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J. Biol. Chem., Vol. 281, Issue 8, 4887-4893, February 24, 2006
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1
¶2

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From the
Laboratory of Physiological Chemistry and
Hormone and Metabolic Research Unit, Christian de Duve Institute of Cellular Pathology, the ¶Division of Cardiology and the ||Department of Hematology, Cliniques Universitaires Saint-Luc, Université Catholique de Louvain, 1200 Brussels, Belgium
Received for publication, November 10, 2005 , and in revised form, December 16, 2005.
| ABSTRACT |
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| INTRODUCTION |
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-D-arabinosylcytosine (cytarabine), 9-
-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 (24). The anti-human immunodeficiency virus drugs 2',3'-dideoxycytidine (zalcitabine) and 2'-deoxy-3'-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 (69). Moreover, a number of in vitro and in vivo studies indicated a positive correlation between dCK activity and nucleoside analogue sensitivity (1015). 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).
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,
-irradiation, and nucleoside analogues such as CdA, have been shown to activate dCK in human normal or leukemic lymphocytes (1823). 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
-protein phosphatase (23) or protein phosphatase 2A (24). Also, further studies indicated that dCK might be dephosphorylated in vivo by protein phosphatase 2A (24). Moreover, dCK activity was surprisingly enhanced by several cell-permeable protein kinase inhibitors in various types of leukemic cells (24) and decreased by hyperosmotic stress, known to induce extensive changes in several cell signaling pathways (23).
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 [32P]orthophosphate and purification of dCK, four phosphorylation sites were identified by mass spectrometric analysis of tryptic peptides. Phosphorylation of Ser-74, the most 32P-labeled site in vivo, was found to be crucial for dCK activity in HEK 293T cells. In addition, data obtained in CCRF-CEM cells showed that Ser-74 phosphorylation could also be important for dCK activity in leukemic cells.
| EXPERIMENTAL PROCEDURES |
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Cell CultureHEK 293T cells, kindly provided by Prof. J. Van Lint (Department of Biochemistry, Katholiek Universiteit, Leuven, Belgium), were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum, 1% penicillin/streptomycin, and 2 mM ultraglutamine in an atmosphere of 5% CO2 in air. CCRF-CEM cells, a human T-acute lymphocytic leukemia cell line, were cultured as described elsewhere (24).
Plasmid Construction and Site-directed MutagenesisThe coding region of dCK from human CCRF-CEM lymphoblasts was amplified by PCR and first cloned in pBluescript II SK(+) vector for DNA sequencing and site-directed mutagenesis. Plasmid DNA was prepared using Qiagen midiprep kit according to the manufacturer's instructions. Wild type constructs were used as a template (20 ng) to create single mutations by PCR (T3A/T3E, S11A/S11E, S15A/S15E, and S74A/S74E). The complete dCK coding region of all plasmids was sequenced on a CEQ2000 sequencer (Beckman Coulter) to verify the newly introduced mutations and the absence of random mutations. The dCK cDNA was then subcloned into the eukaryotic pEF6/His vector between the BamHI and XbaI restriction sites for expression of normal or mutated polyhistidine fusion proteins with a His6 tag at the N-terminal position.
Transfection and Purification of Recombinant dCKFor transient transfections, 1.5 x 106 HEK 293T cells were plated in 8.5-cm dishes and transfected the following day by the jetPEI procedure (PolyPlus Transfection, Illkirch, France) according to the manufacturer's instructions and by using 8 µg of plasmid DNA. After 48 h, cells were washed with cold phosphate-buffered saline (PBS) and resuspended in lysis buffer (buffer A) containing 50 mM HEPES, pH 7.5, 50 mM NaF, 1 mM K2HPO4, 0.1% (w/v)
-mercaptoethanol, 5 mM
-glycerophosphate, 5 mM Na4P2O7, 1% (w/v) Triton X-100, protease inhibitors (1 mM p-toluenesulfonyl fluoride, 5 mM benzamidine, 5 µg/ml leupeptin, and anti-pain), and 1 mM sodium orthovanadate. Cells were lysed by one cycle of freeze-thawing. Cell lysates were centrifuged at 16,100 x g for 10 min, and supernatants were loaded on a Talon (Co2+) affinity column (Clontech). After three washings with buffer B containing 50 mM HEPES, pH 7.5, 0.1% (w/v)
-mercaptoethanol, protease inhibitors, and 300 mM NaCl, followed by two washings with buffer B supplemented with 10 mM imidazole, recombinant dCK was eluted with buffer B devoid of NaCl but containing 150 mM imidazole. Depending on the experiment, the purified protein was stored at -80 °C in the presence of 20% glycerol, concentrated by ultrafiltration, precipitated with chloroform/methanol (25), or boiled in Laemmli SDS-PAGE buffer.
