Questioning the Role of Checkpoint Kinase 2 in the p53 DNA Damage Response
2003; Elsevier BV; Volume: 278; Issue: 23 Linguagem: Inglês
10.1074/jbc.m213185200
ISSN1083-351X
AutoresJin-Woo Ahn, Marshall Urist, Carol Prives,
Tópico(s)Microtubule and mitosis dynamics
ResumoCdc25C and p53 have been reported to be physiological targets of checkpoint kinase 2 (Chk2). Surprisingly, although Chk2 purified from DNA damage sustaining cells has dramatically increased ability to phosphorylate Cdc25C when compared with untreated cells, its ability to phosphorylate p53 is weak before treatment, and there is no increase in its activity toward p53 after DNA damage by γ irradiation or the radiomimetic agent neocarzinostatin. Furthermore, introduction of Chk2 short interfering RNA into three different human tumor cell lines leads to marked reduction of Chk2 protein, but p53 is still stabilized and active after DNA damage. The results with Chk1 short interfering RNA indicate as well that Chk1 does not play a role in human p53 stabilization after DNA damage. Thus, Chk1 and Chk2 are unlikely to be regulators of p53 in at least some human tumor cells. We discuss our results in the context of previous findings demonstrating a requirement for Chk2 in p53 stabilization and activity. Cdc25C and p53 have been reported to be physiological targets of checkpoint kinase 2 (Chk2). Surprisingly, although Chk2 purified from DNA damage sustaining cells has dramatically increased ability to phosphorylate Cdc25C when compared with untreated cells, its ability to phosphorylate p53 is weak before treatment, and there is no increase in its activity toward p53 after DNA damage by γ irradiation or the radiomimetic agent neocarzinostatin. Furthermore, introduction of Chk2 short interfering RNA into three different human tumor cell lines leads to marked reduction of Chk2 protein, but p53 is still stabilized and active after DNA damage. The results with Chk1 short interfering RNA indicate as well that Chk1 does not play a role in human p53 stabilization after DNA damage. Thus, Chk1 and Chk2 are unlikely to be regulators of p53 in at least some human tumor cells. We discuss our results in the context of previous findings demonstrating a requirement for Chk2 in p53 stabilization and activity. The p53 tumor suppressor protein has been well studied as a target of numerous stress-induced signaling pathways in mammalian cells. Various forms of DNA damage bring about stabilization and activation of the p53 tumor suppressor protein presumably through prevention of the interaction of p53 with its negative regulator Mdm2. 1The abbreviations used are: Mdm2, murine double minute 2; ATM, Ataxia telangiectasia-mutated protein; Chk1, Checkpoint Kinase 1; Chk2, Checkpoint Kinase 2; NCS, neocarzinostatin; GST, glutathione S-transferase; siRNA, short interfering RNA; HA, hemagglutinin; Gy, gray; DTT, dithiothreitol; WT, wild type; DNA-PK, DNA-activated protein kinase. Induction of p53, a sequence-specific transcriptional activator, results in expression of a number of gene products whose function is to either arrest cell growth or promote apoptosis. As such, p53 has been defined as a checkpoint factor (reviewed in Refs. 1Morgan S.E. Kastan M.B. Adv. Cancer Res. 1997; 71: 1-25Crossref PubMed Google Scholar, 2Shen Y. White E. Adv. Cancer Res. 2001; 82: 55-84Crossref PubMed Scopus (309) Google Scholar, 3Wahl G.M. Carr A.M. Nat Cell Biol. 2001; 3: E277-E286Crossref PubMed Scopus (326) Google Scholar). ATM is a protein kinase with homology to members of the phosphatidylinositol 3-kinase family, and of the various DNA damage pathways to p53, that involving ATM has been the best documented; ATM-null cells fail to induce p53 after some forms of DNA damage (4Kastan M.B. Zhan Q. el-Deiry W.S. Carrier F. Jacks T. Walsh W.V. Plunkett B.S. Vogelstein B. Fornace Jr., A.J. 