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• W2157384083 abstract "ATM is mutated in the human genetic disorder ataxia telangiectasia, which is characterized by ataxia, immune defects, and cancer predisposition. Cells that lack ATM exhibit delayed up-regulation of p53 in response to ionizing radiation. Serine 15 of p53 is phosphorylated in vivo in response to ionizing radiation, and antibodies to ATM immunoprecipitate a protein kinase activity that, in the presence of manganese, phosphorylates p53 at serine 15. Immunoprecipitates of ATM also phosphorylate PHAS-I in a manganese-dependent manner. Here we have purified ATM from human cells using nine chromatographic steps. Highly purified ATM phosphorylated PHAS-I, the 32-kDa subunit of RPA, serine 15 of p53, and Chk2 in vitro. The majority of the ATM phosphorylation sites in Chk2 were located in the amino-terminal 57 amino acids. In each case, phosphorylation was strictly dependent on manganese. ATM protein kinase activity was inhibited by wortmannin with an IC50 of approximately 100 nm. Phosphorylation of RPA, but not p53, Chk2, or PHAS-I, was stimulated by DNA. The related protein, DNA-dependent protein kinase catalytic subunit, also phosphorylated PHAS-I, RPA, and Chk2 in the presence of manganese, suggesting that the requirement for manganese is a characteristic of this class of enzyme. ATM is mutated in the human genetic disorder ataxia telangiectasia, which is characterized by ataxia, immune defects, and cancer predisposition. Cells that lack ATM exhibit delayed up-regulation of p53 in response to ionizing radiation. Serine 15 of p53 is phosphorylated in vivo in response to ionizing radiation, and antibodies to ATM immunoprecipitate a protein kinase activity that, in the presence of manganese, phosphorylates p53 at serine 15. Immunoprecipitates of ATM also phosphorylate PHAS-I in a manganese-dependent manner. Here we have purified ATM from human cells using nine chromatographic steps. Highly purified ATM phosphorylated PHAS-I, the 32-kDa subunit of RPA, serine 15 of p53, and Chk2 in vitro. The majority of the ATM phosphorylation sites in Chk2 were located in the amino-terminal 57 amino acids. In each case, phosphorylation was strictly dependent on manganese. ATM protein kinase activity was inhibited by wortmannin with an IC50 of approximately 100 nm. Phosphorylation of RPA, but not p53, Chk2, or PHAS-I, was stimulated by DNA. The related protein, DNA-dependent protein kinase catalytic subunit, also phosphorylated PHAS-I, RPA, and Chk2 in the presence of manganese, suggesting that the requirement for manganese is a characteristic of this class of enzyme. ataxia-telangiectasia mutated phosphatidylinositol-3 DNA-dependent protein kinase catalytic subunit DNA-dependent protein kinase dithiothreitol glutathione S-transferase gray replication protein, A Ataxia telangiectasia is a human genetic disorder characterized by ataxia, immunodeficiency, cell cycle checkpoint defects, and predisposition to cancer (1.Lavin M.F. Shiloh Y. Annu. Rev. Immunol. 1997; 15: 177-202Crossref PubMed Scopus (542) Google Scholar, 2.Canman C.E. Lim D.-S. Oncogene. 1998; 17: 3301-3308Crossref PubMed Scopus (140) Google Scholar, 3.Nakamura Y. Nat. Genet. 1998; 11: 1231-1232Crossref Scopus (36) Google Scholar). The gene that is mutated in this disorder, ATM,1encodes a nuclear polypeptide of approximately 350 kDa, which shares amino acid homology in its COOH terminus with the phosphatidylinositol-3 (PI-3) kinase family of proteins (4.Savitsky K. Bar-Shira A. Gilad S. Rotman G. Ziv Y. Vanagaite L. Tagle D.A. Smith S. Uziel T. Sfez S. Ashkenazi M. Pecker I. Frydman M. Harnik R. Patanjali S.R. Simmons A. Clines G.A. Sartiel A. Gatti R.A. Chessa L. Sanal O. Lavin M.F. Jaspers N.G.J. Taylor M.R. Arlett C.F. Miki T. Weissman S.M. Lovett M. Collins F.S. Shiloh Y. Science. 