DOI: 10.1126/science.281.5383.1677. Activation of the ATM Kinase by Ionizing Radiation and Phosphorylation of p53

Activation of the ATM Kinase by Ionizing Radiation and Phosphorylation of p53

10.1126/science.281.5383.1677

Crossref journal-article

American Association for the Advancement of Science (AAAS)

Science (221)

Abstract

The p53 tumor suppressor protein is activated and phosphorylated on serine-15 in response to various DNA damaging agents. The gene product mutated in ataxia telangiectasia, ATM, acts upstream of p53 in a signal transduction pathway initiated by ionizing radiation. Immunoprecipitated ATM had intrinsic protein kinase activity and phosphorylated p53 on serine-15 in a manganese-dependent manner. Ionizing radiation, but not ultraviolet radiation, rapidly enhanced this p53-directed kinase activity of endogenous ATM. These observations, along with the fact that phosphorylation of p53 on serine-15 in response to ionizing radiation is reduced in ataxia telangiectasia cells, suggest that ATM is a protein kinase that phosphorylates p53 in vivo.

Bibliography

Canman, C. E., Lim, D.-S., Cimprich, K. A., Taya, Y., Tamai, K., Sakaguchi, K., Appella, E., Kastan, M. B., & Siliciano, J. D. (1998). Activation of the ATM Kinase by Ionizing Radiation and Phosphorylation of p53. Science, 281(5383), 1677â1679.

Authors 9

Christine E. Canman (first)
Dae-Sik Lim (additional)
Karlene A. Cimprich (additional)
Yoichi Taya (additional)
Katsuyuki Tamai (additional)
Kazuyasu Sakaguchi (additional)
Ettore Appella (additional)
Michael B. Kastan (additional)
Janet D. Siliciano (additional)

References 36 Referenced 1,581

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Morgan S. E., Kastan M. B., Adv. Cancer Res. 71, 1 (1997). (10.1016/S0065-230X(08)60095-0) / Adv. Cancer Res. by Morgan S. E. (1997)
Canman C. E., Wolff A. C., Chen C. Y., Fornace A. J., Kastan M. B., Cancer Res. 54, 5054 (1994); / Cancer Res. by Canman C. E. (1994)
Khanna K. K., Lavin M. F., Oncogene 8, 3307 (1993). / Oncogene by Khanna K. K. (1993)
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10.1101/gad.11.24.3471
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The full-length cDNA encoding NH 2 -terminal FLAG-tagged wild-type ATM [
10.1038/sj.onc.1201319
] was excised from pFB-YZ3 and subcloned into the Xho I site of pcDNA3 (Invitrogen) generating pcDNA-FLAG-ATMwt. To generate catalytically inactive ATM a cDNA fragment encoding the PI-3-kinase–related domain of ATM [
Morgan S. E., et al., Mol. Cell. Biol. 17, 2020 (1997); (10.1128/MCB.17.4.2020) / Mol. Cell. Biol. by Morgan S. E. (1997)
] was mutated by overlap polymerase chain reaction (PCR) substituting Asp 2870 with Ala and Asn 2875 with Lys. A cDNA fragment encoding the kinase domain was excised from wild-type ATM and replaced with a Bpu1101 I–Xho I fragment containing the mutations described above generating pcDNA-FLAG-ATMkd.
We transiently transfected 293T cells with 10 μg of either pcDNA-FLAG-ATMwt pcDNA-FLAG- ATMkd pBJF-FRPwt or pBJF-FRPkd (16) using calcium phosphate and harvested them 2 days later. Cells were lysed through sonication in TGN buffer [50 mM tris (pH 7.5) 50 mM glycerophosphate 150 mM NaCl 10% glycerol 1% Tween 20 1 mM NaF 1 mM NaVO 4 1 mM phenylmethylsulfonyl fluoride 2 μg/mg pepstatin A 5 μg/ml leupeptin 10 μg/ml aprotinin and 1 mM dithiothreitol (DTT)] as described [
10.1126/science.277.5322.99
]. After centrifugation at 13 000 g 2 mg of extract was incubated with mouse immunoglobulin G and protein A/G Sepharose beads (Calbiochem). FLAG-tagged proteins were then immunoprecipitated with anti-FLAG M2 monoclonal antibody (Eastman Kodak) and protein A/G Sepharose beads. Immunoprecipitates were washed twice with TGN buffer once with 100 mM tris (pH 7.5) plus 0.5 M LiCl and twice with kinase buffer [10 mM Hepes (pH 7.5) 50 mM glycerophosphate 50 mM NaCl 10 mM MgCl 2 10 mM MnCl 2 5 μM ATP and 1 mM DTT]. Kinase reactions were initiated by resuspending washed beads in 30 μl of kinase buffer containing 10 μCi [γ- 32 P]ATP and 1 μg GSTp53 1–101 and incubated for 30 min at 30°C. Proteins were electrophoretically separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) transferred to nitrocellulose and analyzed on a PhosphorImager. FLAG-tagged proteins and GSTp53 1–101 were subjected to immunoblotting with either anti-FLAG M2 antibody or monoclonal antibodies directed toward the NH 2 -terminus of p53 (Ab-2 and Ab-6 Calbiochem) as described (8). GSTp53 1–101 fusion protein was made by amplifying cDNA encoding the first 101 amino acids of human p53 by PCR and subcloning the fragment into the Eco RI and Bam HI sites of pGEX-2T (Pharmacia). Serines 6 9 or 15 were substituted with alanine using the QuikChange Site-Directed Mutagenesis kit according to manufacturer's suggestions (Stratagene). GSTp53 1–101 wild-type and mutant proteins were individually expressed in bacteria and purified on glutathione-conjugated agarose beads.
C. E. Canman et al. data not shown.
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Normal (2184) and AT (1526) Epstein-Barr virus immortalized human lymphoblasts were irradiated with a 137 Cs source or treated with 20 μm ALLN (Sigma) for 90 min. Cells were then harvested and lysed and p53 was immunoprecipitated as described (8). Immunoprecipitates were resolved by nonreducing SDS-PAGE transferred to nitrocellulose and immunoblotted with monoclonal antibody to phosphoserine-15. Blots were stripped and reprobed with p53-specific monoclonal antibodies (Ab-2 and Ab-6).
Cells were irradiated with either IR or UV as described (6). Endogenous ATM was immunoprecipitated from 3 mg of lysate with ATM-specific rabbit polyclonal antibody (Ab-3 Calbiochem) and subjected to the in vitro kinase assay (14). The beads and reaction mixtures were separated resolved by SDS-PAGE and transferred onto Immobilon-P (Millipore) for ATM or nitrocellulose for GSTp53 1–101 . Radiolabeled proteins were visualized and quantitated on a PhosphorImager using ImageQuant software (Molecular Dynamics). Membranes were then immunoblotted with antibodies to ATM (Ab-3) or phosphoserine-15.
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; M. H. G. Kubbutat S. N. Jones K. H. Vousden ibid. p. 299.
We thank D. Hill at Oncogene Research Products for the ATM antibodies and Y. Shiloh and Y. Ziv for providing a cDNA for full-length ATM. Supported by NIH grants CA71387 and ES05777 and an American Society of Hematology Scholar Fellowship award (C.E.C.). M.B.K. is the Steven Birnbaum Scholar of The Leukemia Society of America.

