ATM-SPARK: A GFP phase separation-based activity reporter of ATM
The kinase ataxia telangiectasia mutated (ATM) plays a crucial role in the DNA damage response (DDR), making it vital to observe the spatiotemporal dynamics of ATM activity during DDR. In this study, we developed a robust ATM activity reporter, ATM-SPARK (Separation of Phases-Based Activity Reporter of Kinase), which is based on phosphorylation-inducible green fluorescent protein phase separation. Upon ATM activation, the reporter undergoes phase separation through multivalent interactions, forming highly visible droplets. ATM-SPARK allows real-time visualization of endogenous ATM activity in living cells, with its signal correlating directly to the extent of DNA damage. Additionally, ATM-SPARK enables high-throughput screening of both biological and small-molecule regulators. Through screening, we identified protein phosphatase 4, which inhibits ATM activity, and BGT226, a potent ATM inhibitor with a median inhibitory concentration of approximately 3.8 nanomolar. Notably, BGT226 also sensitizes cancer cells to the radiomimetic drug neocarzinostatin, suggesting its potential use in combination with radiotherapy. ATM-SPARK offers a large dynamic range, bright fluorescence, and clear signal patterns, making it a powerful tool for studying ATM activity.