Inhibition of Polo-like Kinase-1 by DNA Damage Occurs in an ATM- or ATR-dependent Fashion

Polo-like kinases play multiple roles in different phases of mitosis. We have recently shown that the mammalian polo-like kinase, Plk1, is inhibited in response to DNA damage and that this inhibition may lead to cell cycle arrests at multiple points in mitosis. Here we have investigated the role of the checkpoint kinases ATM (ataxia telangiectasia mutated) and ATR (ATM- and Rad3-related) in DNA damage-induced inhibition of Plk1. We show that inhibition of Plk1 kinase activity is efficiently blocked by the radio-sensitizing agent caffeine. Using ATM−/− cells we show that under certain circumstances, inhibition of Plk1 by DNA-damaging agents critically depends on ATM. In addition, we show that UV radiation also causes inhibition of Plk1, and we present evidence that this inhibition is mediated by ATR. Taken together, our data demonstrate that ATM and ATR can regulate Plk1 kinase activity in response to a variety of DNA-damaging agents. Polo-like kinases play multiple roles in different phases of mitosis. We have recently shown that the mammalian polo-like kinase, Plk1, is inhibited in response to DNA damage and that this inhibition may lead to cell cycle arrests at multiple points in mitosis. Here we have investigated the role of the checkpoint kinases ATM (ataxia telangiectasia mutated) and ATR (ATM- and Rad3-related) in DNA damage-induced inhibition of Plk1. We show that inhibition of Plk1 kinase activity is efficiently blocked by the radio-sensitizing agent caffeine. Using ATM−/− cells we show that under certain circumstances, inhibition of Plk1 by DNA-damaging agents critically depends on ATM. In addition, we show that UV radiation also causes inhibition of Plk1, and we present evidence that this inhibition is mediated by ATR. Taken together, our data demonstrate that ATM and ATR can regulate Plk1 kinase activity in response to a variety of DNA-damaging agents. phosphate-buffered saline l-α-lysophosphatidylcholine To monitor genomic integrity, cells are equipped with a variety of checkpoint mechanisms (1Elledge S.J. Science. 1996; 274: 1664-1672Crossref PubMed Scopus (1760) Google Scholar). One such checkpoint is the G2DNA damage checkpoint, preventing mitotic entry when DNA is damaged (2O'Connell M.J. Walworth N.C. Carr A.M. Trends Cell Biol. 2000; 10: 296-303Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar). This checkpoint is highly conserved from Schizosaccharomyces pombe to mammalian cells and requires the function of a family of checkpoint kinases that share homology with phosphatidylinositol 3-kinase. In yeast, this family of checkpoint kinases is comprised of scTel1, scMec1, spTel1, and spRad3 (2O'Connell M.J. Walworth N.C. Carr A.M. Trends Cell Biol. 2000; 10: 296-303Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar). The mammalian checkpoint kinases that belong to this family are ATM, the gene mutated in ataxia telangiectasia, and ATR (ATM- andRad3-related). The ATM kinase plays a very important role in the linkage of DNA damage detecting and the induction of a subsequent cell cycle arrest. This is illustrated by the fact that ataxia telangiectasia cells were described to be defective for the G1, S, and G2-M checkpoints (3Beamish H. Lavin M.F. Int. J. Radiat. Biol. 1994; 65: 175-184Crossref PubMed Scopus (186) Google Scholar). Also, ataxia telangiectasia patients show extreme sensitivity to radiation and increased predisposition to tumor formation. In the DNA damage checkpoint, activation of ATM has been shown to result in the activation of other checkpoint kinases, such as Chk1 and Chk2/hCds1 (4Walworth N.C. Bernards R. Science. 