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Updated: Jul 8, 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, CA, 94720 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 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 monitor the fluorescence of kinases fused to a fluorescent protein relative to that of a co-expressed reference fluorescent protein. 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 (SH2) and Src-homology 3 (SH3) 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
We developed a tool to measure protein-kinase stability in cells, revealing how oncogenic mutations and HSP90 affect kinase function. This sensor aids in understanding cancer mutations and drug development.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Oncogenic mutations can disrupt signaling protein stability, leading to uncontrolled activity and disease.
- Understanding the link between mutations, protein stability, and cellular signaling is crucial for cancer research and therapeutic development.
- The HSP90 chaperone system plays a vital role in maintaining the stability of many client proteins, including kinases.
Approach:
- Developed a novel live-cell fluorescence-based sensor to monitor protein-kinase stability.
- Utilized a co-expression system comparing kinase-fluorescent protein fusions to a reference fluorescent protein.
- Applied the sensor to investigate the stability of Src- and Raf-family kinases and their dependence on HSP90.
Key Points:
- The developed sensor accurately reports on protein-kinase destabilization caused by oncogenic mutations.
- Demonstrated the role of the HSP90 chaperone system in maintaining the stability of Src- and Raf-family kinases.
- Identified that Src-homology 2 (SH2) and Src-homology 3 (SH3) domains stabilize Src-family kinases by promoting autoinhibition.
Conclusions:
- The expression-calibrated sensor provides a facile method to characterize the impact of mutations on protein-kinase stability.
- This tool facilitates the study of oncogenic mutations and the screening of small-molecule drugs targeting protein stability.
- Findings contribute to a deeper understanding of kinase regulation and potential therapeutic strategies in oncology.
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