Protein Phosphatase Treatment of Cell ExtractsFor dephosphorylation experiments, cell lysis was carried out in buffer A devoid of protein phosphatase inhibitors. Samples containing
15 µg of cell protein were incubated at 30 °C with or without
-protein phosphatase, as described previously (24). After 1 h, 10-µl aliquots were taken for the measurement of dCK activity. Western blotting with anti-poly(His) antibody was also performed on 1 µg of cell protein extract.
dCK AssayHEK 293T cell extracts were prepared as described above. Extraction of CCRF-CEM cells and dCK assay was performed as reported previously (24), using 440 µg of cell extract protein or 20200 ng of purified dCK for the measure of activity. Except for determining the Km value for deoxycytidine, dCK activity was measured under Vmax conditions, with 10 µM deoxycytidine and 5 mM ATP in the assays. The protein content of samples was measured by the method of Bradford (26) by using bovine serum albumin as standard.
Antibodies and ImmunoblottingAnti-dCK antibody raised against the C-terminal peptide of human dCK (amino acids 246260) was generated by Eurogentec, according to the procedure of Hatzis et al. (27). Anti-poly(His) monoclonal antibody was from GE Healthcare. Anti-phospho-Ser-74 antibody was raised in New Zealand White rabbits injected with a synthetic phosphopeptide corresponding to amino acids 6879 of human dCK with an N-terminal cysteine (CEELTMpSQKNGG), for coupling to keyhole limpet hemocyanin.
For immunoblot analysis, HEK 293T cell lysates (15 µg) or purified dCK (50100 ng) were subjected to SDS-PAGE in gels containing 12% (w/v) acrylamide and transferred to Hybond C-extra membranes (GE Healthcare, Roosendaal, The Netherlands). Membranes were blocked in PBS with 5% (w/v) fat-free milk powder and probed with either anti-poly(His) monoclonal antibody (1/2000), anti-phospho-Ser-74 antibody (1/1000), or anti-dCK antibody (1/1000) in PBS-T (Tween 0.1%, w/v) for 1 h at room temperature or overnight at 4 °C. After extensive washing in PBS-T, the membranes were incubated for 1 h at room temperature with the appropriate secondary antibody coupled to horseradish peroxidase (1/10,000). After further extensive washing in PBS-T, the blots were developed using enhanced chemiluminescence (GE Healthcare). Quantification of dCK expression or phosphorylation was carried out by using National Institutes of Health image software.
In Vivo Labeling with [32P]OrthophosphateHEK 293T cells were cultured and transfected as described above. Two days after transfection, the cells were washed with phosphate-free DMEM and incubated for 3.5 h with the same medium containing [32P]orthophosphate (carrier-free; GE Healthcare) (4 mCi/dish, 670 µCi/ml). Four dishes were used for each experiment. Labeling was stopped by washing the cells twice with ice-cold PBS. Cells were lysed in 0.8 ml of buffer A. dCK was purified from cell extracts by affinity chromatography as described above, concentrated by ultrafiltration, and subjected to SDS-PAGE/autoradiography. Alternatively, dCK was immunoprecipitated from cell extracts with anti-poly(His) antibody coupled to protein A-Sepharose (GE Healthcare). Immune complexes were washed six times with 1 ml of buffer A containing 0.5 M NaCl, and three times with 50 mM Tris-HCl, pH 7.5, containing 0.1 mM EGTA and 0.1% (w/v)
-mercaptoethanol. The beads were mixed with Laemmli buffer, boiled, and subjected to SDS-PAGE/autoradiography.