1996; 271: 353-356Crossref PubMed Scopus (348) Google Scholar,5Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1082) Google Scholar). At least for Chk2, this was shown to occur through direct phosphorylation by ATM in vivo (6Matsuoka S. Rotman G. Ogawa A. Shiloh Y. Tamai K. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10389-10394Crossref PubMed Scopus (683) Google Scholar). In addition, ATM was also shown to be responsible for damage-induced phosphorylation of p53, MDM-2, BRCA1, and NBS1 (7Banin S. Moyal L. Shieh S. Taya Y. Anderson C.W. Chessa L. Smorodinsky N.I. Prives C. Reiss Y. Shiloh Y. Ziv Y. Science. 1998; 281: 1674-1677Crossref PubMed Scopus (1697) Google Scholar, 8Cortez D. Wang Y. Qin J. Elledge S.J. Science. 1999; 286: 1162-1166Crossref PubMed Scopus (868) Google Scholar, 9Khosravi R. Maya R. Gottlieb T. Oren M. Shiloh Y. Shkedy D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14973-14977Crossref PubMed Scopus (352) Google Scholar, 10Lim D.S. Kim S.T. Xu B. Maser R.S. Lin J. Petrini J.H. Kastan M.B. Nature. 2000; 404: 613-617Crossref PubMed Scopus (672) Google Scholar, 11Canman C.E. Lim D.S. Cimprich K.A. Taya Y. Tamai K. Sakaguchi K. Appella E. Kastan M.B. Siliciano J.D. Science. 1998; 281: 1677-1679Crossref PubMed Scopus (1700) Google Scholar). Thus, ATM appears to sit at the top of the checkpoint signaling cascades that trigger cell cycle arrests in response to DNA damage. The cell cycle arrest that is provoked when the G2 DNA damage pathway is activated is achieved at least in part by inhibition of the cyclin B·cdc2 complex (12Rhind N. Furnari B. Russell P. Genes Dev. 1997; 11: 504-511Crossref PubMed Scopus (223) Google Scholar, 13Jin P. Gu Y. Morgan D.O. J. Cell Biol. 1996; 134: 963-970Crossref PubMed Scopus (249) Google Scholar). Activation of this complex is critically required to enter mitosis, and this activation is brought about via dephosphorylation of cdc2, which is mediated by the Cdc25C phosphatase (14Dunphy W.G. Kumagai A. Cell. 1991; 67: 189-196Abstract Full Text PDF PubMed Scopus (446) Google Scholar, 15Gautier J. Solomon M.J. Booher R.N. Bazan J.F. Kirschner M.W. Cell. 1991; 67: 197-211Abstract Full Text PDF PubMed Scopus (684) Google Scholar). This dual specificity phosphatase is the target of the G2 DNA damage checkpoint, and dephosphorylation of Tyr15 on cdc2 is efficiently impaired when DNA is damaged (16Furnari B. Rhind N. Russell P. Science. 1997; 277: 1495-1497Crossref PubMed Scopus (473) Google Scholar). Inactivation of Cdc25C was suggested to be achieved by phosphorylation of Cdc25C by the serine/threonine kinases Chk1 and Chk2 (5Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1082) Google Scholar, 17Sanchez Y. Wong C. Thoma R.S. Richman R. Wu Z. Piwnica-Worms H. Elledge S.J. Science. 1997; 277: 1497-1501Crossref PubMed Scopus (1120) Google Scholar). Phosphorylation of Cdc25C by Chk1 or Chk2 subsequently inhibits cyclin B·cdc2 activation (18Furnari B. Blasina A. Boddy M.N. McGowan C.H. Russell P. Mol. Biol. Cell. 1999; 10: 833-845Crossref PubMed Scopus (178) Google Scholar, 19Peng C.Y. Graves P.R. Thoma R.S. Wu Z. Shaw A.S. Piwnica-Worms H. Science. 1997; 277: 1501-1505Crossref PubMed Scopus (1181) Google Scholar). In contrast to the inhibitory phosphorylation by Chk1 and Chk2, Cdc25C also requires phosphorylation to become activated. Activating phosphorylation is thought to require both cyclin B·cdc2 complexes and the action of Polo-like kinases (Plks) (20Hoffmann I. Clarke P.R. Marcote M.J. Karsenti E. Draetta G. EMBO J. 1993; 12: 53-63Crossref PubMed Scopus (563) Google Scholar, 21Kumagai A. Dunphy W.G. Science. 