Molecular and Cell Biological Analysis of Lipid Kinases in Relation to Signaling and Vesicle Traffic.

从大鼠再生肝的cDNA文库中克隆并鉴定了一个新的II类磷脂酰肌醇(PI)3-激酶。这种由1505个氨基酸组成的酶分子含有一个C2结构域，对PtdIns和PtdIns 4-P表现出限制性底物专一性，因此被称为PI3K-IIGamma。在体外过表达的细胞中，该分子定位于核旁高尔基体区域。在整个发育过程中，mRNA的表达局限于肝脏，在部分肝切除后的肝脏再生过程中，其表达增加。磷脂酸(PA)磷酸酶亚型Dri42的基因表达仅限于胚脑的脑室生发区。PAP催化PA去磷酸化生成二酰甘油(DG)，这是DG激酶磷酸化的一个反向过程，我们对其进行了深入的分子鉴定。原位杂交组织化学也揭示了突触素(肌醇5-磷酸酶)在脑内的定位。Synaptojanin 1mRNA在几乎所有发育和成熟的脑神经元中都有表达，而Synaptojanin 2mRNA最早在出生后早期的神经元中检测到，随着出生后发育的进行，在白质假定的少突胶质细胞中表达明显。分析了参与磷脂酰肌醇代谢两个过程的CDP-DG合成酶和磷脂酰肌醇合成酶的基因表达定位，发现发育和成熟脑中CDP-DG合成酶和磷脂酰肌醇合成酶的表达在时空上存在差异。磷脂酶D在脑内的基因表达定位也在用原位杂交组织化学方法进行。

The cloning and characterization of a novel class II phosphoinositide (PI) 3-kinase was succeeded from the cDNA library of regenerating rat liver. This enzyme molecule composed of 1505 amino acids contained a C2 domain and displayed a restricted substrate specificity for PtdIns and PtdIns 4-P and, thus termed PI3K-IIgamma. This molecule was localized in the juxtanuclear Golgi region in the over-expressed in vitro cells. The mRNA expression was confined to the liver throughout the development and its expression increased during liver regeneration after partial hepatectomy.The gene expression for a subtype of phosphatidate (PA) phosphatase termed Dri42 was confined to the ventricular germinal zone of embryonic brain. PAP catalyses a dephosphorylation of PA to produce diacylglycerol (DG), an reverse process of phosphorylation by DG kinase whose molecular identification has been intensively performed by us. The localization of mRNAs for synaptojanin, inositol 5-phosphatase, in the brain was also revelaed by in situ hybridization histochemistry. Synaptojanin 1 mRNA was expressed in almost all neurons of the brain throughout developing and mature stages while synaptojanin 2 mRNA was first detected in neurons on early postnatal stages and the expression was evident in the white matters presumptive oligodendrocytes as the postnatal development proceeded. All these findings on dephosphorylation were instructive for understanding the significance of phosphorylation by lipid kinases.The gene expression localization of CDP-DG synthase and phosphatidylinositol synthase, both of which are involved in the two sequential PI-metabolic processes, was analyzed and some discrepant expression patterns were found spatio-temporally in developing and mature brain. The gene expression localization of phospholipase D in the brain is also under way by in situ hybridization histochemistry.