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.
