Interaction with Checkpoint Kinase 1 Modulates the Recruitment of

Aug 20, 2009 - the rate of replication fork elongation during unperturbed S phase13 and the ... conditions of replicative stress.15 To gain further in...
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Interaction with Checkpoint Kinase 1 Modulates the Recruitment of Nucleophosmin to Chromatin Songbi Chen,§ Apolinar Maya-Mendoza,§ Kang Zeng, Chi W. Tang, Paul F. G. Sims, Josip Loric, and Dean A. Jackson* Manchester Interdisciplinary Biocentre, University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom Received May 5, 2009

The Checkpoint kinase 1 (Chk1) plays a central role in the cellular response to DNA damage and also contributes to the efficacy of DNA replication in the absence of genomic stress. However, we have only limited knowledge regarding the molecular mechanisms that regulate differential Chk1 function in the absence and presence of DNA damage. To address this, we used vertebrate cells with compromised Chk1 function to analyze how altered Chk1 activity influences protein interactions in chromatin. Avian and mammalian cells with compromised Chk1 activity were used in combination with genomic stress, induced by UV, and DNA-associated proteomes were analyzed using 2-DE/MS proteomics and Western-blot analysis. Only one protein, the histone chaperone nucelophosmin, was altered consistently in line with changes in chromatin-associated Chk1 and increased in response to DNA damage. Purified Chk1 and NPM were shown to interact in vitro and strong in vivo interactions were implied from immunoprecipitation analysis of chromatin extracts. During chromatin immunoprecipitation, coassociation of the major cell cycle regulator proteins p53 and CDC25A with both Chk1 and NPM suggests that these proteins are components of complex interaction networks that operate to regulate cell proliferation and apoptosis in vertebrate cells. Keywords: Checkpoint kinase 1 (Chk1) • nucleophosmin (NPM) • chromatin • DNA damage • proteomics

Introduction The maintenance of genetic integrity in vertebrate cells involves a complex network of cell cycle checkpoints that block cell cycle progression when DNA is damaged or DNA synthesis is compromised.1-3 Checkpoint kinases 1 (Chk1, expressed from CHEK1) and 2 (Chk2, expressed from CHEK2) are two critical regulators of cell cycle arrest in response to checkpoint activation.4 Chk1 is the major checkpoint kinase that controls cell cycle in response to DNA damage, whereas Chk2 functions mainly as a complementary modulator of cell cycle progression.5 Upstream of Chk1/2, two phospho-inositide kinase-related proteins, ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and RAD3-related (ATR), act as damage sensors to activate the DNA damage response.3 Chk1 and Chk2 are activated by ATM/R mediated phosphorylation to function as downstream effector kinases.6 When activated, Chk1 and Chk2 target numerous substrates that act as key cell cycle regulators. As an example, Chk1/2-dependent phosphorylation of the tumor suppressor protein p53 leads to its stabilization and activation of both the G1 and G2 checkpoint pathways.7,8 In * To whom correspondence should be addressed. Dean A. Jackson, The Faculty of Life Sciences, Manchester Interdisciplinary Biocentre, University of Manchester, 131 Princess Street, Manchester M1 7DN, U.K. E-mail: [email protected]. Phone: 0044 161 3064255. Fax: 0044 161 3065201. § Contributed equally to this work. 10.1021/pr900396d CCC: $40.75

 2009 American Chemical Society

addition, in response to DNA damage, Cdc25A, a positive regulator of cyclin-dependent kinases, is hyperphosphorylated by Chk1/2 and degraded through an ubiquitin-mediated pathway so that cell cycle progression is blocked.9,10 The essential checkpoint function of Chk1 in mammalian cells is clearly demonstrated by the requirement for Chk1 kinase activity to activate the ionizing radiation-induced degradation of Cdc25A, which is essential to prevent S phase progression in cells with damaged DNA.11 However, while Chk1 and other proteins that function in checkpoint activation are clearly required to protect eukaryotic genomes from unwanted mutations and damage, there is growing evidence that many of the same proteins also play fundamental roles in DNA synthesis that occurs in the absence of damage. For example, there is compelling evidence that ATR and ATM contribute to the regulation of timing of replication origin firing in vertebrate cells12 and recent evidence that Chk1 also operates during the unperturbed S phase to maintain the natural rate of replication fork elongation.13 These and many other lines of evidence14 emphasize that Chk1 operates during normal proliferation as well as contributes to checkpoint regulation when DNA is damaged. In recent studies, we used the specific inhibitor of Chk1 kinase activity UCN-01 and avian (DT40) CHEK1 gene knockouts to define the molecular role of Chk1 activity in modulating the rate of replication fork elongation during unperturbed S phase13 and the density of active replication origins under Journal of Proteome Research 2009, 8, 4693–4704 4693 Published on Web 08/20/2009

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conditions of replicative stress. To gain further insight into the molecular mechanisms of Chk1 activity, the present study monitors changes in the chromatin proteome in cells with compromised Chk1 kinase activity. We used 2-DE/MS proteomics combined with Western-blot analysis to monitor the composition of chromatin in avian and human cells with altered Chk1 function. Comparative analysis of the chromatin proteome by 2-DE analysis revealed that only one cluster of protein spots was consistently altered in cells with compromised Chk1 activity. This protein was identified as the chromatin chaperone nucleophosmin (NPM). In vitro pull-down analysis supported a direct interaction between Chk1 and NPM. Moreover, coimmonuprecipitation of Chk1 and NPM with other proteins involved in cell cycle regulation suggests that Chk1 and NPM act together in a chromatin complex, which contributes to the regulation of DNA synthesis and the preservation of genome integrity.

