Ataxia-telangiectasia-mutated (ATM) and NBS1-dependent Phosphorylation of Chk1 on Ser-317 in Response to Ionizing Radiation

Abstract

In mammals, the ATM (ataxia-telangiectasia-mutated) and ATR (ATM and Rad3-related) protein kinases function as critical regulators of the cellular DNA damage response. The checkpoint functions of ATR and ATM are mediated, in part, by a pair of checkpoint effector kinases termed Chk1 and Chk2. In mammalian cells, evidence has been presented that Chk1 is devoted to the ATR signaling pathway and is modified by ATR in response to replication inhibition and UV-induced damage, whereas Chk2 functions primarily through ATM in response to ionizing radiation (IR), suggesting that Chk2 and Chk1 might have evolved to channel the DNA damage signal from ATM and ATR, respectively. We demonstrate here that the ATR-Chk1 and ATM-Chk2 pathways are not parallel branches of the DNA damage response pathway but instead show a high degree of cross-talk and connectivity. ATM does in fact signal to Chk1 in response to IR. Phosphorylation of Chk1 on Ser-317 in response to IR is ATM-dependent. We also show that functional NBS1 is required for phosphorylation of Chk1, indicating that NBS1 might facilitate the access of Chk1 to ATM at the sites of DNA damage. Abrogation of Chk1 expression by RNA interference resulted in defects in IR-induced S and G2/M phase checkpoints; however, the overexpression of phosphorylation site mutant (S317A, S345A or S317A/S345A double mutant) Chk1 failed to interfere with these checkpoints. Surprisingly, the kinase-dead Chk1 (D130A) also failed to abrogate the S and G2 checkpoint through any obvious dominant negative effect toward endogenous Chk1. Therefore, further studies will be required to assess the contribution made by phosphorylation events to Chk1 regulation. Overall, the data presented in the study challenge the model in which Chk1 only functions downstream from ATR and indicate that ATM does signal to Chk1. In addition, this study also demonstrates that Chk1 is essential for IR-induced inhibition of DNA synthesis and the G2/M checkpoint. In mammals, the ATM (ataxia-telangiectasia-mutated) and ATR (ATM and Rad3-related) protein kinases function as critical regulators of the cellular DNA damage response. The checkpoint functions of ATR and ATM are mediated, in part, by a pair of checkpoint effector kinases termed Chk1 and Chk2. In mammalian cells, evidence has been presented that Chk1 is devoted to the ATR signaling pathway and is modified by ATR in response to replication inhibition and UV-induced damage, whereas Chk2 functions primarily through ATM in response to ionizing radiation (IR), suggesting that Chk2 and Chk1 might have evolved to channel the DNA damage signal from ATM and ATR, respectively. We demonstrate here that the ATR-Chk1 and ATM-Chk2 pathways are not parallel branches of the DNA damage response pathway but instead show a high degree of cross-talk and connectivity. ATM does in fact signal to Chk1 in response to IR. Phosphorylation of Chk1 on Ser-317 in response to IR is ATM-dependent. We also show that functional NBS1 is required for phosphorylation of Chk1, indicating that NBS1 might facilitate the access of Chk1 to ATM at the sites of DNA damage. Abrogation of Chk1 expression by RNA interference resulted in defects in IR-induced S and G2/M phase checkpoints; however, the overexpression of phosphorylation site mutant (S317A, S345A or S317A/S345A double mutant) Chk1 failed to interfere with these checkpoints. Surprisingly, the kinase-dead Chk1 (D130A) also failed to abrogate the S and G2 checkpoint through any obvious dominant negative effect toward endogenous Chk1. Therefore, further studies will be required to assess the contribution made by phosphorylation events to Chk1 regulation. Overall, the data presented in the study challenge the model in which Chk1 only functions downstream from ATR and indicate that ATM does signal to Chk1. In addition, this study also demonstrates that Chk1 is essential for IR-induced inhibition of DNA synthesis and the G2/M checkpoint. ataxia-telangiectasia-mutated