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In fission yeast, as yet unidentified DNA damage sensor proteins may signal directly or indirectly via downstream kinases or adaptor proteins to the homologous mediator kinases Rad3 and Tel1, which in turn regulate through phosphorylation two effector kinases Cds1 and Chk1 (8Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2645) Google Scholar). A key target for these kinases is the Cdc25 dual specificity phosphatase that in unstressed cycling cells removes repressing phosphates from the cyclin-dependent kinase Cdc2 and thereby allows it to promote passage through G2/M (reviewed in Ref. 9Nurse P. Cell. 1997; 91: 865-867Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). When the human homologues of Chk1 or Cds1 were identified and cloned, it became clear that they could phosphorylate different members of the human Cdc25 family (10Sanchez Y. Wong C. Thoma R.S. Richman R. Wu Z. Piwnica-Worms H. Elledge S.J. 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The relationship between the ATM pathway and Chk1 is less well understood. It is currently believed that Chk1 kinase is downstream of ATR (ataxia-telangiectasia and Rad3-related kinase), another member of the phosphatidylinositol 3-kinase family (20Liu Q. Guntuku S. Cui X.S. Matsuoka S. Cortez D. Tamai K. Luo G. Carattini-Rivera S. DeMayo F. Bradley A. Donehower L.A. Elledge S.J. Genes Dev. 2000; 14: 1448-1459Crossref PubMed Scopus (199) Google Scholar, 21Guo Z. Kumagai A. Wang S.X. Dunphy W.G. Genes Dev. 2000; 14: 2745-2756Crossref PubMed Scopus (367) Google Scholar, 22Zhao H. Piwnica-Worms H. Mol. Cell. Biol. 2001; 21: 4129-4139Crossref PubMed Scopus (871) Google Scholar). Although it is possible to generate cells and animals lacking ATM and Chk2, deletion of ATR or Chk1 causes early embryonic lethal events (20Liu Q. Guntuku S. Cui X.S. Matsuoka S. Cortez D. Tamai K. Luo G. Carattini-Rivera S. DeMayo F. Bradley A. Donehower L.A. Elledge S.J. 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Phosphorylation of p53 at N-terminal sites such as Ser15, Thr18, Ser20, and Ser37 within the vicinity of the region where it interacts with Mdm2 can disrupt its interaction with Mdm2 in vitro (27Shieh S.Y. Ikeda M. Taya Y. Prives C. Cell. 1997; 91: 325-334Abstract Full Text Full Text PDF PubMed Scopus (1760) Google Scholar, 28Dornan D. Hupp T.R. EMBO Rep. 2001; 2: 139-144Crossref PubMed Scopus (79) Google Scholar, 29Lai Z. Auger K.R. Manubay C.M. Copeland R.A. Arch. Biochem. Biophys. 2000; 381: 278-284Crossref PubMed Scopus (58) Google Scholar, 30Chehab N.H. Malikzay A. Appel M. Halazonetis T.D. Genes Dev. 2000; 14: 278-288Crossref PubMed Google Scholar, 31Sakaguchi K. Saito S. Higashimoto Y. Roy S. Anderson C.W. Appella E. J. Biol. Chem. 2000; 275: 9278-9283Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar) and may thereby allow for p53 stabilization (32Haupt Y. Maya R. Kazaz A. Oren M. 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Moreover, expression of either Chk1 kinase-defective or Chk1 antisense constructs leads to diminution of levels of co-transfected p53 protein (39Shieh S.Y. Ahn J. Tamai K. Taya Y. Prives C. Genes Dev. 2000; 14: 289-300Crossref PubMed Google Scholar). In a parallel study Chehab et al. (30Chehab N.H. Malikzay A. Appel M. Halazonetis T.D. Genes Dev. 2000; 14: 278-288Crossref PubMed Google Scholar) reported that Chk2 can phosphorylate Ser20 and that a kinase defective form of Chk2 prevents stabilization and phosphorylation at Ser20 of co-transfected p53 after DNA damage. Overexpression of Chk2 in U2OS cells, which carry wild type p53, augmented G1 arrest following irradiation. Further supporting a role for Chk2 as being upstream of p53, Hirao et al. (40Hirao A. Kong Y.Y. Matsuoka S. Wakeham A. Ruland J. Yoshida H. Liu D. Elledge S.J. Mak T.W. Science. 2000; 287: 1824-1827Crossref PubMed Scopus (1051) Google Scholar) reported that thymocytes and fibroblasts generated from Chk2 knock-out mice are defective in accumulating p53 after γ but not UV irradiation. Intriguingly, a second group using mouse embryo-fibroblasts derived from these mice found that even at low doses of γ irradiation, G1 arrest and p21 induction were intact (41Jack M.T. Woo R.A. Hirao A. Cheung A. Mak T.W. Lee P.W. