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REGULATION AND INTRACELLULAR LOCALISATION OF PHOSPHOIN OSITIDE-BINDING PROTEINS.

磷酸肌苷结合蛋白的调节和细胞内定位。

基本信息

批准号：
10490023
负责人：
YAGISAWA Hitoshi
金额：
$ 8.26万
依托单位：
Himeji Institute of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2001
项目状态：
已结题

项目摘要

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.

磷脂酰肌醇结合蛋白的配体识别，细胞内定位和生理重要性已被研究。已发现普列克底物蛋白同源（PH）结构域中的某些碱性氨基酸对于特异性配体识别是重要的，并且因此负责含PH蛋白与质膜的相互作用。尤其是磷脂酶C-δ1（PLCδ1）。我们证明，渗透胁迫导致表达GFP标记的PLCδ1或其PH结构域的肾上皮细胞的质膜上的荧光快速解离。当细胞膜上的PtdIns（4，5）P2被破坏时，PtdIns（4，5）P2的解离速率增加，提示PtdIns（4，5）P2的水解活性受细胞膜结构的调节。释放的GFP-PLCδ1易位到核周区域，推测为ER，但靶向机制可能不同于PLA 2（和PKC），其C2结构域可能起作用。虽然在正常条件下细胞核中几乎没有PLC δ 1，但PLCδ1确实在细胞质（加上质膜）和细胞核之间穿梭。我们发现PLCδ1具有一个功能性的核输出信号序列，破坏它或抑制crm 1/exportin依赖的核输出可以使PLC δ 1留在核内。这一发现将提供进一步的见解核磷酸肌醇信号，因为新的作用肌醇多磷酸和他们的激酶已被假定。然后，我们探索PtdIns（4，5）P2/PLC在细胞运动中的作用，使用自由生活的阿米巴作为模型。当用PLCδ1-PH或PLCδ1处理阿米巴细胞膜时，肌动蛋白丝的网状结构逐渐消失。此外，PLC的特异性抑制剂可逆地阻断了伪足的形成和体内细胞的运动，这表明细胞骨架的重塑是由水解PtdIns（4，5）P2的Ca^<2+>敏感PLC调节的。