A cell based screening approach for identifying protein degradation regulators
| Autor: | Nagi G. Ayad, Scott Simanski, Valerie Cavett, Jennifer Busby, Trey K. Sato, Marie E. Maloof |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Recombinant Fusion Proteins Cyclin A Mitosis Cell Cycle Proteins Protein degradation 03 medical and health sciences Antigens CD Humans Cell division control protein 4 Cyclin B1 Luciferases Molecular Biology biology Ubiquitin F-Box Proteins Cell Cycle Protein turnover Gene Expression Regulation Developmental Nuclear Proteins Cell Biology Protein-Tyrosine Kinases Cell cycle Cadherins Ubiquitin ligase Cell biology Wee1 030104 developmental biology Proteolysis biology.protein HeLa Cells Reports Developmental Biology |
| Zdroj: | Cell Cycle. 16:940-946 |
| ISSN: | 1551-4005 1538-4101 |
| DOI: | 10.1080/15384101.2017.1301333 |
| Popis: | Cellular transitions are achieved by the concerted actions of regulated degradation pathways. In the case of the cell cycle, ubiquitin mediated degradation ensures unidirectional transition from one phase to another. For instance, turnover of the cell cycle regulator cyclin B1 occurs after metaphase to induce mitotic exit. To better understand pathways controlling cyclin B1 turnover, the N-terminal domain of cyclin B1 was fused to luciferase to generate an N-cyclin B1-luciferase protein that can be used as a reporter for protein turnover. Prior studies demonstrated that cell-based screens using this reporter identified small molecules inhibiting the ubiquitin ligase controlling cyclin B1-turnover. Our group adapted this approach for the G2-M regulator Wee1 where a Wee1-luciferase construct was used to identify selective small molecules inhibiting an upstream kinase that controls Wee1 turnover. In the present study we present a screening approach where cell cycle regulators are fused to luciferase and overexpressed with cDNAs to identify specific regulators of protein turnover. We overexpressed approximately 14,000 cDNAs with the N-cyclin B1-luciferase fusion protein and determined its steady-state level relative to other luciferase fusion proteins. We identified the known APC/C regulator Cdh1 and the F-box protein Fbxl15 as specific modulators of N-cyclin B1-luciferase steady-state levels and turnover. Collectively, our studies suggest that analyzing the steady-state levels of luciferase fusion proteins in parallel facilitates identification of specific regulators of protein turnover. |
| Databáze: | OpenAIRE |
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