REGULATION OF CTP--PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE

卵磷脂是真核细胞中的主要磷脂 膜。磷脂酰胆碱的生物合成部分受 CTP：磷酸胆碱胞苷转移酶(CT)。这种酶长期以来一直是 已知在哺乳动物细胞中以两种形式存在：一种可溶性的，通常 非活性形式和与膜相关的活性形式。细胞的处理 以各种方式刺激磷脂酰胆碱的生物合成导致 可溶性CT转移到膜上，伴随着激活 酶的活性。近年来，我们发现CT是一种磷酸酶 当它被激活并与之关联时，它会被去磷酸化 带着膜。此外，我们已经证明了非活动形式是 核膜的活性形式与核膜有关。 这项赠款提案的目的是研究通过什么机制 CT活动和位置受到控制。问哪一种磷酸化 部位参与决定CT的活性和位置，动力学 各个位点的磷酸化和去磷酸化将是 量过了。以确定个体磷酸化的功能 CT的位置，这些位置会被突变和突变的CT改变 分子将在CT缺陷的CHO细胞中表达和鉴定 排队。鉴定和鉴定调节CT的酶。 细胞，核抽提物将被检测出 作用于CT作为底物。这些酶活性将部分地 纯化和表征，以及它们的性质与那些 已知的激酶和磷酸酶。CT中的明确目标信号将是 通过对CT进行一系列删除以及通过融合 将CT片段转化为β-半乳糖苷酶，并询问这些片段是否可以 把CT传输到核内。当核目标信号已经被 识别后，该信号将以这样一种方式突变以保持 催化活性以及表达结果的细胞的表型 细胞质CT将被确定。这些实验将确定 调节CT活性的机制，并将作为一种 为未来识别调制CT的信号的实验奠定基础 活动。

Phosphatidylcholine is the major phospholipid in eukaryotic cell membranes. Biosynthesis of phosphatidylcholine is regulated in part by CTP:phosphocholine cytidylyltransferase (CT). This enzyme has long been known to exist in two forms in mammalian cells: a soluble, usually inactive form and a membrane-associated, active form. Treatment of cells in various ways to stimulate phosphatidylcholine biosynthesis results in translocation of soluble CT to the membrane, concomitant with activation of the enzyme. We have found in recent years that CT is a phosphoenzyme that becomes dephosphorylated when it is activated and becomes associated with the membrane. Moreover, we have shown that the inactive form is nuclear and that the active form is associated with the nuclear envelope. The objective of this grant proposal is to study the mechanisms by which CT activity and location are controlled. To ask which phosphorylation sites are involved in determining CT activity and location, the kinetics of phosphorylation and dephosphorylation of the individual sites will be measured. To determine the functions of the individual phosphorylation sites of CT, the sites will be altered by mutagenesis and the mutant CT molecules will be expressed and characterized in a CT-deficient CHO cell line. To identify and characterize the enzymes that modulate CT in the cells, nuclear extracts will be assayed for kinases and phosphatases that act on CT as a substrate. These enzymatic activities will be partially purified and characterized, and their properties compared to those of known kinases and phosphatases. The clear targeting signal in CT will be identified by performing a series of deletions of CT as well as by fusing fragments of CT to beta-galactosidase and asking if the fragments can transport CT to the nucleus. When the nuclear targeting signal has been identified, that signal will be mutated in such a way as to retain catalytic activity, and the phenotype of cells expressing the resulting cytoplasmic CT will be determined. These experiments will identify the mechanisms by which CT activity is modulated, and will serve as a foundation of future experiments to identify the signals that modulate CT activity.