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Substrate specificities and identification of putative substrates of ATM kinase family members
1Department of Hematology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
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 Mn(2+), but not DNA ends or Ku proteins, for optimal in vitro activity while DNA-PKCs requires Mg(2+), 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 NH(2)-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 glutathione S-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.
Insights
This study identifies key phosphorylation patterns for DNA repair kinases ATM, ATR, and DNA-PK. It reveals a consensus sequence (S/TQ) essential for their activity, uncovering new potential substrates for ATM.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Ataxia telangiectasia mutated (ATM) is crucial for DNA damage response, phosphorylating p53 after ionizing radiation.
- The complex phenotype of AT cells suggests ATM has additional cellular substrates beyond p53.
- Identifying these substrates is vital for understanding DNA repair pathways.
Purpose of the Study:
- To identify novel substrates for ATM and related kinases ATR and DNA-PK.
- To determine the general phosphorylation consensus sequences for these kinases.
- To characterize the in vitro activity requirements for ATM, ATR, and DNA-PK.
Main Methods:
- Optimized in vitro kinase assays for ATM, ATR, and DNA-PK.
- Developed a rapid peptide screening method to define phosphorylation consensus sequences.
- Utilized p53 peptide mutagenesis and glutathione S-transferase (GST) peptides as substrates.
Main Results:
- ATM and ATR require Mn(2+), while DNA-PK requires Mg(2+), DNA ends, and Ku proteins for optimal activity.
- A minimal essential phosphorylation sequence S/TQ was identified for all three kinases.
- Hydrophobic and negatively charged amino acids enhance phosphorylation, while positively charged amino acids hinder it.
- Putative ATM substrates include p95/nibrin, Mre11, Brca1, Rad17, WRN, and ATM itself.
- ATR specifically phosphorylates Brca2, PI3K, and DNA-5B; DNA Ligase IV is a DNA-PK substrate.
Conclusions:
- The study elucidates the substrate specificity and requirements of ATM, ATR, and DNA-PK.
- A conserved phosphorylation motif (S/TQ) and flanking amino acid preferences were determined.
- Several novel putative substrates for ATM were identified, expanding the known DNA damage response network.