Identification of in Vivo Phosphorylation Sites on Human Deoxycytidine Kinase

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 32Porthophosphate. 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. 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 32Porthophosphate. 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. Deoxycytidine kinase (dCK 4The abbreviations used are: dCK, deoxycytidine kinase; HEK, human embryonic kidney; HPLC, high pressure liquid chromatography; 32P, 32Porthophosphate; ESI-MS/MS, electrospray ionization tandem mass spectrometry; PKC, protein kinase C; PBS, phosphate-buffered saline; DMEM, Dulbecco’s modified Eagle’s medium; CdA, 2-chloro-2′-deoxyadenosine; λ-PP, protein phosphatase; UV, ultraviolet; WT, wild-type. ; EC 2.7.1.74) catalyzes the phosphorylation of deoxycytidine, deoxyguanosine, and deoxyadenosine, with ATP or UTP as phosphoryl donor. This reaction is the rate-limiting step of the deoxyribonucleoside salvage pathway that supplies cells with precursors of DNA as an alternative to de novo synthesis (1Arner E.S. Eriksson S. Pharmacol. Ther. 1995; 67: 155-186Crossref PubMed Scopus (514) Google Scholar). In addition, dCK initiates the activation of several chemotherapeutic nucleoside analogues, such as 1-β-d-arabinosylcytosine (cytarabine), 9-β-d-arabinosyl-2-fluoroadenine (fludarabine), and 2-chloro-2′-deoxyadenosine (CdA, cladribine), commonly used in the treatment of hematological malignancies, and 2′,2′-difluorodeoxycytidine (gemcitabine), active against solid malignant tumors (2Plunkett W. Gandhi V. Cheson B.D. Keating M.J. Plunkett W. Nucleoside Analogs in Cancer Therapy. Marcel Dekker, Inc., New York1997: 1-35Google Scholar, 3Galmarini C.M. Mackey J.R. Dumontet C. Lancet Oncol. 2002; 3: 415-424Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar, 4Pettitt A.R. Br. J. Haematol. 2003; 121: 692-702Crossref PubMed Scopus (82) Google Scholar). The anti-human immunodeficiency virus drugs 2′,3′-dideoxycytidine (zalcitabine) and 2′-deoxy-3′-thiacytidine (lamivudine) are also phosphorylated by dCK (5De Clercq E. J. Clin. 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Ther. 1995; 67: 155-186Crossref PubMed Scopus (514) Google Scholar), which explains the clinical success of nucleoside analogues against lymphoproliferative disorders, such as hairy cell leukemia and B-cell chronic lymphocytic leukemia (16Goodman G.R. Bethel K.J. Saven A. Curr. Opin. Hematol. 2003; 10: 258-266Crossref PubMed Scopus (67) Google Scholar, 17Robak T. Transfus. Apher. Sci. 2005; 32: 33-44Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Because dCK plays an essential role in the therapeutic efficacy of nucleoside analogues, identification of mechanisms that control dCK activity is of particular interest. In recent years, several genotoxic agents, including DNA polymerase and topoisomerase II inhibitors, UV light, γ-irradiation, and nucleoside analogues such as CdA, have been shown to activate dCK in human normal or leukemic lymphocytes (18Sasvari-Szekely M. Spasokoukotskaja T. Szoke M. Csapo Z. Turi A. Szanto I. Eriksson S. Staub M. Biochem. Pharmacol. 1998; 56: 1175-1179Crossref PubMed Scopus (43) Google Scholar, 19Spasokoukotskaja T. Sasvari-Szekely M. Keszler G. Albertioni F. Eriksson S. Staub M. Eur. J. Cancer. 1999; 35: 1862-1867Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 20Cardoen S. Van Den Neste E. Smal C. Rosier J.-F. Delacauw A. Ferrant A. Van den B erghe G. Bontemps F. Clin. Cancer Res. 2001; 7: 3559-3566PubMed Google Scholar, 21Csapo Z. Sasvari-Szekely M. Spasokoukotskaja T. Talianidis I. Eriksson S. Staub M. Biochem. Pharmacol. 2001; 61: 191-197Crossref PubMed Scopus (41) Google Scholar, 22Van Den Neste E. Smal C. Cardoen S. Delacauw A. Frankard J. Ferrant A. Van den Berghe G. Bontemps F. Biochem. Pharmacol. 2003; 65: 573-580Crossref PubMed Scopus (30) Google Scholar, 23Csapo Z. Keszler G. Safrany G. Spasokoukotskaja T. Talianidis I. Staub M. Sasvari-Szekely M. Biochem. Pharmacol. 