Experimental Procedures Cell Culture and Treatment of Cells. HeLa cells and HEK293T cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with antibiotics and 10% fetal bovine serum (FBS). MRC5 cells were grown in MEM supplemented with antibiotics and 10% FBS. DT40 cells15 were grown in RPMI-1640 (Sigma) with 10% FBS, 1% chicken serum, 10-5 M β-mercaptoethanol and antibiotics. Chk1 knockdown (Chk1-/-) DT40 cells were provided by Dr. D. A. Gillespie (Beatson Institute for Cancer Research, Cancer Research UK, Glasgow, U.K.). Replicative stress was induced in different cell lines using 3 treatments: (1) 300 nM UCN-01 for 1.5 h; (2) 20 J/m2 UVC radiation (intensities of the UV lamp were determined with a UVX radiometer); (3) 300 nM UCN-01 for 1.5 h followed by 20 J/m2 UVC radiation. Cells were harvested for analysis 1 h after treatment. Chromatin Protein Extraction and 2-DE Analysis. Chromatin protein extraction was performed as described.16 Twodimensional electrophoresis was performed using immobilized pH gradients (IPGs) as described by the manufacturer (GE Healthcare), with minor modifications. For analytical and preparative gels, 18-cm IPG strips (pH 4-7) were rehydrated overnight with 425 µL of IPG rehydration buffer (10 M Urea, 2% (w/v) CHAPS, 0.15% (w/v) DTT, 2.5 mM EDTA and 2.5 mM EGTA), containing 180 µg of protein, at room temperature. Isoelectric focusing (IEF) was conducted at 20 °C using a Multiphor II apparatus (GE Healthcare). Focusing was performed with several stepwise increases in voltage up to 8000 V for a total of 74.4 kVh. The focused IPG strips were equilibrated twice (2 × 15 min) in DTT and iodoacetamide as described previously.17 The second-dimension electrophoresis was performed by SDS-PAGE in a vertical slab of acrylamide (12% polyacrylamide gel) using a Protean II Multi-cell electrophoresis unit (Bio-RAD). The protein spots in analytical gels were visualized by staining with Colloidal Coomassie Brilliant blue G-250.18 Image and Data Analysis. Gels were scanned using an Epson GT-9600 scanner at a resolution of 1000 dots/in., and raw scans automatically processed using Delta2D 3.0 software (DECODON GmbH, Greifswald, Germany). The gel patterns were matched automatically, with some manual editing, and the matched spots in gels from cells treated under different conditions were compared. The abundance of protein within spots was estimated by the percentage volume (%Vol) and 4694

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quantification of protein spots in analytical gels performed after staining with Colloidal Coomassie. Tryptic In-Gel Digestion. Protein spots were excised from dry gels, and the gel pieces were rehydrated in Milli-Q water for 30 min. Gel pieces were detached from the cellophane film, cut into 1-2 mm pieces and washed twice in MillQ water for 15 min. The washed gel pieces were subjected to two 15 min cycles of dehydration with 50% acetonitrile followed by 50 mM ammonium biocarbonate and digested overnight at 37 °C in 20 µL of sequencing grade trypsin (Sigma-Aldrich), according to the manufacture’s instructions (1 µg in 100 µL of 50 mM ammonium bicarbonate). The supernatants were stored and 30 µL of Milli-Q water was added to the gel pieces at room temperature for 1 h before the two supernatant fractions were pooled.19 LC-MS/MS and Q-TOF MS/MS Analysis and Database Search. Protein spots were excised from 2-D gels and trypsin digested, and mass spectrometric analyses were conducted by nanoflow-liquid chromatography/electrospray ionization using either a Bruker Esquire 3000plus Ion Trap (Bruker Daltonics, Coventry, U.K.), LC-MS/MS or an ESI-Q-TOF Ultima global (Micromass, Manchester, U.K.), Q-TOF-MS/MS.19 Briefly, for ion trap analysis, peptides were separated by chromatography on a 75 µm × 15 cm PepMap nanocolumn (LC Packings) at a flow rate of 250 nL/min using a 60 min linear gradient of acetonitrile (5-95%) in 0.1% formic acid. The column effluent was sprayed directly into the mass spectrometer, which was set to scan the m/z ranging from 400 to 1500 in positive ion mode, capturing MS and MS2 data automatically. Instrument operation, data acquisition, and analysis were performed using HyStar V2.3 and DataAnalysis V3.1 software. For Q-TOF analysis, samples were trypsin digested, dried in a vacuum centrifuge, taken up in 15 µL of 2% acetonitrile and 0.5% formic acid solution, and then analyzed by Q-TOF-MS/MS. Peptides were separated by chromatography on an Ultimate 3000 HPLC (Dionex) using an identical PepMap nanocolumn at a flow rate of 200 nL/min and using a gradient of 5-60% solution B (95% acetonitrile, 0.05% formic acid) in solution A (2% acetonitrile and 0.06% formic acid) over 55 min. Instrument operation, data acquisition and analysis were performed using MassLynx V4.1 software. Data captured by either LC-MS/MS or Q-TOF MS/MS were matched using the MASCOT version 2.2.03 (Matrix Science, U.K.; http://www.matrixscience.com) against MSDB (MSDB database update Sep-08-2006, 3 239 079 sequences; Taxonomy: Homo sapiens 148 148 sequences) and IPI chick database (v3_41; 25 686 sequences). In both cases, protein N-terminal acetylation, carbamidomethyl (Cys) and oxidation (Met) were considered as variable modifications and a single missed cleavage was permitted. For LC-MS/MS data, peptide mass tolerance was set as 1.2 Da and MS/MS ion mass tolerance was set at 0.6 Da. Peptide charge states (+1, +2, +3) were taken into account. Criteria used for routine protein identification required sequence-confirmed data (confidence >95%) for a minimum of two peptides with recognition as the top ranking match in the Mascot Standard scoring system. Western Blotting and Phosphorylation Assay. Whole cells and chromatin extracts were solubilized with lysis buffer [50 mM Tris · Cl (pH 6.8), 100 mM dithiothreitol, 2% SDS, 10% glycerol, 1.0 tablet/50 mL of Complete1 protease inhibitor cocktail (Roche Diagnostics)], and suspensions were centrifuged (5 min at 13 000 rpm) to remove debris. Fixed amounts of protein were separated by gel electrophoresis,20 blotted onto

