Altered mouse leukemia L1210 thymidylate synthase, associated with cell resistance to 5-fluoro-dUrd, is not mutated but rather reflects posttranslational modification

• Authors: Joanna Cieśla , Tomasz Frączyk , Zbigniew Zieliński , Jacek Sikora , Wojciech Rode
• Languages of publication: EN

Abstracts

EN

Thymidylate synthase purified from 5-fluoro-dUrd-resistant mouse leukemia L1210 cells (TSr) was less sensitive to slow-binding inhibition by 5-fluoro-dUMP than the enzyme from the parental cells (TSp), both enzyme forms differing also in sensitivity to several other dump analogues, apparent molecular weights of monomer and dimer, and temperature dependence of the catalyzed reaction. Direct sequencing of products obtained from RT-PCR, performed on total RNA isolated from the parental and 5-fluoro-dUrd-resistant cells, proved both nucleotide sequences to be identical to the mouse thymidylate synthase coding sequence published earlier (NCBI protein database access no. NP_067263). This suggests that the altered properties of TSr are caused by a factor different than protein mutation, presumably posttranslational modification. As a possibility of rat thymidylate synthase phosphorylation has been recently demonstrated (Samsonoff et al. (1997) J Biol Chem 272: 13281), the mouse enzyme amino-acid sequence was analysed, revealing several potential phosphorylation sites. In order to test possible influence of the protein phosphorylation state on enzymatic properties, endogenous TSp and TSr were purified in the presence of inhibitors of phosphatases. Although both enzyme forms were phosphorylated, as shown by electrophoretical separation followed by phosphoprotein detection, the extent of phosphorylation was apparently similar. However, the same two purified enzyme preparations, compared to the corresponding preparations purified in the absence of phosphatase inhibitors, showed certain properties, including sensitivity to the slow-binding inhibition by FdUMP, altered. Thus properties dependence on phosphorylation was indicated.

Keywords

EN

protein phosphorylation; L1210; thymidylate synthase; posttranslational modification; FdUrd resistance

• Publisher: Polish Biochemical Society
• Journal: Acta Biochimica Polonica
• Year: 2006
• Volume: 53
• Issue: 1
• Pages: 189-198

Dates

published

2006

received

2005-12-19

revised

2006-01-31

accepted

2006-02-10

(unknown)

2006-02-27

Contributors

author

Joanna Cieśla

• Nencki Institute of Experimental Biology, Warszawa, Poland

author

Tomasz Frączyk

• Nencki Institute of Experimental Biology, Warszawa, Poland

author

Zbigniew Zieliński

• Nencki Institute of Experimental Biology, Warszawa, Poland

author

Jacek Sikora

• Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland

author

Wojciech Rode

• Nencki Institute of Experimental Biology, Warszawa, Poland

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