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

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