Abstract
Various nucleocytoplasmic proteins are modified with O-linked N-acetylglucosamine (O-GlcNac). It is known that O-GlcNac modification is involved in protein degradation, localization, protein-protein interaction, and regulation of protein phosphorylation. Ataxia-telansiectasia mutated (ATM) is activated by double strand breaks (DSB), and phosphorylates various signaling molecules involved in checkpoint regulation, apoptosis, and DNA repair. However, it is unclear whether ATM is modified with O-GlcNAc and how O-GlcNAcylation regulates the functions of ATM. We have reported that ATM derived from primary cultured neurons in mice was modified with O-GlcNAc. Therefore, in the present study we investigated the regulation of O-GlcNAc modification in the function of ATM.
Primary cultured neurons and Hela cells were exposed to X-irradiation at the dose of 5 Gy. After irradiation, whole cell lysates were immunoprecipitated using anti-ATM and anti-O-GlcNAc antibody, followed by analyses of O-GlcNAcylation of ATM and protein interactions of ATM with other signaling molecules.
From the results of immunoprecipitation with anti-ATM or anti- O-GlcNAc antibody, ATM derived from Hela cells was also modified with O-GlcNAc. After 5 Gy-irradiation, phosphorylated ATM (Ser 1981) (p-ATM) was increased significantly, while p-ATM was not detected in the immunoprecipitated samples with anti-O-GlcNAc antibody. It was suggested that O-GlcNAc modification was decreased in p-ATM.
