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Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor LATS Biosensor
Published on: September 13, 2018
A biosensor to monitor dynamic regulation and function of tumour suppressor PTEN in living cells
Evelyne Lima-Fernandes1, Stanislas Misticone1, Cédric Boularan1
11] Department of Endocrinology, Metabolism and Diabetes, Inserm, U1016, Institut Cochin, 27 rue du Faubourg St Jaques, Paris 75014, France [2] CNRS, UMR8104, Paris 75014, France [3] University Paris Descartes, Sorbonne Paris Cité, Paris 75014, France [4].
Abstract:
Tumour suppressor PTEN is a phosphatase that negatively regulates the PI3K/AKT pathway. The ability to directly monitor PTEN conformation and function in a rapid, sensitive manner is a key step towards developing anti-cancer drugs aimed at enhancing or restoring PTEN-dependent pathways. Here we developed an intramolecular bioluminescence resonance energy transfer (BRET)-based biosensor, capable of detecting signal-dependent PTEN conformational changes in live cells. The biosensor retains intrinsic properties of PTEN, enabling structure-function and kinetic analyses. BRET shifts, indicating conformational change, were detected following mutations that disrupt intramolecular PTEN interactions, promoting plasma membrane targeting and also following physiological PTEN activation. Using the biosensor as a reporter, we uncovered PTEN activation by several G protein-coupled receptors, previously unknown as PTEN regulators. Trastuzumab, used to treat ERBB2-overexpressing breast cancers also elicited activation-associated PTEN conformational rearrangement. We propose the biosensor can be used to identify pathways regulating PTEN or molecules that enhance its anti-tumour activity.
Insights
A novel biosensor tracks tumour suppressor PTEN’s shape changes in real-time. This tool aids in discovering new cancer drug targets by monitoring PTEN activity and regulation.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Tumour suppressor phosphatase and tensin homolog (PTEN) is a critical negative regulator of the phosphoinositide 3-kinase/AKT signaling pathway.
- Dysregulation of PTEN is implicated in various cancers, making it a key target for anti-cancer drug development.
- A rapid and sensitive method to monitor PTEN conformation and function is crucial for developing therapeutics that enhance PTEN activity.
Purpose of the Study:
- To develop and validate an intramolecular bioluminescence resonance energy transfer (BRET)-based biosensor for detecting PTEN conformational changes in live cells.
- To utilize the biosensor to identify novel regulators of PTEN activity and conformational state.
- To assess the utility of the biosensor in drug discovery for PTEN-modulating anti-cancer agents.
Main Methods:
- Development of an intramolecular BRET biosensor engineered to report PTEN conformational dynamics.
- Application of the biosensor in live-cell imaging to detect BRET shifts indicative of conformational changes.
- Utilizing the biosensor to screen for PTEN activation by various stimuli, including G protein-coupled receptors and therapeutic antibodies.
Main Results:
- The BRET biosensor successfully detected signal-dependent PTEN conformational changes in response to mutations and physiological activation.
- Mutations disrupting intramolecular PTEN interactions and promoting plasma membrane targeting induced BRET shifts.
- The biosensor identified several G protein-coupled receptors as novel PTEN regulators and confirmed Trastuzumab-induced PTEN conformational rearrangement.
Conclusions:
- The developed BRET biosensor is a sensitive tool for monitoring PTEN conformation and function in real-time within live cells.
- This biosensor can identify novel pathways regulating PTEN and discover molecules that enhance its anti-tumour activity.
- The tool holds significant potential for accelerating the development of novel anti-cancer therapeutics targeting the PTEN pathway.
