Identification of DNA damage-dependent checkpoint kinase 1 (Chk1) posttranslational modifications and associated proteins

801 DNA damage checkpoints are regulatory signal transduction pathways that allow cells to control DNA damage-induced cell cycle arrest, DNA repair, and cell death by apoptosis. Most human tumors arise from multiple genetic changes, including defective DNA damage checkpoints that provide potential targets for therapeutic use of DNA-damaging agents. One strategy to enhance the impact of these agents is to inhibit the checkpoint signaling in cancer cells. The checkpoint kinase 1 (Chk1) is one of the effector kinases that regulates cell cycle progression after DNA damage. Upon DNA damage, Chk1 is phosphorylated on Ser 345 and 317, and this phosphorylation is dependent on ATM (ataxia-telangiectasia mutated) or ATR (ATM and Rad 3-related). In addition to phosphorylation, DNA damage-dependent Chk1 activity could be modulated by other posttranslational modifications and protein-protein interaction. We have developed experimental procedures for identification of novel Chk1 posttranslational modifications and associated proteins. Using immunoprecipitation followed by mass spectroscopy analysis, we have found novel candidate Chk1-associated proteins. Potentially, interaction of these proteins with Chk1 could inhibit DNA damage-dependent Chk1 activation, leading to abrogation of checkpoint response and sensitization of cancer cells to DNA-damaging drugs.