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 [ 32 P]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 32 P-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 4 ; 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 (1). In addition, dCK initiates the activation of several chemotherapeutic nucleoside analogues, such as 1--D-arabinosylcytosine (cytarabine), 9--D-arabinosyl-2-fluoroadenine (fludarabine), and 2-chloro-2Ј-deoxyadenosine (CdA, cladribine), commonly used in the treatment of hematological malignancies, and 2Ј,2Ј-difluorodeoxycytidine (gemcitabine), active against solid malignant tumors (2-4). The anti-human immunodeficiency virus drugs 2Ј,3Ј-dideoxycytidine (zalcitabine) and 2Ј-deoxy-3Ј-thiacytidine (lamivudine) are also phosphorylated by dCK (5). Phosphorylation of these inactive pro-drugs by dCK is a prerequisite for their pharmacological action, as demonstrated by the resistance of cells lacking dCK activity to nucleoside analogues (6 -9). Moreover, a number of in vitro and in vivo studies indicated a positive correlation between dCK activity and nucleoside analogue sensitivity (10 -15). The enzyme is preferentially expressed in lymphoid cells (1), which explains the clinical success of nucleoside analogues against lymphoproliferative disorders, such as hairy cell leukemia and B-cell chronic lymphocytic leukemia (16,17...
Deoxycytidine kinase (dCK) is a key enzyme in the salvage of deoxyribonucleosides and in the activation of several anticancer and antiviral nucleoside analogues. We have recently shown that dCK is a phosphoprotein. Four in vivo phosphorylation sites were identified: Thr-3, Ser-11, Ser-15, and Ser-74. Site-directed mutagenesis demonstrated that phosphorylation of Ser-74, the major phosphorylated residue, strongly influences dCK activity in eucaryotic cells. Here, we show that phosphorylation of the three other sites, located in the N-terminal extremity of the protein, does not significantly modify dCK activity, but phosphorylation of Thr-3 could promote dCK stability.
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