Fluorescent protein biosensor for probing CDK/cyclin activity in vitro and in living cells

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.