Analysis of proteins phosphorylated at the early response of DNA double-strand breaks

The serine/threonine protein kinase, ATM, recognizes DNA double-strand breaks (DSBs) at a very early stage, and plays a central role in transmitting damage signals to the cell cycle control machinery. To further understand the early events in DSBs recognition, we are undertaking two approaches: 1) identification of novel substrates which are phosphorylated by ATM using phospho-specific antibody, and 2) analysis of phosphorylation profile employing the proteomics-based 2-DE gel electrophoresis technique. There is a well-known consensus sequence for phosphorylation by ATM characterized by serine followed by glutamine (SQ), and their surrounding amino acids. Using one of the consensus sequences, we synthesized a phosphorylated peptide for immunizing rabbit and generated a phospho-specific antibody. This novel antibody recognized phosphorylated proteins in mouse fibroblast cultures after 5 min of exposure to ionizing radiation. This high and constant level of phosphorylation persisted until the 2nd hour and started decreasing to basal levels from the fourth hour. This antibody also detected phospho-proteins in mouse fibroblast cultures exposed to chemical reagents that induce DSBs such as NCS, bleomycin and etoposide, and is thought to be specific for DNA DSBs-related proteins. Furthermore, this antibody did not recognize phospho-proteins in ATM-deficient cells, suggesting that this phosphorylation is dependent on ATM kinase or downstream kinases controlled by ATM. As a second approach, we identified several spots that are phosphorylated in ATM wild type cells but not in ATM-deficient cells by 2-DE proteomic analysis. We are conducting further studies to generate comprehensive, ATM-dependent phosphorylation profiles as a novel approach to understand early events in DNA damage recognition by ATM