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Identification and functional analysis of proteins involved in novel pathway for PLC activation.

PLC 激活新途径中涉及的蛋白质的鉴定和功能分析。

基本信息

批准号：
08458184
负责人：
HOMMA Yoshimi
金额：
$ 4.16万
依托单位：
Fukushima Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

We recently cloned a novel signaling molecule, p122, that shows GAP activity specific for Rho and the ability to enhance the PIP_<> hydrolyzing activity of PLCdelta1 in vitro. Here we identified molecules interacting with p122 and analyzed the in vivo function of p122. Following results were obtained from this research project. 1) p122 interacted with a number of intracellular signaling molecules such as vinculin and p55.2) Microinjection of the GAP domain of p122 suppressed the formation of stress fibers and focal adhesions induced by lysophosphatidic acid. 3) Transfection of p122 also induced the morphological rounding of various adherent cells, followed by detachment from the substrate within 24 h. No stress fibers or focal adhesions were observed in the course of these changes. 4) The morphological changes caused by p122 were inhibited by coexpression of V14-RhoA, which is defective in an intrinsic GTPase activity. Analyses using deletion and point mutants demonstrated that the GAP domain of p122 is responsible for the morphological changes and detachment, and that arginine residues at positions 668 and 710 and a lysine residue at position 706 in the GAP domain of p122, all conserved among several Rho GAPs, are essential for stimulating the GTPase activity of Rho. 5) Using the Ca^<2+>-sensitive dye fluo-3, we found that microinjection of p122 evoked a rapid elevation of intracellular Ca^<2+> levels, suggesting that p122 stimulates the PIP_2 hydrolyzing activity of PLCdelta1 in vivo.

我们最近克隆了一个新的信号分子p122，它显示出对Rho特异的GAP活性和在体外增强PLC δ 1的PIP_<>水解活性的能力。在这里，我们确定了与p122相互作用的分子，并分析了p122的体内功能。本研究项目取得了以下成果。1)p122与黏着斑蛋白和p55等细胞内信号分子相互作用。2）显微注射p122的差距结构域可抑制溶血磷脂酸诱导的应力纤维和粘着斑的形成。3)转染p122还诱导各种粘附细胞的形态变圆，随后在24 h内从基底上脱离。在这些变化过程中未观察到应力纤维或局灶性粘连。4)由p122引起的形态学变化被V14-RhoA的共表达抑制，V14-RhoA在固有的GT3活性中是有缺陷的。使用缺失和点突变体的分析表明，差距结构域的p122是负责的形态学变化和脱离，并在位置668和710的精氨酸残基和位置706的赖氨酸残基在差距域的p122，所有保守的几个Rho GAP，是必不可少的刺激GTdR活性的Rho。5)利用Ca^2+敏感染料fluo-3，我们发现微量注射p122引起细胞内Ca^2+水平迅速升高，这表明p122在体内刺激PLC δ 1的PIP_2水解活性。