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

从再生大鼠肝脏cDNA文库中成功克隆并鉴定了一种新的II类磷酸肌苷（PI） 3-激酶。该酶分子由1505个氨基酸组成，包含一个C2结构域，对PtdIns和PtdIns 4-P和显示出有限的底物特异性，因此被称为PI3K-IIgamma。在过表达的体外细胞中，该分子定位于核旁高尔基区。mRNA的表达在整个发育过程中局限于肝脏，在部分肝切除术后肝脏再生过程中表达增加。磷酸（PA）磷酸酶的一个亚型Dri42的基因表达仅限于胚胎脑的心室生发区。PAP催化PA去磷酸化生成二酰基甘油（DG），这是DG激酶磷酸化的一个反向过程，我们已经进行了大量的分子鉴定。通过原位杂交组织化学也揭示了肌醇5-磷酸酶synaptojanin mrna在脑中的定位。Synaptojanin 1 mRNA在整个发育和成熟阶段几乎在所有大脑神经元中表达，而Synaptojanin 2 mRNA在出生后早期的神经元中首次被检测到，并且随着出生后发育的进行，在白质推定的少突胶质细胞中表达明显。这些关于去磷酸化的发现对理解脂质激酶磷酸化的意义具有指导意义。分析了参与pi连续代谢过程的CDP-DG合成酶和磷脂酰肌醇合成酶的基因表达定位，发现在发育和成熟的大脑中存在一些时空差异的表达模式。脑内磷脂酶D基因表达的原位杂交组织化学定位也在进行中。

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.