Perturbation of ATP-induced tetramerization of human cytosolic thymidine kinase by substitution of serine-13 with aspartic acid at the mitotic phosphorylation site

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Abstract

Human cytosolic thymidine kinase (TK1) is tightly regulated in the cell cycle by multiple mechanisms. Our laboratory has previously shown that in mitotic-arrested cells human TK1 is phosphorylated at serine-13, accompanied by a decrease in catalytic efficiency. In this study we investigated whether serine-13 phosphorylation regulated TK1 activity and found that substitution of serine-13 with aspartic acid (S13D), which mimics phosphorylation, not only diminished the ATP-activating effect on the enzyme, but also decreased its thymidine substrate affinity. Our experimental results further showed that the S13D mutation perturbed ATP-induced tetramerization of TK1. Given that the dimeric form of TK1 is less active than the tetrameric, we propose that mitotic phosphorylation of serine-13 is of physiological importance, in that it may counteract ATP-dependent activation of TK1 by affecting its quaternary structure, thus attenuating its enzymatic function at the G2/M phase.

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Materials and methods

Plasmids. The BamHI/EcoRI fragment of TK1 cDNA from plasmid pGEX-2T-LyTK1Val-106[20] was subcloned into pCDNA3 (Invitrogen) to generate pCDNA3-TK1V106. S13D and S13A mutations of TK1 were generated as described previously [21]. pET-28c-TK1 was constructed by inserting the PCR fragment containing the TK1 coding region, amplified from pCDNA3-TK1V106, into the NdeI/EcoRI-digested pET-28c vector (Novagen). Two primers 5′-GGGAATTCCATATGAGCTGCATTAACCTG-3′ and 5′-GGAATTCTC AGTTGGCAGGGCTGCA-3′ were

ATP-activating effect on wild-type and S13D TK1

In our past investigation, we used the cDNA construct originally from Bradshaw and Deininger [26] to study phosphorylation of TK1. However, sequence analysis from healthy cells, leukemic or transformed cell lines, revealed that cytosolic TK1 has a valine at amino acid 106 [20], the position at which a methionine codon is present in the TK1 cDNA clone from Bradshaw and Deininger [26]. Therefore, Val-106 is most likely the naturally occurring amino acid at position 106 in human TK1. Through in

Acknowledgements

We are grateful to Dr. Birgitte Munch-Petersen at Roskilde University, Denmark, for providing the plasmid containing cDNA of TK1V106. This study was supported by Grant NSC92-3112-B-002-016 from the National Science Council, Taiwan, ROC.

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