1995; 268: 1749-1753Crossref PubMed Scopus (2377) Google Scholar). Other members of this family include the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), FRAP, ATR, and the yeast gene products Mec-1, Tel-1, and Rad3 (for review, see Refs. 5.Hunter T. Cell. 1995; 83: 1-4Abstract Full Text PDF PubMed Scopus (261) Google Scholar and6.Keith C.T. Schreiber S.L. Science. 1995; 270: 50-51Crossref PubMed Scopus (451) Google Scholar). Highly purified DNA-PKcs has weak inherent DNA-stimulated serine/threonine protein kinase activity that is enhanced by interaction with DNA-bound Ku (7.Gottlieb T.M. Jackson S.P. Cell. 1993; 72: 131-142Abstract Full Text PDF PubMed Scopus (1027) Google Scholar, 8.Lees-Miller S.P. Godbout R. Chan D.W. Weinfeld M.W. Day III., R.S. Barron G.M. Allalunis-Turner J. Science. 1995; 267: 1183-1185Crossref PubMed Scopus (504) Google Scholar, 9.Chan D.W. Mody C.H. Ting N.S.Y. Lees-Miller S.P. Biochem. Cell Biol. 1996; 74: 67-73Crossref PubMed Scopus (83) Google Scholar, 10.Hammarsten O. Chu G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 525-530Crossref PubMed Scopus (202) Google Scholar, 11.Yaneva M. Kowalewski T. Lieber M.R. EMBO J. 1997; 16: 5098-5112Crossref PubMed Scopus (269) Google Scholar, 12.West R.B. Yaneva M. Lieber M.R. Mol. Cell. Biol. 1998; 18: 5908-5920Crossref PubMed Scopus (145) Google Scholar). DNA-PKcs does not have PI-3 kinase activity (13.Hartley K.O. Gell D. Zhang H. Smith G.C.M. Divecha N. Connelly M.A. Admon A. Lees-Miller S.P. Anderson C.W. Jackson S.P. Cell. 1995; 82: 849-856Abstract Full Text PDF PubMed Scopus (672) Google Scholar), and it is likely that, like DNA-PK, ATM also behaves as a serine/threonine protein kinase. Indeed, several reports have shown that immunoprecipitates of ATM from whole cells have serine/threonine protein kinase activity toward PHAS-I and p53 in vitro (14.Banin S. Moyal L. Shieh S. Taya Y. Anderson C.W. Chessa L. Smorodinsky N.I. Prives C. Reis Y. Shiloh Y. Ziv Y. Science. 1998; 281: 1674-1677Crossref PubMed Scopus (1711) Google Scholar, 15.Canman 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 (1712) Google Scholar). In immunoprecipitation assays, ATM protein kinase activity was absolutely dependent on the presence of manganese, and activity toward p53 and PHAS-I was not stimulated by DNA (14.Banin S. Moyal L. Shieh S. Taya Y. Anderson C.W. Chessa L. Smorodinsky N.I. Prives C. Reis Y. Shiloh Y. Ziv Y. Science. 1998; 281: 1674-1677Crossref PubMed Scopus (1711) Google Scholar, 15.Canman 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 (1712) Google Scholar). In contrast, others have reported that immunoprecipitated ATM has DNA-dependent kinase activity toward the 32-kDa subunit of single-stranded DNA-binding protein, RPA (16.Gately D.P. Hittle J.C. Chan G.K.T. Yen T.J. Mol. Biol. Cell. 1998; 9: 2361-2374Crossref PubMed Scopus (164) Google Scholar). A recent report has shown that, in crude extracts, ATM binds to DNA that has been damaged by irradiation with x-rays (17.Suzuki K. Kodama S. Watanabe M. J. Biol. Chem. 1999; 274: 25571-25575Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). These sometimes conflicting results underscore the need for biochemical characterization of ATM. Although recombinant ATM protein has been expressed in baculovirus (18.Ziv 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, 19.Scott S.P. Zhang N. Khanna K.K. Khromykh A. Hobson K. Watters D. Lavin M.F. Biochem. Biophys. Res. Commun. 1998; 245: 144-148Crossref PubMed Scopus (14) Google Scholar), the very low protein yield has made direct biochemical study very difficult. Cells that lack ATM are defective in their ability to activate DNA damage response pathways that result in cell cycle arrest at both G1/S and G2/M. ATM acts upstream of p53 and is required for the activation of p53 and the subsequent up-regulation of the cyclin-dependent kinase inhibitor, p21 (for review, see Refs. 1.Lavin M.F. Shiloh Y. Annu. Rev. Immunol. 