New insights into the regulation of deoxycytidine kinase activity via Ser-74 phosphorylation : toward improved activation of anticancer nucleoside analogs
(2014)
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- Abstract
- Deoxycytidine kinase (dCK) catalyzes the phosphorylation of deoxycytidine, deoxyadenosine and deoxyguanosine, which constitutes the first and rate-limiting step in the synthesis of DNA precursors by the deoxynucleoside salvage pathway. In addition, dCK phosphorylates and activates numerous chemotherapeutic nucleoside analogs. As low dCK activity is often associated with reduced nucleoside analog efficacy, it is of particular interest to gain more insights into the mechanisms that regulate its activity. Prior to this thesis project, research conducted in our laboratory had demonstrated that dCK activity is regulated by post-translational modification, and more precisely that its activity can be increased in vivo through Ser-74 phosphorylation, a finding that could be exploited to improve therapeutic efficacy of nucleoside analogs. The first objective of this thesis was to investigate the impact of Ser-74 phosphorylation on nucleoside analog activation and efficacy in cancer cells. We started by a kinetic study using recombinant dCK in which Ser-74 phosphorylation was mimicked by a S74E mutation. Although it was well demonstrated that S74E mutation increases dCK activity toward deoxycytidine and pyrimidine analogs, such as gemcitabine, its effect toward purine analogs had not yet been investigated. We found that S74E mutation enhanced the catalytic activity (Kcat) of dCK toward cladribine and clofarabine, but not fludarabine, with ATP or UTP as phosphoryl donor. However, Km values were also increased so that the catalytic efficiencies (Kcat/Km) of dCK for cladribine and clofarabine were not, or only slightly increased, and even decreased for fludarabine. In addition, increase of endogenous dCK activity, which was easily detected with deoxycytidine or gemcitabine as substrates after in vivo-induced increase of Ser-74 phosphorylation, was not observed with deoxyadenosine or purine analogs. Accordingly, treatment of CLL (chronic lymphocytic leukemia) cells with aphidicolin, which induces dCK activation through Ser-74 phosphorylation, did not enhance the conversion of cladribine or fludarabine into their active triphosphate form. Nevertheless, the same treatment induced higher activation of gemcitabine in CLL as well as in colon cancer cells and produced synergic cytotoxicity. From this study, it was concluded that increasing phosphorylation of dCK on Ser-74 might constitute a valuable strategy to enhance the clinical efficacy of particular nucleoside analogs, like gemcitabine. The second objective of my PhD work was to identify the protein phosphatase (PP) responsible for Ser-74 dephosphorylation. Studies in intact cells with cell-permeable PP inhibitors suggested that this PP could be the phosphoprotein phosphatase 2A (PP2A). Investigation of the dephosphorylation of dCK by purified PP as well as experiments performed in whole or PP2A-immunodepleted cell lysates confirmed this hypothesis. Use of siRNA allowed definitively concluding that PP2A constitutively dephosphorylates dCK at Ser-74 and negatively regulates dCK activity in vivo. This identification adds new information about the signaling pathways that control dCK activity and provides a new potential target for combination therapy.
- Affiliations
UCLouvain SSS/DDUV - Institut de Duve
Citations
Amsaïlale, R. (2014). New insights into the regulation of deoxycytidine kinase activity via Ser-74 phosphorylation : toward improved activation of anticancer nucleoside analogs. https://hdl.handle.net/2078.5/195893