protein kinase ATM and Rad3-related protein kinase ionizing radiation hydroxyurea ataxia-telangiectasia Nijmegan breakage syndrome small interfering RNA gray The detection of DNA damage in mammalian cells induces an array of responses that result in cell cycle arrest, DNA repair, gene transcription, and, in some instances, cell death (1Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2633) Google Scholar). In mammals, the ataxia-telangiectasia-mutated (ATM)1 and ATM and Rad3-related (ATR) protein kinases function as critical regulators of the cellular DNA damage response. ATM and ATR are Ser/Thr-Gln-directed protein kinases with overlapping substrate specificities that are activated in response to distinct as well as partially overlapping types of genotoxic stimuli (2Abraham R.T. Genes Dev. 2001; 15: 2177-2196Crossref PubMed Scopus (1664) Google Scholar, 3Khanna K.K. Jackson S.P. Nat. Genet. 2001; 27: 247-254Crossref PubMed Scopus (1921) Google Scholar). Despite their structural similarity and overlapping substrate specificities, ATR and ATM are functionally non-redundant protein kinases. Cells from A-T patients orATM-nullizygous mice are exquisitely sensitive to ionizing radiation (IR) and other agents that induce double strand breaks and are defective in activating IR-induced G1/S, S phase, and G2/M checkpoints (4Barlow C. Hirotsune S. Paylor R. Liyangage M. Eckhaus M. Collins F. Shiloh Y. Crawley J.N. Ried T. Tagle D. Wynshaw-Boris A. Cell. 1996; 86: 159-171Abstract Full Text Full Text PDF PubMed Scopus (1259) Google Scholar, 5Xu Y. Baltimore D. Genes Dev. 1996; 10: 2401-2410Crossref PubMed Scopus (350) Google Scholar), whereas checkpoint responses to UV light and base-damaging agents are normal in A-T cells. In contrast to ATM−/− mice, which are viable, ATR-deficient mice die early during embryogenesis, and the conditional knockout ofATR gene function in human cells leads to a loss of cellular viability (6de Klein A. Muijtjens M. van Os R. Verhoeven Y. Smit B. Carr A.M. Lehmann A.R. Hoeijmakers J.H. Curr. Biol. 2000; 10: 479-482Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 7Brown E.J. Baltimore D. Genes Dev. 2000; 14: 397-402PubMed Google Scholar, 8Cortez D. Guntuku S. Qin J. Elledge S.J. Science. 2001; 294: 1713-1716Crossref PubMed Scopus (747) Google Scholar). ATR is required for the G2/M DNA damage checkpoint as well as the DNA replication checkpoint, which suppresses mitosis in the presence of unreplicated DNA (9Cliby W.A. Roberts C.J. Cimprich K.A. Stringer C.M. Lamb J.R. Schreiber S.L. Friend S.H. EMBO J. 1998; 2: 159-169Crossref Scopus (483) Google Scholar). ATR mediates responses to a broad spectrum of genotoxic stimuli, including IR, DNA replication inhibitors (e.g. hydroxyurea or HU), UV light, and agents such as cis-platinum that induce DNA inter-strand crosslinks (9Cliby W.A. Roberts C.J. Cimprich K.A. Stringer C.M. Lamb J.R. Schreiber S.L. Friend S.H. EMBO J. 1998; 2: 159-169Crossref Scopus (483) Google Scholar, 10Wright J.A. Keegan K.S. Herendeen D.R. Bentley N.J. Carr A.M. Hoekstra M.F. Concannon P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7445-7450Crossref PubMed Scopus (198) Google Scholar). A common denominator of these agents is the ability to induce transient or prolonged DNA replication fork stalling during S phase. This stalled replication fork may be a critical trigger for ATR signaling potential. The checkpoint functions of ATR and ATM are mediated, in part, by a pair of checkpoint effector kinases termed Chk1 and Chk2/Cds1 (1Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2633) Google Scholar, 2Abraham R.T. Genes Dev. 2001; 15: 2177-2196Crossref PubMed Scopus (1664) Google Scholar,3Khanna K.K. Jackson S.P. Nat. Genet. 