1997; 15: 177-202Crossref PubMed Scopus (542) Google Scholar, 2.Canman C.E. Lim D.-S. Oncogene. 1998; 17: 3301-3308Crossref PubMed Scopus (140) Google Scholar, 3.Nakamura Y. Nat. Genet. 1998; 11: 1231-1232Crossref Scopus (36) Google Scholar). In addition, ATM is required for Chk2-dependent cell cycle arrest at G2/M (20.Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1089) Google Scholar), and Chk2 is phosphorylated in vivo in an ATM-dependent manner in response to ionizing radiation (20.Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1089) Google Scholar,21.Chaturvedi P. Eng W.K. Zhu Y. Mattern M.R. Mishra R. Hurle M.R. Zhang X. Annan R.S. Lu Q. Faucette L.F. Scott G.F. Li X. Carr S.A. Johnson R.K. Winkler J.D. Zhou B.B. Oncogene. 1999; 18: 4047-4054Crossref PubMed Scopus (360) Google Scholar). Recombinant ATM (18.Ziv 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, 19.Scott S.P. Zhang N. Khanna K.K. Khromykh A. Hobson K. Watters D. Lavin M.F. Biochem. Biophys. Res. Commun. 1998; 245: 144-148Crossref PubMed Scopus (14) Google Scholar) and immunoprecipitates of ATM from DNA-damaged cells (14.Banin S. Moyal L. Shieh S. Taya Y. Anderson C.W. Chessa L. Smorodinsky N.I. Prives C. Reis Y. Shiloh Y. Ziv Y. Science. 1998; 281: 1674-1677Crossref PubMed Scopus (1711) Google Scholar, 15.Canman 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 (1712) Google Scholar) phosphorylate p53 on serine 15, and ATM can interact directly with p53 (22.Khanna K.K. Keating K.E. Kozlov S. Scott S. Gatei M. Hobson K. Taya Y. Gabrielli B. Chan D.W. Lees-Miller S.P. Lavin M.F. Nat. Genet. 1998; 20: 398-400Crossref PubMed Scopus (407) Google Scholar). Serine 15 of p53 is phosphorylatedin vivo in response to ionizing radiation (23.Siliciano J.D. Canman C.E. Taya Y. Sakaguchi K. Appella E. Kastan M.B. Genes Dev. 1997; 11: 3471-3481Crossref PubMed Scopus (711) Google Scholar), and phosphorylation at serine 15 plays an important role in the ionizing radiation-induced damage response pathway (for reviewed, see Refs. 24.Prives C. Cell. 1998; 95: 5-8Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholarand 25.Giaccia A.J. Kastan M.B. Genes Dev. 1998; 12: 2973-2983Crossref PubMed Scopus (1177) Google Scholar). However, the precise role of ATM in the activation of p53 remains to be determined. Serine 15 of p53, which occurs in the amino acid sequence PLSQE is also phosphorylated, in vitro, by DNA-PK (26.Lees-Miller S.P. Sakaguchi K. Ullrich S. Appella E. Anderson C.W. Mol. Cell. Biol. 1992; 12: 5041-5049Crossref PubMed Scopus (465) Google Scholar), suggesting that ATM and DNA-PK recognize similar sequences in their target proteins. Here we describe the purification of ATM from normal human tissue. The final protein fraction contains a polypeptide of approximately 350 kDa which cross-reacts with various antibodies to ATM but not to DNA-PKcs. The ATM-containing fraction phosphorylates serine 15 of p53, Chk2, the 32-kDa subunit of RPA, and PHAS-I in a strictly manganese-dependent manner. The protein kinase activity of purified ATM toward p53, PHAS-I, and Chk2 was not stimulated by double-stranded DNA or by Ku. Phosphorylation of RPA was stimulated by calf thymus DNA and calf thymus DNA in the presence of M13 single-stranded circular plasmid DNA, but not by irradiated plasmid DNA. We also show that the related protein, DNA-PKcs, is active in the presence of manganese in the absence of Ku, suggesting that a requirement for manganese may be characteristic of the PI-3 kinase family of enzymes. ATM was purified from human placenta following a similar procedure to that described previously for DNA-PKcs and Ku (9.Chan D.W. Mody C.H. Ting N.S.Y. Lees-Miller S.P. Biochem. Cell Biol. 1996; 74: 67-73Crossref PubMed Scopus (83) Google Scholar). All steps utilizing low pressure chromatography were performed at 4 °C. Chromatographic steps using the Biologic Protein Purification system (Bio-Rad) were performed at room temperature. All buffers (except buffer E, below) contained 0.2 mmphenylmethylsulfonyl fluoride, 0.1 mm benzamidine, 1 μg/ml pepstatin, and 0.1 mm DTT. The presence of ATM and DNA-PKcs was monitored at all stages of purification by Western blotting using a rabbit polyclonal antibody to ATM, 4BA (22.Khanna K.K. Keating K.E. Kozlov S. Scott S. Gatei M. Hobson K. Taya Y. Gabrielli B. Chan D.W. Lees-Miller S.P. Lavin M.F. Nat. Genet. 