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Chk1 and NPM Interaction nitrocellulose, and proteins were detected by immunostaining with the following antibodies: HeLa and MRC5 NPM, monoclonal antibody from Abcam (ab10530); HeLa, MRC5 and DT 40 Chk1, monoclonal antibody from Santa Cruz Biotechnology (sc-8408); HeLa and DT40 phospho-Chk1 (Ser345) (133D3), using phospho-specific antibody from Cell signaling Technology (2348S); HeLa and DT40 p53, monoclonal antibody from Santa Cruz Biotechnology (sc-126); HeLa and DT40 Cdc25A, monoclonal antibody from Santa Cruz Biotechnology (sc-7389); HeLa H2B, monoclonal antibody from Biocompare (ab52599). Western blots were developed using ECL-Plus (GE Healthcare) Western blotting reagents and changes in protein expression were determined from at least 3 independent experiments. Phosphorylation of NPM and Chk1 on DT40 and HeLa chromatin was assayed in 2-DE gels and immunoprecipitates according to the manufacturer’s instructions using the Pro-Q Diamond phosphoprotein gel stain kit (Molecular Probes, Invitrogen). Immunofluorescence. Immunofluorescence was performed on cytospin preparations (Cytospin 4, Thermo Scientific; 500 rpm for 15 min) as described previously.20 The NPM and lamin B1 antibodies were from Acris Antibodies GmbH (Nucleophosmin, BM5056) and Abcam (lamin B1 ab6048). Nuclei were stained with DAPI (Vector Laboratories Inc., Burlingame, CA). Plasmid Constructs. Chk1 and NPM full-length cDNAs were amplified by RT-PCR kit (Invitrogen) from MRC5 cell total RNA using the following primers: Chk1 specific oligonucleotides, 5′-CGCGAATTCTGGCAGTGCCCTTTGTGGAAG3′ and 5′-TTCCTCGAGTCATGTGGCAGGAAGCCAAATCT-3′); NPM specific oligonucleotides, 5′-GAAGAATTCATGGAAGATTCGATGGACATGG-3′ and 5′-CTTCTCGAGTTAAAGAGACTTCCTCCACTGCCAG-3′. The PCR fragments were ligated into pCRII -TOPO vector (Invitrogen) and verified by DNA sequencing. For GST tag expression, full-length Chk1 cDNA was ligated into the pGEX5X-1 vector (GE Healthcare) using EcoRI and XhoI restriction enzyme sites and transformed into Escherichia coli strain BL21 for protein expression. For His tag expression, the full-length NPM1 cDNA was ligated into pQE30 vector (Qiagen) using BamHI and SalI sites and transformed into E. coli strain JM109 for protein expression. Glutathione S-transferase (GST) Pull-Down Assay. GST pulldown assays were performed according to the manufacturer’s instructions using ProFound pull-down GST protein interaction Kit (Perbio Science UK Ltd., Northumberland, U.K.). Briefly, GST-tagged Chk1, expressed in E. coli LB21 cells, and 6×Histagged NPM, expressed in E. coli JM109 cells, were induced by adding 1 mM isopropyl-β-D-thiogalactopyranoside for 6 h at 18 °C. Pull-down assays were performed by binding purified GST-tagged Chk1 (bait protein) to glutathione-affinity-agarose beads, and then incubating with purified 6×His-tagged NPM (prey protein) overnight at 4 °C. Bait-prey complex was eluted from the washed agarose beads with 100 mM reduced glutathione, and samples were prepared for SDS-PAGE. The purity and quantity of the recombinant proteins were determined by Western blotting. Appropriate binding controls were performed as follows: Bait only, GST-tagged Chk1 control was treated as above and eluted after incubation with binding buffer but without added 6×His-tagged NPM; Prey only, beads without bound Chk1 were incubated with 6×His-tagged NPM and eluted after incubation with binding buffer.

Immunoprecipitation. To verify the association of NPM with Chk1 in human and DT40 cells, NPM and Chk1 were immunoprecipitated from chromatin extracts. HeLa and DT40 chromatin extracts prepared using routine procedures with formaldehydecross-linking16 wereresuspendedinRIPAnondenaturing buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, and 1% NP-40) containing protease inhibitor cocktail (Roche Diagnostics) and incubated (overnight at 4 °C) with anti-NPM (Abcam, ab10530 for HeLa cells; Acris Antibodies GmbH, BM5065 for DT40 cells) or anti-Chk1 (Santa Cruz Biotechnology, sc-8408) at a final dilution of 1:50. Antibody complexes were recovered by incubation with protein-G agarose (GE Healthcare; 1.5 h at 4 °C). Beads were separated and washed three times in RIPA buffer (without protease inhibitors). Then, 80 µg/mL DNAase I (Sigma-Aldrich) or 5 µg/mL RNAase (Roche Diagnostics) was incubated with beads in RIPA buffer (2 h at 4 °C). After washing, affinity bound proteins were eluted using 2× SDS sample buffer (5 min at 90-100 °C) and analyzed by immunoblotting using the appropriate antibodies. Myc-Tagged Chk1 Fragments for Analysis of NPM Binding. Chk1 partial cDNA for truncated protein expressions were amplified by PCR using the following primers: Chk1 1-276 amino acids, 1-276 forward, 5′-ccaggtaccATGGCAGTGCCCTTTGTGGAAG-3′, 1-276 reverse, 5′-caactcgagTCGGGGCCTTTTTGCCCCTTTCT-3′; Chk1 276-476 amino acids, 276-476 forward, 5′-caaggtaccGTCACTTCAGGTGGTGTGTCA-3′, 276-476 reverse, 5′-cctctcgagTGTGGCAGGAAGCCAAATCTTC-3′; Chk1 369-476 amino acids, 369-476 forward, 5′-cctggtaccCAGCGGTTGGTCAAAAGAATG-3′, 369-476 reverse, 5′-cctctcgagTGTGGCAGGAAGCCAAATCTTC-3′. The cDNA fragments were ligated into the mammalian expression vector pcDNA3.1-myc to generate fusion proteins with N-terminal 3×Myc tags. Plasmids were transfected into HEK 293T cells with FuGene 6 Transfection Reagent according to the instructions of the manufacturer (Roche). After 24 h, expression of Chk1 fragments was detected by immunoblotting with mouse monoclonal anti-Myc antibody (Sigma Aldrich; M4439). Interaction between Chk1 fragments and NPM and p53 was then detected by immunoblotting after separation of immunoprecipitates by gel electrophoresis.

