New evidences for a regulation of deoxycytidine kinase activity by reversible phosphorylation

Recent studies indicate that deoxycytidine kinase (dCK), which activates various nucleoside analogues used in antileukemic therapy, can be regulated by post-translational modification, most probably through reversible phosphorylation. To further unravel its regulation, dCK was overexpressed in HEK-293 cells as a His-tag fusion protein. Western blot analysis showed that purified overexpressed dCK appears as doublet protein bands. The slower band disappeared after treatment with protein phosphatase lambda (PP lambda) in parallel with a decrease of dCK activity, providing additional arguments in favor of both phosphorylated and unphosphorylated forms of dCK.     

Identification of Phosphorylation Sites on Human Deoxycytidine Kinase After Overexpression in Eucaryotic Cells

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.     

Immunocytochemical Detection of Deoxycytidine Kinase in Pediatric Malignancies in Relation to In Vitro Cytarabine Sensitivity

Deoxycytidine kinase (dCK) is essential for the phosphorylation of cytarabine (ara‐C), a deoxycytidine analog active against acute leukemias. Resistance to ara‐C has been linked to dCK deficiency. In this study we determined the expression of the dCK protein in pediatric malignancies, using immunocytochemistry and related the expression levels to in vitro ara‐C sensitivity (measured with the MTT‐assay). dCK expression was high in the AML and retinoblastoma samples, in the ALL samples dCK expression ranged from low to very high. The brain tumor samples expressed low levels of dCK. AML was significantly more sensitive in vitro to ara‐C compared to ALL (p = 0.03). Retinoblastoma and brain tumor cells were extremely resistant in vitro, we were unable to detect more than 50% ara‐C induced cell kill in the majority of samples. Samples were combined in groups according to dCK expression. Samples with low dCK expression were significantly more resistant to ara‐C compared to samples with high dCK expression. In conclusion, dCK expression varies between individual samples and between different types of malignancies and may play a role in resistance to ara‐C in particular tumor types.     

Casein kinase 1δ activates human recombinant deoxycytidine kinase by Ser-74 phosphorylation, but is not involved in the in vivo regulation of its activity

Deoxycytidine kinase (dCK) is a key enzyme in the salvage of deoxynucleosides and in the activation of several anticancer and antiviral nucleoside analogues. We recently showed that dCK was activated in vivo by phosphorylation of Ser-74. However, the protein kinase responsible was not identified. Ser-74 is located downstream a Glu-rich region, presenting similarity with the consensus phosphorylation motif of casein kinase 1 (CKI), and particularly of CKI δ. We showed that recombinant CKI δ phosphorylated several residues of bacterially overexpressed dCK: Ser-74, but also Ser-11, Ser-15, and Thr-72. Phosphorylation of dCK by CKI δ correlated with increased activity reaching at least 4-fold. Site-directed mutagenesis demonstrated that only Ser-74 phosphorylation was involved in dCK activation by CKI δ, strengthening the key role of this residue in the control of dCK activity. However, neither CKI δ inhibitors nor CKI δ siRNA-mediated knock-down modified Ser-74 phosphorylation or dCK activity in cultured cells. Moreover, these approaches did not prevent dCK activation induced by treatments enhancing Ser-74 phosphorylation. Taken together, the data preclude a role of CKI δ in the regulation of dCK activity in vivo. Nevertheless, phosphorylation of dCK by CKI δ could be a useful tool for elucidating the influence of Ser-74 phosphorylation on the structure-activity relationships in the enzyme.     

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

Quantitative immunoassay of human deoxycytidine kinase in malignant cells.

Deoxycytidine kinase (dCK) is necessary for the activity of several nucleosides used for the chemotherapy of cancer and AIDS. However, the measurement of dCK catalytic activity in crude cell extracts may be imprecise, due to the presence of phosphatases and nucleotidases that degrade the enzyme products. We describe a simple immunoassay for dCK that can measure accurately as little as 5 ng enzyme protein in crude tissue extracts. The assay enabled us to show (i) that mutant cells deficient in dCK activity lack immunoreactive dCK protein, (ii) that dCK catalytic activity and immunoreactivity correlate closely in human tumors, and (iii) that immunoreactive dCK is particularly high in lymphocytes and lymphoid malignancies, although certain solid tumors may also contain the enzyme. The immunoassay of dCK could prove useful in the selection and monitoring of patients who are being treated with nucleosides that are activated by this enzyme.     

