REGULATION OF CTP--PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE

磷脂酰胆碱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：磷酸胆碱胞苷酰转移酶（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：可溶性的，通常 非活性形式和膜相关的活性形式。处理细胞 以各种方式刺激磷脂酰胆碱的生物合成， 可溶性CT易位至膜，伴随活化 的酶。近年来我们发现CT是一种磷酸酶 当它被激活时就会去磷酸化 与膜。此外，我们还证明了非活性形式是 核和活性形式与核膜有关。 这项拨款建议的目的是研究机制， 控制CT活动和位置。要问哪种磷酸化 位点参与决定CT活性和位置， 各个位点的磷酸化和去磷酸化 测定了为了确定单个磷酸化的功能， CT的位点，该位点将通过诱变而改变，并且突变CT 分子将在CT缺陷型CHO细胞中表达和表征 线为了鉴定和表征调节CT的酶， 细胞，将测定核提取物中的激酶和磷酸酶， 作为底物作用于CT。这些酶活性将部分地 纯化和表征，以及它们的性质与 已知的激酶和磷酸酶。CT中的明确目标信号将是 通过进行一系列CT缺失以及通过融合 CT片段与β-半乳糖苷酶的结合，并询问这些片段是否可以 把CT传送到细胞核当核目标信号 一旦被识别，该信号将以这样的方式突变， 催化活性，以及表达所产生的催化活性的细胞的表型。 将测定细胞质CT。这些实验将确定 调节CT活动的机制，并将作为 为将来的实验奠定基础，以识别调制CT的信号 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.