Phosphorylation Site Identification by Tandem Mass Spectrometry For in vivo phosphorylation site analysis, HEK 293T cells were labeled as described above, except that cells were preincubated for 1 h with 0.5 M sorbitol and washed before labeling and that the latter was performed in the presence of 0.5 µM okadaic acid. After purification by affinity chromatography, SDS-PAGE, and Colloidal Blue staining, bands corresponding to dCK were cut from the gel and chopped into 1-mm cubes. The gel pieces were washed, reduced with dithiothreitol, and treated with iodoacetamide prior the tryptic digestion and a two-step extraction of peptides (28). The yield of radioactivity in the two-step extraction was about 80% as determined by 32P counting by Cerenkov radiation. Labeled tryptic peptides obtained from 4 dishes of 32P-labeled HEK 293T cells were then mixed with peptides from 10 dishes of unlabeled HEK 293T cells using exactly the same protocol. The combined extracts were concentrated to about 20 µl in a SpeedVac® concentrator, and trifluoroacetic acid was added to a concentration of 0.1% (v/v). Peptides were separated by reverse-phase narrow-bore HPLC at a flow rate of 200 µl/min (28). The radioactivity of each fraction was measured by Cerenkov counting. Radioactive peaks were concentrated to about 10 µl, mixed with 0.1% (v/v) trifluoroacetic acid, desalted using a Ziptip C18 pipette tip (Millipore), eluted in 50% (v/v) acetonitrile, 0.3% (v/v) acetic acid, and analyzed by nanoelectrospray ionization tandem mass spectrometry (nano-ESI-MS/MS) in an LCQ Deca XP Plus ion-trap mass spectrometer (ThermoFinnigan, San Jose, CA). Spectra were taken in full MS and Zoom scan mode to determine parent ion monoisotopic masses and their charge states. The source voltage was set at 0.8 kV, and the collision-induced dissociation energy was adjusted to the minimum needed for fragmentation. Phosphopeptides were identified in MS2 mode by the loss of H3PO4 (98 Da) under low collision-induced dissociation energy, and the phosphorylated residue was pinpointed in MS3 mode.
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| RESULTS |
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Extracts of HEK 293T cells were incubated with
-protein phosphatase, which led to a marked (
85%) decrease in dCK activity (Fig. 1A), as observed previously for the native enzyme from normal (21) or leukemic human lymphocytes (24). Moreover, this drop in activity was accompanied by a slight increase in electrophoretic migration of the recombinant protein on SDS-polyacrylamide gel (Fig. 1B), as often observed for nonphosphorylated versus phosphorylated proteins. These results indicated that the activity of dCK expressed in eukaryotic cells could be regulated by phosphorylation/dephosphorylation.
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The phosphorylation of a protein is considered to be physiologically relevant, when modification of its 32P labeling occurs in parallel with a change in function. Therefore, we investigated the effect of hyperosmotic stress, which has been shown to decrease dCK activity in human lymphocytes (23). HEK 293T cells were treated with or without 0.5 M sorbitol for 1 h before the end of the incubation with [32P]orthophosphate, and dCK was isolated by immunoprecipitation with anti-poly(His) antibody followed by SDS-PAGE (Fig. 3A). The amount of immunoprecipitated protein was similar in sorbitol-treated and in untreated cells, as assessed by Colloidal Blue staining. By contrast, autoradiography revealed that the labeling of the band corresponding to recombinant dCK was markedly reduced after sorbitol treatment, which led to a decrease in dCK activity (72% in this experiment; Fig. 3B). These data suggest that dCK activity can be controlled by phosphorylation/dephosphorylation in intact cells.
Identification of in Vivo Phosphorylation Sites in dCKBefore incubation with [32P]orthophosphate, HEK 293T cells were treated for 1 h with 0.5 M sorbitol which was then washed out. In addition, okadaic acid was included during the incubation with [32P]orthophosphate to inhibit protein phosphatases. This strategy would be expected to increase 32P incorporation as the procedure resulted in a recovered dCK activity that was 1.5-fold higher than the initial activity. Radiolabeled dCK was purified by affinity chromatography and digested with trypsin. Peptides were separated by reverse-phase HPLC, and fractions were counted by emission of Cerenkov radiation. Several radioactive peaks were detected, indicating the presence of multiple phosphorylation sites in the recombinant protein (Fig. 4). Five labeled peaks (I to V) were observed, with peak I appearing in the flow-through fraction. Except for peak I, each radioactive peak was screened for phosphopeptides by neutral loss of H3PO4 (98 Da) by nano-ESI-MS/MS. Peak II contained a phosphopeptide consistent with a single phosphate addition to a predicted tryptic fragment of the N-terminal hexahistidine-tagged portion of dCK (Table 1). The first two amino acids of this fragment, aspartate and proline, belong to the expression vector pEF6/His and are followed by Ala-2 of dCK; the initial methionine was absent because of the introduction of a BamHI restriction site in the dCK cDNA. The sequence of the tryptic peptide was confirmed by fragmentation, and the phosphorylated residue was identified as Thr-3. Peak III corresponded to a tryptic peptide of dCK containing two phosphates. Fragmentation of the tryptic peptide identified Ser-11 and Ser-15 as the phosphorylated residues. The tryptic peptide present in peak III was detected with four different monoisotopic masses (Table 1) because of alkylation of Cys-9 by iodoacetamide, covalent modification of this residue by acrylamide, or the presence of one or two phosphates. In peak IV, no phosphopeptide was detected, possibly because of poor ionization or because of ion suppression by other peptides in this fraction. By contrast, a phosphopeptide was detected in peak V, and Ser-74 was identified as the phosphorylated residue (Table 1). This phosphopeptide was also present with different isotopic masses because of acrylamide modification of Cys-59 or its alkylation by iodoacetamide and oxidation of Met-73.
