Regulation of the DNA Damage Response by DNA-PKcs Inhibitory Phosphorylation of ATM

Ataxia-telangiectasia mutated (ATM) regulates the DNA damage response as well as DNA double-strand break repair through homologous recombination. Here we show that ATM is hyperactive when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is chemically inhibited or when the DNA-PKcs gene is deleted in human cells. Pre-incubation of ATM protein with active DNA-PKcs also significantly reduces ATM activity in vitro. We characterize several phosphorylation sites in ATM that are targets of DNA-PKcs and show that phospho-mimetic mutations at these residues significantly inhibit ATM activity and impair ATM signaling upon DNA damage. In contrast, phospho-blocking mutations at one cluster of sites increase the frequency of apoptosis during normal cell growth. DNA-PKcs, which is integral to the non-homologous end joining pathway, thus negatively regulates ATM activity through phosphorylation of ATM. These observations illuminate an important regulatory mechanism for ATM that also controls DNA repair pathway choice. [Display omitted] •DNA-PKcs inhibits ATM activity through phosphorylation at multiple sites•DNA-PKcs phosphorylation of ATM impairs ATM signaling upon DNA damage•DNA-PKcs phosphorylation of ATM does not affect ATM function upon oxidative stress•Loss of DNA-PKcs regulation of ATM affects normal cell growth Zhou et al. show that DNA-PKcs phosphorylates ATM at multiple sites in response to DNA damage, resulting in inhibition of ATM activity. DNA-PKcs phosphorylation of ATM regulates its activity in DNA repair and cell-cycle checkpoint activation, and also reduces apoptosis during normal cell growth.