J Biol Chem 1999 Apr 30;274(18):12544-7
Ligand-mediated induction of thymidylate synthase occurs by
enzyme
stabilization. Implications for
autoregulation of translation.
Kitchens ME, Forsthoefel AM, Rafique Z, Spencer HT, Berger FG.
Department of Biological Sciences, University of South Carolina.
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 are directed. The fluoropyrimidines
5-fluorouracil
and 5-fluoro-2'-deoxyuridine are cytotoxic as a consequence of
inhibition
of TS by the metabolite 5-fluoro-2'-deoxyuridine 5'-monophosphate
(FdUMP).
This inhibition occurs through formation of a stable ternary complex
among
the enzyme, the nucleotide analog, and the co-substrate N5,
N10-methylenetetrahydrofolate.
Numerous studies have shown that cellular concentrations of TS undergo
about a 2-4-fold 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
of this autoregulatory translation model. In contrast to expectations,
we find that fluoropyrimidines do not cause a change in the extent of
ribosome
binding to TS mRNA. Furthermore, mutations within the mRNA that abolish
its ability to bind TS have no effect on the induction. Finally, enzyme
turnover measurements show that the induction is associated with an
increase
in the stability of the TS polypeptide. Our results, in total, indicate
that enzyme stabilization, rather than translational derepression, is
the
primary mechanism of TS induction by fluoropyrimidines and call into
question
the general applicability of the autoregulatory translation model.