2001; 27: 247-254Crossref PubMed Scopus (1921) Google Scholar). Although structurally distinct, Chk1 and Chk2 are functionally related kinases that phosphorylate an overlapping pool of cellular substrates (11O'Neill T. Giarratani L. Chen P. Iyer L. Lee C.H. Bobiak M. Kanai F. Zhou B, B. Chung J.H. Rathbun G.A. J. Biol. Chem. 2002; 277: 16102-16115Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). In mammalian cells, evidence has been presented that Chk2 and Chk1 have apparently evolved to channel DNA damage signals from ATM and ATR, respectively. This is based on reports that Chk1 appears to be devoted to the ATR signaling pathway and is modified by ATR in response to replication inhibition and UV-induced damage, whereas Chk2 functions primarily through ATM in response to IR (12Zhao H. Piwnica-Worms H. Mol. Cell. Biol. 2001; 21: 4129-4139Crossref PubMed Scopus (861) Google Scholar, 13Guo Z. Kumagai A. Wang S.X. Dunphy W.G. Genes Dev. 2000; 14: 2745-2756Crossref PubMed Scopus (366) Google Scholar, 14Liu Q. Guntuku S. Cui X.S. Matsuoka S. Cortez D. Tamai K. Luo G. Carattini-Rivera S. De Maya F. Bradley A. Donehower L.A. Elledge S.J. Genes Dev. 2000; 14: 1448-1459Crossref PubMed Scopus (193) Google Scholar). Here, we have investigated the contribution of ATM in the regulation of Chk1 after IR. We demonstrate that Chk1 is a downstream target of ATM and is phosphorylated on Ser-317 in response to IR. Reduction of Chk1 expression by RNA interference results in a defect in regulation of the S and G2 phase checkpoints; however cells overexpressing the phosphorylation site mutants Chk1 were not compromised in their ability to arrest in S and G2 following IR. Control, A-T, and NBS lymphoblastoid cell lines were grown in RPMI 1640 with 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were exposed to genotoxic agents (gamma, UV radiation, or HU) and, unless otherwise stated, harvested at the indicated time points. Cell extracts were prepared by lysis in universal immunoprecipitation buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 2 mm EDTA, 25 mmsodium fluoride, 25 mm β-glycerophosphate, 0.1 mm sodium orthovanadate, 0.1 mmphenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, 1 μg/ml aprotinin, 0.2% Triton X-100, and 0.3% Nonidet-P40). Twenty-five micrograms of whole cell extract was loaded per lane on 7.5% SDS-PAGE gels. Samples were transferred to nitrocellulose using carbonate buffer at 30 V for 3 h, and the membranes were probed with appropriate antibodies. Rabbit polyclonal anti-phosphopeptide antisera against ATM/ATR phosphorylation sites in Chk1 (Ser-317 and Ser-345) were raised by immunizing rabbits with the keyhole limpet hemocyanin-conjugated phosphopeptides NVKYSS-PhosphoSer-QPEPRT (for Ser-317) and VQGISF-PhosphoSer-QPTCPD (for Ser-345). Bleeds were assayed by enzyme-linked immunosorbent assay, and the serum was collected on days corresponding to the peak antibody response and affinity purified against the same peptide. Anti-Chk1 monoclonal and anti-cyclin B antibodies were from Santa Cruz Biotechnology, and anti-S15p53 was from New England Biolabs. ATM-complemented cells (15Ziv Y. Bar-Shira A. Pecker I. Russell P. Jorgensen T.J. Tsarfati I. Shiloh Y. Oncogene. 1997; 15: 159-167Crossref PubMed Scopus (223) Google Scholar) and NBS1-complemented (16Gatei M. Young D. Cerosaletti K.M. Desai-Mehta A. Spring K. Kozlov S. Lavin M.F. Gatti R.A. Concannon P. Khanna K.K. Nat. Genet. 2000; 25: 115-119Crossref PubMed Scopus (410) Google Scholar) cells have been described previously. Kinase-dead Chk1 (D130A, D148E), phosphorylation site Chk1 mutants (S317A, S345A, S317A/S345A, S317D, S345D, S317D/S345D) were constructed using the Quick Change mutagenesis kit (Stratagene) by using FLAG-tagged Chk1 in PCI-Neo vector (Promega) as a template. The details of the primers are available from the authors on request. RNA interference of Chk1 was performed using 5′-UCGUGAGCGUUUGUUGAAC-3′. siRNA specific to HSP70 served as a negative control (GCACAUCAGCCAGAACAAG). The oligos were from A siRNA was using the by the and was using the to the Cell were prepared after Cells were by double S. F. B. Lee Piwnica-Worms H. Mol. Biol. Cell. PubMed Scopus Google Scholar). mm was for h, cells were with and in and for Cells were in mm for this the cells were with and normal This was the at which of cells are in by and cells were harvested at S and time the cells were or with of IR for 30 cell extracts were prepared as described and with the antibodies. Cells were or with Chk1 or Chk1 cells were or exposed to 3 of IR. Cells were harvested 1 after IR and in at the cell was in 100 of of antibody and serum by with a antibody as described B. Mol. Cell. Biol. 2001; 21: PubMed Scopus Google Scholar). 