Deoxycytidine Kinase is Reversibly Phosphorylated in Normal Human Lymphocytes

The activity of deoxycytidine kinase (dCK) has been shown to be enhanced upon genotoxic stress in human lymphocytes, and reversible phosphorylation of the enzyme has been implicated in the activation process. Here, we provide compelling evidence that dCK is a cytosolic phosphoprotein. Two-dimensional gel electrophoresis revealed that dCK has several differentially charged isoforms in cells. One-third of total cellular dCK was bound to a phosphoprotein-binding column irrespective of its activity levels, indicating that other mechanisms rather than phosphorylation alone might also be involved in the stimulation of enzyme activity. We excluded the possibility that activated dCK is translocated to the nucleus, but identified a dCK isoform of low abundance with a higher molecular weight in the nuclear fractions.     

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

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.     

Fluorescence Studies of Substrate Binding to Human Recombinant Deoxycytidine Kinase

Deoxycytidine kinase (dCK), is responsible for the phosphorylation of deoxynucleosides to the corresponding monophosphates using ATP or UTP as phosphate donors. Steady‐state intrinsic fluorescence measurements were used to study interaction of dCK with substrates in the absence and presence of phosphate donors. Enzyme fluorescence quenching by its substrates exhibited unimodal quenching when excited at 295 nm. Binding of substrates induced conformational changes in the protein, suggesting that dCK can assume different conformational states with different substrates and may account for the observed differences in their specificity. dCK bound the substrates more tightly in the presence of phosphate donors and UTP is the preferred phosphate donor. Among the substrates tested, the antitumour drugs gemcitabine and cladribine were bound very tightly by dCK, yielding K_d values of 0.75 and 0.8 µM, respectively, in the presence of UTP.     

Quantitative Real Time PCR of Deoxycytidine Kinase mRNA by Light Cycler PCR; in Relation to Enzyme Activity

Deoxycytidine kinase (dCK) is essential for the phosphorylation of several deoxyribonucleosides and various analogues such as gemcitabine (2′,2′‐difluorodeoxycytidine). We developed and optimized a sensitive real time Light Cycler (LC) PCR assay for dCK with SYBR green detection. The enzymatic activity measured in the same human xenografts of dCK correlated excellently with dCK mRNA expression levels measured by the LC. This assay can be used for evaluation of dCK expression in tumors.     

Selective Increase of dATP Pools upon Activation of Deoxycytidine Kinase in Lymphocytes: Implications in Apoptosis

Stimulation of the activity of deoxycytidine kinase (dCK), the principal deoxynucleoside salvage enzyme, has been recently considered as a protective cellular response to a wide range of agents interfering with DNA repair and apoptosis. In light of this, the potential contribution of dCK activation to apoptosis induction—presumably by supplying dATP or its analogues for the apoptosome formation—deserves consideration. Two‐hour exposure of human tonsillar lymphocytes to 2‐chloro‐deoxyadenosine (CdA) led to a two‐fold activation of dCK. This activation process was inhibited by pifithrin‐α, a potent inhibitor of p53. When the dNTP pools were determined, both deoxypyrimidine triphosphate and dGTP pools were reduced after the treatments, while dATP levels elevated by 62%, 77% and 50% in the CdA, aphidicolin and etoposide‐treated cells, respectively. We assume that dCK activation elicited by cellular damage might be a proapoptotic factor in terms of generating dATP well before the release of cytochrome c and deoxyguanosine kinase from mitochondria.     

The Apoptotic Effects of Toosendanin Are Partially Mediated by Activation of Deoxycytidine Kinase in HL-60 Cells

Triterpenoid toosendanin (TSN) exhibits potent cytotoxic activity through inducing apoptosis in a variety of cancer cell lines. However, the target and mechanism of the apoptotic effects by TSN remain unknown. In this study, we captured a specific binding protein of TSN in HL-60 cells by serial affinity chromatography and further identified it as deoxycytidine kinase (dCK). Combination of direct activation of dCK and inhibition of TSN-induced apoptosis by a dCK inhibitor confirmed that dCK is a target for TSN partially responsible for the apoptosis in HL-60 cells. Moreover, the activation of dCK by TSN was a result of conformational change, rather than auto-phosphorylation. Our results further imply that, in addition to the dATP increase by dCK activation in tumor cells, dCK may also involve in the apoptotic regulation.     