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9825-9829Crossref PubMed Scopus (104) Google Scholar). More recently, the results from a second independently generated Chk2 null mouse have indicated that Chk2 loss protects mice from γ irradiation-induced death consistent with diminished apoptosis in several tissues including the spleen, intestine, and central nervous system and that p53 in these cells is transcriptionally inactive (42Takai H. Naka K. Okada Y. Watanabe M. Harada N. Saito S. Anderson C.W. Appella E. Nakanishi M. Suzuki H. Nagashima K. Sawa H. Ikeda K. Motoyama N. EMBO J. 2002; 21: 5195-5205Crossref PubMed Scopus (355) Google Scholar). Finally, data from human patients have revealed that a subset of Li-Fraumeni cancer-prone families with wild type p53 has Chk2 germ line mutations (43Bell D.W. Varley J.M. Szydlo T.E. Kang D.H. Wahrer D.C. Shannon K.E. Lubratovich M. Verselis S.J. Isselbacher K.J. Fraumeni J.F. Birch J.M. Li F.P. Garber J.E. Haber D.A. Science. 1999; 286: 2528-2531Crossref PubMed Scopus (766) Google Scholar, 44Vahteristo P. Tamminen A. Karvinen P. Eerola H. Eklund C. Aaltonen L.A. Blomqvist C. Aittomaki K. Nevanlinna H. Cancer Res. 2001; 61: 5718-5722PubMed Google Scholar), and such mutant forms of Chk2 are defective as protein kinases (45Wu X. Webster S.R. Chen J. J. Biol. Chem. 2001; 276: 2971-2974Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 46Matsuoka S. Nakagawa T. Masuda A. Haruki N. Elledge S.J. Takahashi T. Cancer Res. 2001; 61: 5362-5365PubMed Google Scholar). Strikingly, this natural experiment is not completely recapitulated in murine models. Hirao et al. (47Hirao A. Cheung A. Duncan G. Girard P.M. Elia A.J. Wakeham A. Okada H. Sarkissian T. Wong J.A. Sakai T. De Stanchina E. Bristow R.G. Suda T. Lowe S.W. Jeggo P.A. Elledge S.J. Mak T.W. Mol. Cell. Biol. 2002; 22: 6521-6532Crossref PubMed Scopus (320) Google Scholar) observed no increase in spontaneous tumors in Chk2–/– mice, whereas Takai et al. (42Takai H. Naka K. Okada Y. Watanabe M. Harada N. Saito S. Anderson C.W. Appella E. Nakanishi M. Suzuki H. Nagashima K. Sawa H. Ikeda K. Motoyama N. EMBO J. 2002; 21: 5195-5205Crossref PubMed Scopus (355) Google Scholar) reported preliminary evidence of an increase in lymphoma development by 71 weeks in Chk2–/– animals. Given that there is somewhat contradictory evidence surrounding the proposed Chk2-p53 connection, we set out to further characterize Chk2 derived from human tumor cells that have an intact DNA damage response. The goal was to study Chk2 before and after activation by either γ irradiation or the radiomimetic compound neocarzinostatin (NCS). To our surprise, the results from both biochemical and short interfering RNA (siRNA) experiments argue against a role for Chk2 in DNA damage-mediated stabilization of p53 in cancer cells following γ radiation or similar stimuli. We discuss the basis for the differences between our present results and those that have been previously published, including our own. Mammalian Cell Lines and Culture Conditions—An HCT116-deryived cell line stably expressing N-terminally HA-tagged wild type hChk2 (HA-Chk2) was kindly provided by Dr. J. Chen (Mayo Clinic, Rochester, MN) (45Wu X. Webster S.R. Chen J. J. Biol. Chem. 2001; 276: 2971-2974Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). These cells were cultured in RPMI 1640 medium containing 400 μg/ml of G416 and 10% fetal bovine serum. For RNAi experiments, HCT116 and RKO (p53 wild type human colorectal adenocarcinoma cells) and MCF-7 (p53 wild type human breast adenocarcinoma cells) were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. HT-29 (p53 mutant human colorectal cancer cells) were maintained in McCoy's 5A medium with 10% fetal bovine serum. The cells were obtained from the American Type Culture Collection (Manassas, VA). Purification of Proteins—Where indicated, the cells were treated with a radiomimetic compound NCS (500 ng/ml) (Kayaku Co., Tokyo, Japan) for 2 h before purification of HA-Chk2 as described below. Alternately, the cells were irradiated