1 of at in the the cells were with and cellular was by The data was using Cell of DNA synthesis after DNA damage was as described J. J. J. Nat. Genet. 2002; PubMed Scopus Google Scholar). Cells were with of for The was during the and the cells were with siRNA oligos Chk1 or with and Chk1 at the of and for Cells were with and, was after the of cells were harvested and in or after with as described J. J. J. Nat. Genet. 2002; PubMed Scopus Google Scholar), and was in an The DNA synthesis was by the of and as a of control the ATM and Chk1, we Chk1 in control and cells with IR. In control lymphoblastoid cells and a small of the Chk1 protein from cells a in with cells, whereas of the cell lines failed to show that in Chk1 UV was in and control lymphoblastoid cell lines studies have that this of Chk1 after DNA damage is of Therefore, we the in of Chk1 in and control cells with or to and control cells were with for 2 after to of Chk1 was from the cell and by 10% and Chk1 was by Chk1 in control and A-T cells appears to be and this phosphorylation was 2 after IR in control cells. In was a Chk1 from cells studies have sites (Ser-317 and Ser-345) of phosphorylation in Chk1, and ATR has been to phosphorylate Chk1 on and Ser-317 primarily in response to replication inhibition and UV (12Zhao H. Piwnica-Worms H. Mol. Cell. Biol. 2001; 21: 4129-4139Crossref PubMed Scopus (861) Google Scholar, 14Liu Q. Guntuku S. Cui X.S. Matsuoka S. Cortez D. Tamai K. Luo G. Carattini-Rivera S. De Maya F. Bradley A. Donehower L.A. Elledge S.J. Genes Dev. 2000; 14: 1448-1459Crossref PubMed Scopus (193) Google Scholar). Therefore, to Chk1 phosphorylation we polyclonal antibodies against a of phosphorylated Ser-317 or We were to phosphorylation of Chk1 in response to IR in lymphoblastoid or cell lines from control phosphorylation of Ser-317 in Chk1 was in response to agents in control cells. A-T cells were defective in phosphorylation of Chk1 on Ser-317 in response to IR or but at a high of IR or after and Chk1 phosphorylation in and control cells was The of phosphorylation of Chk1 was with the phosphorylation of a target of ATM The time that Chk1 phosphorylation was in control cells 30 whereas in cells by 2 and was with at after IR IR-induced Chk1 phosphorylation in A-T cells was by the of ATM is Ser-317 phosphorylation of Chk1 was also defective in lymphoblastoid cells from or for ATM which is to a high of T. A.M. A J. Genet. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, G. M. H. A. Chen K. M. D. C. T. R. J. K. J. Khanna K.K. J. Natl. 2002; PubMed Scopus Google Scholar). We have that this mutant of ATM is but defective as a kinase G. M. H. A. Chen K. M. D. C. T. R. J. K. J. Khanna K.K. J. Natl. 2002; PubMed Scopus Google Scholar). The mutant has been to as a dominant negative that the ATM is to function in the presence of mutant The defective Ser-317 phosphorylation in a of is related to this dominant negative effect from and has that the of Chk1 and Chk2 a of gene that in the and of checkpoint signals Carr A.M. Cell Biol. 2000; 10: Full Text Full Text PDF PubMed Scopus Google Scholar). phosphorylation of ATM substrates such as has been to be defective in NBS cells B. Genes Dev. 2002; PubMed Scopus Google Scholar, Wang Y. S. Lee Qin J. Genes Dev. 2002; PubMed Scopus (410) Google Scholar), whereas phosphorylation of and has been to in NBS cell lines (16Gatei M. Young D. Cerosaletti K.M. Desai-Mehta A. Spring K. Kozlov S. Lavin M.F. Gatti R.A. Concannon P. Khanna K.K. Nat. Genet. 2000; 25: 115-119Crossref PubMed Scopus (410) Google Scholar, M. R. S. