1995 Fiscal Year Annual Research Report

低分子量G蛋白質とその関連蛋白質の機能修飾物質の開発およびその有効利用

Research Project

Project/Area Number	06557012
Research Institution	Osaka University
Principal Investigator	高井義美大阪大学, 医学部, 教授 (60093514)
Co-Investigator(Kenkyū-buntansha)	河田純男大阪大学, 医学部, 助教授 (90183285) 佐々木卓也大阪大学, 医学部, 助手 (40241278) 田中一馬大阪大学, 医学部, 講師 (60188290)
Keywords	低分子量G蛋白質 / Ras / Rap1 / B-Raf / SmgGDS / SMAP / Rho / Rabphilin-3A
Research Abstract	本研究では、私共の発見した低分子量G蛋白質およびその関連蛋白質とそれらの変異体のcDNAやペプチド、特異抗体を作製し、それらを用いて低分子量G蛋白質の活性化機構、機能と作用機構について検討すると共に、低分子量G蛋白質の制御異常による病態の解析や新しい診断法、治療法の開発に応用することを目的としている。私共は、すでに、無細胞系でRasによりMAPキナーゼを活性化するアッセイ系を用いて、Rasの標的蛋白質を精製し、B-Rafと14-3-3蛋白質の複合体であることを明らかにしている。本年度の本研究において、RasファミリーのRaplが、Rasと同様に、B-Rafを活性化することを明らかにし、Rap1がRasの機能に対して促進的に作用する機構が存在することを明らかにした。また、酵母のtwo hybrid法を用いて、私共がすでに見い出しているSmgGDSに結合する新しい蛋白質を見い出し、SMAP(SmgGDS-Associated protein)と命名した。SmgGDSとSMAPは、共にアルマジロ(Arm)配列を有し、他のArm配列を有する蛋白質の機能から、細胞接着や核輸送に機能している可能性がある。一方、私共を含めたいくつかのグループによって、Rhoがアクチン系を介して種々の細胞機能を制御していることが明らかにされている。本年度、私共は、RhoをADPリボシル化して不活性化するC3を用いて、Rhoが細胞外Ca^<2+>によって誘起されるadherens junctionとtight junctionの形成にも関与していることを明らかにした。さらに、Rhoは、アクチン細胞骨格系と細胞膜との結合を制御するERM-CD44系を介してアクチン細胞骨格系の再編成を制御していることを明らかにした。また、酵母をモデル系に用いて、酵母RHO1の標的蛋白質がPKC1(Cキナーゼホモログ)、BNI1(細胞質分裂関連蛋白質)、およびグルカン合成酵素であることを明らかにした。他方、Rab3Aは神経伝達物質の放出を制御しており、私共は、Rab3Aの標的蛋白質としてRabphilin3Aを見い出している。本研究において、私共は、Rabphilin-3Aに類似し、C2様ドメインを二つ持つ蛋白質を見い出してDoc2と命名した。さらに、Rabphilin-3AおよびDoc2とそれらの変異体の遺伝子やリコンビナント蛋白質を用いて、両蛋白質が神経伝達物質の放出を制御していることを明らかにした。以上の結果を得たことにより、本年度の研究計画はほぼ達成できたと考えている。

Research Products
(25 results)

All Other

All Publications (25 results)

