Ku70/80 Modulates ATM and ATR Signaling Pathways in Response to DNA Double Strand Breaks
2007; Elsevier BV; Volume: 282; Issue: 14 Linguagem: Inglês
10.1074/jbc.m611880200
ISSN1083-351X
AutoresNozomi Tomimatsu, Candice Tahimic, Akihiro Otsuki, Sandeep Burma, Akiko Fukuhara, Kenzo Sato, Goshi Shiota, Mitsuo Oshimura, David J. Chen, Akihiro Kurimasa,
Tópico(s)Carcinogens and Genotoxicity Assessment
ResumoDouble strand break (DSB) recognition is the first step in the DSB damage response and involves activation of ataxia telangiectasia-mutated (ATM) and phosphorylation of targets such as p53 to trigger cell cycle arrest, DNA repair, or apoptosis. It was reported that activation of ATM- and Rad3-related (ATR) kinase by DSBs also occurs in an ATM-dependent manner. On the other hand, Ku70/80 is known to participate at a later time point in the DSB response, recruiting DNA-PKcs to facilitate non-homologous end joining. Because Ku70/80 has a high affinity for broken DNA ends and is abundant in nuclei, we examined their possible involvement in other aspects of the DSB damage response, particularly in modulating the activity of ATM and other phosphatidylinositol (PI) 3-related kinases during DSB recognition. We thus analyzed p53Ser18 phosphorylation in irradiated Ku-deficient cells and observed persistent phosphorylation in these cells relative to wild type cells. ATM or ATR inhibition revealed that this phosphorylation is mainly mediated by ATM-dependent ATR activity at 2 h post-ionizing radiation in wild type cells, whereas in Ku-deficient cells, this occurs mainly through direct ATM activity, with a secondary contribution from ATR via a novel ATM-independent mechanism. Using ATM/Ku70 double-null cell lines, which we generated, we confirmed that ATM-independent ATR activity contributed to persistent phosphorylation of p53Ser18 in Ku-deficient cells at 12 h post-ionizing radiation. In summary, we discovered a novel role for Ku70/80 in modulating ATM-dependent ATR activation during DSB damage response and demonstrated that these proteins confer a protective effect against ATM-independent ATR activation at later stages of the DSB damage response. Double strand break (DSB) recognition is the first step in the DSB damage response and involves activation of ataxia telangiectasia-mutated (ATM) and phosphorylation of targets such as p53 to trigger cell cycle arrest, DNA repair, or apoptosis. It was reported that activation of ATM- and Rad3-related (ATR) kinase by DSBs also occurs in an ATM-dependent manner. On the other hand, Ku70/80 is known to participate at a later time point in the DSB response, recruiting DNA-PKcs to facilitate non-homologous end joining. Because Ku70/80 has a high affinity for broken DNA ends and is abundant in nuclei, we examined their possible involvement in other aspects of the DSB damage response, particularly in modulating the activity of ATM and other phosphatidylinositol (PI) 3-related kinases during DSB recognition. We thus analyzed p53Ser18 phosphorylation in irradiated Ku-deficient cells and observed persistent phosphorylation in these cells relative to wild type cells. ATM or ATR inhibition revealed that this phosphorylation is mainly mediated by ATM-dependent ATR activity at 2 h post-ionizing radiation in wild type cells, whereas in Ku-deficient cells, this occurs mainly through direct ATM activity, with a secondary contribution from ATR via a novel ATM-independent mechanism. Using ATM/Ku70 double-null cell lines, which we generated, we confirmed that ATM-independent ATR activity contributed to persistent phosphorylation of p53Ser18 in Ku-deficient cells at 12 h post-ionizing radiation. In summary, we discovered a novel role for Ku70/80 in modulating