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-protein phosphatase strongly reduced wild type dCK activity (see Fig. 1), similar treatment did not affect dCK activity of the S74E mutant (30.7 ± 5.4 nmol/min/mg of protein after a 60-min treatment versus 28.9 ± 4.9 nmol/min/mg of protein for the untreated enzyme, n = 3), suggesting that the other phosphorylation sites of dCK are not, or not directly, implicated in the control of its catalytic activity. To verify this hypothesis, we constructed dCK mutants in which Thr-3, Ser-11, and Ser-15 were replaced by Ala or Glu. None of these mutations significantly modified dCK activity (results not shown).
HEK 293T cells overexpressing either the S74A or S74E mutants were incubated for 1 h with 0.5 M sorbitol. As illustrated in Fig. 6, both mutations prevented the ability of sorbitol to reduce dCK activity, as observed for wild type dCK, suggesting that sorbitol might act by promoting the dephosphorylation of Ser-74. To verify this hypothesis, a polyclonal anti-Ser-74 phosphopeptide antibody was generated and used to probe lysates of HEK 293T cells overexpressing wild type dCK treated with or without sorbitol. Fig. 7A, upper panel, corresponds to the immunoblot for the phosphospecific antibody, and Fig. 7B, lower panel, corresponds to a parallel blot probed with anti-poly(His) antibody as a control for dCK protein expression. The immunoblot in Fig. 7A, left panel, shows that dCK was recognized by the phosphospecific antibody. As expected, the signal was strongly decreased in lysates pretreated with
-protein phosphatase, in parallel with a decrease in dCK activity (Fig. 7C). The immunoblot in Fig. 7A, right panel, shows that dCK phosphorylation in cells treated with sorbitol was markedly decreased, suggesting that Ser-74 dephosphorylation was induced by sorbitol and was involved in the decrease in dCK activity measured in the same cells (Fig. 7C). When extracts from sorbitol-treated cells were incubated with
-protein phosphatase, the phosphorylation was undetectable, and dCK activity was further decreased.
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| DISCUSSION |
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A widely used method to demonstrate the in vivo phosphorylation of a particular protein is to incubate intact cells with [32P]orthophosphate and to examine whether this protein is labeled. Because dCK phosphorylation protein could be not detected in leukemic cells incubated with [32P]orthophosphate because of low levels of expression, we overexpressed dCK in HEK 293T cells. The ability of
-protein phosphatase to decrease dCK activity in lysates of HEK 293T cells (Fig. 1) suggested that the recombinant dCK might be constitutively phosphorylated and that HEK 293T cells overexpressing dCK looked as a good model to study dCK phosphorylation. This approach allowed labeling dCK with [32P]orthophosphate, and after purification of dCK, tryptic digestion, and mass spectrometry analysis, identification of four phosphorylation sites on the protein, namely Thr-3, Ser-11, Ser-15, and Ser-74. Using the NetPhos 2.0 computer program (32), probability scores for phosphorylation of Ser-15 and Ser-74 were 0.993 and 0.982, respectively, 0.698 for Ser-11, and below the limit for potential phosphorylation of Thr-3 (0.404). Other phosphorylation sites probably exist as suggested by the labeling of peaks I and IV in the HPLC profile (Fig. 4), in which the phosphorylated residue was not identified.
