1. Fluorescent protein biosensor for probing CDK/cyclin activity in vitro and in living cells.
- Author
-
Van TN, Pellerano M, Lykaso S, and Morris MC
- Subjects
- Cell Survival, Fluorescence, HeLa Cells, Humans, Luminescent Proteins chemistry, MCF-7 Cells, Tumor Cells, Cultured, Biosensing Techniques, Cyclin-Dependent Kinases metabolism, Cyclins metabolism, Luminescent Proteins analysis
- 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., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2014
- Full Text
- View/download PDF