ATM-dependent ATR activation during DSB damage response and demonstrated that these proteins confer a protective effect against ATM-independent ATR activation at later stages of the DSB damage response. The genome of eukaryotic cells are exposed to a variety of DNA-damaging agents, such as ionizing radiation (IR), 4The abbreviations used are: IR, ionizing radiation; PI, phosphatidylinositol; DSB, double strand break; ATM, ataxia telangiectasia-mutated; ATR, ATM- and Rad3-related; Gy, gray; siRNA, small interfering RNA. UV light, reactive chemicals, and reactive oxygen species (1Abraham R.T. Genes Dev. 2001; 15: 2177-2196Crossref PubMed Scopus (1676) Google Scholar, 2Barzilai A. Rotman G. Shiloh Y. DNA Repair (Amst.). 2002; 1: 3-25Crossref PubMed Scopus (316) Google Scholar). The fallibility of the DNA replication machinery may also result in inaccurately replicated DNA and stalled replication forks that may eventually induce DNA double strand breaks (DSBs) (1Abraham R.T. Genes Dev. 2001; 15: 2177-2196Crossref PubMed Scopus (1676) Google Scholar). The cell's first option is to halt the cell cycle and repair the damage. Should such efforts fail, permanent cell cycle arrest (senescence), apoptosis, or other forms of cell death will occur. Failure of these processes result in the perpetuation of cells with altered DNA and possibly carcinogenesis (3Bernstein C. Bernstein H. Payne C.M. Garewal H. Mutat. Res. 2002; 511: 145-178Crossref PubMed Scopus (463) Google Scholar, 4Thompson L.H. Schild D. Mutat. Res. 2002; 509: 49-78Crossref PubMed Scopus (349) Google Scholar). The most widely accepted model for the DSB damage response puts the MRN (Mre11-Rad50-Nbs1) complex at the top of the pathway as the earliest proteins to be recruited at the sites of DNA damage (5Khanna K.K. Jackson S.P. Nat. Genet. 2001; 27: 247-254Crossref PubMed Scopus (1940) Google Scholar, 6Stavridi E.S. Halazonetis T.D. Nat. Cell Biol. 2005; 7: 648-650Crossref PubMed Scopus (12) Google Scholar). This complex then recruits the PI 3-related kinase member, ataxia telangiectasia-mutated (ATM) kinase, to the damaged DNA ends (7Carson C.T. Schwartz R.A. Stracker T.H. Lilley C.E. Lee D.V. Weitzman M.D. EMBO J. 2003; 22: 6610-6620Crossref PubMed Scopus (421) Google Scholar, 8Niida H. Nakanishi M. Mutagenesis. 2006; 21: 3-9Crossref PubMed Scopus (321) Google Scholar, 9Uziel T. Lerenthal Y. Moyal L. Andegeko Y. Mittelman L. Shiloh Y. EMBO J. 2003; 22: 5612-5621Crossref PubMed Scopus (839) Google Scholar). This enables the activated kinase to phosphorylate its protein targets, such as p53, Chk1, Chk2, Brca1, and NBS1, thereby triggering signal transduction cascades that initiate cell cycle arrest or apoptosis (8Niida H. Nakanishi M. Mutagenesis. 2006; 21: 3-9Crossref PubMed Scopus (321) Google Scholar, 10Kurz E.U. Lees-Miller S.P. DNA Repair (Amst.). 2004; 3: 889-900Crossref PubMed Scopus (398) Google Scholar). It has been shown that in the absence of ATM, phosphorylation of these proteins is diminished or delayed; the residual phosphorylation is thought to be mediated by the ATM- and Rad3-related (ATR) kinase, which is another member of the PI 3-related kinase family (11Wang X. Khadpe J. Hu B. Iliakis G. Wang Y. J. Biol. Chem. 2003; 278: 30869-30874Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). It has recently been revealed that the activation of ATR by DSBs is also ATM-dependent (12Adams K.E. Medhurst A.L. Dart D.A. Lakin N.D. Oncogene. 