Results Effect of Chk1 Inhibitor UCN-01 and Chk1-/- on Protein Binding in Chromatin. We have recently shown that Chk1 is required to maintain the natural rates of replication fork progression on undamaged chromatin.13 To identify molecular mechanisms through which Chk1 functions during DNA synthesis, we performed a proteomic analysis of chromatin extracts under different conditions of compromised Chk1 activity. We first compared the DNA-associated proteome of untreated DT40 cells with cells treated for 1.5 h with 300 nm UCN-01 to inhibit Chk1 kinase signaling (Figure 1). After extraction, chromatin proteins were separated by 2-DE and analyzed using Delta2D software. Protein samples (180 µg) separated on 18 cm two-dimensional gels (pI 4-7) displayed ∼500 detectable protein spots. With the use of 4 gels and 3 biological replicates, Delta2D software was used to define protein spots with significant differential content (p < 0.05; Student’s t test), with 1.5-fold or more increased or decreased intensity. Inhibition of Chk1 correlated with a dramatic, ∼5fold reduction in presence of a single cluster of protein spots (Figure 1A, spot 1). Perhaps remarkably, no other significant changes in the chromatin proteome were seen at this level of Journal of Proteome Research • Vol. 8, No. 10, 2009 4695

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Figure 1. 2-DE images of chromatin from UCN-01-treated DT40 and DT40 Chk1-/- cells. Chromatin isolated from DT40 cells was separated by 2-DE and visualized after staining with Colloidal Coomassie (A). Chromatin from the following samples was compared: (a) control, untreated DT40 cells; (b) DT40 cells treated with UCN-01 (300 nM UCN-01 for 1.5 h); (c) DT40 Chk1-/- cells and (d) DT40 Chk1-/- cells treated with UCN-01. A total of 180 µg of protein was loaded onto each gel. The gel pairs shown are representative of 4 replicate gels collected from 3 independent experiments. Inset boxes (below) for each gel show 4× magnified images of two regions: 1, shows a protein with chromatin association linked to Chk1 expression; this protein was identified by Q-TOF-MS/MS (Supplementary Figure S1) as NPM; 2, shows a protein that binds to chromatin independently of Chk1 expression; this protein was identified by LC-MS/MS (Supplementary Figure S2) as actin. Delta2D gel analysis software was used to determine changes of NPM expression (Ba) using the ratio of normalized spot intensities between treated samples and controls; (-) is down-regulated. Error bars indicate standard deviation of the mean for 4 replicates from 3 independent experiments. Gels like those shown in panel A were also stained using a phosphospecific reagent to reveal phosphorylated proteins (Bb). The spot clusters highlighted show the phosphorylated isoforms of NPM; equal amounts of protein were loaded onto each gel.

sensitivity. With the use of Q-TOF-MS/MS and LC-MS/MS for characterization (Supplementary Figure S1), the protein with reduced DNA-association was shown to be the chromatin chaperone nucleophosmin (NPM). Under our conditions of electrophoresis, NPM typically separates as a series of 4 isoforms that reflect different post-translational protein modifications. The protein isoforms migrated with pI’s in the range 4.81-4.90 and a molecular weight of 39.55 ( 0.22 kDa, measured using ChemiImager 4400 software in 2-DE gels. With actin (Supplementary Figure S2) as a reference for normalization, chromatin-associated NPM in DT40 cells treated with 4696

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UCN-01 was seen to decrease 5.12-fold compared to untreated control cells (Figure 1B). To confirm that Chk1 expression influences NPM recruitment to chromatin, we next analyzed the interaction of NPM with chromatin in CHEK1 knockout DT40 cells.15 Relative to isogenically matched DT40 used as control cells, the Chk1-/cells showed a 5.49-fold decrease in the level of chromatinbound NPM, consistent with Chk1 being a positive regulator of NPM-chromatin interactions in DT40 cells (Figure 1). However, under conditions of genomic stress induced by treatment with UCN-01, slightly higher levels of chromatin-

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Figure 2. Effect of UV irradiation and UCN-01 treatment on the levels of NPM and Chk1 in DT40 chromatin. Chromatin isolated from DT40 cells after treating to induce replicative stress were separated by 2-DE and visualized after staining with Colloidal Coomassie (A). Samples from untreated controls (a) were compared with cells treated with UV (20 J/m2 UVC; b) or UV after UCN-01 (300 nM UCN-01 for 1.5 h; c); samples were prepared 1 h after UV. The gels shown are representative of 4 replicate gels collected from 3 independent experiments. Inset boxes are 4× magnifications of the regions shown. Delta2D gel analysis software was used to determine changes of NPM expression (Ba) using the ratio of normalized spot intensities between treated samples and controls. Error bars indicate standard deviation of the mean for 4 replicate experiments. Gels like those shown in panel A were also stained using a phospho-specific reagent to reveal phosphorylated proteins (Bb). The spot clusters highlighted show the phosphorylated isoforms of NPM; equal amounts of protein were loaded onto each gel. Western-blot analysis (C) was used to measure expression of Chk1 and phosphorylation of Chk1ser345 during checkpoint activation. After treatment, chromatin proteins were separated by SDS-PAGE and Chk1 and Chk1-ser345 identified using appropriate antibodies. Levels of expression were measured using β-actin as a loading control.

bound NPM were seen in Chk1-/- cells (Figure 1). As the behavior of NPM is regulated by post-translational levels of phosphorylation, we also analyzed gels like those shown in Figure 1A after staining to reveal protein phosphorylation (Figure 1Bb). As expected, the isoforms of chromatin-associated NPM were differentially phosphorylated, while levels of phosphorylation were clearly reduced following UCN-01 treatment or CHEK1 deletion. However, the ∼5-fold decline in total chromatin associated NPM following UCN-01 treatment and corresponding ∼2-fold decline in NPM phosphorylation implies that other kinases contribute to NPM phosphoryaltion. Indeed, this is confirmed by the low levels of chromatin associated NPM phosphorylation in DT40 Chk1-/- cells (Figure 1Bb). These data show that in DT40 cells in the absence of genomic stress Chk1 kinase activity is a major pathway for recruiting NPM to chromatin. Notably, the dramatic reduction in chromatin-bound NPM in the absence of Chk1 suggests that the major fraction (>80%) of chromatin-associated NPM is dependent on Chk1 for binding. Residual levels of chromatinassociated NPM, which are seen in wild-type DT40 cells in the

presence of UCN-01 or in Chk1-/- cells, must be recruited to chromatin by a Chk1-independent pathway. A good candidate for this residual activity is ATR, which, in human cells at least, has been shown to contribute to the level of chromatinassociated NPM independently of activation of the DNA damage response.21 Effect of UV-Induced DNA Damage on the Level of NPM and Chk1 in Chromatin of DT40 Cells. The cellular response to UV radiation involves a 1- to 2-fold induction of NPM expression,22 implying that NPM might contribute to DNA repair and cell survival following DNA damage. To further explore the effect of UV-induced DNA damage on the amount of NPM in chromatin, we next analyzed chromatin proteins isolated from UV-irradiated DT40 cells (Figure 2). Following UV treatment, a 2.05-fold (p < 0.05; Student’s t test) increase in NPM bound to DT40 chromatin was seen in 2-DE gels and this increased slightly, to 2.61-fold, when UCN-01 was added to cells prior to UN treatment (Figure 2A,B). As expected, increased levels of chromatin-associated NPM in DT40 cells Journal of Proteome Research • Vol. 8, No. 10, 2009 4697

