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Hiding human thymidine kinase 1 from APC/C-mediated destruction by thymidine binding

Graduate Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taiwan, Republic of China

²Correspondence: Graduate Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, No.1, Section 1, Jen-Ai Rd., Taipei, Taiwan (R.O.C.). E-mail: zfchang{at}ha.mc.ntu.edu.tw

Thymidine kinase 1 (TK1) is a key cytosolic enzyme in the salvage pathway for dTTP synthesis. In mitotic exit, human TK1 (hTK1) is degraded via the anaphase-promoting complex/cyclosome (APC/C)-Cdh1 pathway to limit dTTP production. In this study, we show that thymidine binding stabilizes hTK1 during growth arrest. By in vitro degradation, ubiquitination, and Cdh1 binding analyses, we provide direct evidence that thymidine binding protects wild-type hTK1 protein from APC/C-Cdh1-mediated destruction. In contrast, mutant-type hTK1 protein defective in thymidine binding ability could still be polyubiquitinated by APC/C-Cdh1 in the presence of thymidine. These results suggest that the status of thymidine binding to hTK1 protein determines its susceptibility to degradation due to APC/C targeting. Our in vivo experimental data also demonstrated that thymidine treatment abolished Cdh1/proteasome-responsive suppression of hTK1 expression. Moreover, exposure of mitotic-arrested K562 cells to thymidine (100 µM) stabilized endogenous TK1, causing nucleotide imbalance in the early G₁ phase and an increase of S phase accumulation. In conclusion, thymidine is not only a substrate of TK1 but also acts as its expression regulator by modulating its proteolytic control during mitotic exit, conferring a feed-forward regulation of dTTP formation.—Ke, P.-Y., Hu, C.-M., Chang, Y.-C., Chang, Z.-F. Hiding human thymidine kinase 1 from APC/C-mediated destruction by thymidine binding.

Key Words: cell cycle • proteolysis • ubiquitination • dTTP synthesis