Kinetics of PKCε Activating and Inhibiting Llama Single Chain Antibodies and Their Effect on PKCε Translocation in HeLa Cells

Dysregulation of PKC epsilon is involved in several serious diseases such as cancer, type II diabetes and Alzheimer's disease. Therefore, specific activators and inhibitors of PKC epsilon hold promise as future therapeutics, in addition to being useful in research into PKC epsilon regulated pathways. We have previously described llama single chain antibodies (VHHs) that specifically activate (A10, C1 and D1) or inhibit (E6 and G8) human recombinant PKC epsilon. Here we report a thorough kinetic analysis of these VHHs. The inhibiting VHHs act as non-competitive inhibitors of PKC epsilon activity, whereas the activating VHHs have several different modes of action, either increasing V-max and/or decreasing K-m values. We also show that the binding of the VHHs to PKC epsilon is conformation-dependent, rendering the determination of affinities difficult. Apparent affinities are in the micromolar range based on surface plasmon resonance studies. Furthermore, the VHHs have no effect on the activity of rat PKC epsilon nor can they bind the rat form of the protein in immunoprecipitation studies despite the 98% identity between the human and rat PKC epsilon proteins. Finally, we show for the first time that the VHHs can influence PKC epsilon function also in cells, since an activating VHH increases the rate of PKC epsilon translocation in response to PMA in HeLa cells, whereas an inhibiting VHH slows down the translocation. These results give insight into the mechanisms of PKC epsilon activity modulation and highlight the importance of protein conformation on VHH binding.