Optogenetic control of Protein Kinase C-epsilon activity reveals its intrinsic signaling properties with spatiotemporal resolution

The regulation of PKC epsilon (PKC{varepsilon}) and its downstream effects is still not fully understood, making it challenging to develop targeted therapies or interventions. A more precise tool that enables spatiotemporal control of PKC{varepsilon} activity is thus required. Here, we describe a photo-activatable optogenetic PKC{varepsilon} probe (Opto-PKC{varepsilon}) consisting of an engineered PKC{varepsilon} catalytic domain and a blue-light inducible dimerization domain. Molecular dynamics and AlphaFold simulations enable rationalization of the dark-light activity of the optogenetic probe. We first characterize the binding partners of Opto-PKC{varepsilon}, which are similar to those of PKC{varepsilon}. Subsequent validation of the Opto-PKC{varepsilon} tool is performed with phosphoproteome analysis, which reveals that only PKC{varepsilon} substrates are phosphorylated upon light activation. Opto-PKC{varepsilon} could be engineered for recruitment to specific subcellular locations. Activation of Opto-PKC{varepsilon} in isolated hepatocytes reveals its sustained activation at the plasma membrane is required for its phosphorylation of the insulin receptor at Thr1160. In addition, Opto-PKC{varepsilon} recruitment to the mitochondria results in its lowering of the spare respiratory capacity through phosphorylation of complex I NDUFS4. These results demonstrate that Opto-PKC{varepsilon} may have broad applications for the studies of PKC{varepsilon} signaling with high specificity and spatiotemporal resolution. SummaryWe have developed a photo-activatable optogenetic PKC{varepsilon} probe, which demonstrates differential activity in light versus dark. The tool is subsequently validated with protein association studies and phosphoproteome analysis. It enables dissection of signaling events arising from its activation at defined subcellular locations. For instance, sustained activation of PKC{varepsilon} at the plasma membrane is required for its phosphorylation of the insulin receptor at Thr1160.