Perturbation of ATP-induced tetramerization of human cytosolic thymidine kinase by substitution of serine-13 with aspartic acid at the mitotic phosphorylation site.
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.
Li
Chia-Lung
CL
Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, No 1 Jen Ai Road First Section, Taipei, Taiwan ROC.
Lu
Ching-Yi
CY
Ke
Po-Yuan
PY
Chang
Zee-Fen
ZF
D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000494:pharmacology; D001224:Aspartic Acid / Q000737:chemistry; D002384:Catalysis; D002453:Cell Cycle; D002460:Cell Line; D002845:Chromatography; D002850:Chromatography, Gel; D003600:Cytosol / Q000201:enzymology*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D016195:G2 Phase; D006801:Humans; D007700:Kinetics; D008938:Mitosis; D008954:Models, Biological; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D020836:Protein Structure, Quaternary; D012694:Serine / Q000737:chemistry*; D013936:Thymidine / Q000494:pharmacology; D013937:Thymidine Kinase / Q000737:chemistry*
D001224:Aspartic Acid; D012694:Serine; D000255:Adenosine Triphosphate; D013937:Thymidine Kinase; C092717:thymidine kinase 1; D013936:Thymidine