1. Academic Validation
  2. Development of a live cell assay for real-time monitoring the interactions between the Hippo pathway components 14-3-3 and TAZ

Development of a live cell assay for real-time monitoring the interactions between the Hippo pathway components 14-3-3 and TAZ

  • SLAS Discov. 2024 Dec;29(8):100191. doi: 10.1016/j.slasd.2024.100191.
Blaž Andlovic 1 Alexander Wolf 2 Malgorzata Hiltmann 2 Bert M Klebl 2 Jan Eickhoff 3 Christian Ottmann 4
Affiliations

Affiliations

  • 1 Lead 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.
  • 2 Lead Discovery Center GmbH, Otto-Hahn-Str. 15, 44227 Dortmund, Germany.
  • 3 Lead Discovery Center GmbH, Otto-Hahn-Str. 15, 44227 Dortmund, Germany. Electronic address: eickhoff@lead-discovery.de.
  • 4 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. Electronic address: c.ottmann@tue.nl.
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.

Keywords

Cell-based assay; Compound screening; Hippo pathway; Protein-protein interactions; Split-luciferase complementation.

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