Assessing the role of Ku70 vWA domain phosphorylation in the inhibition of Aurora B and activation of the DNA damage response

Ku is a key component of the Non-Homologous End Joining DNA repair pathway. Recently, a function for Ku in DNA damage response (DDR) signalling was identified through studies exploring a Ku70 S155D phosphomimetic mutant. We hypothesize that Ku70 S155D mimics phosphorylation of Ku70 in response to DNA damage, and that Ku S155 phosphorylation inhibits Aurora B leading to sustained DDR activation. In this study we show that the S155D mutant is competent for heterodimerization, and its expression does not induce DNA damage. Phosphorylated Ku70 associates with Aurora B by co-immunoprecipitation and this association was demonstrated in situ with Ku70 S155D. Additionally, we demonstrate that the Ku70 S155D vWA domain is sufficient to inhibit Aurora B in an in vitro kinase assay. Finally, Aurora B inhibitor treatment of Ku70 S155D cells does not increase the prevalence of a DDR marker γH2AX. This work suggests that Ku70 S155 phosphorylation inhibits Aurora B which in turn leads to DDR activation.