with 14 Gy (using a 137Cs source) and harvested after 2 h for purification of HA-Chk2. Typically, cells in 20 × 140-mm plates were collected and treated with lysis buffer A containing 50 mm Hepes KOH, pH 7.8, 150 mm KCl, 10 mm NaCl, 0.1 mm EDTA, 1.5 mm MgSO4, 1 mm DTT, 0.2% Nonidet P-40, 0.25 mm phenylmethylsulfonyl fluoride, 60 nm okadaic acid, 240 pm cypermethrin, 1 mm NaF, 100 μm NaVO4, and 20% glycerol. The extracts were precleared with 400 μl of protein A conjugated to agarose (Amersham Biosciences) for 4 h at 4 °C and incubated with 100 μl of protein A cross-linked with anti-HA antibody for 12 h at 4 °C. The beads were collected in a disposable column and extensively washed with lysis buffer a containing 20 mm Hepes KOH, pH 7.8 (15 × 1 ml). The proteins were eluted with 100 μg/ml of HA peptide (SynPep, Dublin, CA) and then dialyzed against buffer B containing 20 mm Tris-HCl, pH 8.0, 100 mm KCl, 0.1 mm EDTA, 1 mm DTT, and 20% glycerol. C-terminally FLAG-tagged wild type and mutant (D347A) Chk2 (Chk2-FLAG) mutant proteins were immunopurified from recombinant baculovirus infected sf-9 insect cells. Typically, 20 × 140-mm plates were collected and treated with the buffer. The extracts collected after centrifugation at 14,000 × g at 4 °C for 30 min were incubated with 0.25 ml of anti-FLAG antibody conjugated to agarose (Sigma). The beads were collected in a 5-ml syringe and washed with lysis buffer A. The proteins were eluted with 100 μg of FLAG peptide (Sigma) and dialyzed against buffer B. Wild type and kinase-defective mutant (D130A) GST-Chk1 proteins were purified from baculovirus-infected insect cells as follows. The cells were incubated with lysis buffer A, and the extracts were collected with 0.5 ml of glutathione-Sepharose 4BL beads (Amersham Biosciences). GST-Chk1 proteins were eluted with 10 mm glutathione after washing away any unbound protein with lysis buffer A and then dialyzed as described above with buffer B. The plasmid encoding GST-Cdc25C (200–256) was kindly provided by Dr. Junjie Chen. GST fusion proteins, GST-Cdc25C (200–256), GST-p53 (1–82), GST-p53 (97–363), and GST-p53 (WT) were expressed in Escherichia coli BL21 cells after induction with isopropyl-β-d-thiogalactoside for 2.5 h (final concentration, 1 mm)at20 °C. The proteins were purified by binding to glutathione-Sepharose 4BL beads in lysis buffer A followed by elution with 10 mm of glutathione and dialyzed as described above. His-p53 (WT) was purified as described by Zhou et al. (48Zhou J. Ahn J. Wilson S.H. Prives C. EMBO J. 2001; 20: 914-923Crossref PubMed Scopus (280) Google Scholar). In Vitro Kinase Assays and Western Blotting—Typically 5–20 ng of kinase was incubated with 0.1–4 μg of GST fusion protein substrates. The reaction mixtures were incubated at 30 °C for 30 min in 20 μl of Buffer C containing 20 mm Hepes KOH, pH 7.8, 100 mm KCl, 10 mm MgCl2, 1 mm DTT, 60 nm okadaic acids, 240 pm Cypermethrin, 1 mm NaF, 100 μm NaVO4, and 100 μm ATP supplemented with 1 μCi of [γ-32P]ATP. The reactions were terminated by adding 20 μl of SDS-PAGE sample loading buffer. The proteins were separated by SDS-PAGE, transferred to nitrocellulose, and subsequently identified and quantitated by immunoblotting with the appropriate antibodies. The radiolabeled proteins were visualized with autoradiography and quantitated with PhosphorImager (Molecular Dynamics). The antibodies were obtained as follows: anti-GST and anti-FLAG (M2) were from Sigma; anti-HA was from Covance (Princeton, NJ); and anti-His was from Santa Cruz Biotechnology (Santa Cruz, CA). Immunoprecipitation Kinase Assays—HCT 116 parental cells in 4 × 140-mm plates were collected and treated with lysis buffer A as described above. The extracts were incubated with 50 μl of agarose beads conjugated with protein A (Amersham Biosciences) and 2 μg of anti-Chk2 antibody (Santa Cruz Biotechnology) for 4 h at 4 °C. The beads were washed with buffer A (15 × 0.5 ml). The indicated amount of beads were incubated with GST-fused substrates in kinase buffer C, and the data were analyzed as described