2006; 25: 3894-3904Crossref PubMed Scopus (129) Google Scholar, 13Cuadrado M. Martinez-Pastor B. Murga M. Toledo L.I. Gutierrez-Martinez P. Lopez E. Fernandez-Capetillo O. J. Exp. Med. 2006; 203: 297-303Crossref PubMed Scopus (190) Google Scholar, 14Jazayeri A. Falck J. Lukas C. Bartek J. Smith G.C. Lukas J. Jackson S.P. Nat. Cell Biol. 2006; 8: 37-45Crossref PubMed Scopus (882) Google Scholar, 15Myers J.S. Cortez D. J. Biol. Chem. 2006; 281: 9346-9350Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). Current knowledge assigns a role for the Ku70/80 heterodimer in the later stages of the DNA damage response, particularly in one of the two processes for DSB repair, non-homologous end joining (16Blier P.R. Griffith A.J. Craft J. Hardin J.A. J. Biol. Chem. 1993; 268: 7594-7601Abstract Full Text PDF PubMed Google Scholar, 17Meek K. Gupta S. Ramsden D.A. Lees-Miller S.P. Immunol. Rev. 2004; 200: 132-141Crossref PubMed Scopus (179) Google Scholar). In this process, the Ku70/80 heterodimer binds to the free DNA ends at a DSB and recruits the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), the third member of the PI 3-related kinase family. The formation of the DNA-PK complex at the site of the DSBs results in the recruitment and phosphorylation of XRCC4, DNA Ligase IV, Cernunnos/XLF, and Artemis to ligate the broken DNA ends (18Buck D. Malivert L. de Chasseval R. Barraud A. Fondaneche M.C. Sanal O. Plebani A. Stephan J.L. Hufnagel M. le Deist F. Fischer A. Durandy A. de Villartay J.P. Revy P. Cell. 2006; 124: 287-299Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar, 19Burma S. Chen B.P. Chen D.J. DNA Repair (Amst.). 2006; 5: 1042-1048Crossref PubMed Scopus (310) Google Scholar, 20Downs J.A. Jackson S.P. Nat. Rev. Mol. Cell Biol. 2004; 5: 367-378Crossref PubMed Scopus (304) Google Scholar, 21Drouet J. Frit P. Delteil C. de Villartay J.P. Salles B. Calsou P. J. Biol. Chem. 2006; 281: 27784-27793Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 22Lieber M.R. Ma Y. Pannicke U. Schwarz K. Nat. Rev. Mol. Cell Biol. 2003; 4: 712-720Crossref PubMed Scopus (793) Google Scholar). Because Ku70/80 possess a high affinity for DNA broken ends and is highly abundant in the nucleus, we were interested in determining whether these proteins might also influence the signaling aspects of the DNA damage response, particularly the activation of ATM and other PI 3-related kinases during initial DSB recognition. To explore this possibility, we performed PI 3-kinase inhibition in Ku70- and Ku80-deficient cells and then examined the phosphorylation of p53Ser18 following irradiation. Furthermore, we successfully established Ku70 and ATM double deficient cells and used these cells to analyze the possible relationship of the Ku70/80 heterodimer with ATM and ATR signaling in the early stages of the DSB response. Our findings suggest a novel role for the Ku70/80 heterodimer in the early stages of the DNA damage response, particularly in modulating ATM-dependent ATR activation in response to DSB damage. Moreover, we have proven the existence of an ATM-independent mechanism for ATR activation following DSB damage in Ku-deficient cells. Cell Culture and Induction of DNA Damage—The following spontaneously immortalized fibroblast cell lines were derived from mice with genotypes indicated in parentheses: PK34N (wild type), PK33N (DNA-PKcs–/–) (23Kurimasa A. Ouyang H. Dong L.J. Wang S. Li X. Cordon-Cardo C. Chen D.J. Li G.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1403-1408Crossref PubMed Scopus (159) Google Scholar), PK/80-1A (DNA-PKcs+/– Ku80–/–), PK/80–193A (DNA-PKcs–/–Ku80–/–) (23Kurimasa A. Ouyang H. Dong L.J. Wang S. Li X. Cordon-Cardo C. Chen D.J. Li G.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1403-1408Crossref PubMed Scopus (159) Google Scholar, 24Nussenzweig A. Chen C. da Costa Soares V. Sanchez M. Sokol K. Nussenzweig M.C. Li G.C. Nature. 