[Publications] Kuroda,S.: "Purification and characterization of REKS from Xenopus eggs Identification of REKS as a Ras-dependent mitogen-activated protein kinase kinase kinase" J.Biol.Chem.270. 2460-2465 (1995)
[Publications] Bourgoin,S.: "Low molecular weight GTP-binding proteins in HL-60 granulocytes." J.Biol.Chem.270. 3172-3178 (1995)
[Publications] Yamamori,B.: "Purification of a Ras-dependent mitogen-activated protein kinase kinase kinase from bovine brain cytosol and its identification as a complex of B-raf and 14-3-3 proteins." J.Biol.Chem.270. 11723-11726 (1995)
[Publications] Chung,S-H.: "Evidence that the Rab3A-binding protein,Rabphilin-3A,enhances regulated secretion-studies in adrenal chromaffin cells." J.Biol.Chem.270. 16714-16718 (1995)
[Publications] Kuribara,H.: "Synergistic activation of rat brain phospholipase D by ADP-ribosylation factor and rhoA p21,and its inhibition by clostridium botulinum C3 exoenzyme." J.Biol.Chem.270. 25667-25671 (1995)
[Publications] McGeady,P.: "The farnesyl group of H-ras facilitates the activation of a soluble upstream activator of mitogen-activated protein kinase." J.Biol.Chem.270. 26347-26351 (1995)
[Publications] Gotoh,T.: "Identification of Rap1 as a target for the Crk SH3 Domain-binding guanine nucleotide-releasing factor C3G." Mol.Cell.Biol.15. 6746-6753 (1995)
[Publications] Nonaka,H.: "A downstream target of RHO1 small GTP-binding protein is PKC1,a homolog of protein kinase C, which leads to activation of the MAP kinase cascade in Saccharomyces cerevisiae." EMBO J.14. 5931-5938 (1995)
[Publications] Takaishi,k.: "Translocation of activated Rho from the cytoplasm to membrane ruffling area,cell-cell adhesion sites and cleavage furrows." Oncogene. 11. 39-48 (1995)
[Publications] Nishimura,N.: "Cloning of a brain-type isoform of human rab GDI and its expression inhuman neroblastoma cell lines and tumor specimens." Cancer Res.55. 5445-5450 (1995)
[Publications] Orita,S.: "Doc2:a novel brain protein having two repeated C2-like domains." Biochem.Biophys.Res.Commun.206. 439-448 (1995)
[Publications] Araki,K.: "Purification and characterization of Rab GDIβ from rat brain." Biochem.Biophys.Res.Commun.211. 296-305 (1995)
[Publications] Takahashi,K.: "Heterotetramer formation of prenylated Rab3A with two Rabphilin-3A molecules." Biochem.Biophys.Res.Commun.217. 979-986 (1995)
[Publications] Sakaguchi,G.: "Molecular cloning of an isoform of Doc2 having two C2-like domains." Biochem.Biophys.Res.Commun.217. 1053-1061 (1995)
[Publications] Miyazaki,M.: "Rabphilin-3A binds to a Mr 115,000 polypeptide in a phosphatidylserine-and Ca2+-dependent manner." Mo1.Brain.Res.28. 29-36 (1995)
[Publications] Regazzi,R.: "Prenylcysteine analogs mimicking the C-terminus of GTP-binding proteins stimulate exocytosis from permeabilized HIT-T15 cellsーcomparison with the effect of Rab3AL peptide." Biochim.Biophys.Acta. 1268. 269-278 (1995)
[Publications] Takai,Y.: "Rho as a regulator of the cytoskeleton." Trends in Biochemical sciences. 20. 227-231 (1995)
[Publications] Ohtsuka,T.: "Activation of brain B-Raf protein kinase by Rap1B small GTP-binding protein." J.Biol.Chem.271. 1258-1261 (1996)
[Publications] Orita,S.: "Doc2 enhances Ca2+-dependent exocytosis from PC12 cells." J.Biol.Chem.(in press).
[Publications] Fujita,Y.: "Phosphorylation of Munc-18/n-Sec1/rbSec1 by protein kinase cーIts implication in regulating the interaction of Munc-18/n-Sec1/rbSec1 with Syntaxin." J.Bio1.Chem.(in press).
[Publications] Drgonova,J.: "Rho1p,a yeast protein at the juncture between cell polarization and morphogenesis." Science. (in press).
[Publications] Ozaki,K.: "Rom1p and Rom2p are GDP/GTP exchange proteins(GEPs)for the Rho1p small GTP-binding protein in Saccharomyces cerevisiae." EMBO J.(in press).
[Publications] Matsuda,S.: "A membrane-associated GDP/GTP exchange protein specific for Rho small GTP-binding proteinーpartial purification and characterization from rat brain." Oncogene. (in press).
[Publications] Taniguchi,T.: "Cholecystokinin-B/gastrin receptors mediate rapid formation of actin stress fibers." Oncogene. (in press).
[Publications] Komuro,R.: "Involvement of rabphilin-3A in Ca2+-dependent exocytosis from PC12 cells." Biochem.Biophys.Res.Commun.(in press).

1995 Fiscal Year Annual Research Report

低分子量G蛋白質とその関連蛋白質の機能修飾物質の開発およびその有効利用

Principal Investigator

高井 義美 大阪大学, 医学部, 教授 (60093514)

Research Products

[Publications] Kuroda,S.: "Purification and characterization of REKS from Xenopus eggs Identification of REKS as a Ras-dependent mitogen-activated protein kinase kinase kinase" J.Biol.Chem.270. 2460-2465 (1995)

[Publications] Bourgoin,S.: "Low molecular weight GTP-binding proteins in HL-60 granulocytes." J.Biol.Chem.270. 3172-3178 (1995)

[Publications] Yamamori,B.: "Purification of a Ras-dependent mitogen-activated protein kinase kinase kinase from bovine brain cytosol and its identification as a complex of B-raf and 14-3-3 proteins." J.Biol.Chem.270. 11723-11726 (1995)

