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Updated: Jun 8, 2025
Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor LATS Biosensor
Published on: September 13, 2018
Development of a live cell assay for real-time monitoring the interactions between the Hippo pathway components
Blaž Andlovic1, Alexander Wolf2, Malgorzata Hiltmann2
1Lead Discovery Center GmbH, Otto-Hahn-Str. 15, 44227 Dortmund, Germany; Laboratory of Chemical Biology, Department of Biomedical Engineering and Institute for Complex Molecular Systems, Eindhoven University of Technology, Den Dolech 2, 5612 AZ Eindhoven, The Netherlands.
Abstract:
The Hippo pathway plays an important role in organ size control and tissue homeostasis. Dysregulation is involved in many pathologies, including cancer, which has attracted interest in targeting the Hippo pathway. Since the upstream components are bona fide tumor suppressors, it is feasible to target oncogenic downstream targets such as TAZ, a key downstream effector in the Hippo pathway. Its activity is regulated by phosphorylation on multiple sites, with Ser89 playing a critical role in regulation of TAZ activity. Phosphorylation of TAZ at Ser89 promotes binding to 14-3-3 scaffolding proteins, preventing nuclear translocation and abolishing target gene transcription. Here we describe the development of a cell-based assay suitable for high-throughput screening, based on a split NanoLuc luciferase, for monitoring interactions between 14 3-3 and TAZ in living cells. We have validated the assay by screening of a kinase-biased library. The assay can be quickly adapted for higher throughput and thus offers a valuable tool to study new signal inputs involved in regulation of TAZ activity as well as for identification of molecules that modulate the Hippo pathway.
Insights
Researchers developed a high-throughput assay to monitor interactions between 14-3-3 proteins and TAZ, a key Hippo pathway effector. This tool aids in discovering molecules that modulate the Hippo pathway for cancer therapy.
Area of Science:
- Cell biology
- Molecular biology
- Biochemistry
Background:
- The Hippo pathway is crucial for organ size control and tissue homeostasis.
- Hippo pathway dysregulation is implicated in various pathologies, notably cancer.
- Targeting oncogenic downstream effectors like TAZ is a promising therapeutic strategy.
Purpose of the Study:
- To develop a cell-based assay for high-throughput screening (HTS) of molecules modulating the Hippo pathway.
- To monitor interactions between 14-3-3 proteins and TAZ, a key downstream effector.
- To facilitate the identification of novel therapeutic agents targeting TAZ activity.
Main Methods:
- Development of a split NanoLuc luciferase-based cell assay.
- Monitoring of 14-3-3 and TAZ interactions in living cells.
- Validation of the assay through screening of a kinase-biased library.
Main Results:
- A robust cell-based assay for monitoring TAZ-14-3-3 interactions was successfully developed.
- The assay demonstrated suitability for high-throughput screening.
- Initial validation confirmed the assay's utility in identifying modulators of TAZ activity.
Conclusions:
- The developed assay provides a valuable tool for studying TAZ regulation and the Hippo pathway.
- This HTS-compatible assay can accelerate the discovery of novel Hippo pathway modulators.
- The assay facilitates research into new signal inputs regulating TAZ and potential cancer therapeutics.
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