1996; 382: 551-555Crossref PubMed Scopus (576) Google Scholar), A82-1 (ATM–/–) (25Barlow C. Hirotsune S. Paylor R. Liyanage 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 (1264) Google Scholar), D14-3 (ATM–/– Ku702LoxP/2LoxP). Only STEFKu70 (Ku70–/–) (26Ouyang H. Nussenzweig A. Kurimasa A. Soares V.C. Li X. Cordon-Cardo C. Li W. Cheong N. Nussenzweig M. Iliakis G. Chen D.J. Li G.C. J. Exp. Med. 1997; 186: 921-929Crossref PubMed Scopus (240) Google Scholar) is a Papillomavirus E6-, E7-transformed mouse fibroblast cell line. These cells were maintained in a humidified atmosphere with 5% CO2 in Dulbecco's modified Eagle's medium containing 5% calf serum supplemented with penicillin and streptomycin. Cells were grown to ∼70–90% confluence in 12-well plates and were irradiated with X-rays at the rate of 1.0 Gy/min (150 kV, 5 mA) (MBR-1505R2, Hitachi Medico, Japan) to achieve a cumulative dose of 8 Gy for all experiments unless otherwise mentioned. PI 3-related Kinase Inhibition Experiments—Cells were incubated with the indicated concentrations of wortmannin (Sigma), caffeine (Kanto Chemical), or KU55933 for 1 h and then treated with 8 Gy of IR. After 2 and 12 h, cell extracts were prepared for Western blotting. Wortmannin and KU55933 were dissolved in Me2SO at 10 and 1 mm, respectively, as a stock solution. Caffeine was dissolved in water at a concentration of 100 mm. Protein Extraction and Western Blotting—Western blotting experiments were performed using whole cell extracts following standard techniques. Cells were resuspended and lysed in 1× SDS buffer (67.5 mm Tris, pH 6.8, 25 mm NaCl, 0.5 mm EDTA, 12.5% glycerol, 2.5% SDS, and 100 mm dithiothreitol). Lysates were boiled for 2 min and sonicated. For Western blotting, cell extracts were electrophoresed on 8% SDS-polyacrylamide gels to detect medium-sized proteins or low-bis 8% SDS-acrylamide gels for high molecular weight proteins. Proteins were then transferred to a polyvinylidene difluoride membrane (GE Healthcare). Membranes were incubated in TBS-T (137 mm NaCl, 2.7 mm KCl, 25 mm Tris, pH 7.4, and 0.1% Tween 20) and 5% skim milk (Snow Brand) added to TBS-T. Membranes were then stained with Ponceau S dye to check for equal loading and homogeneous transfer. Primary antibodies used in this study were Ku70 (C-19, M-19), ATM (2C1), ATR (M-19) (Santa Cruz Biotechnology), phospho-p53Ser18 (Cell Signaling Technology) and α-tubulin (ICN). Anti-mouse and anti-rabbit secondary antibodies were obtained from GE Healthcare, and an anti-goat antibody was obtained from Jackson ImmunoResearch Laboratories. Proteins were visualized using ECL Western blotting detection systems (GE Healthcare). After probing with the phosphospecific antibodies, immunoblots were stripped and reprobed with tubulin to check for equal loading. Small Interfering RNA (siRNA) Transfections—All siRNA transfections were performed using Lipofectamine 2000 (Invitrogen) following the manufacturer's recommendations. Approximately 0.3–0.6 × 105 cells/well were seeded in 12-well plates with 1 ml of antibiotic-free Dulbecco's modified Eagle's medium with 5% calf serum. The next day, the cells were treated with Lipofectamine 2000 and 20 pmol of control, ATR, or ATM siRNA (Qiagen) (siRNA sequences available upon request). After 24 h, this procedure was repeated. The cells were analyzed 48 h after the last siRNA transfection. Selective Targeting of Ku70—We initially generated a tetracycline-inducible Ku70 conditional allele (supplemental Fig. 2a). Ku70 sequences were either directly derived or amplified from genomic DNA obtained from CJ7 embryonic stem cells or a cosmid clone carrying the Ku70 locus. The 5′ end of the targeting vector consisted of a 1.2-kb region possessing homology to intron 1 and was generated by high fidelity PCR (26Ouyang H. Nussenzweig A. Kurimasa A. Soares