1998; 20: 398-400Crossref PubMed Scopus (407) Google Scholar), or a rabbit polyclonal antibody, DPK1 to DNA-PKcs (8.Lees-Miller S.P. Godbout R. Chan D.W. Weinfeld M.W. Day III., R.S. Barron G.M. Allalunis-Turner J. Science. 1995; 267: 1183-1185Crossref PubMed Scopus (504) Google Scholar). Antibodies to the amino-terminal, Rad3 and carboxyl-terminal domains of ATM (27.Chan D.W. Gately D.P. Urban S. Galloway A.M. Lees-Miller S.P. Yen T. Allalunis-Turner J. Int. J. Radiat. Biol. 1998; 74: 217-224Crossref PubMed Scopus (67) Google Scholar) were used in preliminary studies. The initial stages of purification were similar to those described previously for the purification of DNA-PKcs and Ku (9.Chan D.W. Mody C.H. Ting N.S.Y. Lees-Miller S.P. Biochem. Cell Biol. 1996; 74: 67-73Crossref PubMed Scopus (83) Google Scholar). Human placenta was obtained from the Foothills Hospital, Calgary, in accordance with the safety and ethical requirements of the university. Freshly obtained human placenta was homogenized in buffer containing 0.5 msalt buffer and 10 mm magnesium chloride as described previously (9.Chan D.W. Mody C.H. Ting N.S.Y. Lees-Miller S.P. Biochem. Cell Biol. 1996; 74: 67-73Crossref PubMed Scopus (83) Google Scholar), and ATM was precipitated by the addition of ammonium sulfate to 40% saturation. After dialysis into buffer B (50 mm Tris-HCl, pH 8, 5% glycerol, 0.2 mm EDTA) containing 100 mm KCl, the sample was applied to a 5 × 30-cm column of DEAE-Fast Flow Sepharose (Amersham Pharmacia Biotech) equilibrated in the same buffer and eluted with buffer B containing 1 m KCl. In preliminary preparations, the protein sample was dialyzed into buffer B containing 100 mmKCl and applied to a 5 × 15-cm column of SP-Fast Flow Sepharose (Amersham Pharmacia Biotech). However, subsequently, this step was replaced by a 5 × 15-cm column of phenyl-Sepharose, Fast Flow (Amersham Pharmacia Biotech), equilibrated in buffer B containing 1m KCl. ATM was eluted with buffer B containing 50 mm KCl. This column provided better yields of ATM protein than the SP-Sepharose column because of higher binding capacity for ATM. Also, ATM was separated from the majority of the DNA-PK because under these conditions, DNA-PKcs and Ku70/80 did not bind to the phenyl-Sepharose column. The ATM-containing fractions from the phenyl-Sepharose column were dialyzed into buffer B containing 100 mm KCl, applied to a 5 × 5-cm column of MacroPrep DEAE (Bio-Rad), and eluted with buffer B containing 100 mmKCl and 100 mm magnesium chloride. A similar step involving elution of DEAE-anion exchange resin using magnesium chloride was used for the purification of DNA-PKcs and Ku from HeLa cells (28.Lees-Miller S.P. Chen Y.-R. Anderson C.W. Mol. Cell. Biol. 1990; 10: 6472-6481Crossref PubMed Scopus (359) Google Scholar) and placenta (9.Chan D.W. Mody C.H. Ting N.S.Y. Lees-Miller S.P. Biochem. Cell Biol. 1996; 74: 67-73Crossref PubMed Scopus (83) Google Scholar). Fractions containing ATM were then dialyzed into buffer B containing 100 mm KCl and passed over single-stranded DNA-cellulose (Sigma), either in batch mode or by column chromatography (1.5 × 4-cm column). Unlike DNA-PKcs and Ku, in our hands, ATM did not bind to either single-stranded DNA-cellulose or double-stranded DNA-cellulose. ATM from the flow-through fractions of the DNA-cellulose column was then applied to a 5-ml heparin HiTrap column (Amersham Pharmacia