1996; 273: 1377-1380Crossref PubMed Scopus (468) Google Scholar). Because depletion of Plx1, the Xenopus homologue of Polo-like kinases, from oocyte extracts, results in a block of Cdc25C activation, an essential role for Plks in the initiation of the activation-loop of Cdc25C has been suggested (22Qian Y.W. Erikson E. Li C. Maller J.L. Mol. Cell. Biol. 1998; 18: 4262-4271Crossref PubMed Scopus (212) Google Scholar). Recently, Plks were also shown to be regulated in response to DNA damage. Cdc5, the Saccharomyces cerevisiae homologue of Plk, was shown to be post-translationally modified in response to DNA damage (23Cheng L. Hunke L. Hardy C.F. Mol. Cell. Biol. 1998; 18: 7360-7370Crossref PubMed Scopus (95) Google Scholar). Furthermore, Cdc5 appeared to act downstream of Rad53, the S. cerevisiae homologue of Chk2, because a loss-of-function mutant of Cdc5 could revert a Rad53 defect, and overexpression of Cdc5 resulted in a DNA damage checkpoint override (24Sanchez Y. Bachant J. Wang H. Hu F. Liu D. Tetzlaff M. Elledge S.J. Science. 1999; 286: 1166-1171Crossref PubMed Scopus (454) Google Scholar). Recent work from our laboratory has demonstrated that the mammalian Polo-like kinase, Plk1, is also a target of the G2-M DNA damage checkpoint (25Smits V.A. Klompmaker R. Arnaud L. Rijksen G. Nigg E.A. Medema R.H. Nat. Cell Biol. 2000; 2: 672-676Crossref PubMed Scopus (395) Google Scholar). Here we have investigated the role of the ATM and ATR checkpoint kinases in the inhibition of Plk1 by DNA damage. We show that inhibition of Plk1 occurs in an ATM-dependent fashion but that under certain conditions ATR can also signal inhibition of Plk1. Moreover, the data presented here demonstrate that Plk1 is also inhibited by UV irradiation, in addition to the DNA-damaging agents described previously. The human osteosarcoma cell line U2OS and the ATM−/− (ATBR1 and GM05849) and ATM+/+(GM00498) fibroblasts were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum. ATM−/− (GM05849) and ATM+/+ (GM00498) fibroblasts were purchased from the Coriell Institute (Camden, NJ). Media for all cell lines were supplemented with 2 mml-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. To inflict DNA damage with adriamycin, the cells were incubated with different concentrations of adriamycin in Dulbecco’s modified Eagle’s medium for 1 h and subsequently extensively washed. For bleomycin treatment, the cells were washed twice with PBS1 containing 1 mm CaCl2 and permeabilized with 4 μg/ml ofl-α-lysophosphatidylcholine (LPC) in PBS-CaCl2 for 2 min. Subsequently, the cells were treated with indicated concentrations of bleomycin for 30 min. LPC alone was taken along as a control. After treatment, the cells were extensively washed. Mouse monoclonal anti-MPM-2 and rabbit polyclonal anti-plk-1 were from Upstate Biotechnology, Inc. The mouse monoclonal anti-cyclin B1 and protein A/G-agarose were from Santa Cruz. Protein A-Sepharose was from Amersham Pharmacia Biotech.Fluorescein isothiocyanate-labeled goat anti-mouse secondary antibody was from Becton Dickinson. Histone H1 was from Roche Molecular Biochemicals. Adriamycin, bleomycin, LPC, wortmannin, caffeine, nocodazole, propidium iodide, and dephosphorylated α-casein were from Sigma. For Plk1 kinase assays, the cells were lysed in E1A lysis buffer (ELB, 150 mm NaCl, 50 mm Hepes, pH 7.5, 5 mmEDTA, 0.1% Nonidet P-40, 5 mm NaF, 1 mmphenylmethylsulfonyl fluoride, 50 kallikrein-inactivating units of aprotinin, 10 μg/ml leupeptin, 10 μg/ml trypsin inhibitor, 0.5 mm sodium orthovanadate, 20 mmβ-glycerophosphate) for 1 h at 4 °C. Total cell lysate was incubated with protein A-Sepharose beads and anti-Plk1 for overnight immunoprecipitation. Protein A-Sepharose beads were extensively washed, and subsequently kinase reactions were performed in kinase buffer (20 mm Hepes, pH 7.4, 150 mm KCl, 10 mmMgCl2, 1 mm EGTA, 0.5 mmdithiothreitol, 5 mm NaF) in the presence of 0.5 mg/ml dephosphorylated α-casein, 10 μm ATP, and 4 μCi of γ-32PATP. For cyclin B-associated kinase reactions, the cells were lysed in lysis buffer (20 mm Tris, pH 8.0, 1 mm EDTA, 400 mm NaCl, 0.5% Nonidet P-40, 5 mm NaF, 1 mm phenylmethylsulfonyl fluoride, 50 kallikrein-inactivating units of aprotinin, 10 μg/ml leupeptin, 10 μg/ml trypsin inhibitor, 0.5 mm sodium orthovanadate, 20 mm β-glycerophosphate). 