Imaging Oscillations of Protein Kinase C Activity in Cells

The protein kinase C (PKC) family contains ten members that share a catalytic core but differ in the composition of their regulatory modules. The conventional PKCs are a subfamily whose regulatory domains bind to Ca²⁺ and to the lipid second messenger diacylglycerol and thus they are activated by coincidence detection of Ca²⁺ and diacylglycerol at the plasma membrane. In HeLa cells, oscillations in Ca²⁺ evoke oscillations in membrane PKC activity. The only method to date that offers the time resolution to observe these rapid changes in PKC activity is the utilization of a genetically encoded membrane-targeted PKC activity reporter, MyrPalm-CKAR (C Kinase Activity Reporter). CKAR is a fluorescence resonance energy transfer (FRET)-based, modular protein that undergoes a conformational change upon phosphorylation resulting in a change in FRET, thereby serving as a direct readout of cellular kinase activity. As a genetically encoded reporter, it is easily introduced into cells and can be targeted to distinct intracellular compartments through the addition of short targeting sequences. MyrPalm-CKAR is targeted to plasma membranes through the amino-terminal addition of seven amino acids that encode a sequence that is myristoylated and palmitoylated in the cell. This chapter details the method of utilizing MyrPalm-CKAR to monitor acute changes in PKC signaling at the plasma membrane that are a consequence of acute changes in Ca²⁺ levels.