2003; 65: 2031-2039Crossref PubMed Scopus (37) Google Scholar). Activation of dCK by these agents could not be explained by allosteric effects or by a change in dCK protein levels, suggesting that dCK activity might be regulated by reversible covalent modification, e.g. via phosphorylation/dephosphorylation. Accordingly, the activity of dCK in extracts of normal or leukemic lymphocytes was markedly decreased on treatment with λ-protein phosphatase (23Csapo Z. Keszler G. Safrany G. Spasokoukotskaja T. Talianidis I. Staub M. Sasvari-Szekely M. Biochem. Pharmacol. 2003; 65: 2031-2039Crossref PubMed Scopus (37) Google Scholar) or protein phosphatase 2A (24Smal C. Cardoen S. Bertrand L. Delacauw A. Ferrant A. Van den Berghe G. Van Den Neste E. Bontemps F. Biochem. Pharmacol. 2004; 68: 95-103Crossref PubMed Scopus (25) Google Scholar). Also, further studies indicated that dCK might be dephosphorylated in vivo by protein phosphatase 2A (24Smal C. Cardoen S. Bertrand L. Delacauw A. Ferrant A. Van den Berghe G. Van Den Neste E. Bontemps F. Biochem. Pharmacol. 2004; 68: 95-103Crossref PubMed Scopus (25) Google Scholar). Moreover, dCK activity was surprisingly enhanced by several cell-permeable protein kinase inhibitors in various types of leukemic cells (24Smal C. Cardoen S. Bertrand L. Delacauw A. Ferrant A. Van den Berghe G. Van Den Neste E. Bontemps F. Biochem. Pharmacol. 2004; 68: 95-103Crossref PubMed Scopus (25) Google Scholar) and decreased by hyperosmotic stress, known to induce extensive changes in several cell signaling pathways (23Csapo Z. Keszler G. Safrany G. Spasokoukotskaja T. Talianidis I. Staub M. Sasvari-Szekely M. Biochem. Pharmacol. 2003; 65: 2031-2039Crossref PubMed Scopus (37) Google Scholar). Despite all the evidence suggesting that dCK is regulated by reversible phosphorylation, direct demonstration was lacking. To verify that dCK is indeed phosphorylated, human dCK was expressed in human embryonic kidney (HEK) 293T cells as a His-tagged fusion protein. Following incubation of the cells with 32Porthophosphate and purification of dCK, four phosphorylation sites were identified by mass spectrometric analysis of tryptic peptides. Phosphorylation of Ser-74, the most 32P-labeled in was to be crucial for dCK activity in HEK 293T In addition, in cells showed that Ser-74 phosphorylation could also be for dCK activity in leukemic was and were DMEM, and DNA were for and mutagenesis were and polymerase were polymerase and were and were or 293T cells, provided by J. Van of were in Dulbecco’s modified Eagle’s with and in an of in cells, a human lymphocytic leukemia cell were as (24Smal C. Cardoen S. Bertrand L. Delacauw A. Ferrant A. Van den Berghe G. Van Den Neste E. Bontemps F. Biochem. Pharmacol. 2004; 68: 95-103Crossref PubMed Scopus (25) Google Scholar). and Site-directed of dCK human was by and in II for DNA and DNA was to the were used as a to by and The dCK of all was on a to verify the and the of The dCK was the between the and sites for of normal or fusion with a the and of HEK 293T cells were in and the following by the to the and by of After cells were with phosphate-buffered and in inhibitors and and were by of were for and were on a After with B inhibitors, and by with B with dCK was with B of on the the protein was in the of by with Biochem. PubMed Scopus Google Scholar), or in of cell was in of protein phosphatase of cell protein were with or λ-protein as (24Smal C. Cardoen S. Bertrand L. Delacauw A. Ferrant A. Van den Berghe G. Van Den Neste E. Bontemps F. Biochem. Pharmacol. 2004; 68: 95-103Crossref PubMed Scopus (25) Google Scholar). After were for the of dCK activity. with antibody was also on of cell protein dCK 293T cell extracts were as of cells and dCK was as reported (24Smal C. Cardoen S. Bertrand L. Delacauw A. Ferrant A. Van den Berghe G. Van Den Neste E. Bontemps F. Biochem. Pharmacol. 2004; 68: 95-103Crossref PubMed Scopus (25) Google Scholar), of cell protein or of dCK for the of activity. for the for deoxycytidine, dCK activity was with deoxycytidine and ATP in the The protein of was by the of Biochem. PubMed Scopus Google Scholar) by as and antibody against the of human dCK was by to the of P. M. Eriksson S. Talianidis I. