| Project/Area Number |
10490023
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| Research Category |
Grant-in-Aid for Scientific Research (B).
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
広領域
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| Research Institution | Himeji Institute of Technology |
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Principal Investigator |
YAGISAWA Hitoshi Himeji Institute of Technology, Department of Life Science, Associate Professor, 理学部, 助教授 (40192380)
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| Co-Investigator(Kenkyū-buntansha) |
HIRATA Masato Kyushu University, Graduate School of Dental Science, Molecular & Cellular Biochemistry, Professor, 大学院・歯学研究院, 教授 (60136471)
KAMATA Hideaki Himeji Institute of Technology, Department of Life Science, Research Assistant, 理学部, 助手 (10233925)
HIRATA Hajime Himeji Institute of Technology, Department of Life Science, Professor, 理学部, 教授 (40049052)
HIGUCHI Yoshiki Kyoto University, Graduate School of Science, Department of Chemistry, Associate Professor, 大学院・理学系, 助教授 (90183574)
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| Project Period (FY) |
1998 – 2001
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥12,900,000 (Direct Cost: ¥12,900,000)
Fiscal Year 2000: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1999: ¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 1998: ¥7,000,000 (Direct Cost: ¥7,000,000)
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| Keywords | phospholipase C / nuclear export / nuclear import / osmotic stress / ameba movement / cytoskeleton / phophoinositide / pleckstrin homology(PH) / PHドメイン / 核移行シグナル(NLS) / 核外移行シグナル(NES) / 緑色蛍光タンパク(GFP) / 細胞運動 / プレクストリン相同性領域 / 浸透圧ショック / 核外移行シグナル / トランスロケーション / 脂質・タンパク相互作用 / プレクストリン相同性領域(ph領域 / 細胞内局在 / 浸透圧 / 緑色けい光タンパク(GFP) |
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| Research Abstract |
Ligand recognition, intracellular localization and physiological importance of phosphoinositide-binding proteins have been examined. It was found that certain basic amino acids in the pleckstrin homology (PH) domain are important for specific ligand recognition, and therefore, responsible for interaction of PH-containing proteins with the plasma membrane. This was especially the case for phospholipase C-δ1 (PLCδ1). We demonstrated that osmotic stress caused a rapid dissociation of the fluorescence from the plasma membrane of renal epithelial cells expressing GFP-tagged PLCδ1 or its PH domain. The dissociation rate increased when cells were treated with cytoskeleton-disrupting reagents, suggesting the PtdIns(4,5)P2 hydrolysing actuvity is regulated by juxtamembrane structures. Released GFP-PLCδ1 translocated to perinuclear regions, presumably ER, but the mechanism of targeting may be different from those of PLA2 (and PKC) whose C2 domain may play a role. Although little is present in the nucleus under normal conditions, PLCδ1 does shuttle between the cytosol (plus the plasma membrane) and the nucleus. We have shown that PLCδ1 has a functional nuclear export signal sequence, and disruption of it or inhibition of the crm1/exportin-dependent nuclear exp ort could let the enzyme stay in the nucleus. This finding would provide further insights into the nuclear phosphoinositide signalling, since new roles of inositol polyphosphates and their kinases have been postulated. We then explored roles of PtdIns(4,5)P2/PLC in cell movements using a free living amoeba as a model. When membrane sheets prepared from amoebae were treated with PLCδ1-PH or PLCδ1, a mesh work structure of actin filaments gradually disappeared. Furthermore, a specific inhibitor of PLC reversibly blocked the pseudopod formation and the cell movement in vivo, suggesting that remodelling of cytoskeleton is regulated by Ca^<2+>-sensitive PLC that hy droly ses PtdIns(4,5)P2.
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