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Figure 3. Effect of UCN-01 and UV on the levels of NPM and Chk1 in chromatin of HeLa cells. Chromatin prepared from HeLa cells was separated by 2-DE and visualized after staining with Colloidal Coomassie (A). Chromatin proteins isolated from untreated cells (Aa) were compared with those in chromatin isolated from cells treated with UCN-01 (Ab). The gel pair shown is representative of 4 replicate gels collected from 3 independent experiments. Inset boxes are 4× magnifications of the regions shown, to highlight clearly detectable spots. All spots in region 1 were shown by LC-MS/MS (Supplementary Figure S3) to be human nucleophosmin. Western-blot analysis (B) was used to confirm the expression of NPM in chromatin of control and UCN-01-treated HeLa cells. Chromatin proteins were separated by SDS-PAGE and NPM was detected by Western blot using mouse anti-NPM monoclonal antibody (ab10530). Experiments like those shown in panel (A) were also performed to monitor the effect of UV treatment on HeLa chromatin (C). Chromatin 2-DE images were prepared from untreated controls (Ca) and after treatment with UV (20 J/m2 UVC; Cb) or UV after UCN-01 (300 nM UCN01 for 1.5 h; Cc); samples were prepared 1 h after UV. Gels shown are representative of 4 replicate gels collected from 3 independent experiments. Inset boxes are 4× magnifications of the regions shown. The levels of binding of NPM to chromatin after the different treatments used in panel C were confirmed using Western blot analysis (D). Delta2D gel analysis software was used to determine changes of chromatin-bound NPM (E) using the ratio of normalized spot intensities (NPM expression normalized to actin) between treated samples and controls. Error bars indicate standard deviation of the mean for 4 replicate experiments. Gels like those shown were also stained using a phospho-specific reagent to reveal phosphorylated proteins (F). The spot clusters highlighted show the phosphorylated isoforms of NPM; equal amounts of protein were loaded onto each gel. Chromatin-associated Chk1 was also monitored using Western blot analysis (G).

following UV-irradiation correlated with a ∼2-fold increase in phosphorylation to NPM in chromatin (Figure 2Bb). In response to DNA damage, Chk1 is phosphorylated and activated by the orthologues of ATR and/or ATM. Chk1 proteins have several SQ/TQ motifs in their regulatory C-terminal domain that are targets for ATR and ATM kinases phosphorylation. However, studies to date indicate that two conserved 4698

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sites, S345/Q346 and S317/Q318, are major targets during the DNA damage response.6 Hence, to monitor activation of Chk1, we next used immunoblotting with antibodies to Chk1 and phospho(s345)-Chk1 to monitor changes in chromatin associated Chk1 during treatment of cells with the Chk1 kinase inhibitor UCN-01 and UV to induce the DNA damage response (Figure 2C). UV treatment correlated with a ∼1.3-fold increase

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in chromatin-associated Chk1 and corresponding activation of the Chk1 response as shown by the reduced electrophoretic mobility of ∼1/3 of Chk1 (Figure 2Ca Chk1 blot UV treated) and Chk1 phosphorylation on serine 345 (Figure 2C). However, Chk1 binding to chromatin following UV was unaffected by inhibition of Chk1 kinase activity with UCN-01 (Figure 2C). These experiments show that during the DNA damage response NPM is recruited to chromatin under conditions where Chk1 is activated by phosphorylation at ser345 (Figure 2C), but phosphorylation of downstream Chk1 targets is prevented using UCN-01. Together these experiments show that, while Chk1 is the major regulator of chromatin-associated NPM in unperturbed cells, the increased binding of NPM following UV-induced DNA damage is not dependent on the function of proteins that lie downstream of Chk1 in the DNA damage response. Effect of UCN-01 and UV-Induced DNA Damage on the Levels of NPM and Chk1 in Mammalian Chromatin. The only experimental system that allows complete deletion of Chk1 protein has been developed using a gene deletion in chicken DT40 cells. However, the use of DT40 cells to explore the interactions between NPM, Chk1 and chromatin raises obvious questions about the possibility of equivalent pathways in mammalian cells. To more carefully examine the effect of UCN-01 and UV on the expression of NPM and Chk1, experiments described above were repeated using HeLa and diploid human fibroblast (MRC5) cells. Following 2-DE analysis of HeLa cell chromatin (Figure 3), NPM (spot 1) was identified by LC-MS/MS (Supplementary Figure S3). Compared to actin (spot 2), identified by LC-MS/MS (Supplementary Figure S4), a significant (p < 0.05; Student’s t test) 1.5-fold decrease in chromatin-bound NPM expression was seen following UCN-01 treatment, when compared to levels in untreated controls (Figure 3A). Consistent changes in chromatin-associated NPM were seen in all gels from 3 biological replicates (Figure 3A,E) and confirmed by quantitative Western blot analysis (Figure 3B). This 1.5-fold decrease in NPM binding to chromatin is clearly less dramatic than the 5.12-fold decrease seen in DT40 cells under equivalent conditions (Figure 1). Even so, qualitatively, changes seen in cells treated with UCN-01 are consistent with Chk1 contributing to the recruitment of NPM to chromatin in both mammalian and avian cells. Functional similarities were also seen in response to UVinduced DNA damage. By 2-DE (Figure 3C) and Western blot analysis using 3 independent biological replicates (Figure 3D), UV-treated HeLa cells showed a 1.23-fold increase in chromatinassociated NPM and further increase, to 1.36-fold, in cells treated with UV and UCN-01 (Figure 3E). While this patterns reflects changes seen in DT40 cells under equivalent conditions (Figure 2), the biological significance of such a small change is difficult to assess. Even so, equivalent changes in NPM phosphorylation were seen (Figure 3F), with a ∼2-fold decrease in chromatin associated phospho-NPM following UCN-01 treatment and 1.5- to 2-fold increase after UV-irradiation. As in DT40 cells, changes in chromatin-associated NPM in response to DNA damage and checkpoint activation correlate with minimal changes in chromatin-associated Chk1, with the only significant change being a ∼1.3-fold binding of Chk1 to chromatin following UV treatment (Figure 3G). The same pattern of NPM association with chromatin was also seen in normal human diploid fibroblasts (MRC5 cells), confirming that

