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Deoxycytidine kinase (dCK) catalyses the rate-limiting reaction in the salvage of deoxyribonucleosides, supplying cells with dNTPs for DNA synthesis. The enzyme phosphorylates deoxycytidine , désoxyguanosine and deoxyadenosine, with ATP as phosphate donor. In addition, dCK activates number of nucleoside analogues used in the treatment of lymphoproliferative disorders and viral infections. Studies of the mechanisms that control the activity of this enzyme are thus of particular interest. Recently, C. Smal, a graduate student of our team, has identified four phosphorylation sites on human dCK overexpressed on HEK 293T cells : Thr3, Ser11, Ser15 and Ser74. By site-directed mutagenesis, she has demonstrated that Ser74 phosphorylation in crucial for dCK activity.
The aim of my work was to investigate whether Thr3 and Ser11 phosphorylation influences dCK activity. First, we improved the purification of dCK expressed as a His-tag fusion protein. Then, we confirmed that the kinetic characteristics of the recombinant dCK were similar to those of the native enzyme, despite the presence of the His tag. Moreover, we showed that dCK treatment with λ protein phosphatase profoundly decreased dCK activity, corroborating that the recombinant enzyme was phosphorylated.
The following step was a site-directed mutagenesis study, HEK 293T cells were transferred with mutated versions of His-tagged dCK. Thr3 and Ser11 were mutated both to Ala (T3A and S11A mutants) to eliminated the possibility of phosphorylation of this residue. We found that these mutations provoked a decrease of dCK activity, which was particularly pronounced for the T3A mutant. However, the decreases in dCK activity were approximately parallel to the decrease in protein expression. Affinity of dCK mutants for désoxycytidine was not modified. Theses results indicate that Thr3 and Ser11 phosphorylation does not control dCK activity, but could influence its expression or stability La désoxycytidine kinase (dCK) catalyse la phosphorylation de la désoxycytidine, de la désoxyadénosine et de la désoxyguanosine, et initie ainsi la formation de dNTPs, indispensables à la synthèse de l’ADN. En plus de ce rôle physiologique, la dCK active plusieurs analogues de nucléosides utilisés dans le traitement de désordres lymphoprolifératifs et d’infections virales, d’où l’intérêt porté à cette enzyme par les biochimistes. Récemment, une doctorante du laboratoire ; C. Smal, a identifié quatre sites de phosphorylation sur la dCK humaine, après surexpression dans les cellules HEK 293T : la Thr3, et les Ser11, 15 et 74. Elle a montré aussi par mutagenèse dirigée que la phosphorylation de la Ser74 était cruciale pour l’activité de la dCK.
L’objectif principal de mon travail était d’examiner si la phosphorylation de la Thr3 et de la Ser11 influençait l’activité de la dCK. Nous avons commencé par optimiser la méthode de purification de la dCK surexprimée avec une étiquette polyhistidine. Nous avons ensuite confirmé que les caractéristiques cinétiques de l’enzyme recombinante étaient comparables à celles rapportées dans la littérature, malgré la présence de l’étiquette polyhistidine. Finalement, nous avons montré que le traitement de l’enzyme recombinante avec la protéine phosphatase lambda diminuait fortement son activité, ce qui confirmait que l’enzyme recombinante était bien phosphorylée.
Nous avons poursuivi notre étude de mutagenèse dirigée. Des mutations ont été introduites au niveau des Thr3 et Ser11 de la dCK. Ces deux acides aminés ont été remplacés soit par une alanine, ne pouvant être phosphorylée, soit par un acide glutamique mimant la charge négative du groupe phosphate. Les quatre mutants ont été exprimés dans les cellules HEK 293T. Leur analyse a montré que l’activité de la dCK était abaissée à des degrés divers par les différentes mutations, et particulièrement par la mutation T3A. toutefois, les baisses d’activité observées étaient parallèles à une diminution de l’expression protéique. Le Km de la dCK, quant à lui, n’était pas modifié par les mutations. Ces résultats suggèrent que la phosphorylation de la Thr3 et de la Ser11 n’influence pas les propriétés cinétiques de la dCK, mais qu’elle pourrait influencer l’expression ou la stabilité de l’enzyme.

Phosphothreonine --- Serine --- Mutagenesis, Site-Directed --- Deoxycytidine

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