Thymidylate synthase (TS) is an indispensable enzyme in the de novo synthesis of dTMP in dividing cells. As such, it is an excellent target at which anti-cancer drugs are directed. A number of pharmacological agents, such as 5-fluoropyrimidines (e.g., 5-fluorouracil and 5-fluoro-2'-deoxyuridine) and folic acid analogs (e.g., raltitrexed and BW1843U89), are cytotoxic to proliferating cells as a consequence of their ability to generate metabolites that inhibit TS. It has long been recognized that treatment of cells or tumors with TS inhibitors results in induction of the enzyme's concentration. Such induction has been viewed as a primary mechanism for the emergence of cellular resistance to these inhibitors, and, therefore, as an obstacle to effective chemotherapeutic response. Our laboratory has shown that the induction of TS in drug-exposed cells is due to stabilization of the enzyme by the binding of inhibitory ligands. Building on crystal structures of human TS, along with recent experiments indicating that the enzyme is degraded by the 26S proteasome, we have proposed a molecular mechanism for ligand-mediated stabilization of the TS polypeptide. We postulate that the ligand-free enzyme, which exhibits a disordered conformation in the region between residues 107-128, is readily ubiquitinated and targeted to the 26S proteasome; it is therefore unstable and expressed at low levels. The ligand-bound enzyme, on the other hand, becomes ordered in the 107-128 region, which decreases ubiquitination and allows "escape" from the proteasome; this results in stabilization of the enzyme, and higher levels of expression. In the present grant, we propose experiments that will rigorously test this model. We will determine if TS is ubiquitinated in a ligand-dependent manner (AIM 1). In addition, we will establish whether or not ubiquitination is critical to proteasome-mediated degradation of the TS polypeptide (AIMS 2, 3). Finally, we will assess the role(s) of the disordered region and nearby lysine residues (AIMS 4-6). The proposed experiments will provide novel information on the role of TS turnover in regulating the enzyme's function as a drug target. As such, they will have application in the use of TS inhibitors as anti-cancer agents, and will be useful in designing high expressing TS genes for protection of normal tissues against the toxic effects of these inhibitors.