V.C. Li X. Cordon-Cardo C. Li W. Cheong N. Nussenzweig M. Iliakis G. Chen D.J. Li G.C. J. Exp. Med. 1997; 186: 921-929Crossref PubMed Scopus (240) Google Scholar). The early part of exon 2 containing the untranslated region (referred to as exon 2x) was fused to the tetracycline transactivator gene tTA having a terminal codon and poly(A) sequence. The later half of exon 2 (referred to as exon 2y) beginning from the ATG start site of Ku70 was placed under the control of the tetracycline-responsive promoter (tetracycline-responsive element). A pair of LoxP sites flanked this TRE-exon 2y sequence. A phosphoglycerate kinase-driven neomycin selection marker was positioned between the first loxP site and the TRE-2y region. The 3′ arm of the targeting vector consisted of a 7.7-kb EcoRI fragment derived from the region spanning introns 2–5. Generation of Ku70-conditional Knock-out Mice—A correctly targeted embryonic stem cell clone, confirmed by Southern blot analysis, was injected into 3.5-day-postcoitus C57BL/6J blastocysts. Approximately 10 embryonic stem cells were injected per blastocyst, and twenty blastocysts were transferred to each pseudopregnant recipient. The resulting chimeric offspring were crossed with 40 mice to generate F1 progeny. To generate ATM-deficient/Ku70-conditional mice, we crossed Ku70 heterozygotes with ATM heterozygotes (25Barlow C. Hirotsune S. Paylor R. Liyanage 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 (1264) Google Scholar). The resulting Ku70/ATM double heterozygotes were crossed with each other. Progeny that had an ATM–/– Ku702Loxp/2LoxP genotype were identified by PCR screening. Primary fibroblast cells were obtained from one of the ATM homozygous null Ku70 homozygous conditional (ATM–/– Ku702LoxP/2LoxP) mice, D14-3, and further cultured to obtain a spontaneously transformed cell line. Generation of ATM and Ku70 Double Deficient Cell Lines—To generate the Ku70-null allele, spontaneously transformed D14-3 fibroblasts were infected with a Cre recombinase-expressing adenovirus vector (AxCANCre, obtained from RIKEN Bioresource Center) to achieve excision of the PGK-neo cassette and exon 2y, which contained the Ku70 start codon. Infection was performed on monolayers of fibroblast cells in Dulbecco's modified Eagle's medium supplemented with 5% calf serum and 10% PEG6000 at 80 multiplicity of infection (27Ohsawa T. Nakamura T. Mihara M. Sato K. J. Dermatol. 2000; 27: 244-251Crossref PubMed Scopus (7) Google Scholar). ATM and Ku70 expression was confirmed by Western blotting (supplemental Fig. 2b). After transient Cre expression, Ku70 protein levels progressively decreased and were undetectable 3 days after transfection. Two independently isolated floxed clones, D14-3Fx1 and D14-3Fx2, were expanded and used in this study. Prolonged Phosphorylation of p53Ser18 in Ku-deficient Cells—The most well known ATM and ATR substrate is the tumor suppressor protein p53 (28Banin 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 (1711) Google Scholar, 29Tibbetts R.S. Brumbaugh K.M. Williams J.M. Sarkaria J.N. Cliby W.A. Shieh S.Y. Taya Y. Prives C. Abraham R.T. Genes Dev. 1999; 13: 152-157Crossref PubMed Scopus (868) Google Scholar). This protein plays a major role in cellular responses to DNA damage and other genomic aberrations (30Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6759) Google Scholar). Activation of p53 leads to either cell cycle arrest to allow DNA repair or apoptosis. DSBs induce ATM-dependent phosphorylation of p53Ser18, and this results in reduced interaction of p53 with its negative regulator, oncoprotein MDM2 (31Shieh S.Y. Ikeda M. Taya Y. Prives C. Cell. 1997; 91: 325-334Abstract Full Text Full Text PDF PubMed Scopus (1760) Google Scholar). To investigate whether the Ku70/80 status of a cell might influence DNA damage signaling, we examined p53Ser18 phosphorylation and response to IR over a time course. We utilized four fibroblast cell lines, PK34N (wild type) and three other cell lines that were null mutants for components of the DNA-PK complex, namely PK33N (DNA-PKcs–/–) (23Kurimasa A. Ouyang H. Dong L.J. Wang S. Li X. Cordon-Cardo C. Chen D.J. Li G.