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Updated: Jun 30, 2025
Author Spotlight: Integrating BRET-Based Assays and Rare Mutation Analysis to Decipher RAF Kinase Regulation in Live Cells
Published on: March 1, 2024
Bioluminescence Resonance Energy Transfer (BRET)-based Assay for Measuring Interactions of CRAF with 14-3-3 Proteins
1Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina; Hollings Cancer Center, Medical University of South Carolina; Laboratory of Cell and Developmental Signaling, Center for Cancer Research, National Cancer Institute-Frederick; spenceru@musc.edu.
Abstract:
CRAF is a primary effector of RAS GTPases and plays a critical role in the tumorigenesis of several KRAS-driven cancers. In addition, CRAF is a hotspot for germline mutations, which are shown to cause the developmental RASopathy, Noonan syndrome. All RAF kinases contain multiple phosphorylation-dependent binding sites for 14-3-3 regulatory proteins. The differential binding of 14-3-3 to these sites plays essential roles in the formation of active RAF dimers at the plasma membrane under signaling conditions and in maintaining RAF autoinhibition under quiescent conditions. Understanding how these interactions are regulated and how they can be modulated is critical for identifying new therapeutic approaches that target RAF function. Here, I describe a bioluminescence resonance energy transfer (BRET)-based assay for measuring the interactions of CRAF with 14-3-3 proteins in live cells. Specifically, this assay measures the interactions of CRAF fused to a Nano luciferase donor and 14-3-3 fused to a Halo tag acceptor, where the interaction of RAF and 14-3-3 results in donor-to-acceptor energy transfer and the generation of the BRET signal. The protocol further shows that this signal can be disrupted by mutations shown to prevent 14-3-3 binding to each of its high-affinity RAF docking sites. This protocol describes the procedures for seeding, transfecting, and replating the cells, along with detailed instructions for reading BRET emissions, performing data analysis, and confirming protein expression levels. In addition, example assay results, along with optimization and troubleshooting steps, are provided.
Insights
A new bioluminescence assay measures CRAF and 14-3-3 protein interactions in live cells. This method aids in understanding RAF kinase regulation for cancer and developmental disorder therapies.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- CRAF is a key effector in RAS-driven cancers and linked to Noonan syndrome via germline mutations.
- RAF kinases interact with 14-3-3 proteins at phosphorylation-dependent sites, regulating RAF dimer formation and autoinhibition.
- Understanding CRAF-14-3-3 interactions is crucial for developing targeted therapies for RAF-related diseases.
Purpose of the Study:
- To develop and describe a novel bioluminescence resonance energy transfer (BRET)-based assay for quantifying CRAF and 14-3-3 protein interactions in live cells.
- To validate the assay's utility by demonstrating its ability to detect disruptions in CRAF-14-3-3 binding caused by specific mutations.
Main Methods:
- A BRET assay was established using CRAF fused to Nano luciferase (donor) and 14-3-3 fused to Halo tag (acceptor).
- Live-cell imaging measured BRET signal generated by energy transfer upon CRAF-14-3-3 interaction.
- Mutations known to disrupt 14-3-3 binding sites on RAF were introduced to confirm assay specificity.
Main Results:
- The BRET assay successfully detected and quantified interactions between CRAF and 14-3-3 proteins in living cells.
- The assay signal was significantly reduced by mutations targeting high-affinity 14-3-3 docking sites on CRAF, confirming the assay's specificity.
- Detailed protocols for cell culture, transfection, BRET signal detection, data analysis, and protein expression validation were provided.
Conclusions:
- The described BRET assay provides a robust method for studying CRAF-14-3-3 interactions in real-time within live cells.
- This assay is a valuable tool for investigating the role of RAF-14-3-3 binding in cancer and developmental disorders.
- The assay facilitates the discovery and validation of therapeutic strategies targeting RAF kinase signaling pathways.
