1999)

西塚泰美神戸大学, 学長 (10025546)