Dates

Type	When
Created	23 years ago (July 27, 2002, 5:37 a.m.)
Deposited	1 year, 7 months ago (Jan. 12, 2024, 10:27 p.m.)
Indexed	3 days, 15 hours ago (Aug. 20, 2025, 8:58 a.m.)
Issued	26 years, 11 months ago (Sept. 11, 1998)
Published	26 years, 11 months ago (Sept. 11, 1998)
Published Print	26 years, 11 months ago (Sept. 11, 1998)

Funders 0

None

BibTeX

@article{Canman_1998, title={Activation of the ATM Kinase by Ionizing Radiation and Phosphorylation of p53}, volume={281}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.281.5383.1677}, DOI={10.1126/science.281.5383.1677}, number={5383}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Canman, Christine E. and Lim, Dae-Sik and Cimprich, Karlene A. and Taya, Yoichi and Tamai, Katsuyuki and Sakaguchi, Kazuyasu and Appella, Ettore and Kastan, Michael B. and Siliciano, Janet D.}, year={1998}, month=sep, pages={1677–1679} }

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      "unstructured": "]. After centrifugation at 13 000 g 2 mg of extract was incubated with mouse immunoglobulin G and protein A/G Sepharose beads (Calbiochem). FLAG-tagged proteins were then immunoprecipitated with anti-FLAG M2 monoclonal antibody (Eastman Kodak) and protein A/G Sepharose beads. Immunoprecipitates were washed twice with TGN buffer once with 100 mM tris (pH 7.5) plus 0.5 M LiCl and twice with kinase buffer [10 mM Hepes (pH 7.5) 50 mM glycerophosphate 50 mM NaCl 10 mM MgCl 2 10 mM MnCl 2 5 \u03bcM ATP and 1 mM DTT]. Kinase reactions were initiated by resuspending washed beads in 30 \u03bcl of kinase buffer containing 10 \u03bcCi [\u03b3- 32 P]ATP and 1 \u03bcg GSTp53 1\u2013101 and incubated for 30 min at 30\u00b0C. Proteins were electrophoretically separated by SDS\u2013polyacrylamide gel electrophoresis (SDS-PAGE) transferred to nitrocellulose and analyzed on a PhosphorImager. FLAG-tagged proteins and GSTp53 1\u2013101 were subjected to immunoblotting with either anti-FLAG M2 antibody or monoclonal antibodies directed toward the NH 2 -terminus of p53 (Ab-2 and Ab-6 Calbiochem) as described (8). GSTp53 1\u2013101 fusion protein was made by amplifying cDNA encoding the first 101 amino acids of human p53 by PCR and subcloning the fragment into the Eco RI and Bam HI sites of pGEX-2T (Pharmacia). Serines 6 9 or 15 were substituted with alanine using the QuikChange Site-Directed Mutagenesis kit according to manufacturer's suggestions (Stratagene). GSTp53 1\u2013101 wild-type and mutant proteins were individually expressed in bacteria and purified on glutathione-conjugated agarose beads."
    },
    {
      "key": "e_1_3_1_16_2",
      "unstructured": "C. E. Canman et al. data not shown."
    },
    {
      "key": "e_1_3_1_17_2",
      "doi-asserted-by": "crossref",
      "first-page": "2423",
      "DOI": "10.1101/gad.10.19.2423",
      "volume": "10",
      "author": "Keegan K. S.",
      "year": "1996",
      "unstructured": "Keegan K. S., et al., Genes Dev. 10, 2423 (1996).",
      "journal-title": "Genes Dev."
    },
    {
      "key": "e_1_3_1_18_2",
      "doi-asserted-by": "crossref",
      "first-page": "159",
      "DOI": "10.1093/emboj/17.1.159",
      "volume": "17",
      "author": "Cliby W. A.",
      "year": "1998",
      "unstructured": "Cliby W. A., et al., EMBO J. 17, 159 (1998).",
