title: Phosphorylation of deoxycytidine kinase on Ser-74: impact on kinetic properties and nucleoside analog activation in cancer cells.
abstract: Deoxycytidine kinase (dCK) (EC 2.7.1.74) is a key enzyme in the activation of several therapeutic nucleoside analogs (NA). Its activity can be increased in vivo by Ser-74 phosphorylation, a property that could be used for enhancing NA activation and clinical efficacy. In line with this, studies with recombinant dCK showed that mimicking Ser-74 phosphorylation by a S74E mutation increases its activity toward pyrimidine analogs. However, purine analogs had not been investigated. Here, we show that the S74E mutation increased the k(cat) for cladribine (CdA) by 8- or 3-fold, depending on whether the phosphoryl donor was ATP or UTP, for clofarabine (CAFdA) by about 2-fold with both ATP and UTP, and for fludarabine (F-Ara-A) by 2-fold, but only with UTP. However, the catalytic efficiencies (k(cat)/Km) were not, or slightly, increased. The S74E mutation also sensitized dCK to feed-back inhibition by dCTP, regardless of the phosphoryl donor. Importantly, we did not observe an increase of endogenous dCK activity toward purine analogs after in vivo-induced increase of Ser-74 phosphorylation. Accordingly, treatment of CLL cells with aphidicolin, which enhances dCK activity through Ser-74 phosphorylation, did not modify the conversion of CdA or F-Ara-A into their active triphosphate form. Nevertheless, the same treatment enhanced activation of gemcitabine (dFdC) into dFdCTP in CLL as well as in HCT-116 cells and produced synergistic cytotoxicity. We conclude that increasing phosphorylation of dCK on Ser-74 might constitute a valuable strategy to enhance the clinical efficacy of some NA, like dFdC, but not of CdA or F-Ara-A.
authors.affiliation: Laboratory of Physiological Chemistry, de Duve Institute & Université catholique de Louvain, B-1200 Brussels, Belgium.
authors.forename: Rachid
authors.initials: R
authors.lastname: Amsailale
authors.forename: Eric
authors.initials: E
authors.lastname: Van Den Neste
authors.forename: Angélique
authors.initials: A
authors.lastname: Arts
authors.forename: Eliza
authors.initials: E
authors.lastname: Starczewska
authors.forename: Françoise
authors.initials: F
authors.lastname: Bontemps
authors.forename: Caroline
authors.initials: C
authors.lastname: Smal
mesh_terms: D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D016590:Aphidicolin / Q000494:pharmacology; D001711:Biotransformation; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D017338:Cladribine / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D003842:Deoxycytidine Kinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D045325:HCT116 Cells; D019073:HT29 Cells; D006801:Humans; D007700:Kinetics; D009154:Mutation; D010766:Phosphorylation; D011684:Purine Nucleosides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011741:Pyrimidine Nucleosides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014740:Vidarabine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D000093542:Gemcitabine
chemical_list: D000970:Antineoplastic Agents; D011684:Purine Nucleosides; D011741:Pyrimidine Nucleosides; D003841:Deoxycytidine; D016590:Aphidicolin; D012694:Serine; D017338:Cladribine; D003842:Deoxycytidine Kinase; D014740:Vidarabine; C024352:fludarabine; D000093542:Gemcitabine