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). antibody was antibody was in New with a to of human dCK with an for to HEK 293T cell or dCK were to in and to The were in with and with antibody anti-phospho-Ser-74 antibody or antibody in for or After extensive in the were for with the antibody to After further extensive in the were enhanced of dCK or phosphorylation was by of In with 293T cells were and as after the cells were with and for with the 32Porthophosphate were used for was by the cells with were in of A. dCK was cell extracts by as by and to dCK was cell extracts with antibody to protein were with of and with and The were with and to Phosphorylation by in vivo phosphorylation HEK 293T cells were labeled as that cells were for with and labeling and that the latter was in the of After purification by and to dCK were the and The were with and with the tryptic digestion and a of peptides A. R. J. U. T. J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The of in the was as by by tryptic peptides of 32P-labeled HEK 293T cells were with peptides of HEK 293T cells the The extracts were to in a and was to a of were by a of A. R. J. U. T. J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The of was by were to with a in and by ionization tandem mass in an mass were in and to and their The was and the was to the for were identified in by the of and the phosphorylated was in have been or The results of are as the was by the of dCK in HEK 293T activity of dCK after as a fusion protein in HEK 293T cells was in purification of by a major in that was following with antibody not This that of by E. Mitchell B.S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) for the dCK is in with the mass of the fusion protein a of dCK activity with a mass of that the active of dCK is a not as reported for the enzyme C. Eriksson S. PubMed Scopus Google Scholar). The of for deoxycytidine, with ATP as was in with reported for dCK expressed in M. A. Biochem. Pharmacol. 1995; PubMed Scopus Google Scholar). of HEK 293T cells were with λ-protein which to a decrease in dCK activity as for the enzyme normal Z. Sasvari-Szekely M. Spasokoukotskaja T. Talianidis I. Eriksson S. Staub M. Biochem. Pharmacol. 2001; 61: 191-197Crossref PubMed Scopus (41) Google Scholar) or leukemic human lymphocytes (24Smal C. Cardoen S. Bertrand L. Delacauw A. Ferrant A. Van den Berghe G. Van Den Neste E. Bontemps F. Biochem. Pharmacol. 2004; 68: 95-103Crossref PubMed Scopus (25) Google Scholar). Moreover, in activity was by a in of the protein on as for phosphorylated These results indicated that the activity of dCK expressed in cells could be regulated by phosphorylation/dephosphorylation. In Phosphorylation of verify that dCK was phosphorylated in HEK 293T cells dCK were with 32Porthophosphate for to the ATP and also of dCK by by and labeling of a This labeled to the major by and to the by the that the labeled was indeed dCK. The phosphorylation of a protein is to be of its labeling in with a change in we the of hyperosmotic stress, which been shown to decrease dCK activity in human lymphocytes (23Csapo Z. Keszler G. Safrany G. Spasokoukotskaja T. Talianidis I. Staub M. Sasvari-Szekely M. Biochem. Pharmacol. 2003; 65: 2031-2039Crossref PubMed Scopus (37) Google Scholar). HEK 293T cells were with or for the of the incubation with and dCK was by with antibody by The of protein was in and in cells, as by that the labeling of the to dCK was markedly after which to a decrease in dCK activity in These that dCK activity can be controlled by in intact of in Phosphorylation in incubation with HEK 293T cells were for with which was In addition, was the incubation with 32Porthophosphate to protein This be to as the in a dCK activity that was the activity. dCK was by and with were by HPLC, and were by of radioactive were the of phosphorylation sites in the protein labeled to were with in the for radioactive was for by of by II a with a to a tryptic of the of dCK The of and to the and are by of the was of the of a in the dCK The of the tryptic was by and the phosphorylated was identified as to a tryptic of dCK of the tryptic identified and as the phosphorylated The tryptic in was detected with four of of by covalent of by or the of or In was of ionization or of by other peptides in a was detected in and Ser-74 was identified as the phosphorylated This was also with of of or its by and of of dCK by were by were identified by of and the phosphorylated was further