Biotech), equilibrated in buffer B containing 100 mm KCl and 0.02% (v/v) Tween 20. This and all subsequent steps were performed on a Biologic Protein Purification system. ATM was eluted with a linear gradient of buffer B containing 100 mmKCl and 0.02% Tween 20, to buffer B containing 1m KCl and 0.02% Tween 20 over 75 min. 1-ml fractions were collected. ATM eluted at approximately 200 mm KCl, whereas DNA-PKcs applied to the same column under the same conditions eluted at approximately 300–400 mm KCl. ATM-containing fractions from the heparin HiTrap column were pooled and dialyzed into buffer C (10 mmsodium phosphate, 50 mm KCl, 5% glycerol, pH 7.2). The sample was applied to an Econo-Pac hydroxyapatite cartridge (Bio-Rad) at a flow rate of 0.7 ml/min. The column was washed with buffer C before applying a 32-ml linear gradient of increasing buffer D (400 mm sodium phosphate, 50 mm KCl, 5% glycerol, pH 6.8). ATM eluted at approximately 20% buffer D, corresponding to approximately 90 mm sodium phosphate. Fractions containing ATM were pooled and dialyzed into buffer B containing 100 mm KCl and 0.02% Tween 20 and applied to Mono Q HR5/5 fast protein liquid chromatography column that had been equilibrated in the same buffer. Bound proteins were eluted with a linear gradient of buffer B containing 100 mm KCl, 0.02% Tween 20, and MgCl2 at a flow rate of 1 ml/min. The magnesium concentration was increased at 1 mm/min. ATM eluted off the Mono Q column between 22 and 30 mm MgCl2. In preliminary preparations, ATM-containing fractions off the Mono Q column were applied to ATP-Sepharose, gamma-phosphate linked (Upstate Biotechnology Inc.) that had been equilibrated in buffer E (25 mm Hepes, pH 7.5, 1 mm DTT, 60 mmMgCl2, 0.5 mm phenylmethylsulfonyl fluoride) containing 150 mm NaCl and eluted with buffer E containing 1m NaCl. In subsequent preparations ATM from the Mono Q step was dialyzed into buffer F (50 mm Tris-HCl, pH 8.0, 5% glycerol, without EDTA) containing 100 mm NaCl and applied to a 5-ml HiTrap chelation Sepharose column (Amersham Pharmacia Biotech) that had been loaded with nickel sulfate according to the manufacturer's recommendations. The column was equilibrated in buffer F containing 100 mm NaCl and after sample loading was washed sequentially with 15 ml of buffer F containing 100 mm NaCl followed by 15 ml of buffer F containing 500 mm NaCl. ATM was eluted with a linear gradient of buffer F containing 500 mm NaCl to buffer F containing 500 mm NaCl, 150 mm imidazole, and 150 mm ammonium chloride at 1.5 mm imidazole and ammonium chloride per minute. ATM-containing fractions were dialyzed and applied onto a Mono Q HR5/5 column that had been equilibrated in buffer B containing 100 KCl and 0.02% Tween-20 and eluted with a gradient of buffer B containing 1m KCl and 0.02% Tween 20. ATM-containing fractions were desalted and concentrated on a Centricon 100 microconcentrator (Amicon), washed with buffer B containing 100 mm KCl, 0.02% Tween 20, 0.5 mmphenylmethylsulfonyl fluoride, 1 mm DTT, and stored in aliquots at −80 °C. ATM kinase assays contained 25 mm Tris-HCl, pH 8, 50 mm KCl, 5% glycerol, 0.5 mm DTT, 5 μCi of [γ-32P]ATP, 10 μm cold ATP, 10 mm MnCl2, 0.25–0.5 μg of PHAS-I (Stratagene) or other substrate as indicated, and 2–4 ng of purified ATM. The reactions were incubated at 30 °C for 30 min, stopped with SDS sample buffer, and analyzed by SDS-polyacrylamide gel electrophoresis on 15% acrylamide gels. Gels were stained with Coomassie Blue, destained extensively, dried, and exposed to Fuji x-ray film with intensifying screen at −80 °C. DNA-PK was purified and assayed as described previously (9.Chan D.W. Mody C.H. Ting N.S.Y. Lees-Miller S.P. Biochem. Cell Biol. 1996; 74: 67-73Crossref PubMed Scopus (83) Google Scholar, 40.Chan D.W. Lees-Miller S.P. J. Biol. Chem. 1996; 271: 8936-8941Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). Sonicated calf thymus DNA was prepared as described previously (28.Lees-Miller S.P. Chen Y.