Figure 4. Effect of UCN-01 and UV treatment on the levels of Chk1 and NPM in whole cell extracts from HeLa and MRC5 cells. The global expression of Chk1 and NPM in HeLa and MRC5 cells was monitored using Western blot analysis. Cells were treated as shown (below), whole cell extracts were separated by SDS-PAGE and Chk1 and NPM were detected using appropriate antibodies (sc-8408 for Chk1 and ab10530 for NPM). β-Actin was used as a loading control.

the behavior is not a product of cell transformation (Supplementary Figure S5). Effect of UCN-01 and UV Radiation on the Level of NPM and Chk1 in the Whole Cells. We next wanted to confirm if the levels of chromatin-associated Chk1 and NMP reflected global changes in expression that might be revealed using total protein extracts. HeLa and MRC5 cells were treated with UCN01 and UV and whole cell extracts analyzed using Western blots and immunostaining (Figure 4). In both cells types, the global expression of Chk1 showed no significant change following treatment with UCN-01 and slight but variable increases in expression, in the range 1- to 1.2-fold, following UV treatment (Figure 4). NPM showed no significant variations in expression under any of the conditions used (Figure 4). Unfortunately, quantitative immunoblotting of NPM in DT40 cells was not possible as in our hands the commercial anti-NPM antibody (Nucleophosmin, N038, BN5056) was unsuitable for Western blot analysis. Indirect immunofluorescence was, however, possible (Supplementary Figure S6), and while less reliable for quantitative analysis, this approach did confirm that the overall expression of NPM in both wild-type and Chk1-/- DT40 cells was similar and stable following UV treatment. These results show that under the conditions of UCN-01 or UV treatment used the global expression of Chk1 and NPM is essentially unaltered. We conclude that the UV-induced accumulation of NPM in chromatin of HeLa and MRC5 cells is part of a damage response that involves the nuclear redistribution of NPM and is dependent on direct interaction between NPM and proteins of the damage response pathways. Identifying Protein-Protein Interactions between Chk1 and NPM in Vitro. The role of Chk1 in regulating the binding of NPM to chromatin implies that the two proteins might interact in vivo. Moreover, as binding is not substantially dependent on checkpoint activation, which results in Chk1 phosphorylation and kinase activation, chromatin binding is likely to operate through protein-protein interaction rather than checkpoint induced post-translational modification of NPM. The possibility of direct protein-protein association between Chk1 and NPM was evaluated in vitro using purified proteins and a GST pull-down assay. With this technique, one Journal of Proteome Research • Vol. 8, No. 10, 2009 4699

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Figure 5. Chk1 interacts with NPM. Chk1 was shown to interact directly with NPM in vitro (A). GST-tagged Chk1 (full-length) and 6×Histagged NPM (full length) were separated by SDS-PAGE (4-15%) and visualized after staining with Colloidal Coomassie (Aa). After purification, GST-tagged Chk1 was captured on glutathione agarose beads and incubated with purified 6×His-tagged NPM. Beads were washed extensively and protein complexes were identified using Western blot analysis to detect Chk1 (Ab) and NPM (Ac) using appropriate antibodies (sc-8408 for Chk1 and ab10530 for NPM). Control mixtures used to define the specificity of binding are indicated on the respective panels (Ab,c). Chk1 was also shown to interact with NPM in vivo using immunoprecipitation (IP) of chromatin extracts (B). Cleared chromatin extracts were incubated with control IgG, anti-NPM or anti-Chk1 antibody and antibody complexes were bound to protein-G agarose and purified by washing. Bound proteins were separated by SDS-PAGE and visualized after staining with Colloidal Coomassie (Ba). Proteins were blotted onto nitrocellulose and specific proteins were detected by immunostaining using appropriate antibodies as shown (Bb-e). Experiments were performed using both HeLa and DT40 chromatin with different experimental conditions shown on individual panels (Bb-e). Appropriate control experiments were performed to ensure the specificity of binding interactions. For example, use of H2B as an immunostaining control (Bb) shows that interactions between Chk1 and NPM are not formed indirectly via independent binding to chromatin.

of the putative binding partners, the bait protein, is tagged with GST to facilitate binding to agarose beads. The other partner, the prey protein, is then incubated with the beads and direct binding is monitored after bead isolation (Figure 5A). Tagged full-length human Chk1 and NPM were generated using PCR and expressed in bacteria. Purified GST-tagged Chk1 (bait) was immobilized on glutathione-agarose beads and incubated overnight at 4 °C with purified 6×His-tagged NPM (prey). Appropriate experimental controls were used to minimize the role of nonspecific binding (Figure 5A). Western blot analysis of protein complexes isolated from the washed beads (Figure 4700

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5Ab,c) supports the suggestion that NPM and Chk1 interact in this in vitro pull-down assay. Chromatin Immunoprecipitation Supports Interaction between Chk1 and NPM in Vivo. We next performed immunoprecipitation (IP) on HeLa and DT40 chromatin extracts to monitor Chk1 and NPM interaction in vivo (Figure 5B). IP experiments were performed on chromatin extracts using antiNPM and anti-Chk1 antibodies and protein G-conjugated beads. Importantly, as NPM has recognized nucleic acid binding properties,23 DNAase and RNAase treatment were used to remove any nucleic acid that might link NPM and Chk1.

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Figure 6. Using fragments of Chk1 to map interactions with NPM and p53. (A) Chk1 functional domains as detailed by Zachos, et al.40 (B) The expression of selected Chk1 fragments 1-276 aa (clone no. 7), 276-476 aa (clone no. 12) and 369-476 aa (clone no. 15) were expressed after transfection into HEK293T cells. Total cell extracts were separated by gel electrophoresis and fragments detected by Western blot analysis using a mouse monoclonal anti-Myc antibody. The endogenous myc protein runs at ∼37 kDa under these conditions and the 3 myc-tagged Chk1 fragments at ∼36.5, ∼36, and ∼20 kDa, respectively. (C and D) Interaction of myc-tagged Chk1 fragments with NPM was monitored by immunoprecipitation. Myc-tagged Chk1 fragments were expressed in 293 cells and IP was performed using antibodies to NPM (B23) and p53 (P53). Proteins were separated and myc-tagged Chk1 fragments were detected by Western blotting using a mouse monoclonal anti-myc antibody. NPM binding with Chk1 fragments (arrowed) indicate the specificity of interaction between Chk1 and NPM (C). Specific interaction of Chk1 fragments with p53 is also shown (D).

