リン脂質代謝関連蛋白及びSH2/SH3領域による細胞増殖制御

• Project/Area Number: 06283202
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas (A)
• Allocation Type: Single-year Grants
• Research Institution: The University of Tokyo
• Principal Investigator: 竹縄 忠臣 東京大学, 医科学研究所, 教授 (40101315)
• Co-Investigator(Kenkyū-buntansha): 三木 裕明 東京大学, 医科学研究所, 助手 (80302602) ; 深見 希代子 東京大学, 医科学研究所, 講師 (40181242)
• Project Period (FY): 1999
• Project Status: Completed (Fiscal Year 1999)
• Budget Amount: ¥80,000,000 (Direct Cost: ¥80,000,000); Fiscal Year 1999: ¥10,000,000 (Direct Cost: ¥10,000,000); Fiscal Year 1998: ¥10,000,000 (Direct Cost: ¥10,000,000); Fiscal Year 1997: ¥10,000,000 (Direct Cost: ¥10,000,000); Fiscal Year 1996: ¥18,000,000 (Direct Cost: ¥18,000,000); Fiscal Year 1995: ¥16,000,000 (Direct Cost: ¥16,000,000); Fiscal Year 1994: ¥16,000,000 (Direct Cost: ¥16,000,000)
• Keywords: アクチン / N-WASP / Arp2 / 3 / 細胞運動 / 膜ラッフリング / プロフィリン / Rac / WAVE / フィロポジア / Cdc42 / チロシンキナーゼ / Ash / Grb-2 / SH_2 / SH_3 / Ras / SH2 / SH3

Research Abstract

N-waspは静止期にはAsh/Grb2などのアダプター蛋白質と複合体を形成して存在する。細胞が活性化され、チロシンキナーゼが活性化されると、チロシンリン酸化部位にリクルートされ膜近辺でCdc42とPlP_2がN-WASPに結合する。するとN-WASP分子が開裂して、VCA領域が露出する。その結果、ARP2/3複合体がCA領域に結合してアクチン重合を促進し、フィロポジア形成に至る、と考えられた。N-WASPのverprolin、confilin様ドメインは直接アクチンフィラメントと結合し、その部位の欠失機能を失ってしまうので、重要な役割を果たしていると考えられた。そこで、verprolin、confilin様ドメインを持つ新しい蛋白質を検索した。すでにデーターベース上に機能は分からないが、verprolin、confilin様ドメインを持つ蛋白質の記載があった。この蛋白質を解析したところ、膜ラッフリング形成にかかわっている蛋白質であったので、WAVEと名付けた。WAVEをRacとともに発現させると膜ラッフルの形成が見られ、WAVEはラッフリング部位に局在した。しかしCdc42との共発現においてはフィロポジア形成は生じなかった。WAVEのdoinant negative体を発現した細胞をPDGFで処理すると、通常見られる膜ラッフリングの抑制をおこした。WAVEはRacとは直接結合しなかったが、両者を発現させた細胞において、WAVEはRacと複合体を形成した。これらの結果からWAVEはRacの下流にあって、膜ラッフリング形成に関与する蛋白質であると結論した.

