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Identification of phosphorylation sites on human deoxycytidine kinase after overexpression in eucaryotic cells.

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Smal, Caroline [UCL] Vertommen, Didier [UCL] Bertrand, Luc [UCL] Rider, Mark H. [UCL] Van Den Neste, Eric [UCL] Bontemps, Françoise [UCL]

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.

Document type Article de périodique (Journal article) – Journal Article, Research Support, Non-U.S. Gov't
Access type Accès restreint
Publication date 2006
Journal information "Nucleosides, nucleotides & nucleic acids" - Vol. 25, no. 9-11, p. 1141-6 (2006)
Peer reviewed yes
issn 1525-7770
Publication status Publié
Affiliations UCL - MD/BICL - Département de biochimie et de biologie cellulaire
UCL - MD/MINT - Département de médecine interne
UCL - (SLuc) Service d'hématologie
MESH Subject Animals ; Binding Sites ; Cell Line ; DNA, Complementary - metabolism ; Deoxycytidine Kinase - biosynthesis ; Eukaryotic Cells - metabolism ; Gene Expression Regulation, Enzymologic ; Humans ; Mass Spectrometry ; Mutation ; Phosphates - pharmacology ; Phosphorylation ; Spectrometry, Mass, Electrospray Ionization
Links
  1. Csapó Zsolt, Sasvári-Székely Mária, Spasokoukotskaja Tatjana, Talianidis Iannis, Eriksson Staffan, Staub Mária, Activation of deoxycytidine kinase by inhibition of DNA synthesis in human lymphocytes11Abbreviations: dCK, deoxycytidine kinase; dTK, thymidine kinase; CdA, 2-chloro-2′-deoxyadenosine; araC, 1-β-D-arabinofuranosylcytosine; 3HdC, 2′-deoxy-[5-3H] cytidine; 3HdT, 2′-deoxy-[5-methyl-3H] thymidine; HL 60, human promyelocytic cell line; AML, acute myeloid leukaemia; PBMC, peripherial blood mononuclear cell; APC, aphidicolin; and DEAE, diethylaminoethyl., 10.1016/s0006-2952(00)00534-7
  2. Csapo Zsolt, Keszler Gergely, Safrany Geza, Spasokoukotskaja Tatjana, Talianidis Iannis, Staub Maria, Sasvari-Szekely Maria, Activation of deoxycytidine kinase by gamma-irradiation and inactivation by hyperosmotic shock in human lymphocytes, 10.1016/s0006-2952(03)00182-5
  3. Van Den Neste Eric, Smal Caroline, Cardoen Sabine, Delacauw Anne, Frankard Joëlle, Ferrant Augustin, Van den Berghe Georges, Bontemps Françoise, Activation of deoxycytidine kinase by UV-C-irradiation in chronic lymphocytic leukemia B-lymphocytes, 10.1016/s0006-2952(02)01559-9
  4. Smal Caroline, Cardoen Sabine, Bertrand Luc, Delacauw Anne, Ferrant Augustin, Berghe Georges Van den, Neste Eric Van Den, Bontemps Françoise, Activation of deoxycytidine kinase by protein kinase inhibitors and okadaic acid in leukemic cells, 10.1016/j.bcp.2004.02.031
  5. Smal C., Bertrand L., Van Den Neste E., Cardoen S., Veiga‐da‐Cunha M., Marie S., Race V., Ferrant A., Van den Berghe G., Bontemps F., New Evidences for a Regulation of Deoxycytidine Kinase Activity by Reversible Phosphorylation, 10.1081/ncn-200027620
  6. Woods Angela, Vertommen Didier, Neumann Dietbert, Türk Roland, Bayliss Jayne, Schlattner Uwe, Wallimann Theo, Carling David, Rider Mark H., Identification of Phosphorylation Sites in AMP-activated Protein Kinase (AMPK) for Upstream AMPK Kinases and Study of Their Roles by Site-directed Mutagenesis, 10.1074/jbc.m303946200
  7. Sabini Elisabetta, Ort Stephan, Monnerjahn Christian, Konrad Manfred, Lavie Arnon, Structure of human dCK suggests strategies to improve anticancer and antiviral therapy, 10.1038/nsb942
  8. Smal Caroline, Vertommen Didier, Bertrand Luc, Ntamashimikiro Sandrine, Rider Mark H., Van Den Neste Eric, Bontemps Françoise, Identification ofin VivoPhosphorylation Sites on Human Deoxycytidine Kinase : ROLE OF SER-74 IN THE CONTROL OF ENZYME ACTIVITY, 10.1074/jbc.m512129200
  9. Keszler Gergely, Spasokoukotskaja Tatjana, Csapo Zsolt, Talianidis Iannis, Eriksson Staffan, Staub Maria, Sasvari-Szekely Maria, Activation of deoxycytidine kinase in lymphocytes is calcium dependent and involves a conformational change detectable by native immunostaining, 10.1016/j.bcp.2003.10.019
  10. Wang Li-Ming, Kucera Gregory L, Deoxycytidine kinase is phosphorylated in vitro by protein kinase Cα, 10.1016/0167-4889(94)90186-4
  11. Spasokoukotskaja Tatjana, Csapo Zsolt, Sasvari-Szekely Maria, Virga Szula, Talianidis Iannis, Eriksson Staffan, Staub Maria, Effect of Phosphorylation on Deoxycytidine Kinase Activity, Purine and Pyrimidine Metabolism in Man X ISBN:0306465159 p.281-285, 10.1007/0-306-46843-3_55
Bibliographic reference Smal, Caroline ; Vertommen, Didier ; Bertrand, Luc ; Rider, Mark H. ; Van Den Neste, Eric ; et. al. Identification of phosphorylation sites on human deoxycytidine kinase after overexpression in eucaryotic cells.. In: Nucleosides, nucleotides & nucleic acids, Vol. 25, no. 9-11, p. 1141-6 (2006)
Permanent URL http://hdl.handle.net/2078.1/26099