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1403-1408Crossref PubMed Scopus (159) Google Scholar), STEFKu70 (Ku70–/–) (26Ouyang H. Nussenzweig A. Kurimasa A. Soares V.C. Li X. Cordon-Cardo C. Li W. Cheong N. Nussenzweig M. Iliakis G. Chen D.J. Li G.C. J. Exp. Med. 1997; 186: 921-929Crossref PubMed Scopus (240) Google Scholar), PK/80-1A (DNA-PKcs+/–Ku80–/–), and PK/80–193A (DNA-PKcs–/–Ku80–/–) (23Kurimasa A. Ouyang H. Dong L.J. Wang S. Li X. Cordon-Cardo C. Chen D.J. Li G.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1403-1408Crossref PubMed Scopus (159) Google Scholar, 24Nussenzweig A. Chen C. da Costa Soares V. Sanchez M. Sokol K. Nussenzweig M.C. Li G.C. Nature. 1996; 382: 551-555Crossref PubMed Scopus (576) Google Scholar). Cells were either mock-treated or subjected to 8 Gy of IR and harvested at the indicated time points. Processed samples were analyzed for p53Ser18 phosphorylation by Western blotting, with tubulin as a loading control (Fig. 1). Ku70-deficient mice exhibit decreased levels of Ku80, whereas Ku80-deficient mice show decreased levels of Ku70, revealing the functional synergy of these proteins (24Nussenzweig A. Chen C. da Costa Soares V. Sanchez M. Sokol K. Nussenzweig M.C. Li G.C. Nature. 1996; 382: 551-555Crossref PubMed Scopus (576) Google Scholar). Previous studies have shown that Ku70- and Ku80-deficient cells have similar phenotypes. Thus, when discussed together, we shall collectively refer to these three cell lines as Ku-deficient cells. In wild type cells and DNA-PKcs-deficient cells, phosphorylation of p53Ser18 was transient and reached maximum levels 2 h after irradiation. At 8 h post-IR, phosphorylation levels dropped significantly, with p53Ser18 levels returning to background levels at 12 h post-IR. On the other hand, phosphorylation of p53Ser18 was persistent, with minimal change within the 16-h time period after IR for both Ku80- and Ku70-deficient cells. Our results indicate that loss of DNA-PKcs does not result in aberrant p53Ser18 phosphorylation. However, loss of Ku results in persistent p53 phosphorylation. In the absence of Ku, further loss of DNA-PKcs function does not enhance the persistent p53Ser18 phosphorylation. Relative Contribution of ATM and ATR Kinases to p53Ser18 Phosphorylation in Ku-deficient Cells—Following DSB damage, ATM is activated to phosphorylate target proteins such as p53Ser18. It is currently thought that activation of ATR following DSB damage requires ATM activity (ATM-dependent ATR activation) (12Adams K.E. Medhurst A.L. Dart D.A. Lakin N.D. Oncogene. 2006; 25: 3894-3904Crossref PubMed Scopus (129) Google Scholar, 13Cuadrado M. Martinez-Pastor B. Murga M. Toledo L.I. Gutierrez-Martinez P. Lopez E. Fernandez-Capetillo O. J. Exp. Med. 2006; 203: 297-303Crossref PubMed Scopus (190) Google Scholar, 14Jazayeri A. Falck J. Lukas C. Bartek J. Smith G.C. Lukas J. Jackson S.P. Nat. Cell Biol. 2006; 8: 37-45Crossref PubMed Scopus (882) Google Scholar). To determine whether ATM and/or ATR kinase(s) were involved in the persistent phosphorylation of p53Ser18 in Ku-deficient cells, we first performed knockdown experiments using siRNAs. PK34N, STEFKu70, PK/80-1A, and PK/80–193A cells were individually transfected with siRNAs against ATR (ATR1 or ATR3) or ATM (ATM1). As shown in Fig. 