5 μg of total cell lysate, in the presence of protein A/G-Sepharose and anti-cyclin B, was used for overnight immunoprecipitations. Protein A/G-Sepharose beads plus were extensively washed, and subsequently kinase reactions were performed in kinase buffer (50 mm Hepes, pH 7.5, 5 mmMgCl2, 2.5 mm MnCl2, 1 mm dithiothreitol) in the presence of 10 μg of histone H1, 50 μm ATP, and 2.5 μCi of γ-32PATP. The cells were fixed in 70% ethanol for 2 h at 4 °C. Ethanol was washed away with PBS, and subsequently the cells were incubated with anti-MPM-2 antibody for 1 h at 4 °C. Then the cells were washed with PBS and subsequently incubated with fluorescein isothiocyanate-labeled goat anti-mouse secondary antibody for 30 min at 4 °C, protected from light. Next, the cells were washed with PBS and then were incubated with RNase (0.25 mg/ml) and propidium iodide for 15 min at 37 °C. DNA content and MPM-2 positivity were analyzed using flow cytometry using Cell Quest software (Becton Dickinson). To investigate the role of ATM in Plk1 regulation, we examined the influence of the radio-sensitizing agent caffeine on DNA damage-induced inhibition of Plk1. Work from a number of laboratories has shown that the radio-sensitizing effect of caffeine is due to specific inhibition of the ATM/ATR checkpoint kinases (26Blasina A. Price B.D. Turenne G.A. McGowan C.H. Curr. Biol. 1999; 9: 1135-1138Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 27Hall-Jackson C.A. Cross D.A. Morrice N. Smythe C. Oncogene. 1999; 18: 6707-6713Crossref PubMed Scopus (193) Google Scholar, 28Sarkaria J.N. Busby E.C. Tibbetts R.S. Roos P. Taya Y. Karnitz L.M. Abraham R.T. Cancer Res. 1999; 59: 4375-4382PubMed Google Scholar, 29Zhou B.B. Chaturvedi P. Spring K. Scott S.P. Johanson R.A. Mishra R. Mattern M.R. Winkler J.D. Khanna K.K. J. Biol. Chem. 2000; 275: 10342-10348Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). Because we had obtained data that indicated that the damage-induced inhibition of Plk1 was reverted by the addition of caffeine (25Smits V.A. Klompmaker R. Arnaud L. Rijksen G. Nigg E.A. Medema R.H. Nat. Cell Biol. 2000; 2: 672-676Crossref PubMed Scopus (395) Google Scholar), we decided to investigate the involvement of ATM/ATR and the effects of caffeine in more detail. As shown in Fig. 1, the activity of Plk1 is high in U2OS cells trapped in mitosis with the microtubuli-destabilizing drug nocodazole, but this activity is severely inhibited by the DNA-damaging agent adriamycin, consistent with our previous findings. The addition of caffeine completely prevented DNA damage-induced Plk1 inhibition (Fig. 1 a), suggesting a role for ATM and/or ATR in the effects of DNA damage on Plk1. To extend these findings, we investigated the effects of adriamycin and caffeine on mitotic entry in the presence of DNA-damaging agents. To this end, we analyzed the appearance of the MPM-2 phospho-epitope. The monoclonal anti-MPM-2 antibody recognizes mitosis-specific phospho-epitopes and thus can be used to specifically detect mitotic cells (30Davis F.M. Tsao T.Y. Fowler S.K. Rao P.N. Proc. Natl. Acad. Sci. U. S. A. 1983; PubMed Scopus Google Scholar). trapped in mitosis with high MPM-2 cells a of MPM-2 positivity (Fig. 1 consistent with a block in mitotic caffeine was to this effect of DNA because a of the MPM-2 positivity was in the presence of DNA-damaging agents. data demonstrate that caffeine can a very checkpoint in these cells and that inhibition of Plk1 by DNA damage