-R. Anderson C.W. Mol. Cell. Biol. 1990; 10: 6472-6481Crossref PubMed Scopus (359) Google Scholar). Irradiated pUC18 plasmid DNA was prepared as described previously (29.Weinfeld M. Chaudry M.A. D'Amours D. Pelletier J.D. Poirier G.G. Povirk L.F. Lees-Miller S.P. Radiat. Res. 1997; 148: 22-28Crossref PubMed Scopus (56) Google Scholar). Single-stranded M13 circular plasmid DNA was obtained from Life Technologies, Inc. Amino acids 1–40 of p53 were expressed in bacteria as a GST fusion protein, purified over glutathione Sepharose, and dialyzed into buffer B containing 100 mm KCl before use. The amino-terminal 222 amino acids of human Chk2 were expressed in pGEX-5X-1 as a GST fusion, and protein was expressed and purified as for p53. A GST fusion protein expressing amino acids 58–222 of Chk2 (Chk2(D57N)) was created by deleting the first 174 base pairs from the Chk2 coding sequence. The protein was expressed and purified as above. A plasmid expressing His-tagged RPA32 and 14-kDa subunits was a kind gift from Dr. Aled Edwards (Ontario Cancer Institute). ATM and DNA-PKcs were analyzed on 8% SDS-acrylamide gels, as described previously (9.Chan D.W. Mody C.H. Ting N.S.Y. Lees-Miller S.P. Biochem. Cell Biol. 1996; 74: 67-73Crossref PubMed Scopus (83) Google Scholar). The antibodies used for detection of ATM were a rabbit polyclonal to the Rad3 domain of ATM, 4BA (22.Khanna K.K. Keating K.E. Kozlov S. Scott S. Gatei M. Hobson K. Taya Y. Gabrielli B. Chan D.W. Lees-Miller S.P. Lavin M.F. Nat. Genet. 1998; 20: 398-400Crossref PubMed Scopus (407) Google Scholar). A rabbit polyclonal antibody to amino acids 819–894 of ATM (Oncogene Scientific, ATM Ab-3, PC116) was also used. The p53 monoclonal antibody, DO1, was obtained from Oncogene Scientific. The rabbit polyclonal antibody to DNA-PKcs (DPK1) was as described previously (8.Lees-Miller S.P. Godbout R. Chan D.W. Weinfeld M.W. Day III., R.S. Barron G.M. Allalunis-Turner J. Science. 1995; 267: 1183-1185Crossref PubMed Scopus (504) Google Scholar). A rabbit polyclonal antibody specific for serine 15 of p53 was as described previously (22.Khanna K.K. Keating K.E. Kozlov S. Scott S. Gatei M. Hobson K. Taya Y. Gabrielli B. Chan D.W. Lees-Miller S.P. Lavin M.F. Nat. Genet. 1998; 20: 398-400Crossref PubMed Scopus (407) Google Scholar). The cDNA sequence of ATM predicts a polypeptide of approximately 350 kDa with amino acid similarity to the PI-3 kinase family of proteins in the carboxyl-terminal domain. Attempts to express active ATM in baculovirus have not been very successful, probably because of the large size of the protein. To date, most of the information regarding the properties of ATM has been obtained from immunoprecipitates. Because immunoprecipitates may contain contaminating or interacting proteins and because antibody binding could interfere with the biochemical properties of the putative protein, we considered it important to purify the protein by biochemical means to characterize the biochemical properties of ATM fully. Here we have used conventional biochemical techniques to purify ATM from human placenta. We previously described a procedure to purify the related protein DNA-PK from the same source (9.Chan D.W. Mody C.H. Ting N.S.Y. Lees-Miller S.P. Biochem. Cell Biol. 1996; 74: 67-73Crossref PubMed Scopus (83) Google Scholar) and therefore used these procedures as a guide to purify ATM. Because immunoprecipitates of ATM phosphorylate p53 on serine 15 and because the same residue is phosphorylated by DNA-PK in vitro, it was important to separate DNA-PK from ATM completely during the purification procedure. The presence of ATM and DNA-PKcs was determined by Western blot at each stage of the purification, and fractions containing ATM were judiciously pooled. ATM was precipitated by 40% ammonium sulfate and bound to DEAE-Sepharose