title: Identification of phosphorylation sites on human deoxycytidine kinase after overexpression in eucaryotic cells.
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.
authors.affiliation: Laboratory of Physiological Chemistry, Christian de Duve Institute of Cellular Pathology, Brussels, Belgium.
authors.forename: C
authors.initials: C
authors.lastname: Smal
authors.forename: D
authors.initials: D
authors.lastname: Vertommen
authors.forename: L
authors.initials: L
authors.lastname: Bertrand
authors.forename: M H
authors.initials: MH
authors.lastname: Rider
authors.forename: E
authors.initials: E
authors.lastname: van den Neste
authors.forename: F
authors.initials: F
authors.lastname: Bontemps
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D003842:Deoxycytidine Kinase / Q000096:biosynthesis*; D005057:Eukaryotic Cells / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D013058:Mass Spectrometry; D009154:Mutation; D010710:Phosphates / Q000494:pharmacology; D010766:Phosphorylation; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D018076:DNA, Complementary; D010710:Phosphates; D003842:Deoxycytidine Kinase