depends on the ATM and/or ATR To ATM and ATR as an of Plk1 the activity of Plk1 was examined in cell lines ATM, from different we a in inhibition of Plk1 kinase activity in response to the DNA-damaging agent adriamycin in both ATM−/− cell lines with (Fig. 2 Plk1 activity was inhibited very efficiently in the ATM+/+ cells using μm adriamycin, inhibition was at this of adriamycin in the This a role for ATM in the DNA damage-induced inhibition of Plk1 To these the effects of bleomycin, were examined (Fig. 2 was in with LPC to the of bleomycin A.S. Res. PubMed Scopus Google Scholar). to the effects of adriamycin, bleomycin efficiently blocked Plk1 activity in ATM+/+ with ATM−/− cells a inhibition of Plk1 was In both these the addition of LPC alone Plk1 activity Next, we investigated the inhibition of Plk1 with a inhibition of cyclin kinase activity (Fig. 2 cells cyclin B-associated kinase these were when cells were blocked in mitosis using As DNA damage resulted in a of cyclin kinase This was by caffeine, to the effect of caffeine on Plk1 kinase Moreover, inhibition of cyclin kinase activity by adriamycin a on ATM, as demonstrated by a of inhibition in ATM−/− cells (Fig. 2 in checkpoint response in ATM−/− cells was also at the of mitotic entry in these cells (Fig. 2 In ATM+/+ we a inhibition in mitotic entry in the presence of adriamycin, this effect was by the addition of caffeine. In in ATM−/− the addition of adriamycin could mitotic entry by We subsequently the effect of of adriamycin with to Plk1 inhibition in ATM−/− cells to a in of adriamycin result in inhibition of Plk1 activity in ATM−/− cells a), Thus, high of DNA damage can result in inhibition of Plk1, that other checkpoint kinases could also be We that Plk1 inhibition at high of adriamycin in ATM−/− cells was by the addition of caffeine (Fig. suggesting that the kinase, was responsible for this This in addition to ATM, ATR could also trigger inhibition of Plk1 under certain have shown that ATM is activated in response to For activation of the checkpoint kinase Chk2 is in response to radiation when ATM is present (6Matsuoka S. Rotman G. Ogawa A. Shiloh Y. Tamai K. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10389-10394Crossref PubMed Scopus (683) Google Scholar, C.H. N. Piwnica-Worms H. J.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar). In the ATM homologue ATR was shown to be activated in response to UV S. S. D. Tamai K. G. S. F. A. Elledge S.J. Genes Dev. 2000; PubMed Scopus (193) Google Scholar, M. B.B. K. Scott S. D. Khanna K.K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). ATM and ATR share a specificity for different that are in cell cycle which are BRCA1, p53, and Chk2 (6Matsuoka S. Rotman G. Ogawa A. Shiloh Y. Tamai K. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10389-10394Crossref PubMed Scopus (683) Google Scholar, S. Moyal L. Shieh S. Taya Y. Anderson C.W. Chessa L. Smorodinsky N.I. Prives C. Reiss Y. Shiloh Y. Ziv Y. Science. 1998; 281: 1674-1677Crossref PubMed Scopus (1697) Google Scholar, 8Cortez D. Wang Y. Qin J. Elledge S.J. Science. 1999; 286: 1162-1166Crossref PubMed Scopus (868) Google Scholar, 11Canman C.E. Lim D.S. Cimprich K.A. Taya Y. Tamai K. Sakaguchi K. Appella E. Kastan M.B. Siliciano J.D. Science. 