Identification of in Vivo Phosphorylation Sites on Human Deoxycytidine Kinase

Abstract

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...

Protein phosphatase 2A regulates deoxycytidine kinase activity via Ser‐74 dephosphorylation

“…OA was from Enzo Life Sciences. Anti-phospho-Ser-74 and anti-dCK antibodies were generated as described previously [11]. Anti-poly(His) monoclonal antibody, protein A Sepharose beads and NAP-5 columns were from GE Healthcare.…”

“…CCRF-CEM cells and HEK293T cells that stably express human dCK were cultured as described [11,14]. EBV-transformed lymphoblasts GM0536 (ATM+/+) and GM1526 (ATMÀ/À), purchased from The NIGMS human Mutant Cell Repository, were maintained in RPMI-1640 with Glutamax supplemented with 15% heat-inactivated FCS.…”

“…Thereafter, several studies [7][8][9][10] suggested that dCK activity could be regulated by posttranslational modification, which was definitively confirmed by our group. We established that dCK is a phosphoprotein containing at least four phosphorylation sites: Thr-3, Ser-11, Ser-15 and Ser-74, the latter being the major phosphorylated residue and the only one to play a role in the regulation of dCK activity [11]. Furthermore, we found that increase in dCK activity induced by genotoxic treatments, such as etoposide, aphidicolin, UV-irradiation, was correlated to an increase of Ser-74 phosphorylation, whereas decrease of its activity induced by osmotic stress was related to Ser-74 dephosphorylation [11,12].…”

“…We established that dCK is a phosphoprotein containing at least four phosphorylation sites: Thr-3, Ser-11, Ser-15 and Ser-74, the latter being the major phosphorylated residue and the only one to play a role in the regulation of dCK activity [11]. Furthermore, we found that increase in dCK activity induced by genotoxic treatments, such as etoposide, aphidicolin, UV-irradiation, was correlated to an increase of Ser-74 phosphorylation, whereas decrease of its activity induced by osmotic stress was related to Ser-74 dephosphorylation [11,12]. Insight into the mechanism by which Ser-74 phosphorylation modifies dCK activity has recently been provided through structural studies of Hazra et al [13], who propose that phosphorylation of Ser-74 favors the adoption by dCK of an open conformation more conducive for binding of nucleoside and release of the nucleoside monophosphate product.…”

Noninvasive prediction of tumor responses to gemcitabine using positron emission tomography

“…Membranes were probed with primary antibodies for 1 h at room temperature. The rabbit polyclonal antibody against DCK was provided by Françoise Bontemps (University Catholique de Louvain, Belgium) (44). The mouse anti-actin antibody (A4700; Sigma) was used as loading control.…”