With the use of appropriate experimental controls, NPM and Chk1 IP extracts isolated from HeLa chromatin were separated by SDS-PAGE, stained with Colloidal Coomassie (Figure 5Ba) and individual proteins were detected by Western-blot analysis (Figure 5Bb-f). The stained gels show a small number of visible polypeptides in addition to the antibody peptides (at ∼28 and ∼55 kDa). It is noteworthy, however, that these do not reflect major proteins in the chromatin input and so reflect proteins that are concentrated by precipitation. In addition, as these antibodies are highly specific for the respective antigens, the pull-down experiments are unlikely to reflect nonspecific binding. Hence, coassociation of the proteins by IP confirms that NPM is associated with Chk1 and is consistent with the interaction seen in vitro, using purified components. Moreover, as the same levels of interaction are seen in the presence or absence of UCN-01, Chk1-NPM interactions must exist independently of the Chk1 kinase activity that is activated in response to DNA damage (Figure 5Bd,e). As Chk1 and NPM interact in vivo, we also evaluated if other checkpoint proteins might be associated in Chk1 and NPMcontaining complexes. To address this, extracts prepared from Chk1 and NPM IPs were analyzed by immunostaining using antibodies to different proteins involved in cell cycle control. With this approach, the major Chk1 targets p53 and Cdc25A were seen to coimmunoprecipitate with both chromatin-associated Chk1 and NPM (Figure 5e and Supplementary Figure S5B). These observations suggest that the network of protein-protein interactions that regulate cell cycle progression also facilitates interaction with chromatin chaperones, such as NPM, which play essential roles in maintenance of chromatin structure.

Finally, we wanted to evaluate if specific regions of Chk1 were involved in a direct interaction with NPM. The major functional domains of Chk1 were subcloned into expression vectors that incorporated a myc tag and immunoprecipitation performed using antibodies to NPM and p53 (Figure 6). Importantly, this analysis of binding to discrete Chk1 fragments shows that while binding is not restricted to a specific small region of the protein some regions, notably the domain from aa 369-476, show no binding at all (Figure 6C). On the basis of the known structure of NPM (Supplementary Figure S7) and Chk1 (Figure 6A and Supplementary Figure S8), this analysis support an interaction that incorporates the kinase domain of Chk1. In addition to providing evidence for interactions between Chk1 and NPM both in vitro and in vivo, the fact that proteins such as p53 and Cdc25A can be immunoprecipitated by both Chk1 and NPM antibodies, while p53 also interacts by IP with Chk1 (Figure 6D), is consistent with the formation of protein complexes that contain NPM and proteins linked to cell cycle regulation such as Chk1, Cdc25 and p53.

Discussion Proteomics can be a powerful tool to analyze changes in the organization of nuclear compartments. For example, Lamond and colleagues have made a comprehensive study of the proteome of nucleoli isolated from human cells under different conditions to demonstrate how the protein content of this major nuclear compartment responds to changes in metabolic activity.24,25 With the use of the resolving power of 2-DE as an analytical tool, we set out to identify changes in the DNAJournal of Proteome Research • Vol. 8, No. 10, 2009 4701

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Figure 7. Chk1 and NPM biological network. A biological interaction network related to Chk1 and NPM was generated using Pathway Studio analysis. Chk1, NPM, ATR, ATM, TP53 and CDC25A were matched to the Pathway Studio data (ResNet 5.0) and combined with data generated within this study. The network shows relationships between interaction partners including protein-protein binding, expression, regulation, direct regulation, and protein modification.

associated proteome of avian and mammalian cells after altering the activity of the major DNA damage checkpoint protein Chk1. Surprisingly, we found that the chromatin proteome of cells with compromised Chk1 function contained only one major protein, represented by a cluster of protein spots, which altered significantly, relative to untreated controls. This protein was shown to be the chromatin chaperone protein nucleophosmin. On the basis of this observation, we were able to show for the first time that Chk1 and NPM interact in both avian and mammalian cells. Chk1 has been known for many years to play a central role in the response to DNA damage and genomic stress in vertebrate cells.1-4 It is known, for example, that the G2-M phase transition, which is regulated by Chk1 function, serves to inhibit entry into mitosis if DNA replication is incomplete or if DNA integrity is compromised.26,27 However, it is also clear that functions performed by Chk1 are not restricted to conditions of genomic stress.14 In particular, it is notable that early 4702

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embryonic death of Chk1-/- mice indicates that Chk1 is essential for cell growth and differentiation at early stages of development.28 Like Chk1, NPM is also involved in the cellular response to genomic stress, and perhaps as a result is often overexpressed during cell transformation.23 NPM is a major nuclear protein with a global expression that is proportional to the cell growth rate, suggesting that the protein plays a positive role(s) in cell growth and proliferation.29,30 Overexpression or down-regulation of NPM reportedly alters the cellular status with respect to proliferation, differentiation and apoptosis, although some contradictory results have been reported.31,32 In addition, NPM responds to genomic stress, with rapid redistribution from the nucleolus correlating with the onset of the stress response33,34 and recruitment of NPM to chromatin.35 In the mouse, NPM1 is an essential gene, with knockout mice showing midstage embryonic lethality due to the accumulation of DNA damage, activation of p53, and widespread apoptosis.36,37

