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

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

Publication year: 2012
Authors: Milla Summanen 1,2, Niko Granqvist 3, Raimo K. Tuominen 2, Marjo Yliperttula 3, C.Theo Verrips 1, Johannes Boonstra 1; Christophe Blanchetot 1, Elina Ekokoski 2

1 – Cell Biology, Department of Biology, University of Utrecht, Utrecht, The Netherlands,
2 – Division of Pharmacology and Toxicology, Faculty of Pharmacy, University ofHelsinki, Helsinki, Finland
3 – Division of Biopharmaceutics and Pharmacokinetics, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland

Published in: PLos ONE, 2012, Vol. 7, Issue 4, e3563


activation binding kinetics conformation first order Langmuir model HeLa Llama PKCε protein second order Langmuir model VHH


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