The three-dimensional structure of human dCK was recently solved by Sabini et al. (33). The enzyme is homodimeric with a fold similar to that described for the Drosophila melanogaster deoxynucleoside kinase. Each monomer consists of 10
-helices surrounding a five-stranded parallel
-sheet. The N-terminal extremity of dCK (residues 119) that contains three of the four phosphorylation sites identified is flexible, and its structure could not be solved. However, this region is predicted to lie outside the protein core. Ser-74 is located in a 15-residue mobile insert. Thus, the four phosphorylation sites we identified are located in flexible loops at the surface of the protein and would be accessible to protein kinases.
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-protein phosphatase treatment. Moreover, mutation of Thr-3, Ser-11, and Ser-15 to Ala did not acutely influence dCK activity, suggesting that phosphorylation of these residues is not essential for dCK activity. However, phosphorylation of these sites might be involved in other types of regulation, like enzyme stability or intracellular localization, which are currently under investigation. Hyperosmotic stress induced by sorbitol was shown previously to decrease dCK activity (23) and was used as a tool to induce a change in dCK activity. As expected, we found that sorbitol decreased both dCK activity and 32P labeling of the protein, showing that activity of dCK can be correlated to its phosphorylation state. Moreover, mutation of Ser-74 to alanine or glutamate prevented the ability of sorbitol to reduce dCK activity, suggesting that dephosphorylation of Ser-74 occurred on treatment with sorbitol. This hypothesis was confirmed by use of an anti-phospho-Ser-74 antibody. Indeed, the signal detected with this specific antibody in untreated HEK 293T cells was almost abolished after incubation with sorbitol (Fig. 7). These results not only show that Ser-74 phosphorylation mirrors dCK activity but also provide an insight into the molecular mechanism by which sorbitol induces dCK inactivation. However, the signaling pathway by which sorbitol decreases Ser-74 phosphorylation remains to be elucidated.
Concerning the protein kinase(s) implicated in Ser-74 phosphorylation and the control of dCK activity, Prosite (www.expasy.org/tools/scanprosite/) (34) and Phosite (www.phosite.com) (35) searches indicate that Ser-74 could be a protein kinase C (PKC) site. Indeed, Wang and Kucera (36) found that dCK purified from leukemic blasts could be phosphorylated in vitro by PKC
. By contrast, human recombinant dCK, expressed in bacteria, was a very poor PKC substrate (37). Also, specific inhibitors or activators of PKC did not modify dCK activity in intact leukemic lymphocytes (24), suggesting that PKC does not play important role in the control of dCK activity in vivo. Further studies are thus required to identify the protein kinase(s) responsible for dCK phosphorylation. Regarding the dephosphorylation of dCK, protein phosphatase 2A might be directly or indirectly involved (24).
Recently, Keszler et al. (38) suggested that activation of dCK was accompanied by a conformational change. This hypothesis was based on the observation that extracts from CdA- or etoposide-treated cells were better recognized in native immunoblots by the antibody raised against dCK than extracts from control cells. Post-translational modification was proposed to induce a more open conformation, providing better accessibility for the antibody. Therefore, phosphorylation of Ser-74, which is located far from the active site, might induce long range conformational changes in dCK and promote an open conformation, as suggested by Keszler et al. (38).
Finally, an important aspect of this work is the finding that phosphorylation of Ser-74 was also detected in the leukemic CCRF-CEM cells and that Ser-74 phosphorylation was enhanced in these cells by agents (Fig. 8) known to increase dCK activity. These results strengthen a role for Ser-74 phosphorylation in the control of dCK activity, even in leukemic cells. These findings could be exploited for improving the activation of nucleoside analogues used in anti-cancer and antiviral chemotherapy or for the design of more active dCK mutants for suicide-gene therapy.
| FOOTNOTES |
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2 Research Associate of the Belgian Fonds National de la Recherche Scientifique. ![]()
3 To whom correspondence should be addressed: Laboratory of Physiological Chemistry, Christian de Duve Institute of Cellular Pathology, UCL-ICP 7539, 75 Av. Hippocrate, 1200 Brussels, Belgium. Tel.: 32-2-7647539; Fax: 32-2-7647598; E-mail: bontemps{at}bchm.ucl.ac.be.
4 The abbreviations used are: dCK, deoxycytidine kinase; HEK, human embryonic kidney; HPLC, high pressure liquid chromatography; 32P, [32P]orthophosphate; 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;
-PP, protein phosphatase; UV, ultraviolet; WT, wild-type. ![]()
| ACKNOWLEDGMENTS |
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| REFERENCES |
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