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

从大鼠再生肝cDNA文库中成功克隆了一个新的II类磷脂酰肌醇（PI）3-激酶。该酶分子由1505个氨基酸组成，含有C2结构域，并显示出对PtdIns和PtdIns 4-P的限制性底物特异性，因此称为PI 3 K-II γ。该分子在体外过表达的细胞中定位于非核高尔基体区域。在整个发育过程中，mRNA表达局限于肝脏，在肝部分切除后的肝再生过程中表达增强，磷脂酸（PA）磷酸酶的一个亚型Dri 42的基因表达局限于胚脑的脑室生发区。PAP催化PA去磷酸化产生甘油二酯（DG），这是DG激酶磷酸化的逆过程，我们已经对其进行了大量的分子鉴定。用原位杂交组织化学方法观察了突触连蛋白（synaptojanin）、肌醇5-磷酸酶（inositol 5-phosphatase）mRNA在脑内的分布。Synaptojanin 1 mRNA在整个发育和成熟阶段的大脑中几乎所有的神经元中表达，而Synaptojanin 2 mRNA首先在出生后早期的神经元中检测到，并且随着出生后发育的进行，在白色物质推定的少突胶质细胞中的表达是明显的。这些发现对于理解脂质激酶磷酸化的意义具有指导意义。我们分析了CDP-DG合成酶和磷脂酰肌醇合成酶在发育和成熟脑中的基因表达定位，发现了一些在时空上不同的表达模式。磷脂酶D在脑中的基因表达定位也正在进行原位杂交组织化学。

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.