CLONING / CHARACTERIZATION OF NOVEL DIACYLGLYCEROL KINAS

甘油二酯酶(DGK)催化甘油二酯的转化 (DG)为磷脂酸。DG是一个关键的第二信使，它调节 蛋白激酶C(PKC)活性。PKC信号通路是 对细胞生长和分化功能至关重要。因此，一个 DGK的功能是衰减DG信号，从而衰减PKC 活动。我们已经克隆了一个与已知DGKs同源的独特的cDNAs (P40)。表达的部分基因具有DGK催化活性。建议 研究的目的是分离剩余的编码区并使用 用p40突变构建物研究p40的结构和功能 域结构和组织。在其他研究中，我们描述了 激动剂促进GH4C1大鼠DG生成和PKC同工酶活性 垂体细胞和正常和转化的REF52成纤维细胞。我们将使用 这些特性良好的细胞系来确定p140/DGK的作用 激动剂刺激的DG产生活性、PKC同工酶激活、PKC 底物磷酸化和细胞反应。这些研究将开始 明确p140在调节各种DG池中的作用。通过关联 对DG池的影响以及对PKC的影响，这些研究也将有所帮助 阐明细胞的各种来源和池之间的关系 与细胞生长、基因表达相关的DGS和PKC信号事件 和转型。

Diacylglycerol kinase (DGK) catalyzes the conversion of diacylglycerol (DG) to phosphatidic acid. DG is a key second messenger that regulates the activity of protein kinase C (PKC). PKC signaling pathways are of central importance for cell growth and differentiated function. Thus, one function of DGK is to attenuate DG signals, and consequently, PKC activity. We have cloned a unique cDNA with homology to known DGKs (pl40). The expressed partial cDNA has DGK catalytic activity. Proposed studies are designed to isolate the remaining coding region and to use structure-function studies with pl40 mutant constructs to study pl40 domain structure and organization. In other studies we have characterized agonist stimulated DG production and PKC isozyme activation in GH4C1 rat pituitary cells and normal and transformed REF52 fibroblasts. We will use these well-characterized cell lines to determine the effects of p140/DGK activity on agonist-stimulated DG production, PKC isozyme activation, PKC substrate phosphorylation and cellular responses. These studies will begin to define the role of p140 in regulating various DG pools. By correlating effects on DG pools with effects on PKCs, these studies will also help elucidate the relationship between various sources and pools of cellular DGs and PKC signaling events associated with cell growth, gene expression and transformation.