2, in wild type PK34N cells, ATR knockdown resulted in loss of p53Ser18 phosphorylation, indicating that ATR activity significantly contributes to the phosphorylation of p53Ser18. Moreover, although ATR activity remained intact in these cells, knockdown of ATM completely abolished p53Ser18 phosphorylation at 2 h post-IR. These results demonstrate that ATR is activated under the control of ATM; that is, in the absence of functional ATM, ATR is unable to rescue p53Ser18 phosphorylation. These findings agree with the ATM-dependent ATR activation model for DSB damage response, wherein activation of ATR by ATM is required for ATR to phosphorylate its targets (13Cuadrado M. Martinez-Pastor B. Murga M. Toledo L.I. Gutierrez-Martinez P. Lopez E. Fernandez-Capetillo O. J. Exp. Med. 2006; 203: 297-303Crossref PubMed Scopus (190) Google Scholar, 15Myers J.S. Cortez D. J. Biol. Chem. 2006; 281: 9346-9350Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). Our results suggest that, in the earlier stages (2 h) of the DSB damage response, phosphorylation of p53Ser18 is predominantly mediated by ATM-dependent ATR kinase activity. At 12 h post-IR, PK34N cells treated with either ATR or ATM siRNA exhibited basal phosphorylation levels of p53Ser18 comparable with those of cells treated with control siRNA. Interestingly, in Ku-deficient cells, knockdown of ATR did not significantly decrease p53Ser18 phosphorylation at 2 h post-IR. On the other hand, ATM knockdown resulted in a substantial reduction in p53Ser18 phosphorylation. These results suggest that, in the absence of Ku70/80, ATM contributes significantly to p53Ser18 phosphorylation. Nevertheless, ATR participates in p53Ser18 phosphorylation to a minor extent. In contrast to the predominance of ATM-dependent ATR activity observed in wild type cells, contribution of this mechanism in p53Ser18 phosphorylation is minimal in Ku-deficient cells. The involvement of DNA-PKcs in phosphorylation of p53Ser18 could be excluded, because phosphorylation of p53Ser18 was still observable in the Ku80/DNA-PKcs double deficient cells (PK/80–193A). This leads us to the question as to what kinase phosphorylates p53Ser18 in the absence of ATM activity. With these results, we are led to conclude that residual p53Ser18 phosphorylation after ATM knockdown is mediated by ATR through an ATM-independent mechanism. As expected, p53Ser18 phosphorylation was no longer visible at the 12-h time point in wild type cells. The non-responsiveness of p53Ser18 phosphorylation to ATM or ATR siRNA treatment reflects the return of ATM and ATR activity to basal levels at the later stage of the DSB response. For the PK/80-1A cell line, p53Ser18 phosphorylation patterns in response to ATR or ATM siRNA treatment were similar to those observed at 2 h post-IR. In the case of STEFKu70 and PK/80–193A cell lines, ATR or ATM siRNA treatment resulted in a slight reduction of p53Ser18 phosphorylation levels. This suggests the involvement of ATM in p53Ser18 phosphorylation and at the same time, an increase in the contribution of ATR in this phosphorylation. From these findings, we conclude that the persistent activation of p53Ser18 in Ku-deficient cells is mediated by aberrant activation of ATR via an ATM-independent mechanism. To confirm these results, we subjected cells to various inhibitors of PI 3-related kinases. Wortmannin inhibits PI 3-kinase activity in a dose-dependent manner. Low concentrations mainly suppress ATM and DNA-PKcs, whereas higher concentrations are required for ATR inhibition (32Powis G. Bonjouklian R. Berggren M.M. Gallegos A. Abraham R. Ashendel C. Zalkow L. Matter W.F. Dodge J. Grindey G. Vlahos J.C. Cancer Res. 1994; 54: 2419-2423PubMed Google Scholar, 33Sarkaria