Research Products

• [Publications] Nagano, K.: "A Novel Phospholipase C84 spling Isoform as a negative regulator of PLC"J. Biol. Chem.. 274. 2874-2879 (1999)
• [Publications] Rohatgi. R.: "The Reaction between N-wasp and the Arp2/3 complexed links Cdc42 dependent signals location assembly"Cell. 97. 221-231 (1999)
• [Publications] Takenawa, T.: "Regulation of phosphatidylinositol 4,5-biphosphat levels and its roles in cyroskeletat re-organization and malignant transformation"Chem. Phys. Lipids. 98. 13-22 (1999)
• [Publications] Mochizuki, Y.: "Novel Inositol polyphosphate 5-phosphatase Localize at Membrance Ruffles"J. Biol. Chem.. 274. 36790-36795 (1999)
• [Publications] Miki, H.: "PH domains in WSP-a bug in the system? Reply"Trends Cell Biol.. 9. 212 (1999)
• [Publications] Fukami, K.: "Growth factor-induced promoter activation of murine phospholipase C delta 4 gene"Eur J Biochem. 267. 28-36 (2000)
• [Publications] Miki,H.,et al.: "Induction of filopodium formation by a WASP related actin depolymeriziing protein N-WASP" Nature. 391. 93-97 (1998)
• [Publications] Yu,H.,et al.: "Phosphatidylinositol 4,5-bisphosphate reverses the inhibition of RNA transcription causd by histon H1." Eur.J.Biochem.251. 281-287 (1998)
• [Publications] Itoh,T.,et al.: "Identification of a novel phosphatidylinositol 4-phosphate 5-kinsae ; evidence for its phosphorylation and transcation in response to mitogenic signals" J.Biol.Chem.273. 20292-20299 (1998)
• [Publications] Suetsugu,S.,et al.: "The essential role of profilin in the assembly of actin for microspike formation" EMBO J.17. 6516-6526 (1998)
• [Publications] Miki,H.,et al.: "WAVE,a novel WASP-family protein involved in actin-polymerization induced by Rac." EMBO J.17. 6932-6941 (1998)
• [Publications] Hiroyama,M.,et al.: "Purification and characterization of a lysophosphatidic acid-specific phosphatase" Biochem.J.336. 483-489 (1998)
• [Publications] Miki,H., et al.: "A tyrosine kinase signalling regulates WASP function,which is essential for megakaryocyte differentiation." Cell Grwoth & Differ.8. 195-202 (1997)
• [Publications] Houssa,B., et al.: "Cloniong of a novel human diacylglycerol kinase containing three cystein-rich domains,a proline-rich region and a pleckstrin homology domain with overlapping ras-associating domain." J.Biol.Chem.272. 10422-10428 (1997)
• [Publications] Sakisaka,T., et al.: "Phosphatidylinositol 4,5-bisphosphate phosphatase regulates the rearrangement of actin filaments." Mol.Cell Biol.17. 6246-6253 (1997)
• [Publications] Matuoka,K., et al.: "Phosphatidylinositol 4,5-bisphosphate phosphatase regulates the rearrangement of actin filaments." Biochem.Biophys.Res.Commun.239. 488-492 (1997)
• [Publications] Fukuoka,M., et al.: "dentification of N-WASP homologs in human and rat brain." Gene. 196. 43-48 (1997)
• [Publications] Miki,H., et al.: "N-WASP,not WASP,induces filopodium formation downstream of Cdc42." Nature. 391. 93-96 (1998)
• [Publications] K.Fukami,et al: "Identification of a PIP2 binding site in chicken skeletal muscle α-actinin" J.Biol.Chem.271. 2646-2650 (1996)
• [Publications] S.Moriya,et al: "PDGF activates protein kinase C ε through redundant and indedundant signaling pathway involving phospholipase C γ or phosphatidylinositol 3-kinase" Proc.Natl.Acad.Sci.USA. 93. 151-155 (1996)
• [Publications] K.Miura,et al: "Interaction of Ash/Grb2 via its SH3 domains with neuron-specific p150 and p65" Biochem.J.316. 639-645 (1996)
• [Publications] M.Matsuda,et al: "Interaction between the amino-terminal SH3 domain of Crk and its natural target proteins" J.Biol.Chem.271. 14468-14472 (1996)
• [Publications] H.Miki,et al: "A novel actin-depolymerizing protein N-WASP regulates thecor-tical cytoskeletal rearrangement in a PIP2 dependent manner at the downstream of tyrosine kinase" EMBO J.15. 5326-5335 (1996)
• [Publications] T.Ito,et al: "β-Tubulin binds Src homorogy 2 domains through a region different from the tyrosinephosphorylated protein-recognizing site" J.Biol.Chem.271. 27931-27935 (1996)
• [Publications] N.Liu,et al.: "A new phospholipase Cδ4 is induced at S-phase of cell cycle and appears in the uncleus" J.Biol.Chem.271. 2646-2650 (1996)
• [Publications] K.Watanabe,et al.: "Splicing isoforms of rat Ash/Grb2:Isolation and characterization of the cDNA and genomic DNA Clones and implications for the physiological roles of the isoforms" J.Biol.Chem.270. 13733-13739 (1995)
• [Publications] H.Hosoya,et al.: "Developmental expression of the neural adhesion molecule F3 in the rat brain" Neuroscience Lett.186. 83-86 (1995)
• [Publications] F.Nakamura,et al.: "Characterization of Gq family G proteins.Gl1a(G14a)Gl2a(Gl1a),and Gqa expressed in the baculovirus-insect cell system" J.Biol.Chem.270. 6246-6253 (1995)
• [Publications] J.C.Hay,et al.: "ATP-dependent inositide phosphorylation required for Ca^<2+>-activated secretion" Nature. 374. 173-177 (1995)
• [Publications] S.Shimihama,et al.: "Characterization of the association of phospholipase C-δ with Alzheimer neurofibrillary tangles" Brain Research. 669. 217-224 (1995)
• [Publications] A.R.Saltiel,他: "Growth factors differentially stimulate the phosphorylation of Shc proteins and their association with Grb2 in PC 12 phenochromocytoma cells." J.Biol.Chem.269. 1143-1148 (1994)
• [Publications] T.Takenawa,他: "d-Actinin and vinculin are PIP2-binding proteins involved in signaling by tyrosine kinase." J.Biol.Chem.269. 1518-1522 (1994)
• [Publications] T.Takenawa,他: "Association of ash/grb-2 with dynamin through the src homology 3 domain." J.Biol.Chem.269. 5489-5492 (1994)
• [Publications] T.Takenawa,他: "Phosphatidylinositol 3-kinase binds to α-actinin through the p85 subunit" Biochem.J.302. 551-557 (1994)