User menu

Accès à distance ? S'identifier sur le proxy UCLouvain

Identification of in vivo phosphorylation sites on human deoxycytidine kinase. Role of Ser-74 in the control of enzyme activity.

Primary tabs

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

Deoxycytidine kinase (dCK) catalyzes the rate-limiting step of the deoxyribonucleoside salvage pathway in mammalian cells and plays a key role in the activation of numerous nucleoside analogues used in anti-cancer and antiviral chemotherapy. Although compelling evidence indicated that dCK activity might be regulated by phosphorylation/dephosphorylation, direct demonstration was lacking. Here we showed that dCK overexpressed in HEK 293T cells was labeled after incubating the cells with [32P]orthophosphate. Sorbitol, which was reported to decrease dCK activity, also decreased the labeling of dCK. These results indicated that dCK may exist as a phosphoprotein in vivo and that its activity can be correlated with its phosphorylation level. After purification of 32P-labeled dCK, digestion by trypsin, and analysis of the radioactive peptides by tandem mass spectrometry, the following four in vivo phosphorylation sites were identified: Thr-3, Ser-11, Ser-15, and Ser-74, the latter being the major phosphorylation site. Site-directed mutagenesis and use of an anti-phospho-Ser-74 antibody demonstrated that Ser-74 phosphorylation was crucial for dCK activity in HEK 293T cells, whereas phosphorylation of other identified sites did not seem essential. Phosphorylation of Ser-74 was also detected on endogenous dCK in leukemic cells, in which the Ser-74 phosphorylation state was increased by agents that enhanced dCK activity. Our study provided direct evidence that dCK activity can be controlled by phosphorylation in intact cells and highlights the importance of Ser-74 for dCK activity.

