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. We have reported that ataxia-telansiectasia mutated (ATM) was modified with O-GlcNAc, and that the elevation of O-GlcNAc modifications suppressed the activation of ATM due to X-irradiation. In the present study, in order to clarify the mechanisms of the regulation of O-GlcNAc modification in the activation of AT, we examined the effects of O-GlcNAc modifications on the interaction of ATM with various signaling molecules and O-GlcNAc related proteins.
Hela cells were exposed to X-irradiation at the dose of 5 Gy. To elevate the levels of O-GlcNAc modified proteins in the cells, we added 100µM PUGNAc (O-(2-acetamido-2-deoxy-D- glucopyranosylidene)amino N-phenyl carbamate) to the cell cultures. After irradiation, nuclear fractions were obtained by cell fractionations and immunoprecipitated using anti-ATM antibody. Protein interactions of ATM with other signaling molecules and O-GlcNAc related proteins were analyzed by western blots.
From the results of immunoprecipitations, ATM interacted with O-GlcNAc transferase (OGT), which adds O-GlcNAc to proteins, but not O-GlcNAcase (OGA), which removes O-GlcNAc from proteins. Furthermore, it was suggested that Rad50, Mre11, Tip60, and PP2A interacted with ATM and that the interactions was affected by the addition of PUGNAc.
