Development of a live cell assay for real-time monitoring the interactions between the Hippo pathway components 14-3-3 and TAZ
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 targe...
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| Language: | English |
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Elsevier
2024-12-01
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| Series: | SLAS Discovery |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2472555224000534 |
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| author | Blaž Andlovic Alexander Wolf Malgorzata Hiltmann Bert M. Klebl Jan Eickhoff Christian Ottmann |
| author_facet | Blaž Andlovic Alexander Wolf Malgorzata Hiltmann Bert M. Klebl Jan Eickhoff Christian Ottmann |
| author_sort | Blaž Andlovic |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-25b43e58e9044192aebb9c5e2cea5ea3 |
| institution | Kabale University |
| issn | 2472-5552 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | SLAS Discovery |
| spelling | doaj-art-25b43e58e9044192aebb9c5e2cea5ea32024-12-17T05:00:17ZengElsevierSLAS Discovery2472-55522024-12-01298100191Development of a live cell assay for real-time monitoring the interactions between the Hippo pathway components 14-3-3 and TAZBlaž Andlovic0Alexander Wolf1Malgorzata Hiltmann2Bert M. Klebl3Jan Eickhoff4Christian Ottmann5Lead 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 NetherlandsLead Discovery Center GmbH, Otto-Hahn-Str. 15, 44227 Dortmund, GermanyLead Discovery Center GmbH, Otto-Hahn-Str. 15, 44227 Dortmund, GermanyLead Discovery Center GmbH, Otto-Hahn-Str. 15, 44227 Dortmund, GermanyLead Discovery Center GmbH, Otto-Hahn-Str. 15, 44227 Dortmund, Germany; Corresponding author at: Assay development & screening, 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; Corresponding author at: Christian Ottmann, 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.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.http://www.sciencedirect.com/science/article/pii/S2472555224000534Cell-based assayProtein-protein interactionsSplit-luciferase complementationHippo pathwayCompound screening |
| spellingShingle | Blaž Andlovic Alexander Wolf Malgorzata Hiltmann Bert M. Klebl Jan Eickhoff Christian Ottmann Development of a live cell assay for real-time monitoring the interactions between the Hippo pathway components 14-3-3 and TAZ SLAS Discovery Cell-based assay Protein-protein interactions Split-luciferase complementation Hippo pathway Compound screening |
| title | Development of a live cell assay for real-time monitoring the interactions between the Hippo pathway components 14-3-3 and TAZ |
| title_full | Development of a live cell assay for real-time monitoring the interactions between the Hippo pathway components 14-3-3 and TAZ |
| title_fullStr | Development of a live cell assay for real-time monitoring the interactions between the Hippo pathway components 14-3-3 and TAZ |
| title_full_unstemmed | Development of a live cell assay for real-time monitoring the interactions between the Hippo pathway components 14-3-3 and TAZ |
| title_short | Development of a live cell assay for real-time monitoring the interactions between the Hippo pathway components 14-3-3 and TAZ |
| title_sort | development of a live cell assay for real time monitoring the interactions between the hippo pathway components 14 3 3 and taz |
| topic | Cell-based assay Protein-protein interactions Split-luciferase complementation Hippo pathway Compound screening |
| url | http://www.sciencedirect.com/science/article/pii/S2472555224000534 |
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