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Fluorescent Protein Biosensor for Probing CDK/Cyclin Activity in vitro and in Living Cells
Author(s) -
Van Thi Nhu Ngoc,
Pellerano Morgan,
Lykaso Sophie,
Morris May C.
Publication year - 2014
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201402318
Subject(s) - cyclin dependent kinase , kinase , microbiology and biotechnology , context (archaeology) , fluorescence , cell cycle , live cell imaging , biology , fusion protein , biochemistry , cyclin , chemistry , biophysics , cell , recombinant dna , paleontology , physics , quantum mechanics , gene
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.