1998; 281: 1677-1679Crossref PubMed Scopus (1700) Google Scholar, M. B.B. K. Scott S. D. Khanna K.K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, R.S. D. R. D. Elledge S.J. Abraham R.T. Genes Dev. 2000; PubMed Scopus Google Scholar, R.S. J.N. Shieh Taya Y. Prives C. Abraham R.T. Genes Dev. 1999; PubMed Scopus Google Scholar). For these activation radiation is on ATM, ATR is responsible for To investigate ATR can also regulate Plk1 kinase we decided to investigate the influence of UV on Plk1. As is shown in UV of ATM+/+ cells results in an inhibition of Plk1 kinase Moreover, UV radiation causes a inhibition in Plk1 activity in ATM−/− that inhibition can occur in an inhibition of Plk1 is due to an effect on but due to a in Plk1 activity (Fig. 4 be that UV is as as adriamycin in Plk1 in ATM+/+ This could be due to the fact that adriamycin may both ATR and ATM, UV and subsequently to could a in the of ATM and ATR to Plk1. To the inhibition of Plk1 to we examined the effects of caffeine on Plk1 activity UV of ATM−/− cells (Fig. 4 ATM−/− cells a inhibition in Plk1 kinase the addition of caffeine completely this the role of ATR in Plk1 We also examined the effects of on Plk1 has been shown to ATM function but is in ATR when to cells in J.N. Tibbetts R.S. Busby E.C. Abraham R.T. Cancer Res. 1998; 4375-4382PubMed Google Scholar). the inhibitory effects of adriamycin on Plk1 activity in ATM+/+ effects on inhibition of Plk1 activity were UV in cells (Fig. 4 consistent with a role for ATM in Plk1 activity in response to we the block to mitotic the of inhibition by UV was to that of Plk1 activity (Fig. 4 entry was blocked by UV radiation in ATM−/− and ATM+/+ as by the in MPM-2 Thus, inhibition of Plk1 with the block in mitotic entry in these In this is important to that Plk1 is also inhibited by adriamycin and UV radiation in cells that are blocked in mitosis (Fig. 4 the effects of adriamycin and UV radiation on Plk1 activity are also in cells that have the G2 DNA damage checkpoint, that inhibition of Plk1 is an of a DNA damage-induced G2 arrest. Also, this that DNA damage checkpoints are also at of mitosis, consistent with we described (25Smits V.A. Klompmaker R. Arnaud L. Rijksen G. Nigg E.A. Medema R.H. Nat. Cell Biol. 2000; 2: 672-676Crossref PubMed Scopus (395) Google Scholar). The results shown here that the inhibition of Plk1 kinase which is when the DNA damage checkpoint is requires ATM because the inhibition can be by the addition of caffeine, shown to with the function of ATM and because we were to show that Plk1 inhibition by the agents adriamycin and bleomycin was impaired in cells ATM. at very high of adriamycin we a inhibition of Plk1 activity in ATM−/− We that this is due to activation of ATR under these circumstances, because the inhibition could be by caffeine. using UV to specifically and ATM, we that Plk1 was also inhibition of Plk1 in ATM−/− and ATM+/+ that UV can Plk1 in an Furthermore, the inhibition of Plk1 activity in ATM−/− cells was efficiently by caffeine, a of cells were incubated with wortmannin, a of Plk1 activity was This is in with the inhibitory effects of on ATR in was shown by J.N. Tibbetts R.S. Busby E.C. Abraham R.T. Cancer Res. 1998; 4375-4382PubMed Google Scholar). Taken together, our results show that Plk1 can be regulated in an or in response to DNA damage and that the of damage which of these checkpoint kinases be Because Plk1 was shown to function at different of mitosis, be very to at which of mitosis ATM and ATR can checkpoint function and ATM or ATR can Plk1 p53, and Chk2, are on in by ATM (6Matsuoka S. Rotman G. Ogawa A. Shiloh Y. Tamai K. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10389-10394Crossref PubMed Scopus (683) Google Scholar, S. Moyal L. Shieh S. Taya Y. Anderson C.W. Chessa L. Smorodinsky N.I. Prives C. Reiss Y. Shiloh Y. Ziv Y. Science. 1998; 281: 1674-1677Crossref PubMed Scopus (1697) Google Scholar, 8Cortez D. Wang Y. Qin J. Elledge S.J. Science. 1999; 286: 1162-1166Crossref PubMed Scopus (868) Google Scholar, T. Ziv Y. S.P. Abraham R.H. J.H. D. Shiloh Y. G.A. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). Because Plk1 such a we that inhibition of Plk1 by ATM and ATR requires other checkpoint kinases, such as Chk1 or We R. for the cell line and other of the of Medema for