  1. Arnér Elias S.J., Eriksson Staffan, Mammalian deoxyribonucleoside kinases, 10.1016/0163-7258(95)00015-9
  2. Blood, 81, 597 (1993)
  3. Stegmann A. P. A., Honders M. W., Hagemeijer A., Hoebee B., Willemze R., Landegent J. E., In vitro-induced resistance to the deoxycytidine analogues cytarabine (AraC) and 5-aza-2′-deoxycytidine (DAC) in a rat model for acute myeloid leukemia is mediated by mutations in the deoxycytidine kinase (dck) gene, 10.1007/bf01696231
  4. Arnér Elias S. J., On the Phosphorylation of 2-chlorodeoxy-adenosine (CdA) and Its Correlation with Clinical Response in Leukemia Treatment, 10.3109/10428199209067604
  5. Blood, 96, 1517 (2000)
  6. Clin. Cancer Res., 7, 3263 (2001)
  7. Månsson Emma, Flordal Emma, Liliemark Jan, Spasokoukotskaja Tatiana, Elford Howard, Lagercrantz Svetlana, Eriksson Staffan, Albertioni Freidoun, Down-regulation of deoxycytidine kinase in human leukemic cell lines resistant to cladribine and clofarabine and increased ribonucleotide reductase activity contributes to fludarabine resistance, 10.1016/s0006-2952(02)01484-3
  8. Goodman Grant R., Bethel Kelly J., Saven Alan, Hairy cell leukemia: an update : , 10.1097/00062752-200307000-00002
  9. Robak Tadeusz, Therapy of chronic lymphocytic leukemia with purine analogs and monoclonal antibodies, 10.1016/j.transci.2004.10.004
  10. Sasvári-Székely Mária, Spasokoukotskaja Tatjana, Szóke Melinda, Csapó Zsolt, Turi Ágnes, Szántó Ildikó, Eriksson Staffan, Staub Mária, Activation of deoxycytidine kinase during inhibition of DNA synthesis by 2-chloro-2′-deoxyadenosine (cladribine) in human lymphocytes, 10.1016/s0006-2952(98)00108-7
  11. Spasokoukotskaja T., Sasvári-Székely M., Keszler G., Albertioni F., Eriksson S., Staub M., Treatment of normal and malignant cells with nucleoside analogues and etoposide enhances deoxycytidine kinase activity, 10.1016/s0959-8049(99)00223-3
  12. Plunkett, W., and Gandhi, V. (1997) in Nucleoside Analogs in Cancer Therapy (Cheson, B. D., Keating, M. J., and Plunkett, W., eds) pp. 1-35, Marcel Dekker, Inc., New York
  13. Clin. Cancer Res., 7, 3559 (2001)
  14. 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
  15. 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
  16. 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
  17. 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
  18. Wessel D., Flügge U.I., A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids, 10.1016/0003-2697(84)90782-6
  19. Bradford Marion M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, 10.1016/0003-2697(76)90527-3
  20. Hatzis Pantelis, Al-Madhoon Ashraf Said, Jüllig Mia, Petrakis Thodoris G., Eriksson Staffan, Talianidis Iannis, The Intracellular Localization of Deoxycytidine Kinase, 10.1074/jbc.273.46.30239
  21. 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
  22. Chottiner E. G., Shewach D. S., Datta N. S., Ashcraft E., Gribbin D., Ginsburg D., Fox I. H., Mitchell B. S., Cloning and expression of human deoxycytidine kinase cDNA., 10.1073/pnas.88.4.1531
  23. Galmarini Carlos M, Mackey John R, Dumontet Charles, Nucleoside analogues and nucleobases in cancer treatment, 10.1016/s1470-2045(02)00788-x
  24. Bohman Christina, Eriksson Staffan, Deoxycytidine kinase from human leukemic spleen: preparation and characterization of the homogeneous enzyme, 10.1021/bi00412a009
  25. Johansson Magnus, Karlsson Anna, Differences in kinetic properties of pure recombinant human and mouse deoxycytidine kinase, 10.1016/0006-2952(95)00129-n
  26. Blom Nikolaj, Gammeltoft Steen, Brunak Søren, Sequence and structure-based prediction of eukaryotic protein phosphorylation sites, 10.1006/jmbi.1999.3310
  27. 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
  28. Martensen Todd M., Chemical properties, isolation, and analysis of O-phosphates in proteins, Methods in Enzymology (1984) ISBN:9780121820077 p.3-23, 10.1016/0076-6879(84)07003-8
  29. Koenig M., Grabe N., Highly specific prediction of phosphorylation sites in proteins, 10.1093/bioinformatics/bth455
  30. Wang Li-Ming, Kucera Gregory L, Deoxycytidine kinase is phosphorylated in vitro by protein kinase Cα, 10.1016/0167-4889(94)90186-4
  31. Adv. Exp. Med. Biol., 486, 281 (2000)
  32. 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
  33. Pettitt Andrew R., Mechanism of action of purine analogues in chronic lymphocytic leukaemia, 10.1046/j.1365-2141.2003.04336.x
  34. De Clercq Erik, Antiviral drugs in current clinical use, 10.1016/j.jcv.2004.02.009
  35. Carson D. A., Wasson D. B., Kaye J., Ullman B., Martin D. W., Robins R. K., Montgomery J. A., Deoxycytidine kinase-mediated toxicity of deoxyadenosine analogs toward malignant human lymphoblasts in vitro and toward murine L1210 leukemia in vivo., 10.1073/pnas.77.11.6865
  36. Cancer Res., 41, 4478 (1981)
  37. Clin. Cancer Res., 1, 391 (1995)
  38. Galmarini C M, Mackey J R, Dumontet C, Nucleoside analogues: mechanisms of drug resistance and reversal strategies, 10.1038/sj.leu.2402114
Bibliographic reference Smal, Caroline ; Vertommen, Didier ; Bertrand, Luc ; Ntamashimikiro, Sandrine ; Rider, Mark H. ; et. al. Identification of in vivo phosphorylation sites on human deoxycytidine kinase. Role of Ser-74 in the control of enzyme activity.. In: The Journal of biological chemistry, Vol. 281, no. 8, p. 4887-93 (2006)
Permanent URL http://hdl.handle.net/2078.1/26103