Purification and Characterization of ATM from Human Placenta: A MANGANESE-DEPENDENT, WORTMANNIN-SENSITIVE SERINE/THREONINE PROTEIN KINASE

Doug W Chan; Seong-Cheol Son; Wesley Block; Ruiqiong Ye; Kum Kum Khanna; Marc S Wold; Pauline Douglas; Aaron A Goodarzi; Jennifer Pelley; Yoichi Taya; Martin F Lavin; Susan P Lees-Miller

doi:10.1074/jbc.275.11.7803

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Purification and Characterization of ATM from Human Placenta: A MANGANESE-DEPENDENT, WORTMANNIN-SENSITIVE SERINE/THREONINE PROTEIN KINASE

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Purification and Characterization of ATM from Human Placenta: A MANGANESE-DEPENDENT, WORTMANNIN-SENSITIVE SERINE/THREONINE PROTEIN KINASE

Doug W Chan, Seong-Cheol Son, Wesley Block, Ruiqiong Ye, Kum Kum Khanna, Marc S Wold, Pauline Douglas, Aaron A Goodarzi, Jennifer Pelley, Yoichi Taya, …

The Journal of biological chemistry, Vol.275(11), pp.7803-7810

03/17/2000

DOI: 10.1074/jbc.275.11.7803

PMID: 10713094

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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 IC(50) 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.

Details

Title: Subtitle: Purification and Characterization of ATM from Human Placenta: A MANGANESE-DEPENDENT, WORTMANNIN-SENSITIVE SERINE/THREONINE PROTEIN KINASE
Creators: Doug W Chan
Seong-Cheol Son
Wesley Block
Ruiqiong Ye
Kum Kum Khanna
Marc S Wold
Pauline Douglas
Aaron A Goodarzi
Jennifer Pelley
Yoichi Taya
Martin F Lavin
Susan P Lees-Miller
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.275(11), pp.7803-7810
DOI: 10.1074/jbc.275.11.7803
PMID: 10713094
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Language: English
Date published: 03/17/2000
Academic Unit: Radiation Oncology; Biochemistry and Molecular Biology
Record Identifier: 9984024516302771

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