above. Phosphorylation of p53 by DNA-PK—GST-p53 (1–82) (10 μg) was incubated with DNA-PK (100 ng) in buffer containing 25 mm Hepes, pH 7.9, 50 mm KCl, 10 mm MgCl2, 20% glycerol, 1 mm DTT, 100 μm ATP, and 10 μg/ml of DNA fragments generated from HpaII-digested pBlue-script. Purified DNA-PK was a generous gift of D. Chan (Lawrence Berkeley National Laboratory, Berkley, CA). The mixtures were incubated for 4 h at 30 °C. Phosphorylation at Ser15 was confirmed by separating the reaction mixture on 10% SDS-PAGE, transferring to nitrocellulose, and immunoblotting with anti-Ser(P)15-specific antibody (Cell Signaling, Beverly, MA). The prephosphorylated GST-p53 was purified by binding to glutathione-Sepharose 4CL beads as described above. RNA Interference—siRNA duplexes were synthesized by Xeragon Oligoribonucleotides (Huntsville, AL). The luciferase control sequence has been described previously (49Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8186) Google Scholar). The sequences of the Chk1 oligonucleotides were: 5′-GAAGCAGUCGCAGUGAAGATT-3′ and 5′-UCUUCACUGCGACUGCUUCTT-3′. The sequences of the Chk2 oligonucleotides were: 5′-GAACCUGAGGACCAAGAAC-3′ and 5′-GUUCUUGGUCCUCAGGUUC-3′. The cells were transfected twice 24 h apart with 1.68 μg of the indicated siRNA duplex using Lipo-fectAMINE 2000 (Invitrogen). The cells that were untreated or treated with 500 ng/ml NCS for the indicated time periods were lysed in TEGN buffer (10 mm Tris, pH 7.5, 1 mm EDTA, 10% glycerol, 0.5% Nonidet P-40, 400 mm NaCl, 1 mm DTT, 0.5 mm phenylmethylsulfonyl fluoride, and protease inhibitor mixture) 72 h after the first transfection. Chk1 was detected with a mouse monoclonal antibody (Santa Cruz Biotechnology), and Chk2 was detected with a rabbit polyclonal antibody (ProSci Inc., Poway, CA). p53 was detected using a mixture of monoclonal antibodies DO-1 and 1801. Protein loading was estimated using an anti-actin polyclonal antibody (Sigma). -Cdc25CS216 and p53S20 antibodies (Cell Signaling) phospho-specific antibodies were used. p53 transcriptional targets were detected using anti-p21 (Ab-1) and anti-HDM2 (Ab-1) (Oncogene Research, San Diego, CA) and anti-p53-induced gene 3 (kindly provided by D. Hill, Oncogene Research Products, Cambridge, MA) antibodies. Chk2 Is Not Activated to Phosphorylate p53 after DNA Damage—Chk2 isolated from cells that have sustained DNA strand breaks is greatly stimulated to phosphorylate Cdc25C at Ser216 when compared with Chk2 isolated from untreated cells (17Melchionna R. Chen X.B. Blasina A. McGowan C.H. Nat. Cell Biol. 2000; 2: 762-765Crossref PubMed Scopus (264) Google Scholar, 18Matsuoka 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 (694) Google Scholar, 19Ahn J.Y. Schwarz J.K. Piwnica-Worms H. Canman C.E. Cancer Res. 2000; 60: 5934-5936PubMed Google Scholar). We were therefore interested in determining whether DNA damage to human cells would similarly activate Chk2 to phosphorylate p53. To characterize Chk2 protein kinase activity, we used clonally derived cells from the colorectal cancer cell line HCT116 that were engineered to stably express HA-tagged Chk2 (45Wu X. Webster S.R. Chen J. J. Biol. Chem. 2001; 276: 2971-2974Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). These cells express similar levels of stably expressed exogenous HA-Chk2 and endogenous Chk2 (Ref. 45Wu X. Webster S.R. Chen J. J. Biol. Chem. 2001; 276: 2971-2974Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar and Fig. 1A). HCT116 cells harbor wild type p53 that becomes stabilized after DNA damage (50Waldman T. Kinzler K.W. Vogelstein B. Cancer Res. 1995; 55: 5187-5190PubMed Google Scholar). HA-Chk2 protein could be specifically immunopurified from HCT116 cells using anti-HA antibody cross-linked to protein A-Sepharose beads (Fig. 1B). We compared immunopurified HA-Chk2 from HCT116 cells that had been either not treated or treated with the radiomimetic agent NCS. As expected, the DNA-damaging treatment led to hyper-phosphorylation of HA-Chk2 as evidenced by a mobility shift on SDS-PAGE, and HA-Chk2 from NCS treated