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Fluorescent protein biosensor for probing CDK/cyclin activity in vitro and in living cells
Thi Nhu Ngoc Van1, Morgan Pellerano, Sophie Lykaso
1Interdisciplinary Institute for NeuroScience-UMR 5297, 146 rue Léo-Saignat, 33077 Bordeaux (France).
Abstract:
Cyclin-dependent kinases (CDKs) play an essential role in the coordination of cell cycle progression and transcriptional regulation; hyperactivation is associated with cancer. However there are few means of measuring their activity in a physiological context or their inhibition in response to therapeutics. To this aim we engineered a modular fluorescent protein biosensor that reports on phosphorylation by CDK/cyclins through real-time changes in fluorescence intensity. This allowed a comparison of enzymatic activity of recombinant kinases, monitoring inhibition by small molecules, and probing endogenous activities in lysates from healthy and cancer cell lines in a sensitive and quantitative fashion. This versatile tool was further implemented to probe the oscillatory activity of these kinases throughout the cell cycle by time-lapse imaging and ratiometric fluorescence quantification, following delivery of a red fluorescent protein fusion mediated by cell-penetrating peptides.
Insights
Researchers developed a novel fluorescent biosensor to measure cyclin-dependent kinase (CDK) activity in real-time. This tool enables sensitive monitoring of CDK inhibition and activity in both healthy and cancerous cells.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Cyclin-dependent kinases (CDKs) are crucial regulators of cell cycle and transcription.
- Aberrant CDK activity is linked to cancer development.
- Limited methods exist for measuring CDK activity in physiological settings.
Purpose of the Study:
- To engineer a versatile fluorescent biosensor for real-time monitoring of CDK activity.
- To enable sensitive quantification of CDK inhibition by therapeutic agents.
- To probe endogenous CDK activity in various cell types.
Main Methods:
- Development of a modular fluorescent protein biosensor.
- Real-time fluorescence intensity measurements.
- Enzymatic activity assays and small molecule inhibition studies.
- Cell-based assays using lysates from healthy and cancer cell lines.
- Time-lapse imaging and ratiometric fluorescence quantification for cell cycle analysis.
Main Results:
- The biosensor accurately reports on CDK/cyclin phosphorylation through fluorescence changes.
- It allows sensitive and quantitative comparison of recombinant kinase activity.
- The tool effectively monitors inhibition of CDKs by small molecules.
- Endogenous CDK activity in healthy and cancer cell lines was probed.
- Oscillatory CDK activity throughout the cell cycle was visualized.
Conclusions:
- The developed fluorescent biosensor provides a sensitive and quantitative tool for measuring CDK activity.
- This versatile tool facilitates the study of CDK function in various biological contexts, including cancer.
- It enables real-time monitoring of therapeutic inhibition and cell cycle-dependent kinase dynamics.