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Chk1 and NPM Interaction Our observation that Chk1 and NPM interact in vertebrate cells is consistent with the idea that Chk1 modulates the recruitment of NPM to chromatin. If this is true, it is of particular importance to understand how the recruitment of NPM to chromatin is regulated under conditions of genomic stress, such as DNA damage induced by UV radiation. Once associated with chromatin, we reasoned that the chromatin remodelling functionality of NPM might play an essential role in facilitating the repair of DNA damage as well as allowing activation of genes, such as p53 target genes, which are expressed in response to genomic stress. Deletion or inhibition of Chk1 activity in avian DT40 cells clearly has a dramatic influence on levels of chromatinassociated NPM (Figure 1), suggesting that Chk1 is a positive regulator of NPM binding to chromatin. Similar results were seen in two mammalian cell lines (Figure 3 and Supplementary Figure S5). However, to our surprise, we found that the recruitment of NPM to chromatin following DNA damage was independent of Chk1 kinase activity in both avian (Figure 2) and mammalian cells (Figure 3 and Supplementary Figure S5). In the presence of DNA damage, induction of Chk1 phosphorylation by ATM and ATR is the initial step in the cellular response to genomic insult. ATR is known to regulate Chk1 phosphorylation at ser345 in a UV-dependent manner,38 strongly implicating ser345 phosphorylation of Chk1 as a molecular mediator of radiation-induced G2 arrest.39 One interpretation of our observation is that interactions between Chk1 and NPM will continually recruit NPM to chromatin so that when the amount of chromatin-associated Chk1 increases, as is seen in response to UV (Figure 2C), the steady-state structure of chromatin contains an increased amount of bound NPM. Following UV treatment, phosphorylation of Chk1 by ATR might contribute to the elevated levels of chromatin-bound Chk1 and associated increases in chromatin-bound NPM. However, it is notable that while phosphorylation of Chk1 correlates with its increased binding to chromatin, experiments performed in the presence UCN-01, to inhibit Chk1 kinase activity, demonstrate that elevated binding of NPM to chromatin is not dependent on the damage-dependent activation of Chk1 kinase. On the basis of these results, it is clear that NPM lies downstream of Chk1 in a pathway that recruits NPM to chromatin. Moreover, data presented here show that Chk1 regulates the recruitment of NPM to chromatin under unperturbed growth conditions. This is in line with our recent proposal that, as well as playing a major role in response to DNA damage, Chk1 also operates at replication sites under normal growth conditions to facilitate the natural rates of replication fork elongation.13,15 In this context, a direct interaction between Chk1 and NPM might serve to recruit NPM to replication forks where its protein chaperone functions, for example, during chromatin remodelling and assembly, might be required. Chk1 and NPM Interaction Networks. Both in vitro and in vivo studies described here support the association of Chk1 and NPM. Furthermore, immunoprecipitation experiments performed on chromatin extracts are also consistent with Chk1 and NPM being involved in complex protein networks that involve proteins such as p53 and the cell cycle regulator Cdc25A. The regulatory potential of this wider protein interaction network can be elaborated by employing a Pathway Studio software program (www. ariadnegenomics.com). Chk1, NPM, ATR, ATM, TP53 and CDC25A were matched to the Pathway

Studio data (ResNet 5.0) combined with data from this study to generate the network shown in Figure 7. Relationships between different interaction partners include protein-protein binding, expression, and regulation and protein modification. The network is characterized by processes linked to cell cycle regulation, as denoted by the direct involvement of Chk1, ATM and TP53. While Chk1 and NPM are recruited to chromatin naturally to maintain the natural rate of fork elongation, the induction of genomic stress results in activation of the checkpoint system as well as increased expression and chromatin association of proteins such as Chk1. As a downstream consequence, elevated binding of NPM to chromatin might serve to facilitate chromatin remodelling that is required during DNA repair or for activation of previously suppressed genes that are expressed during the stress response.

Acknowledgment. We thank Dr. Catherine Martin (University of Manchester) for useful discussions and Drs. Chris Storey and Sarah Hart (University of Manchester) for assistance with mass spectrometry. DT40 cells were generously provided by Professor David Gillespie (Beatson Institute for Cancer Research, Cancer Research UK, Glasgow, U.K.). This study was supported by a grant from BBSRC (project grant-D00327X). Supporting Information Available: Identification of chicken NPM, chicken actin, human NPM, and human actin by mass spectrometry; Chk1 and NPM in mammalian cells; NPM in DT40 cells after UCN-01 and UV treatment; comparison of nucleophosmin sequence and 3-D structure in human and chicken; comparison of Chk1 sequence and 3-D structure in human and chicken. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Abraham, R. T. Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev. 2001, 15, 2177–2196. (2) Bartek, J.; Lukas, C.; Lukas, J. Checking on DNA damage in S phase. Nat. Rev. Mol. Cell Biol. 2004, 5, 792–804. (3) Sancar, A.; Lindsey-Boltz, L. A.; Unsal-Kacmaz, K.; Linn, S. Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu. Rev. Biochem. 2004, 73, 39–85. (4) Bartek, J.; Lukas, J. Chk1 and Chk2 kinases in checkpoint control and cancer. Cancer Cell 2003, 3, 421–429. (5) Zhou, B. B.; Bartek, J. Targeting the checkpoint kinases: chemosensitization versus chemoprotection. Nat. Rev. Cancer 2004, 4, 216– 225. (6) Chen, Y.; Sanchez, Y. Chk1 in the DNA damage response: conserved roles from yeasts to mammals. DNA Repair 2004, 3, 1025–1032. (7) Shieh, S. Y.; Ahn, J.; Tamai, K.; Taya, Y.; Prives, C. The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites. Genes Dev. 2000, 14, 289–300. (8) Meek, S. E.; Lane, W. S.; Piwnica-Worms, H. Comprehensive proteomic analysis of interphase and mitotic 14-3-3-binding proteins. J. Biol. Chem. 2004, 279, 32046–32054. (9) Chen, M. S.; Ryan, C. E.; Piwnica-Worms, H. Chk1 kinase negatively regulates mitotic function of Cdc25A phosphatase through 14-3-3 binding. Mol. Cell. Biol. 2003, 23, 7488–7497. (10) Tang, J.; Erikson, R. L.; Liu, X. Checkpoint kinase 1 (Chk1) is required for mitotic progression through negative regulation of polo-like kinase 1 (Plk1). Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 11964–11969. (11) Sorensen, C. S.; Syljuasen, R. G.; Falck, J.; Schroeder, T.; Ronnstrand, L.; Khanna, K. K.; Zhou, B. B.; Bartek, J.; Lukas, J. Chk1 regulates the S phase checkpoint by coupling the physiological turnover and ionizing radiation-induced accelerated proteolysis of Cdc25A. Cancer Cell 2003, 3, 247–258. (12) Shechter, D.; Costanzo, V.; Gautier, J. ATR and ATM regulate the timing of DNA replication origin firing. Nat. Cell Biol. 2004, 6, 648– 655.

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