| The pyrimidine nucleotides (uridine and cytidine) are essential molecules for cell proliferation. Not only are these nucleotides necessary for DNA and RNA synthesis, but also for the synthesis of phospholipids and UDP-sugars (9). Mammalian cells have two major mechanisms for pyrimidine synthesis, either the de novo synthetic pathway, which requires the multifunctional enzyme CAD or the salvage synthesis pathway, which requires the nucleoside transporters described in Project#1. Our lab showed that CAD is phosphorylated by MAP kinase in vitro and is phosphorylated in cells in response to signals that increase MAP kinase activity (i.e. EGFR) (10). However, other kinases and other proteins may also regulate CAD activity and these events are under investigation (11). Our current research is directed at identifying all the phosphorylation sites in CAD as well as CAD interacting proteins using modern methods of mass spectrometry (12). In a related project, we are investigating the regulation of CTP synthetase. This enzyme catalyzes the conversion of uridine nucleotides to cytidine nucleotides that is pivotal for the synthesis of cellular phospholipids. Using specific inhibitors of UTP or CTP synthesis, recent studies from our lab have demonstrated an essential role for CTP in regulating cell cycle progression (13,14). The overall objectives of this project are to determine the cellular signals that regulate the activity of CAD and CTP synthetase and to evaluate the influence of these events on cell cycle progression. |