The regulation mechanism of lipid kinase in the signal transduction

首次从大鼠脑cDNA文库中克隆了一个编码462个氨基酸的CDP-二酰甘油合成酶(CDS)活性基因，作为脊椎动物的酶分子。推测该CDS的分子质量为53 kDa，推测的一级结构包括几个可能的跨膜区域。该酶以1-硬脂酰基-2-花生四烯基磷脂酸酯为底物，其活性被磷脂酰肌醇(PtdIns)4，5-二磷酸强烈抑制。该分子主要定位于与体外高表达细胞的内质网膜密切相关。CDS在成年大鼠小脑浦肯野细胞、松果体、光感受器细胞内段、有丝分裂后精母细胞和精子细胞中均有较强的表达。这一发现与花生四烯酰二甘油激酶(DGK)的发现非常相似，在这方面，我们与最先识别…的J.A.Glomset博士讨论了这一点进一步鉴定了DGK，并试图通过K.Gotok博士的免疫组织化学方法检测两个分子的共存情况。通过从人脑和视网膜cDNA文库中克隆出一个新的DGK，并与W.J.van Blitterswjk博士合作，将其命名为DGKtheta。DGKtheta由951个氨基酸组成，含有3个富含半胱氨酸的结构域，一个富含脯氨酸和甘氨酸的结构域，具有一个可能的SH3结构域，以及一个具有重叠的RAS结合结构域的Pleckstrin同源结构域。在成年大鼠脑内，小脑皮质和海马区均有高表达。从再生大鼠肝脏的cDNA文库中成功克隆并鉴定了一个新的II类肌醇磷脂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具有限制性底物专一性，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，该基因的表达局限于肝脏，在肝部分切除后的肝再生过程中，其表达增强。进一步的分子表征正在与L.C.坎特利博士的合作中。通过原位杂交组织化学方法，在舌下神经切断后2-7天，受影响的舌下核Akt的表达显著增加。在PI3激酶和皮肤型脂肪酸结合蛋白(FABP)的时间进程中也观察到了这种基因表达的增加，这表明PI信号与FABP之间存在功能关系。因此，S-FABP的基因敲除正在与F·斯皮纳博士合作进行。磷脂酶D在大脑中的基因表达定位也正在与Y·Nozawa博士合作进行。与D.M.Hunt博士合作，尝试绘制PtdIns4-Kinase基因组的染色体图谱，并开始进行。然而，在实验过程中，我们不幸地发现，它已经被另一个外国研究小组所做，因此放弃了这一尝试。较少

A cDNA encoded a 462-amino acid protein, which showed CDP-diacylglycerol synthase (CDS) activity was cloned for the first time as the vertebrate enzyme molecule from rat brain cDNA library. The deduced molecular mass of this CDS was 53 kDa, and putative primary structure included several possible membrane-spanning regions. This enzyme molecule preferred 1-stearoyl- 2-arachidonoyl phosphatidate as a substrate and its activity was strongly inhibited by phosphatidylinositol (PtdIns) 4,5-bisphosphate. The molecule was mainly localized in close association with the membrane of the endoplasmic reticulum of over-expressed in vitro cells. The intense mRNA expression of CDS was localized in the cerebellar Purkinje cells, pineal body, and the inner segment of photoreceptor cells, and postmitotic spermatocytes and spermatids of testis of mature rats. This finding is closely similar to that of arachidonoyl-diacylglycerol kinase (DGK) and in this regard we discussed with Dr.J.A.Glomset who first id … More entified the DGK and attempted to examine the colocalization of the two molecules by immunohistochemistry by Drs.K.Goto.A novel DGK was identified by cDNA cloning from human brain and retina cDNA library and termed DGKtheta in co-operation with Dr.W.J.van Blitterswijk. DGKtheta was composed of 951 amino acids and contained three cysteine-rich domain, a proline- and glycine-rich domain with a putative SH3 domain-binding site and a pleckstrin homology domain with an overlapping Ras-associating domain. In adult rat brain high expression was detected in the cerebellar cortex and hippocampus.The cloning and characterization of a novel class II phosphoinositide 3-kinase was succeeded from the cDNA library of regenerating rat liver. This enzyme molecule composed of1505 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. Further molecular characterization is under way in co-operation with Dr.L.C.Cantley.By in situ hybridization histochemistry a dramatic increase in expression of Akt was detected in affected hypoglossal nucleus after 2-7 days following axotomy of the hypoglossal nerve. Such an increase in the gene expression after axotomy was observed in a similar time course for PI3 kinase and skin type-fatty acid binding protein (FABP), suggesting a functional relation between PI-signal and FABP.Thus the gene knockout for s-FABP is under way in co-operation with Dr.F.Spener.The gene expression localization of phospholipase D in the brain is also under way in cooperation with Dr.Y.Nozawa. The chromosome mapping of the genome for PtdIns 4-kinase was attempted in cooperation with Dr.D.M.Hunt and started. However, in the course of experiment we unfortunately found that it has already done by another foreign research group and thus gave up this attempt. Less