      "journal-title": "EMBO J."
    },
    {
      "key": "e_1_3_1_19_2",
      "unstructured": "Normal (2184) and AT (1526) Epstein-Barr virus immortalized human lymphoblasts were irradiated with a 137 Cs source or treated with 20 \u03bcm ALLN (Sigma) for 90 min. Cells were then harvested and lysed and p53 was immunoprecipitated as described (8). Immunoprecipitates were resolved by nonreducing SDS-PAGE transferred to nitrocellulose and immunoblotted with monoclonal antibody to phosphoserine-15. Blots were stripped and reprobed with p53-specific monoclonal antibodies (Ab-2 and Ab-6)."
    },
    {
      "key": "e_1_3_1_20_2",
      "unstructured": "Cells were irradiated with either IR or UV as described (6). Endogenous ATM was immunoprecipitated from 3 mg of lysate with ATM-specific rabbit polyclonal antibody (Ab-3 Calbiochem) and subjected to the in vitro kinase assay (14). The beads and reaction mixtures were separated resolved by SDS-PAGE and transferred onto Immobilon-P (Millipore) for ATM or nitrocellulose for GSTp53 1\u2013101 . Radiolabeled proteins were visualized and quantitated on a PhosphorImager using ImageQuant software (Molecular Dynamics). Membranes were then immunoblotted with antibodies to ATM (Ab-3) or phosphoserine-15."
    },
    {
      "key": "e_1_3_1_21_2",
      "doi-asserted-by": "crossref",
      "first-page": "197",
      "DOI": "10.1007/BF00281059",
      "volume": "75",
      "author": "McKinnon P. J.",
      "year": "1987",
      "unstructured": "McKinnon P. J., Hum. Genet. 75, 197 (1987).",
      "journal-title": "Hum. Genet."
    },
    {
      "key": "e_1_3_1_22_2",
      "first-page": "3242",
      "volume": "55",
      "author": "Shafman T. D.",
      "year": "1995",
      "unstructured": "Shafman T. D., et al., Cancer Res. 55, 3242 (1995).",
      "journal-title": "Cancer Res."
    },
    {
      "key": "e_1_3_1_23_2",
      "first-page": "1815",
      "volume": "14",
      "author": "Nelson W. G.",
      "year": "1994",
      "unstructured": "Nelson W. G., Kastan M. B., Mol. Cell Biol. 14, 1815 (1994).",
      "journal-title": "Mol. Cell Biol."
    },
    {
      "key": "e_1_3_1_24_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1101/gad.10.19.2401"
    },
    {
      "key": "e_1_3_1_24_3",
      "doi-asserted-by": "crossref",
      "first-page": "453",
      "DOI": "10.1038/ng1297-453",
      "volume": "17",
      "author": "Barlow C.",
      "year": "1997",
      "unstructured": "Barlow C., Brown K. D., Deng C.-X., Tagle D. A., Wynshaw-Boris A., Nature Genet. 17, 453 (1997).",
      "journal-title": "Nature Genet."
    },
    {
      "key": "e_1_3_1_25_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/387296a0"
    },
    {
      "key": "e_1_3_1_25_3",
      "unstructured": "; M. H. G. Kubbutat S. N. Jones K. H. Vousden ibid. p. 299."
    },
    {
      "key": "e_1_3_1_26_2",
      "unstructured": "We thank D. Hill at Oncogene Research Products for the ATM antibodies and Y. Shiloh and Y. Ziv for providing a cDNA for full-length ATM. Supported by NIH grants CA71387 and ES05777 and an American Society of Hematology Scholar Fellowship award (C.E.C.). M.B.K. is the Steven Birnbaum Scholar of The Leukemia Society of America."
    }
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