Ligand-mediated induction of thymidylate synthase occurs by enzyme stabilization - Implications for autoregulation of translation

Thymidylate synthase (TS) is indispensable in the de novo synthesis of dTMP . As such, it has been an important target at which anti-neoplastic drugs a re directed. The fluoropyrimidines B-fluorouracil and 5-fluoro-2'-deoxyurid ine are cytotoxic as a consequence of inhibition of TS by the metabolite 5- fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP), This inhibition occurs thr ough formation of a stable ternary complex among the enzyme, the nucleotide analog, and the co-substrate N-5,N-10-methylenetetrahydrofolate. Numerous studies have shown that cellular concentrations of TS undergo about a 2-4-f old induction following treatment with TS inhibitors. An extensive body of in vitro studies has led to the proposal that this induction occurs because of relief of the translational repression brought on by the binding of TS to its own mRNA. In the current study, we have tested several predictions o f this autoregulatory translation model. In contrast to expectations, we fi nd that fluoropyrimidines do not cause a change in the extent of ribosome b inding to TS mRNA. Furthermore, mutations within the mRNA that abolish its ability to bind TS have no effect on the induction. Finally, enzyme turnove r measurements show that the induction is associated with an increase in th e stability of the TS polypeptide, Our results, in total, indicate that enz yme stabilization, rather than translational derepression, is the primary m echanism of TS induction by fluoropyrimidines and call into question the ge neral applicability of the autoregulatory translation model.