Substrate specificities and identification of putative substrates of ATM kinase family members

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.