REGULATION OF PHOSPHOLIPID SYNTHESIS

描述(申请人提供)：在酿酒酵母中，膜磷脂是通过互补的(CDP-二酰甘油和肯尼迪)途径合成的，这些途径中的基因和酶受到遗传和生化机制的调节。本申请中提出的工作的主要假设是磷脂的合成受磷酸化和mRNA稳定性的调节。我们将重点介绍胆碱激酶和转录因子Opi1p的磷酸化。胆碱激酶通过肯尼迪途径的CDP-胆碱分支催化磷脂酰胆碱合成的关键步骤。不受调控的胆碱激酶活性水平是人类各种癌症的共同特征，这一事实强调了对其调控的了解。Opi1p是一个负转录因子，它控制着几个磷脂生物合成基因的表达，以响应肌醇的补充。胆碱激酶被蛋白激酶A磷酸化，我们将验证该酶也被蛋白激酶C磷酸化的假设，蛋白激酶C磷酸化缺陷的突变将被用来检查后果。 蛋白激酶C的磷酸化对胆碱激酶活性的影响，以及对磷脂酰胆碱合成的调节。蛋白激酶A和蛋白激酶C位点的分级磷酸化将被检测。Opi1p被蛋白激酶A和C磷酸化，我们将验证Opi1p也被酪蛋白激酶II磷酸化的假设。我们将构建磷酸化缺陷的突变体，并用来检测酪蛋白激酶II磷酸化对Opi1p阻遏功能的作用。蛋白激酶A和C的靶点突变将包括在这一分析中。本课程将检测蛋白激酶A、蛋白激酶C和酪蛋白激酶II的分级磷酸化。当肯尼迪通路被阻断时，CDP-双甘油通路被激活的一个机制就是信使核糖核酸稳定。利用磷脂代谢缺陷的突变体，我们将检验这样一种假设，即通过mRNA稳定性调节CDP-二酰甘油途径的信号是肯尼迪途径的最终产物、肯尼迪途径的水溶性中间体或通过肯尼迪途径合成的磷脂周转产生的分子。CHO1mRNA(CDP-二酰甘油途径转录本)的降解机制将被研究。

DESCRIPTION (provided by applicant): In the yeast Saccharomyces cerevisiae, membrane phospholipids are synthesized by complementary (CDP-diacylglycerol and Kennedy) pathways, and the genes and enzymes in these pathways are regulated by genetic and biochemical mechanisms. The major hypotheses of the work proposed in this application are that phospholipid synthesis is regulated by phosphorylation and by mRNA stability. We will focus on the phosphorylation of choline kinase and the transcription factor Opi1p. Choline kinase catalyzes the committed step in phosphatdylcholine synthesis via the CDP-choline branch of the Kennedy pathway. Understanding its regulation is emphasized by the fact that unregulated levels of choline kinase activity is a common property of various cancers in humans. Opi1p is a negative transcription factor that controls expression of several phospholipid biosynthetic genes in response to inositol supplementation. Choline kinase is phosphorylated by protein kinase A, and we will examine the hypothesis that the enzyme is also phosphorylated by protein kinase C. Mutants defective in protein kinase C phosphorylation will be used to examine the consequences of protein kinase C phosphorylation on choline kinase activity, and on the regulation of phosphatidylcholine synthesis. Hierarchical phosphorylation of the protein kinase A and protein kinase C sites will be examined. Opi1p is phosphorylated by protein kinases A and C, and we will examine the hypothesis that Opi1p is also phosphoylated by casein kinase II. Mutants defective in phosphorylation will be constructed and used to examine the role of casein kinase II phosphoylation on Opi1p repressor function. Target site mutants for protein kinases A and C will be included in this analysis. Hierarchical phosphorylation of the protein kinase A, protein kinase C, and casein kinase II will be examined. mRNA stability is a mechanism by which the CDP-diaclyglycerol pathway is activated when the Kennedy pathway is blocked. Using mutants defective in phospholipid metabolism, we will examine the hypothesis that the signal for regulation of the CDP-diacylglycerol pathway by mRNA stability is a Kennedy pathway end product, a water-soluble intermediate of the Kennedy pathway, or a molecule derived from the turnover of a phospholipid synthesized via the Kennedy pathway. The mechanism for CHO1 mRNA (a CDP-diacylgylcerol pathway transcript) degradation will be examined.