A Protein Phosphatase 2a-Atm Complex Regulates The Dna Damage Response And Pro-Survival Signaling

Background: Ataxia telangiectasia mutated (ATM), a S/T protein kinase, is a major regulator of the DNA damage response (DDR) and is activated by DNA double-strand breaks (DSB) caused by ionizing radiation (IR) and radiomimetic drugs. ATM controls cell cycle checkpoints and DSB repair leading to cell cycle arrest and recovery, or apoptosis. ATM also regulates pro-survival AKT signaling. We and other groups have previously reported that an ATM-dependent phosphatase negatively controls AKT phosphorylation in response to insulin and IR. Many DDR proteins, phosphorylated after DNA damage are subsequently dephosphorylated by phosphatases such as protein phosphatase 2A (PP2A) to reverse the DDR and reset the cell to normalcy. In addition to dephophorylating AKT, PP2A controls the G2/M checkpoint and dephosphorylates γ-H2AX after IR. Recently, it was reported that ATM negatively regulates PP2A activity by phosphorylating its scaffolding subunit Aα. Objective: We previously reported that a small molecule inhibitor of the ATM kinase results in AKT dephosphorylation. The present study was aimed at determining how ATM and PP2A are co-regulated during the DDR and pro-survival signaling. We hypothesized that ATM phosphorylates PP2A-Aα thereby inhibiting phosphatase activity to increase AKT signaling. Results: We generated specific PP2A-Aα mutations at S401; a S401A mutant which cannot be phosphorylated and a phospho-mimetic S401D mutant. Co-immunoprecipitation studies in HEK293 cells over-expressing PP2A-Aα WT, S401A or S401D showed that ATM, AKT and the PP2A catalytic subunit (PP2A-C) bind to the WT and S401A mutant but not the S401D mutant. We then generated PP2A-Aα conditional knockout mouse embryonic fibroblasts (MEFs) expressing PP2A-Aα-WT, S401A or S401D transgenes by floxing out the endogenous PP2A-Aα alleles with Cre. Treatment with insulin increased pAKT (S473) levels in cells expressing the S401D mutant relative to WT and S401A. Furthermore, H2AX phosphorylation after low dose of IR showed prolonged phosphorylation whereas AKT showed decreased phosphorylation, suggesting that there was a delay in re-establishing the pre-IR, non-phosphorylated state. Additionally, the S401D mutant was retained in the cytoplasm after IR and was less efficient in DSB repair. S401D mutant cells were also more radiosensitive relative to the WT and S401A. Conclusion: Taken together, our findings suggest that ATM-mediated phosphorylation of PP2A-Aα at S401 modulates the DDR and AKT pro-survival signaling and perhaps other proteins modulating DSB repair. These studies are very important as they will help elucidate the intimate regulatory relationship between PP2A and ATM and establish a more detailed mechanisms how a single, critical amino acid (S401) in PP2A-Aα affects the DDR, DSB repair and survival. Citation Format: Amrita D. Sule, Mary Tokarz, Mostafa Ahmed, Glen E. Kellogg, Kristoffer C. Valerie. A protein phosphatase 2A-ATM complex regulates the DNA damage response and pro-survival signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3325. doi:10.1158/1538-7445.AM2015-3325