cells was far more effective in phosphorylating a fragment of Cdc25C that contains Ser216, than HA-Chk2 from untreated cells (Fig. 1C). By contrast, a fragment of p53 that spans several of its Chk2 phosphorylation sites within the N terminus was phosphorylated less well by HA-Chk2 than Cdc25C, and, remarkably, there was virtually no increase in the ability of HA-Chk2 to phosphorylate p53 after NCS treatment (Fig. 1C).Fig. 1ADNA damage activates Chk2 to phosphorylate a fragment of Cdc25C but not the N terminus of p53.A, total cell extracts (100 μg) of HCT116 (HA-Chk2) (first lane) or HCT116 parental (second lane) cells were subjected to SDS-PAGE and then immunoblotted with anti-Chk2 antibody. B, HA-Chk2 was immunopurified from extracts of HCT116-HAChk2 cells (20 × 140-mm plates) using anti-HA antibody cross-linked to protein A-beads (second lane) or mock purified by protein A-beads (first lane). The proteins were eluted from the beads by incubating with HA peptide and then dialyzed as described under "Experimental Procedures." Aliquots of dialysates (25% of total) were separated by SDS-PAGE and visualized by silver staining. C, HA-Chk2 (10, 20, and 40 ng) purified from HCT116 cells without (lanes 1–3) or with (lanes 4–6) NCS treatment were incubated with 500 ng each of GST-p53 (1–82) and GST-Cdc25C (200–256). The reaction mixtures were separated by 8% SDS-PAGE, transferred to nitrocellulose, and analyzed by auto-radiography (lanes 1–6, left panel) or by immunoblotting with either anti-GST or anti-HA antibodies (lanes 7–12, right panel). D, HA-Chk2 (5 ng) purified from HCT116 cells that were treated (lanes 3–6) or not (lanes 7–9) with γ irradiation (13 Gy) were incubated either with 500 ng of either GST-Cdc25C (200–256) (lanes 3, 5, and 8) or GST-p53 (1–82) (lanes 3, 4, and 7) and analyzed as in C. HA-Chk2 was mock-purified from HCT116 parental cells (as in B) with (lanes 10–12) or without (lanes 13–15) γ irradiation. The proteins were incubated with either GST-Cdc25C (lanes 11 and 14) or GST-p53 (lanes 10 and 13). GST-p53 (lane 1) or GST-Cdc25C (lane 2) were incubated with kinase buffer as controls. E, Chk2-FLAG (4, 8, 16, 32, or 100 ng) purified from recombinant baculovirus infected insect cells was incubated with 500 ng of GST-Cdc25C (200–256) (lanes 1–5) or 500 ng of GST-p53 (1–82) (lanes 6–10) in kinase buffer and analyzed as described in A. The proteins used were detected by immunoblotting with anti-GST and anti-FLAG antibodies (lanes 11–20). F, endogenous Chk2 was immunoprecipitated with anti-Chk2 antibody from HCT116 parental cells that were untreated (lane 1) or NCS-treated (500 ng/ml) for 2 h (lanes 2–4). Kinase activity of untreated Chk2 (2 μl of beads from untreated cells) was compared with that of NCS-activated Chk2 (2, 4, 6 μl beads) for phosphorylation of 500 ng of GST-p53 (1–82) and GST-Cdc25C (200–256). The amount of protein used in each assay was determined by immunoblotting as shown in the right panel.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A commonly used inducer of DNA strand breaks is γ irradiation, and so we performed a similar experiment comparing HA-Chk2 from γ irradiated or unirradiated HCT116 cells (Fig. 1D). The limitation in this case was that it was not possible to rapidly irradiate large quantities of these cells with ease, and thus smaller amounts of Chk2 were used. Nevertheless the results were essentially the same as with NCS. HA-Chk2 activity toward Cdc25C was significantly increased after γ irradiation, whereas that toward p53 was not detectably affected by irradiation. In this experiment we tested Cdc25C and p53 alone as well as together to demonstrate that the lack of increased phosphorylation of p53 after DNA damage is not due to competition with Cdc25C within the same reaction mixture for available kinase, a conclusion further supported by experiments shown below. Control purification from extracts of parental HCT116 cells revealed no contaminating kinases that were activated after γ irradiation to phospho
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