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Updated: Jun 25, 2025
Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques
Published on: November 2, 2018
A fluorescence-based sensor for calibrated measurement of protein kinase stability in live cells
Joseph W Paul1,2, Serena Muratcioğlu3, John Kuriyan3,4
1Department of Molecular and Cell Biology, University of California, Berkeley, California, USA.
Abstract:
Oncogenic mutations can destabilize signaling proteins, resulting in increased or unregulated activity. Thus, there is considerable interest in mapping the relationship between mutations and the stability of signaling proteins, to better understand the consequences of oncogenic mutations and potentially inform the development of new therapeutics. Here, we develop a tool to study protein-kinase stability in live mammalian cells and the effects of the HSP90 chaperone system on the stability of these kinases. We determine the expression levels of protein kinases by monitoring the fluorescence of fluorescent proteins fused to those kinases, normalized to that of co-expressed reference fluorescent proteins. We used this tool to study the dependence of Src- and Raf-family kinases on the HSP90 system. We demonstrate that this sensor reports on destabilization induced by oncogenic mutations in these kinases. We also show that Src-homology 2 and Src-homology 3 domains, which are required for autoinhibition of Src-family kinases, stabilize these kinase domains in the cell. Our expression-calibrated sensor enables the facile characterization of the effects of mutations and small-molecule drugs on protein-kinase stability.
Insights
Scientists developed a new tool to measure protein-kinase stability in cells. This sensor reveals how oncogenic mutations and the HSP90 chaperone system impact kinase stability, aiding drug development.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- Oncogenic mutations can alter signaling protein stability, leading to uncontrolled activity.
- Understanding mutation-induced protein instability is crucial for cancer research and therapeutic development.
- The HSP90 chaperone system plays a key role in maintaining protein homeostasis and client protein stability.
Purpose of the Study:
- To develop and validate a novel tool for studying protein-kinase stability in live mammalian cells.
- To investigate the impact of the HSP90 chaperone system on the stability of Src- and Raf-family kinases.
- To assess how oncogenic mutations affect kinase stability and the role of specific protein domains.
Main Methods:
- Developed a fluorescence-based sensor to monitor protein-kinase expression levels in live cells.
- Normalized kinase fluorescence to co-expressed reference fluorescent proteins for accurate expression calibration.
- Utilized the sensor to study the HSP90 dependence of Src- and Raf-family kinases and the effect of oncogenic mutations.
Main Results:
- The developed sensor accurately reports on protein-kinase expression and stability.
- Demonstrated that oncogenic mutations destabilize Src- and Raf-family kinases.
- Showed that Src-homology 2 and Src-homology 3 domains stabilize Src-family kinase domains.
Conclusions:
- The expression-calibrated sensor provides a facile method for characterizing protein-kinase stability.
- This tool can reveal the effects of mutations and small-molecule drugs on kinase stability.
- Findings contribute to understanding oncogenic mutations and developing targeted therapeutics.
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