identified by in or phosphorylated not in a In analysis of peptides by indicated that dCK was phosphorylated in vivo on four Thr-3, Ser-11, Ser-15, and The labeling of the was in of the of indicated that the major in vivo phosphorylation of dCK was Ser-74 by and and and of is that the Ser-74 is between human and other with dCK and whereas the phosphorylation in the of dCK are The Ser-74 a of the phosphorylation the and by a a a and a the and of dCK phosphorylation sites in The following dCK or protein were human number number number number number number The were by The the of of to the identified phosphorylation sites in human dCK. are in phosphorylated are in are in in a of Ser-74 Phosphorylation on dCK in HEK 293T To phosphorylation of Ser-74, the most phosphorylated in was for dCK activity, HEK 293T cells were with His-tagged dCK. Ser-74 was by to phosphorylation or by to its phosphorylation The did not dCK activity whereas the markedly decreased activity suggesting that phosphorylation of Ser-74 is a crucial for dCK activity. of for deoxycytidine were and for the and the dCK to the of dCK Although treatment with λ-protein phosphatase dCK activity treatment did not dCK activity of the of protein after a treatment of protein for the suggesting that the other phosphorylation sites of dCK are or not in the control of its activity. To verify we dCK in which Thr-3, Ser-11, and were by or of these modified dCK activity not HEK 293T cells the or were for with in the of to dCK activity, as for dCK, suggesting that might by the of To verify a antibody was and used to of HEK 293T cells dCK with or to the for the and to a with antibody as a control for dCK protein The in that dCK was by the the was decreased in with λ-protein in with a decrease in dCK activity The in that dCK phosphorylation in cells with was markedly suggesting that Ser-74 was by and was in the decrease in dCK activity in the cells extracts cells were with λ-protein the phosphorylation was and dCK activity was further of on Ser-74 phosphorylation in HEK 293T cells dCK. HEK 293T cells with dCK were with or for were with or λ-protein phosphatase for and to by dCK was detected with the anti-phospho-Ser-74 antibody or with antibody as a control for dCK are The of Ser-74 phosphorylation expressed in to after of and to or to an λ-protein phosphatase was to the dCK in the used for the are the the Phosphorylation of Ser-74 in to of dCK by reversible phosphorylation in leukemic cells T. Sasvari-Szekely M. Keszler G. Albertioni F. Eriksson S. Staub M. Eur. J. Cancer. 1999; 35: 1862-1867Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 22Van Den Neste E. Smal C. Cardoen S. Delacauw A. Frankard J. Ferrant A. Van den Berghe G. Bontemps F. Biochem. Pharmacol. 2003; 65: 573-580Crossref PubMed Scopus (30) Google Scholar, C. Cardoen S. Bertrand L. Delacauw A. Ferrant A. Van den Berghe G. Van Den Neste E. Bontemps F. Biochem. Pharmacol. 2004; 68: 95-103Crossref PubMed Scopus (25) Google Scholar). To Ser-74 phosphorylation might a role in the control of dCK activity in these cells, we the effects of agents that we to dCK activity dCK in leukemic cells Den Neste E. Smal C. Cardoen S. Delacauw A. Frankard J. Ferrant A. Van den Berghe G. Bontemps F. Biochem. Pharmacol. 2003; 65: 573-580Crossref PubMed Scopus (30) Google Scholar, C. Cardoen S. Bertrand L. Delacauw A. Ferrant A. Van den Berghe G. Van Den Neste E. Bontemps F. Biochem. Pharmacol. 2004; 68: 95-103Crossref PubMed Scopus (25) Google Scholar). cells were with the or UV reported to activation of dCK. After cell were with the anti-phospho-Ser-74 antibody or with the antibody could phosphorylation of Ser-74 in Moreover, we that Ser-74 phosphorylation increased that enhanced dCK activity. showed that and UV increased Ser-74 phosphorylation by and whereas dCK were not Our study the direct demonstration that dCK, a key enzyme in the salvage pathway and in the activation of nucleoside analogues used in anti-cancer and antiviral is phosphorylated and regulated in vivo by phosphorylation/dephosphorylation. used to the in vivo phosphorylation of a particular protein is to intact cells with 32Porthophosphate and to protein is Because dCK phosphorylation protein could be not detected