Smal C, Vertommen D, Bertrand L, Rider M H, van den Neste E, Bontemps F
Laboratory of Physiological Chemistry, Christian de Duve Institute of Cellular Pathology, Brussels, Belgium.
Nucleosides Nucleotides Nucleic Acids. 2006;25(9-11):1141-6. doi: 10.1080/15257770600890194.
Compelling evidence suggests that deoxycytidine kinase (dCK), a key enzyme in the salvage of deoxyribonucleosides and in the activation of clinically relevant nucleoside analogues, can be regulated by reversible phosphorylation. In this study, we show that dCK overexpressed in HEK-293T cells was labelled after incubation of the cells with [32P]orthophosphate. Tandem mass spectrometry allowed the identification of 4 in vivo phosphorylation sites, Thr3, Ser11, Ser15, and Ser74. These results provide the first evidence that dCK is constitutively multiphosphorylated in intact cells. In addition, site-directed mutagenesis demonstrated that phosphorylation of Ser74, the major in vivo phosphorylation site, is crucial for dCK activity.
有力证据表明，脱氧胞苷激酶（dCK）是脱氧核糖核苷补救途径及临床相关核苷类似物激活过程中的关键酶，可通过可逆磷酸化进行调节。在本研究中，我们发现，用[32P]正磷酸盐孵育HEK-293T细胞后，其中过表达的dCK会被标记。串联质谱法鉴定出4个体内磷酸化位点，即苏氨酸3、丝氨酸11、丝氨酸15和丝氨酸74。这些结果首次证明dCK在完整细胞中持续发生多磷酸化。此外，定点诱变表明，体内主要磷酸化位点丝氨酸74的磷酸化对dCK活性至关重要。
