title: Fluorescent protein biosensor for probing CDK/cyclin activity in vitro and in living cells.
abstract: Cyclin-dependent kinases (CDKs) play an essential role in the coordination of cell cycle progression and transcriptional regulation; hyperactivation is associated with cancer. However there are few means of measuring their activity in a physiological context or their inhibition in response to therapeutics. To this aim we engineered a modular fluorescent protein biosensor that reports on phosphorylation by CDK/cyclins through real-time changes in fluorescence intensity. This allowed a comparison of enzymatic activity of recombinant kinases, monitoring inhibition by small molecules, and probing endogenous activities in lysates from healthy and cancer cell lines in a sensitive and quantitative fashion. This versatile tool was further implemented to probe the oscillatory activity of these kinases throughout the cell cycle by time-lapse imaging and ratiometric fluorescence quantification, following delivery of a red fluorescent protein fusion mediated by cell-penetrating peptides.
authors.affiliation: Interdisciplinary Institute for NeuroScience-UMR 5297, 146 rue Léo-Saignat, 33077 Bordeaux (France).
authors.forename: Thi Nhu Ngoc
authors.initials: TN
authors.lastname: Van
authors.forename: Morgan
authors.initials: M
authors.lastname: Pellerano
authors.forename: Sophie
authors.initials: S
authors.lastname: Lykaso
authors.forename: May C
authors.initials: MC
authors.lastname: Morris
mesh_terms: D015374:Biosensing Techniques; D002470:Cell Survival; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism*; D005453:Fluorescence; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis* / Q000737:chemistry; D061986:MCF-7 Cells; D014407:Tumor Cells, Cultured
chemical_list: D016213:Cyclins; D008164:Luminescent Proteins; D018844:Cyclin-Dependent Kinases
keywords: CDK/cyclin; biosensors; fluorescence; kinase activity; live-cell imaging