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Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET
Published on: January 27, 2012
Monitoring ATM kinase activity in living cells
Sam A Johnson1, Zhongsheng You, Tony Hunter
1Molecular and Cell Biology Laboratory, Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.
Abstract:
DNA double strand breaks (DSB) in mammalian cells result in the activation of the ATM protein kinase. This leads to phosphorylation of numerous downstream transducer and effector proteins that coordinate a cellular response including DNA repair, cell cycle arrest or apoptosis. We have developed a reporter protein that allows the measurement of ATM kinase activity in single living cells. This CFP-YFP FRET-based biosensor uses an ATM phosphorylation site and an FHA phosphospecific binding domain to produce a phosphorylation-induced change in conformation, which alters the FRET efficiency between CFP and YFP. We show that the reporter provides a measurable output in response to DSBs and is specific for ATM over ATR or DNA-PK. We expect the description of the spatiotemporal dynamics of ATM activity in living cells that this reporter provides will be helpful in providing a more detailed understanding of the DNA damage response.
Insights
Scientists developed a novel biosensor to measure ATM kinase activity in living cells following DNA damage. This tool tracks the DNA damage response, offering insights into cellular repair and survival mechanisms.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- DNA double-strand breaks (DSB) activate ATM protein kinase in mammalian cells.
- ATM activation initiates signaling cascades for DNA repair, cell cycle arrest, or apoptosis.
- Understanding ATM kinase dynamics is crucial for comprehending the DNA damage response.
Purpose of the Study:
- To develop a novel biosensor for measuring ATM kinase activity in real-time within single living cells.
- To investigate the spatiotemporal dynamics of ATM activation in response to DNA damage.
- To provide a tool for deeper insights into the DNA damage response pathways.
Main Methods:
- Development of a Förster Resonance Energy Transfer (FRET)-based biosensor using CFP and YFP fluorescent proteins.
- The biosensor incorporates an ATM phosphorylation site and an FHA phosphospecific binding domain.
- Measurement of FRET efficiency changes indicative of ATM kinase activity upon phosphorylation.
Main Results:
- The developed biosensor successfully measures ATM kinase activity in response to DSBs in living cells.
- The biosensor demonstrates specificity for ATM kinase, distinguishing it from ATR or DNA-PK.
- The reporter enables visualization of the spatiotemporal dynamics of ATM activity.
Conclusions:
- The novel FRET-based biosensor is a valuable tool for quantifying ATM kinase activity in living cells.
- This reporter facilitates a more detailed understanding of the DNA damage response and ATM signaling.
- The findings pave the way for further research into cellular responses to DNA damage.