[Publications] Chung,S-H.: "Evidence that the Rab3A-binding protein,Rabphilin-3A,enhances regulated secretion-studies in adrenal chromaffin cells." J.Biol.Chem.270. 16714-16718 (1995)

[Publications] Kuribara,H.: "Synergistic activation of rat brain phospholipase D by ADP-ribosylation factor and rhoA p21,and its inhibition by clostridium botulinum C3 exoenzyme." J.Biol.Chem.270. 25667-25671 (1995)

[Publications] McGeady,P.: "The farnesyl group of H-ras facilitates the activation of a soluble upstream activator of mitogen-activated protein kinase." J.Biol.Chem.270. 26347-26351 (1995)

[Publications] Gotoh,T.: "Identification of Rap1 as a target for the Crk SH3 Domain-binding guanine nucleotide-releasing factor C3G." Mol.Cell.Biol.15. 6746-6753 (1995)

[Publications] Nonaka,H.: "A downstream target of RHO1 small GTP-binding protein is PKC1,a homolog of protein kinase C, which leads to activation of the MAP kinase cascade in Saccharomyces cerevisiae." EMBO J.14. 5931-5938 (1995)

[Publications] Takaishi,k.: "Translocation of activated Rho from the cytoplasm to membrane ruffling area,cell-cell adhesion sites and cleavage furrows." Oncogene. 11. 39-48 (1995)

[Publications] Nishimura,N.: "Cloning of a brain-type isoform of human rab GDI and its expression inhuman neroblastoma cell lines and tumor specimens." Cancer Res.55. 5445-5450 (1995)

[Publications] Orita,S.: "Doc2:a novel brain protein having two repeated C2-like domains." Biochem.Biophys.Res.Commun.206. 439-448 (1995)

[Publications] Araki,K.: "Purification and characterization of Rab GDIβ from rat brain." Biochem.Biophys.Res.Commun.211. 296-305 (1995)

[Publications] Takahashi,K.: "Heterotetramer formation of prenylated Rab3A with two Rabphilin-3A molecules." Biochem.Biophys.Res.Commun.217. 979-986 (1995)

[Publications] Sakaguchi,G.: "Molecular cloning of an isoform of Doc2 having two C2-like domains." Biochem.Biophys.Res.Commun.217. 1053-1061 (1995)

[Publications] Miyazaki,M.: "Rabphilin-3A binds to a Mr 115,000 polypeptide in a phosphatidylserine-and Ca2+-dependent manner." Mo1.Brain.Res.28. 29-36 (1995)

[Publications] Regazzi,R.: "Prenylcysteine analogs mimicking the C-terminus of GTP-binding proteins stimulate exocytosis from permeabilized HIT-T15 cellsーcomparison with the effect of Rab3AL peptide." Biochim.Biophys.Acta. 1268. 269-278 (1995)

[Publications] Takai,Y.: "Rho as a regulator of the cytoskeleton." Trends in Biochemical sciences. 20. 227-231 (1995)

[Publications] Ohtsuka,T.: "Activation of brain B-Raf protein kinase by Rap1B small GTP-binding protein." J.Biol.Chem.271. 1258-1261 (1996)

[Publications] Orita,S.: "Doc2 enhances Ca2+-dependent exocytosis from PC12 cells." J.Biol.Chem.(in press).

[Publications] Fujita,Y.: "Phosphorylation of Munc-18/n-Sec1/rbSec1 by protein kinase cーIts implication in regulating the interaction of Munc-18/n-Sec1/rbSec1 with Syntaxin." J.Bio1.Chem.(in press).

[Publications] Drgonova,J.: "Rho1p,a yeast protein at the juncture between cell polarization and morphogenesis." Science. (in press).

[Publications] Ozaki,K.: "Rom1p and Rom2p are GDP/GTP exchange proteins(GEPs)for the Rho1p small GTP-binding protein in Saccharomyces cerevisiae." EMBO J.(in press).

[Publications] Matsuda,S.: "A membrane-associated GDP/GTP exchange protein specific for Rho small GTP-binding proteinーpartial purification and characterization from rat brain." Oncogene. (in press).

[Publications] Taniguchi,T.: "Cholecystokinin-B/gastrin receptors mediate rapid formation of actin stress fibers." Oncogene. (in press).

[Publications] Komuro,R.: "Involvement of rabphilin-3A in Ca2+-dependent exocytosis from PC12 cells." Biochem.Biophys.Res.Commun.(in press).

高井義美大阪大学, 医学部, 教授 (60093514)