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, we the of Chk1 phosphorylation on phosphorylation of Chk1 in response to IR was also in cells in NBS1 as with control cells but after and Chk1 phosphorylation in cells and control cells was Phosphorylation of Chk1 was with the of NBS1 in NBS1 is a target of and ATM NBS1 on This phosphorylation is required for of the S phase checkpoint. We the for the phosphorylated of NBS1 in Chk1 phosphorylation by using the NBS cells that were with NBS1 and the ATM phosphorylation site mutant NBS1 Chk1 expression was by overexpression of mutant NBS1 however, the of Ser-317 phosphorylation on NBS1 phosphorylation was not of data indicated that to the of Chk1 was in cells. of Chk1 after IR required a functional ATM we assayed Chk1 in cells with vector or vector cells with radiation effect on Chk1 and, cells ATM of Chk1 with cells. IR in the presence of ATM a small effect on the of Chk1 we Chk1 after IR in NBS1 defective cells with or with NBS1 and failed to the NBS1 of Chk1 after however, the ATM cells, NBS1 cells with vector only cells. the functional of the phosphorylation of Chk1, we Ser-317 and or to or The for these was that the which be might not be Chk1 phosphorylation is for and the of may and be to functions to phosphorylated Chk1. cells were with FLAG-tagged Chk1, phosphorylation site and kinase-dead Chk1 to kinase of Chk1 was as a of after cells were or with of IR. were prepared and with antibody and for Chk1 protein and Chk1 kinase in phosphorylation site mutant of Chk1 and specific with Chk1, whereas kinase (D130A) in this the of the after with IR not results indicate that the phosphorylation site mutants of Chk1 not the of Chk1 to a studies using small inhibitors of Chk1 and have a for Chk1 in the regulation of the G2/M checkpoint L. J. Piwnica-Worms H. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, J.R. A. C. L.A. A. 2000; Google Scholar). is to their to Chk1 as these inhibitors other kinases as We to the for Chk1 in G2 checkpoint signaling using an siRNA knockout of Chk1 cells were with and the Chk1 expression was in cells control and siRNA cells were for their ability to in G2 at 1 after to of IR by of antibody to cells from G2 cells. cells with siRNA a in the of cells mitosis A and The Chk1 in following 1 IR A results that Chk1 is required for the in after IR. phosphorylation events to G2/M checkpoint regulation by Chk1, we an IR-induced G2 checkpoint be by overexpression of phosphorylation site mutants of Chk1. Chk1 and phosphorylation site mutant cells and double Ser-317 and Ser-345) and in the of cells. Cells the kinase Chk1 and were also normal in the ability to in IR. the same expression of kinase-dead ATM only a in the of after IR. these results show Chk1 is required for G2 arrest, the of the at Ser-317 and in G2 arrest to be phosphorylation of Chk1 on Ser-317 during normal cell cycle we cells at the by double and their through the cell cycle after the Chk1 was phosphorylated on Ser-317 at the and S phase of normal S phase however, phosphorylation was in cells in G2 or G2/M In phosphorylation of Chk1 on Ser-317 was at cell cycle results were with cells by serum and the not S phase phosphorylation of Chk1 is also required for an IR-induced S phase checkpoint, we cells, which have been to be in branches of the S phase checkpoint and cells at inhibition of their DNA synthesis 1 IR, as by Cells kinase Chk1 (D130A) or mutant Chk1 (S317A, S345A, and of of DNA synthesis after IR these not show any obvious negative effect of these mutants on the checkpoint function in the presence of endogenous Chk1. the other of endogenous Chk1 by siRNA by IR and of the S phase checkpoint, a DNA synthesis a of checkpoint results the DNA synthesis were the of Chk1 was by the not to the of Chk1 for the of the G2/M checkpoint, Chk1 is also essential for the IR-induced inhibition of DNA The protein kinase Chk1 is phosphorylated at Ser-317 and in response to DNA damage. has been evidence that these phosphorylation events are by ATR (12Zhao H. Piwnica-Worms H. Mol. Cell. Biol. 2001; 21: 4129-4139Crossref PubMed Scopus (861) Google Scholar, 13Guo Z. Kumagai A. Wang S.X. Dunphy W.G. Genes Dev. 2000; 14: 2745-2756Crossref PubMed Scopus (366) Google Scholar, 14Liu Q. Guntuku S. Cui X.S. Matsuoka S. Cortez D. Tamai K. Luo