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

我们最近克隆了一种新的信号分子P122，它具有Rho特异性的GAP活性，并在体外能够增强PLCdelta1的PIP_&gt；水解性。在这里，我们确定了与P122相互作用的分子，并分析了P122在体内的功能。本研究项目取得了以下成果。1)P122与纽蛋白、p552等细胞内信号分子相互作用。5)微量注射P122的间隙结构域可抑制溶血磷脂酸诱导的应力性纤维形成和灶性粘连。3)转染P122后，细胞形态明显变圆，24 h内即从基质上脱落，未见应力纤维或局灶性粘连。4)内源性GTP酶活性缺陷的V14-RhoA共表达抑制了P122引起的细胞形态改变。利用缺失和点突变的分析表明，P122的GAP结构域是导致形态变化和脱落的原因，而P122的GAP结构域中668和710位的精氨酸残基以及706位的赖氨酸残基都是在几个Rho间隙中保守的，对于刺激Rho的GTP酶活性是必不可少的。5)利用钙敏感染料Fluo-3，我们发现微量注射P122可引起细胞内Ca~(2+)和Gt~(2+)水平的快速升高，提示P122在体内刺激了PLCdelta1的PIP_2水解酶活性。

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.