Identification of Phosphorylation Sites on Human Deoxycytidine Kinase After Overexpression in Eucaryotic Cells |
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| Authors: | C Smal D Vertommen L Bertrand M H Rider E Van Den Neste F Bontemps |
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| Institution: | 1. Laboratory of Physiological Chemistry , Christian de Duve Institute of Cellular Pathology , Brussels, Belgium;2. Hormone and Metabolic Research Unit , Christian de Duve Institute of Cellular Pathology , Brussels, Belgium;3. Laboratory of Physiological Chemistry , Christian de Duve Institute of Cellular Pathology , Brussels, Belgium;4. Department of Hematology , Cliniques Universitaires Saint-Luc, Université Catholique de Louvain , Brussels, Belgium |
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| Abstract: | 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. |
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| Keywords: | Deoxycytidine kinase Protein phosphorylation Tandem mass spectrometry |
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