Design and synthesis of a fluorescent reporter of protein kinase activity

There is widespread interest in developing fluorescent reporters of protein kinase activity, species that can furnish a visual readout of both when and where intracellular kinases are activated in response to a stimulus. We have constructed and identified, via a combination of rational design, library synthesis, and screening, a difluorofluorescein-appended peptide-based species that responds to protein kinase C phosphorylation in a fluorescently sensitive fashion. The phosphorylation-induced divalent metal ion-mediated 265% enhancement in fluorescence proceeds with a V(max) of 8.5 micromol/min.mg and a K(m) of 20.5 microM.