The functional significance of ATM phosphorylation of Bub3 on Serine 135 in sensitivity to DNA damaging agents.

Abstract

e15048 Background: Identification of biomarkers to assess and modify the sensitivity of cancer cells to radiotherapy and chemotherapy is critical to improve cancer treatment outcome. Budding uninhibited by benzimidazoles 3 (Bub3) is a mitotic checkpoint protein, and it is frequently overexpressed in many cancers and associated with low survival rates. Bub3 is involved in the repair of DNA damage induced by radiotherapy and chemotherapy. We recently identified that the ATM kinase phosphorylated Bub3 on Serine 135 (S135) via the stable isotope labeled amino acid in cell culture -mass spectrometry analysis. This study aims to explore the mechanism of ATM-phosphorylated Bub3 in the DNA damage response (DDR) and its effect on tumor sensitivity to DNA damaging agents. Methods: The radiosensitivity of the cells was detected by clonal formation assay, the proliferation ability of the cells was detected by the MTS assay. The expression of DDR protein γ-H2AX in the nucleus was detected by immunofluorescence assay. Genomic instability was observed by multinuclear formation. Co-immunoprecipitation and Western Blot were used to explore the internal mechanism of ATM phosphorylation of Bub3 in DDR. Results: We showed that ionizing radiation (IR) could induce Bub3 S/TQ (the specific ATM consensus motif) phosphorylation in an ATM dependent manner. Mutation of Bub3 Serine 135 to alanine (S135A) led to a phosphorylation defect. Phenotypic experiments showed hypersensitivity to IR in cells expressing Bub3 S135A. Bub3 S135A prolonged existence of γ-H2AX foci and increased the proportion of cells containing micronuclei. Further, we found that Ku70 and Ku80 showed a significant increase after IR in their interactions with Bub3, while Bub3 S135A mutation significantly reduced the interaction, leading to impaired DNA repair. Conclusions: We demonstrate that Bub3 S135 phosphorylation mediated by ATM is essential for an optimal DDR and disruption of this pathway increases tumor sensitivity to DNA damaging agents.