Substrate Specificities and Identification of Putative Substrates of ATM Kinase Family Members

doi:10.1074/jbc.274.53.37538

Substrate Specificities and Identification of Putative Substrates of ATM Kinase Family Members*

From the Department of Hematology-Oncology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105

Abstract

Ataxia telangiectasia mutated (ATM) phosphorylates p53 protein in response to ionizing radiation, but the complex phenotype of AT cells suggests that it must have other cellular substrates as well. To identify substrates for ATM and the related kinases ATR and DNA-PK, we optimized in vitro kinase assays and developed a rapid peptide screening method to determine general phosphorylation consensus sequences. ATM and ATR require Mn²⁺, but not DNA ends or Ku proteins, for optimal in vitro activity while DNA-PKCs requires Mg²⁺, DNA ends, and Ku proteins. From p53 peptide mutagenesis analysis, we found that the sequence S/TQ is a minimal essential requirement for all three kinases. In addition, hydrophobic amino acids and negatively charged amino acids immediately NH₂-terminal to serine or threonine are positive determinants and positively charged amino acids in the region are negative determinants for substrate phosphorylation. We determined a general phosphorylation consensus sequence for ATM and identified putative in vitro targets by using glutathioneS-transferase peptides as substrates. Putative ATM in vitro targets include p95/nibrin, Mre11, Brca1, Rad17, PTS, WRN, and ATM (S440) itself. Brca2, phosphatidylinositol 3-kinase, and DNA-5B peptides were phosphorylated specifically by ATR, and DNA Ligase IV is a specific in vitro substrate of DNA-PK.

Footnotes

↵* This work was supported in part by National Institutes Health Grants CA71387, ES0577, and CA21765, Glaxo-Wellcome, and the American Lebanese Syrian Associated Charities (ALSAC) of the St. Jude Children's Research Hospital.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ Contributed equally to the results of this study.
↵§ Steven Birnbaum Scholar of the Leukemia Society of America. To whom correspondence should be addressed: Dept. of Hematology-Oncology, St. Jude Children's Research Hospital, 332 N. Lauderdale St., Memphis, TN 38105. Tel.: 901-495-3968; Fax: 901-495-3966; E-mail: Michael.Kastan@stjude.org.
↵2 D-S. Lim, Kim, S. T., Xu, B., Maser, R. S., Lin, J., Petrini, J. H. J., and Kastan, M. B., submitted for publication.
Abbreviations:

AT

ataxia telangiectasia

IR

ionizing irradiation

GST

glutathioneS-transferase

PTS

putative tumor suppressor

PKcs

PK catalytic subunit
- Received September 17, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.