J.N. Tibbetts R.S. Busby E.C. Kennedy A.P. Hill D.E. Abraham R.T. Cancer Res. 1998; 58: 4375-4382PubMed Google Scholar, 34Ui M. Okada T. Hazeki K. Hazeki O. Trends Biochem. Sci. 1995; 20: 303-307Abstract Full Text PDF PubMed Scopus (521) Google Scholar). In wild type cells, phosphorylation of p53Ser18 at 2 h post-IR was strongly suppressed by 10 μm Wortmannin (Fig. 3a). In agreement with the results from our previous siRNA experiments, inhibition of ATM in wild type PK34N cells completely abolished p53Ser18 phosphorylation at 2 h post-IR. Again, we have demonstrated that p53Ser18 phosphorylation is mainly mediated by direct ATM activity or through a two-step ATM-dependent process involving ATR. At the same time point, in Ku-deficient cells, some p53Ser18 phosphorylation was still observed at low Wortmannin concentrations that selectively inhibit ATM, suggesting that residual p53 phosphorylation is attributed to ATM-independent ATR activity. Complete suppression of this phosphorylation was achieved at higher concentrations known to inhibit ATR activity. In Ku-deficient cells, at 12 h post-IR, the persistent phosphorylation of p53Ser18 was difficult to suppress at low concentrations of Wortmannin. These results are consistent with our previous findings and suggest that p53Ser18 phosphorylation is primarily mediated by ATM kinase activity, with a minor contribution from ATM-independent ATR activity. Our siRNA results point to the existence of ATM-independent ATR activity in both the earlier and later stages of the DSB response in Ku-deficient cells. To confirm this, we also treated cells with KU55933, a potent and specific inhibitor of ATM (IC50 for ATM was 12.9 nm, whereas IC50 for ATR was >100 μm) (35Hickson I. Zhao Y. Richardson C.J. Green S.J. Martin N.M. Orr A.I. Reaper P.M. Jackson S.P. Curtin N.J. Smith G.C. Cancer Res. 2004; 64: 9152-9159Crossref PubMed Scopus (996) Google Scholar, 36Lau A. Swinbank K.M. Ahmed P.S. Taylor D.L. Jackson S.P. Smith G.C. O'Connor M.J. Nat. Cell Biol. 2005; 7: 493-500Crossref PubMed Scopus (123) Google Scholar) (Fig. 3b). At the 2-h time point, phosphorylation in wild type cells was significantly reduced by treatment with 1 μm KU55933. On the other hand, the phosphorylation in Ku-deficient cells was more resistant to KU55933 until concentrations of up to 10 μm. At 12 h post-IR, p53Ser18 phosphorylation was no longer observable in wild type cells. The 2- and 12-h time points exhibited similar patterns of reductions in p53Ser18 phosphorylation in Ku-deficient cells. Similar to the siRNA and Wortmannin experiments, we observed residual p53Ser18 phosphorylation even after ATM-specific inhibition. This remaining p53Ser18 phosphorylation could only reflect ATR activity that occurs independently of ATM, thus supporting the existence of ATM-independent ATR activity in Ku-deficient cells. We also performed caffeine treatment at concentrations that inhibited both ATM and ATR activity (37Cortez D. J. Biol. Chem. 2003; 278: 37139-37145Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 38Hu B. Zhou X.Y. Wang X. Zeng Z.C. Iliakis G. Wang Y. J. Biol. Chem. 2001; 276: 17693-17698Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 39Sarkaria 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) (supplemental Fig. 1). Wild type cells at 2 h post-IR and Ku-deficient cells at 2 and 12 h post-IR exhibited similar patterns of decrease in p53Ser18 phosphorylation. To summarize, we have demonstrated that, (1Abraham R.T. Genes Dev. 2001; 15: 2177-2196Crossref PubMed Scopus (1676) Google Scholar) in wild type cells at the earlier stages of the DSB damage response, phosphorylation of p53Ser18 is predominantly mediated by ATM-dependent ATR activity
Referência(s)