anion exchange matrix. DNA-PKcs and Ku proteins were also found in the same fractions. However, most of the DNA-PKcs and Ku was removed from ATM by binding to phenyl-Sepharose in buffer B containing 1m KCl. Under these conditions, most of the DNA-PKcs and Ku did not bind to the column, whereas ATM bound and was eluted with buffer B containing 50 mm salt. Partially purified ATM that was eluted from DEAE by magnesium-containing buffer did not bind to either double-stranded or single-stranded DNA-cellulose resins. In contrast, DNA-PKcs and Ku bound tightly to both resins, again providing a means for separation of DNA-PK from ATM. ATM and DNA-PKcs also eluted at different salt conditions on heparin HiTrap chromatography, again providing for maximum separation of the two proteins. In preliminary preparations, the final step of the purification of ATM was binding to ATP-Sepharose (gamma-phosphate linked). However, very little ATM was recovered from this column, and despite various elution strategies, the majority of the protein remained bound to the ATP-Sepharose beads. 2D. W. Chan, P. Douglas, and S. P. Lees-Miller, unpublished observations. Preliminary experiments indicated that ATM bound to chelation Sepharose resin that had been loaded with nickel, copper, or zinc but not with magnesium or manganese. 3W. Block and S. P. Lees-Miller, unpublished observations. Chelation Sepharose was therefore used in place of the ATP-Sepharose column. ATM-containing fractions were applied to a nickel-loaded HiTrap chelation Sepharose column and eluted with a gradient of ammonium chloride and imidazole in buffer B containing 500 mm NaCl. Two polypeptides of approximately 350 and 300 kDa were observed on silver-stained SDS-polyacrylamide gels (Fig.1 A, upper arrow andlower arrow, respectively). ATM was detected by Western blotting in fractions 3–21 (Fig. 1 B). The upper band in the Western blot (Fig. 1 B) comigrated with the upper band in the silver stained gel (Fig. 1 A, upper arrow). The lower band in the Western blot ran slightly ahead of the 200-kDa marker and is likely a breakdown product of ATM. The ATM-containing fractions were assayed for their ability to phosphorylate PHAS-I, a substrate that has been shown previously to be phosphorylated by immunoprecipitates of ATM in the presence of manganese (14.Banin S. Moyal L. Shieh S. Taya Y. Anderson C.W. Chessa L. Smorodinsky N.I. Prives C. Reis Y. Shiloh Y. Ziv Y. Science. 1998; 281: 1674-1677Crossref PubMed Scopus (1711) Google Scholar, 15.Canman 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 (1712) Google Scholar). Manganese-dependent phosphorylation of PHAS-I was observed in the ATM-containing fractions (Fig. 1 C). ATM-containing fractions were pooled and applied to a Mono Q fast protein liquid chromatography column and eluted with a salt gradient. The ATM-containing fractions were concentrated and desalted as described under “Experimental Procedures.” The final yield of ATM was estimated by comparison with known amounts of DNA-PKcs on silver-stained gels (Fig. 1 D). The major band in the sample migrated at 350 kDa, coincident with ATM in Western blots. The polypeptide at approximately 300 kDa comigrated with the ATM breakdown product on Western blot. We estimate that approximately 2 μg of highly purified ATM was obtained from one placenta (TableI). In contrast, up to 500 μg of DNA-PKcs and 1 mg of Ku70/80 heterodimer could be purified from the same amount of starting material, suggesting that ATM is present in far less abundance than DNA-PKcs in human tissues. In preliminary studies, we examined rat liver and bovine testis as potential sources of ATM; however, poor antibody recognition and/or low abundance made purification of ATM from non-human sources impractical. 