Genetically encoded FRET probe for PKC activity based on pleckstrin

Abstract

We developed a probe for investigating protein kinase C (PKC) activity in living cells. The probe is based on a fragment of pleckstrin enclosed by two FRET-capable fluorophores. PKC activity modulation was reliably followed by FRET change in vitro and in vivo. The probe responds quickly to PKC activation by phorbol ester. FRET changes were reversible when PKC inhibitors were administered. Stimulation of cellular signaling pathways using histamine or bradykinin triggered PKC in a physiologically relevant way.