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

The regulation of PKC epsilon (PKCε) 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 PKCε activity is thus required. Here, we describe a photo-activatable optogenetic PKCε probe (Opto-PKCε) consisting of an engineered PKCε 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-PKCε, which are similar to those of PKCε. Subsequent validation of the Opto-PKCε tool is performed with phosphoproteome analysis, which reveals that only PKCε substrates are phosphorylated upon light activation. Opto-PKCε could be engineered for recruitment to specific subcellular locations. Activation of Opto-PKCε 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-PKCε recruitment to the mitochondria results in its lowering of the spare respiratory capacity through phosphorylation of complex I NDUFS4. These results demonstrate that Opto-PKCε may have broad applications for the studies of PKCε signaling with high specificity and spatiotemporal resolution.