PKC epsilon (PKC ε) Redistribution Assay

• Designed to assay compounds for their ability to modulate activation of PKC ε
• Coupled to EGFP for easy monitoring of the cellular translocation event
• Robust cell-based assay for use in high content analysis and fluorescence microscope applications

• Biologically relevant data: Compounds tested in a cellular environment
• Validated: Functionally tested cells provided with an optimized assay protocol
• Easy to use: Just plate cells, add compounds, and image

Protein Kinase C is an expanding family of at least 10 enzymes that can be divided into three classes. The conventional kinases (α, βI, βII, γ), the novel (δ, ε, η, θ, μ), and the atypical (ζ, ι, λ). The kinases are localized to the cytoplasm, but upon activation they translocate to an organelle or the plasma membrane. The conventional PKC isoforms are activated by Ca²⁺ and 1,2-diacylglycerol (DAG), the novel isofoms require DAG for activation, and activation of the atypical isorforms is less described. A common mechanism of Ca²⁺ and DAG release is through activation of G_q-coupled GPCRs. A large number of PKC substrates are then phosphorylated and thereby regulated by PKC.

PKC ε has been reported to be involved many different cellular functions, for example apoptosis, heat shock response, macrophage activation, neurite development, and insulin signaling.

Figure 1. Translocation to the plasma membrane of PKCε-EGFP. Untreated U2OS cells expressing the NK1 receptor and PKCε-EGFP (DMSO control, left) and cells treated with 3 nM Substance P for 2 min (right). Activation of the NK1 receptor leads to release of Ca²⁺ and DAG which in turn induces translocation of PKCε-EGFP from cytoplasmic foci to the plasma membrane. Arrows indicate cytoplasmic foci detected by the image analysis algorithm.

Figure 2. Substance P concentration response in the PKC ε assay. Concentration response was measured in 9 point half log dilution series (n=16). The EC₅₀ of Substance P is approximately 230 pM. Cells were treated with Substance P for 2 min. Cells were then fixed and disappearance of cytoplasmic foci (membrane translocation) was measured using the Cellomics ArrayScan® V^TI Reader and the SpotDetectorV3 BioApplication. % activity was calculated relative to the positive (3 nM Substance P) and negative control (0.25% DMSO).