in leukemic cells with 32Porthophosphate of of we overexpressed dCK in HEK 293T The of λ-protein phosphatase to decrease dCK activity in of HEK 293T cells that the dCK might be phosphorylated and that HEK 293T cells dCK as a to study dCK This labeling dCK with and after purification of dCK, tryptic and mass identification of four phosphorylation sites on the Thr-3, Ser-11, Ser-15, and the S. S. J. 1999; PubMed Scopus Google Scholar), for phosphorylation of and Ser-74 were and for Ser-11, and the for phosphorylation of phosphorylation sites exist as by the labeling of and in the in which the phosphorylated was not The of human dCK was by E. S. C. M. A. 2003; 10: PubMed Scopus Google Scholar). The enzyme is with a to that for the of a The of dCK that of the four phosphorylation sites identified is and its could not be is to the protein Ser-74 is in a the four phosphorylation sites we identified are in the of the protein and be to protein Ser-74 was identified as the major phosphorylated in 32P-labeled to a decreased the activity of dCK expressed in HEK 293T cells, whereas of Ser-74 to to phosphorylation was These results that phosphorylation of Ser-74 is for dCK activity. phosphorylation of Ser-74 is for the control of dCK activity is by that activity of the was not decreased by λ-protein phosphatase Moreover, of Thr-3, Ser-11, and to did not dCK activity, suggesting that phosphorylation of these is not essential for dCK activity. phosphorylation of these sites might be in other types of enzyme or which are by was shown to decrease dCK activity (23Csapo Z. Keszler G. Safrany G. Spasokoukotskaja T. Talianidis I. Staub M. Sasvari-Szekely M. Biochem. Pharmacol. 2003; 65: 2031-2039Crossref PubMed Scopus (37) Google Scholar) and was used as a to induce a change in dCK activity. we that decreased dCK activity and labeling of the that activity of dCK can be correlated to its phosphorylation Moreover, of Ser-74 to or the of to dCK activity, suggesting that of Ser-74 on treatment with This was by use of an anti-phospho-Ser-74 the detected with antibody in HEK 293T cells was after incubation with These results not that Ser-74 phosphorylation dCK activity also an the by which dCK the signaling pathway by which Ser-74 phosphorylation to be the protein in Ser-74 phosphorylation and the control of dCK activity, PubMed Scopus Google Scholar) and M. 2004; PubMed Scopus (22) Google Scholar) that Ser-74 could be a protein kinase site. and PubMed Scopus Google Scholar) that dCK leukemic could be phosphorylated in vitro by human dCK, expressed in was a T. Csapo Z. Sasvari-Szekely M. S. Talianidis I. Eriksson S. Staub M. 2000; PubMed Google Scholar). Also, inhibitors or of did not dCK activity in intact leukemic lymphocytes (24Smal C. Cardoen S. Bertrand L. Delacauw A. Ferrant A. Van den Berghe G. Van Den Neste E. Bontemps F. Biochem. Pharmacol. 2004; 68: 95-103Crossref PubMed Scopus (25) Google Scholar), suggesting that not role in the control of dCK activity in studies are to the protein for dCK the of dCK, protein phosphatase 2A might be or (24Smal C. Cardoen S. Bertrand L. Delacauw A. Ferrant A. Van den Berghe G. Van Den Neste E. Bontemps F. Biochem. Pharmacol. 2004; 68: 95-103Crossref PubMed Scopus (25) Google Scholar). Keszler G. Spasokoukotskaja T. Csapo Z. Talianidis I. Eriksson S. Staub M. Sasvari-Szekely M. Biochem. Pharmacol. 2004; 67: PubMed Scopus Google Scholar) that activation of dCK was by a This was on the that extracts or cells were in by the antibody against dCK extracts control was to induce a for the phosphorylation of Ser-74, which is the active might induce changes in dCK and an as by Keszler G. Spasokoukotskaja T. Csapo Z. Talianidis I. Eriksson S. Staub M. Sasvari-Szekely M. Biochem. Pharmacol. 2004; 67: PubMed Scopus Google Scholar). an of is the that phosphorylation of Ser-74 was also detected in the leukemic cells and that Ser-74 phosphorylation was enhanced in these cells by agents known to dCK activity. These results a role for Ser-74 phosphorylation in the control of dCK activity, in leukemic These could be for the activation of nucleoside analogues used in anti-cancer and antiviral or for the of active dCK for S. M. G. Van den and E. Van for or for the of the