title: A cell based screening approach for identifying protein degradation regulators.
abstract: 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.
authors.affiliation: a Department of Cancer Biology , Scripps Florida, The Scripps Research Institute , Jupiter , FL , USA.
authors.forename: Scott
authors.initials: S
authors.lastname: Simanski
authors.affiliation: b Department of Psychiatry and Behavioral Sciences , Center for Therapeutic Innovation, Sylvester Comprehensive Cancer Center , Miami , FL , USA.
authors.forename: Marie E
authors.initials: ME
authors.lastname: Maloof
authors.affiliation: c Department of Molecular Therapeutics , Scripps Florida, The Scripps Research Institute , Jupiter , FL , USA.
authors.forename: Trey K
authors.initials: TK
authors.lastname: Sato
authors.affiliation: c Department of Molecular Therapeutics , Scripps Florida, The Scripps Research Institute , Jupiter , FL , USA.
authors.forename: Valerie
authors.identifier: http://orcid.org/0000-0003-2859-6355
authors.initials: V
authors.lastname: Cavett
authors.affiliation: c Department of Molecular Therapeutics , Scripps Florida, The Scripps Research Institute , Jupiter , FL , USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Caldwell Busby
authors.affiliation: b Department of Psychiatry and Behavioral Sciences , Center for Therapeutic Innovation, Sylvester Comprehensive Cancer Center , Miami , FL , USA.
authors.forename: Nagi G
authors.initials: NG
authors.lastname: Ayad
mesh_terms: D015703:Antigens, CD; D015820:Cadherins / Q000235:genetics*; D002453:Cell Cycle / Q000235:genetics; D018797:Cell Cycle Proteins / Q000235:genetics*; D056744:Cyclin B1 / Q000235:genetics*; D044783:F-Box Proteins / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008938:Mitosis / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000235:genetics; D025801:Ubiquitin / Q000235:genetics
chemical_list: D015703:Antigens, CD; C534699:CCNB1 protein, human; C513384:CDH1 protein, human; D015820:Cadherins; D018797:Cell Cycle Proteins; D056744:Cyclin B1; D044783:F-Box Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D008156:Luciferases; D011505:Protein-Tyrosine Kinases; C499349:WEE1 protein, human
keywords: cell-based screening; degradation; luciferase; substrate; ubiquitin