Mimicking phosphorylation of Ser-74 on human deoxycytidine kinase selectively increases catalytic activity for dC and dC analogues

Intracellular phosphorylation of dCK on Ser-74 results in increased nucleoside kinase activity. We mimicked this phosphorylation by a Ser-74-Glu mutation in bacterially produced dCK and investigated kinetic parameters using various nucleoside substrates. The S74E mutation increases the k_cat values 11-fold for dC, and 3-fold for the anti-cancer analogues dFdC and AraC. In contrast, the rate is decreased for the purine substrates. In HEK293 cells, we found that by comparing transiently transfected dCK(S74E)-GFP and wild-type dCK-GFP, mimicking the phosphorylation of Ser-74 has no effect on cellular localisation. We note that phosphorylation may represent a mechanism to enhance the catalytic activity of the relatively slow dCK enzyme.     

Modulation of human deoxycytidine kinase activity as a response to cellular stress induced by NaF.

Deoxycytidine kinase (dCK) is one of the key enzymes of deoxynucleoside salvage supplying resting lymphocytes with DNA precursors for synthesis and repair. The level of dCK activity is especially important in chemotherapy with the use of deoxynucleoside analogues like arabinosyl cytosine (Citarabid, ara-C), or 2-chloro-deoxyadenosine (Cladribine, CdA). Previous results showed that Cladribine treatment of human lymphocytes increased several fold the activity of dCK without increasing the amount of dCK protein itself (Sasvári-Székely, et al., 1998, Biochem. Pharmacol. 56, 1175), and a possible post-translational modification was suggested. This theory was further investigated using NaF as an inhibitor of protein phosphatases. It was shown that NaF treatment of cells elevated dCK activity while inhibiting DNA synthesis. The possible mechanism of dCK activation/inactivation induced by exposure of cell cultures to different agents is discussed.     

Special function of deoxycytidine kinase (dCK) in the activation of chemotherapeutic nucleoside analogs and in the inhibition of cell proliferation

Deoxycytidine kinase (dCK) plays a central role in the deoxynucleoside salvage processes, phosphorylating dC, dA, and dG to their monophosphates. In mammalian cells, the major source of dTTP comes also from dC via dCMP deaminase. Moreover, based on its broad substrate specificity, this enzyme is responsible for the activation of several nucleoside analogues of therapeutical importance, influencing the sensitivity of malignant tissues towards chemotherapy. The expression of dCK is highest in different lymphoid cells/tissues, in embryonic cells and in most malignant cells (2, 7, 13-15, 18). The activity of dCK is not cell cycle-regulated. In contrast to this, dCK activity was found to be elevated several fold upon short-term treatments of normal human lymphocytes with therapeutic nucleoside analogs, and other genotoxic agents as well as by DNA damaging agents including the DNA polymerase inhibitor aphidicolin, the topoisomerase II inhibitor etoposide and gamma-irradiation, which might be a potentially important phenomenon with respect to the clinical practice, too. These findings indicated that the main trigger of activation could be the damaged DNA itself, and the biological relevance might be to supply the dNTPs for the enhanced DNA repair. Activation of dCK was paralleled by elevated levels of intracellular dATP, raising the possibility that dCK activation is linked to the induction of apoptosis. With regard to the mechanism of enzyme activation, no changes were found in the protein and mRNA levels of dCK upon stimulation, while the activation process was calcium dependent and comprised a protein phosphorylation step. A positive correlation was found between the enzymatic activity and the native immunoreactivity of dCK, strongly arguing that dCK undergoes a conformational change during activation, which results in the formation of a catalytically more active steric structure (8-11, 22, 26, 32-34, 35, 36).     

Fluorescence studies of substrate binding to human recombinant deoxycytidine kinase

Deoxycytidine kinase (dCK), is responsible for the phosphorylation of deoxynucleosides to the corresponding monophosphates using ATP or UTP as phosphate donors. Steady-state intrinsic fluorescence measurements were used to study interaction of dCK with substrates in the absence and presence of phosphate donors. Enzyme fluorescence quenching by its substrates exhibited unimodal quenching when excited at 295 nm. Binding of substrates induced conformational changes in the protein, suggesting that dCK can assume different conformational states with different substrates and may account for the observed differences in their specificity. dCK bound the substrates more tightly in the presence of phosphate donors and UTP is the preferred phosphate donor. Among the substrates tested, the antitumour drugs gemcitabine and cladribine were bound very tightly by dCK, yielding Kd values of 0.75 and 0.8 microM, respectively, in the presence of UTP.     

