ALTERED PROPERTIES OF PHOSPHORYLATED OR NITROSYLATED THYMIDYLATE SYNTHASE

• Published in: Anticancer research Vol. 28; no. 5C
• Main Authors: Fraczyk, T, Ruman, T, Rut, D, Dabrowska-Mas, E, Ciesla, J, Zielinski, Z, Sieczka, K, Sikora, J, Walajtys-Rode, E, Shugar, D, Rode, W
• Format: Journal Article
• Language: English
• Published: 01-10-2008
• Subjects: Caenorhabditis elegans; Trichinella spiralis
• ISSN: 0250-7005

Thymidylate synthase (TS; EC 2.1.1.45), a major target in cancer chemotherapy, catalyzes the N super(5,10)-methylene-tetrahydrofolate (meTHF)-assisted C(5)-methylation of 2'-deoxyuridine-5'-monophosphate (dUMP), leading to formation of 2'-deoxythymidine-5'-monophosphate. Possible phosphorylation of TS in cultured rat cells, previously reported (Samsonoff et al: J Biol Chem 272:13281-13285, 1997), prompted us to examine this in more detail. TS preparations from various sources, all highly purified in the presence of phosphatase inhibitors, included L1210 parental and FdUrd-resistant forms, as well as mouse, rat, human and Trichinella spiralis recombinant enzymes. They were analyzed, following SDS-PAGE, with the Pro-Q registered Diamond Phosphoprotein Gel Stain, and all were found to include a low proportion of phosphorylated forms. However, MS analysis of the SDS-PAGE bands did not reveal any phosphorylated amino acid residues. By contrast, MS analysis of IEF fractions of TS preparations from parental and FdUrd-resistant mouse leukemia L1210 cells, whose differing sensitivity to inactivation by FdUMP and its analogues was previously found not due to mutations (Ciesla et al: Acta Biochim Pol 53: 189-198, 2006), demonstrated phosphorylation of Ser10 and Serl6 in the resistant, but not the parental, enzyme. Each of the four recombinant TS preparations, expressed in bacterial cells, was separated into phosphorylated and non-phosphorylated fractions, using metal oxide/hydroxide affinity chromatography on A1(OH) sub(3) beads, yielding phosphorylated fractions corresponding to approximately 1% of the total. Each phosphorylated form exhibited a 3- to 4-fold lower V super(a) sub(m) super(p) sub(a) super(p) sub(x), but unaltered K super(a) sub(m) super(pp) with either substrate or cofactor relative to the non-phosphorylated form, and ability to repress translation (catalyzed by a rabbit reticulocyte preparation) of its own (as well as luciferase) mRNA. Surprisingly, MS analyses did not reveal the presence of phosphorylated amino acid residues in any of the fractions investigated. In striking contrast, super(31)P NMR spectroscopy clearly demonstrated the presence of phosphorylated residues in the phosphorylated enzyme fractions, and their absence in non-phosphorylated. Further analyses of the super(31)P NMR spectra (including their time-dependent changes following acidification), and comparison with those of synthetic phosphoramide derivatives of basic amino acids (Lys, Arg and His), and commercially available phospho-serine, phospho-threonine and phospho-tyrosine, revealed the presence of phosphorus in a phosphoramide (acid-labile) bond, pointing to modification of histidine residue(s). Biological nitration of protein tyrosine residues, which may lead to modulation of protein function, is associated with many diseases, including cancer, and is correlated with intensified NO biosynthesis. Human, mouse and Caenorhabditis elegans recombinant TS preparations, each incubated in vitro in the presence of NaHCO sub(3), NaNO sub(2) and H sub(2)O sub(2), underwent nitration of tyrosine residues, resulting in a distinctly diminished rate of the enzyme-catalyzed reaction. The V super(a) sub(m) super(P) sub(a) super(P) sub(x) value was 2-fold lower when 1 tyrosine residue per monomer was nirated (with human or C. elegans TS) or 2 tyrosine residues per monomer (with mouse TS). No distinct effect on enzyme interactions with substrate (dUMP), cofactor (meTHF) or inhibitor (5-fluoro-dUMP) was apparent.