G. Carattini-Rivera S. De Maya F. Bradley A. Donehower L.A. Elledge S.J. Genes Dev. 2000; 14: 1448-1459Crossref PubMed Scopus (193) Google Scholar). ATR overlapping substrate specificities with ATM in is in response to studies have indicated in mammalian cells, ATR and ATM might parallel branches of the DNA damage response pathway and that Chk1 and Chk2 in might have evolved to signal DNA damage from ATR and (12Zhao H. Piwnica-Worms H. Mol. Cell. Biol. 2001; 21: 4129-4139Crossref PubMed Scopus (861) Google Scholar, 13Guo Z. Kumagai A. Wang S.X. Dunphy W.G. Genes Dev. 2000; 14: 2745-2756Crossref PubMed Scopus (366) Google Scholar). The pathway is by DNA whereas the pathway primarily to by UV and DNA replication we evidence for cross-talk the pathways by that ATM does target Chk1 in response to IR. We demonstrate that phosphorylation of Chk1 on Ser-317 in response to IR is compromised in cells and that expression of ATM this Chk1 phosphorylation in response to and UV is by ATR, as indicated by studies that have that expression of a of ATR with UV-induced phosphorylation of Chk1 Z. Kumagai A. Wang S.X. Dunphy W.G. Genes Dev. 2000; 14: 2745-2756Crossref PubMed Scopus (366) Google Scholar, 14Liu Q. Guntuku S. Cui X.S. Matsuoka S. Cortez D. Tamai K. Luo G. Carattini-Rivera S. De Maya F. Bradley A. Donehower L.A. Elledge S.J. Genes Dev. 2000; 14: 1448-1459Crossref PubMed Scopus (193) Google Scholar). results that the NBS1 gene is required for IR-induced phosphorylation of Chk1 on Ser-317 but is for phosphorylation in response to and The of the data is that NBS1 ATM in some of This is with ATM and NBS1 of phosphorylation in response to IR B. Genes Dev. 2002; PubMed Scopus Google Scholar, Wang Y. S. Lee Qin J. Genes Dev. 2002; PubMed Scopus (410) Google Scholar). and phosphorylation (16Gatei M. Young D. Cerosaletti K.M. Desai-Mehta A. Spring K. Kozlov S. Lavin M.F. Gatti R.A. Concannon P. Khanna K.K. Nat. Genet. 2000; 25: 115-119Crossref PubMed Scopus (410) Google Scholar, A. J. Cell Biol. 2000; PubMed Scopus Google Scholar) in cells, by and that might the phosphorylation of these substrates by and be phosphorylated by ATM in whereas Chk1 is a ATM substrate in not has been in the as to Chk1 is after DNA however, after we have not been to demonstrate any in after IR. we not a defect in Chk1 kinase in or cells in response to IR. phosphorylation of Chk1 is required for checkpoint we phosphorylation site mutants of Chk1 and or in to study their on G2/M and S phase checkpoint. phosphorylation site mutants were as a protein kinase with or to IR. results demonstrate that the phosphorylation site mutants not interfere with the checkpoint the of some of phosphorylation in Chk1, of the checkpoint is not that these sites of phosphorylation are the only Chk1 sites other including and Z. Kumagai A. Wang S.X. Dunphy W.G. Genes Dev. 2000; 14: 2745-2756Crossref PubMed Scopus (366) Google Scholar). to the we were to demonstrate a dominant negative effect through overexpression of Chk1 or the that Chk1 does not on and that the with substrates are with a of the of the protein may not be of might not be to demonstrate a dominant negative effect of Chk1 mutants in overexpression Therefore, the of the phosphorylation sites only be by the of and mutant Chk1 to the checkpoint functions in cells. The with these is the of of cells, as Chk1 is an essential gene in mice Q. Guntuku S. Cui X.S. Matsuoka S. Cortez D. Tamai K. Luo G. Carattini-Rivera S. De Maya F. Bradley A. Donehower L.A. Elledge S.J. Genes Dev. 2000; 14: 1448-1459Crossref PubMed Scopus (193) Google Scholar, H. K. H. T. K. K. M. K. Genes Dev. 2000; 14: Google Scholar). we have that the of Chk1 expression in mammalian cells by siRNA does to a defect in S and G2/M checkpoint which is with a H. Piwnica-Worms H. Proc. Natl. Acad. Sci. U. S. A. Scopus Google Scholar). cells might be for studies with or Chk1 mutant The challenge here will be to Chk1 to to inhibition by which will be the of We for cell lines from the Shiloh for the A-T cell and ATM cells, Concannon for the NBS1 cells, and Hoekstra for the Chk1 expression