Fluorescence energy transfer studies of human deoxycytidine kinase: role of cysteine 185 in the conformational changes that occur upon substrate binding

Human deoxycytidine kinase (dCK) phosphorylates both pyrimidine and purine deoxynucleosides, including numerous nucleoside analogue prodrugs. Energy transfer studies of transfer between Trp residues of dCK and the fluorescent probe N-(1-pyrene)maleimide (PM), which specifically labels Cys residues in proteins, were performed. Two of the six Cys residues in dCK were labeled, yielding a protein that was functionally active. We determined the average distances between PM-labeled Cys residues and Trp residues in dCK in the absence and presence of various pyrimidine and purine nucleoside analogues with the Trp residues as energy donors and PM-labeled Cys residues as acceptors. The transfer efficiency was determined from donor intensity quenching and the F?rster distance R(0) at which the efficiency of energy transfer is 50%, which was 19.90 A for dCK-PM. The average distance R between the Trp residues and the labeled Cys residues in dCK-PM was 18.50 A, and once substrates bound, this distance was reduced, demonstrating conformational changes. Several of the Cys residues of dCK were mutated to Ala, and the properties of the purified mutant proteins were studied. PM labeled a single Cys residue in Cys-185-Ala dCK, suggesting that one of the two Cys residues labeled in wild-type dCK was Cys 185. The distance between the single PM-labeled Cys residue and the Trp residues in Cys-185-Ala dCK was 20.75 A. Binding of nucleosides had no effect on the pyrene fluorescence of Cys-185-Ala dCK, indicating that the conformational changes observed upon substrate binding to wild-type dCK-PM involved the "lid region" of which Cys 185 is a part. The substrate specificity of Cys-185-Ala dCK was altered in that dAdo and UTP were better substrates for the mutant than for the wild-type enzyme.     

Immunocytochemical detection of deoxycytidine kinase in pediatric malignancies in relation to in vitro cytarabine sensitivity

Deoxycytidine kinase (dCK) is essential for the phosphorylation of cytarabine (ara-C), a deoxycytidine analog active against acute leukemias. Resistance to ara-C has been linked to dCK deficiency. In this study we determined the expression of the dCK protein in pediatric malignancies, using immunocytochemistry and related the expression levels to in vitro ara-C sensitivity (measured with the MTT-assay). dCK expression was high in the AML and retinoblastoma samples, in the ALL samples dCK expression ranged from low to very high. The brain tumor samples expressed low levels of dCK. AML was significantly more sensitive in vitro to ara-C compared to ALL (p = 0.03). Retinoblastoma and brain tumor cells were extremely resistant in vitro, we were unable to detect more than 50% ara-C induced cell kill in the majority of samples. Samples were combined in groups according to dCK expression. Samples with low dCK expression were significantly more resistant to ara-C compared to samples with high dCK expression. In conclusion, dCK expression varies between individual samples and between different types of malignancies and may play a role in resistance to ara-C in particular tumor types.     

Deoxycytidine kinase regulates the G2/M checkpoint through interaction with cyclin-dependent kinase 1 in response to DNA damage

Deoxycytidine kinase (dCK) is a rate limiting enzyme critical for phosphorylation of endogenous deoxynucleosides for DNA synthesis and exogenous nucleoside analogues for anticancer and antiviral drug actions. dCK is activated in response to DNA damage; however, how it functions in the DNA damage response is largely unknown. Here, we report that dCK is required for the G2/M checkpoint in response to DNA damage induced by ionizing radiation (IR). We demonstrate that the ataxia–telangiectasia-mutated (ATM) kinase phosphorylates dCK on Serine 74 to activate it in response to DNA damage. We further demonstrate that Serine 74 phosphorylation is required for initiation of the G2/M checkpoint. Using mass spectrometry, we identified a protein complex associated with dCK in response to DNA damage. We demonstrate that dCK interacts with cyclin-dependent kinase 1 (Cdk1) after IR and that the interaction inhibits Cdk1 activity both in vitro and in vivo. Together, our results highlight the novel function of dCK and provide molecular insights into the G2/M checkpoint regulation in response to DNA damage.