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Sensitive fluorescent biosensor reveals differential subcellular regulation of PKC
Qi Su1, Jing Zhang1,2, Wei Lin1
1Department of Pharmacology, University of California San Diego, La Jolla, CA, USA.
Nature Chemical Biology
|October 11, 2024
Summary
A new reporter tool, ExRai-CKAR2, precisely measures protein kinase C (PKC) activity at specific cell locations. This reveals how different PKC types are activated by cell signals, offering insights into cellular regulation.
Area of Science:
- Cellular Biology
- Biochemistry
- Molecular Signaling
Background:
- Protein Kinase C (PKC) enzymes are crucial for diverse cellular processes.
- Understanding PKC regulation at specific subcellular locations remains a challenge.
Purpose of the Study:
- To develop a sensitive reporter for detecting subcellular PKC activity.
- To investigate the spatial and temporal regulation of PKC isoforms during cellular signaling.
Main Methods:
- Development of a sensitive excitation ratiometric C kinase activity reporter (ExRai-CKAR2).
- Integration of ExRai-CKAR2 with a diacylglycerol (DAG) biosensor.
- Application of the reporter in cellular models and 3D organoids.
Main Results:
- ExRai-CKAR2 detects minute changes in subcellular PKC activity.
- G-protein-coupled receptor stimulation leads to sustained PKC activity at ER and lysosomes.
- Differential regulation of conventional and novel PKCs by Ca2+ and DAG, respectively.
- Detection of previously inaccessible atypical PKC activity in organoids.
Conclusions:
- ExRai-CKAR2 is a highly sensitive tool for studying PKC activity.
- Reveals distinct subcellular localization and activation mechanisms of PKC isoforms.
- Enables interrogation of PKC regulation in complex biological systems like organoids.
