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

我们最近克隆了一个新的信号分子p122，它显示出对Rho特异性的GAP活性，并且能够在体外增强PLCdelta1的PIP_<>水解活性。本研究鉴定了与p122相互作用的分子，并分析了p122在体内的功能。本研究项目得出以下结果：1) p122与多种细胞内信号分子如vinculin和p55.2相互作用)微量注射p122的GAP结构域可抑制溶血磷脂酸诱导的应力纤维的形成和局灶粘连。3)转染p122后，各贴壁细胞形态呈圆形，24 h内脱离底物，在此过程中未观察到应力纤维和局灶性粘连。4) p122引起的形态学改变被GTPase活性缺陷的V14-RhoA的共表达所抑制。使用删除和点突变分析表明p122负责领域的差距形态变化和超然,668年和710年,精氨酸残基位置和赖氨酸残留在706 p122领域的空白位置,所有保存在几个ρ差距,对于刺激ρ的GTPase活性至关重要。5)使用Ca ^ < 2 + >敏感染料fluo-3,我们发现显微镜下注射p122诱发细胞内钙快速海拔^ < 2 + >水平,提示p122在体内刺激PLCdelta1的PIP_2水解活性。

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.