ATM-SPARK: A GFP phase separation-based activity reporter of ATM

The kinase ataxia telangiectasia mutated (ATM) plays a key role in the DNA damage response (DDR). It is thus essential to visualize spatiotemporal dynamics of ATM activity during DDR. Here, we designed a robust ATM activity reporter based on phosphorylation-inducible green fluorescent protein phase separation, dubbed ATM-SPARK (separation of phases-based activity reporter of kinase). Upon ATM activation, it undergoes phase separation via multivalent interactions, forming intensely bright droplets. The reporter visualizes spatiotemporal dynamics of endogenous ATM activity in living cells, and its signal is proportional to the amount of DNA damage. ATM-SPARK also enables high-throughput screening of biological and small-molecule regulators. We identified the protein phosphatase 4 that blocks ATM activity. We also identified BGT226 as a potent ATM inhibitor with a median inhibitory concentration of similar to 3.8 nanomolars. Furthermore, BGT226 sensitizes cancer cells to the radiomimetic drug neocarzinostatin, suggesting that BGT226 might be combined with radiotherapeutic treatment. ATM-SPARK achieves large dynamic range, bright fluorescence, and simple signal pattern.

Automated summary

In this study, a robust ATM activity reporter called ATM-SPARK was designed based on phosphorylation-inducible green fluorescent protein phase separation. The reporter allows visualization of endogenous ATM activity and DNA damage in living cells, and can be used for high-throughput screening. The study identified protein phosphatase 4 as an ATM activity blocker and the compound BGT226 as a potent ATM inhibitor.