4W. Truong and S. P. Lees-Miller, unpublished observations. Table IProtein yields during purification of ATMPurification stepTotal proteinmgAmmonium sulfate precipitation4179DEAE 1m eluate3349Phenyl-Sepharose, 50 mmeluate92DEAE, magnesium eluate40DNA-cellulose flow22Heparin HiTrap pooled fractions8Hydroxyapatite, pooled fractions1.2Mono Q, magnesium elution, pooled fractions0.1Nickel chelation HiTrap, pooled fractionsNDMono Q, salt elution, pooled fractions0.001ATM was purified as described under “Experimental Procedures,” starting with approximately 300 g of placenta. Protein concentrations for steps up to the first Mono Q FPLC column were determined using the Bio-Rad protein assay and bovine serum albumin as standard. The concentration of ATM in the final step was estimated by comparison with known amounts of DNA-PKcs protein on silver-stained gels (see Fig. 1 B). ND, not determined. Open table in a new tab ATM was purified as described under “Experimental Procedures,” starting with approximately 300 g of placenta. Protein concentrations for steps up to the first Mono Q FPLC column were determined using the Bio-Rad protein assay and bovine serum albumin as standard. The concentration of ATM in the final step was estimated by comparison with known amounts of DNA-PKcs protein on silver-stained gels (see Fig. 1 B). ND, not determined. The highly purified ATM fraction was next assayed for its ability to phosphorylate a number of protein substrates in the presence of either magnesium or manganese as metal ion. ATM activity was also assayed in the presence of the PI-3 kinase inhibitor, wortmannin. Because the kinase activity of the related protein, DNA-PKcs, is modulated by DNA and the Ku70/80 heterodimer, ATM was also assayed in the presence of both sonicated calf thymus DNA and Ku. Phosphorylation of PHAS-I was completely dependent on the presence of manganese (Fig.2 A) and was inhibited by wortmannin (Fig. 2 A, lane 3) with an IC50 of approximately 100 nm (data not shown). The addition of sheared calf thymus DNA did not stimulate PHAS phosphorylati" @default.
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• W2157384083 title "Purification and Characterization of ATM from Human Placenta" @default.
• W2157384083 cites W1818313483 @default.
• W2157384083 cites W1959049775 @default.
• W2157384083 cites W1969393623 @default.
• W2157384083 cites W1971850308 @default.
• W2157384083 cites W1974010399 @default.
• W2157384083 cites W1977145510 @default.
• W2157384083 cites W1979303367 @default.
• W2157384083 cites W1988876917 @default.
• W2157384083 cites W1994163195 @default.
• W2157384083 cites W2003196136 @default.
• W2157384083 cites W2007635472 @default.
• W2157384083 cites W2010940856 @default.
• W2157384083 cites W2014170738 @default.
• W2157384083 cites W2018741051 @default.
• W2157384083 cites W2023324317 @default.
• W2157384083 cites W2038424689 @default.
• W2157384083 cites W2043136696 @default.
• W2157384083 cites W2044874191 @default.
• W2157384083 cites W2048059265 @default.
• W2157384083 cites W2049941746 @default.
• W2157384083 cites W2055299116 @default.
• W2157384083 cites W2058939604 @default.
• W2157384083 cites W2062232773 @default.
• W2157384083 cites W2078882775 @default.
• W2157384083 cites W2081183767 @default.
• W2157384083 cites W2086218293 @default.
• W2157384083 cites W2089574530 @default.
• W2157384083 cites W2089888782 @default.
• W2157384083 cites W2091825641 @default.
• W2157384083 cites W2121672275 @default.
• W2157384083 cites W2149013144 @default.
• W2157384083 cites W2156299289 @default.
• W2157384083 cites W2159168998 @default.
• W2157384083 cites W2160401113 @default.
• W2157384083 cites W2163658254 @default.
• W2157384083 cites W2171489185 @default.
• W2157384083 cites W2171775098 @default.
• W2157384083 cites W2172192445 @default.
• W2157384083 cites W2331622751 @default.
• W2157384083 cites W4212962560 @default.
• W2157384083 cites W4250719896 @default.
• W2157384083 cites W67399661 @default.
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