Abstract
Dysregulation of PKCε is involved in several serious diseases such as cancer, type II diabetes and Alzheimer's disease. Therefore, specific activators and inhibitors of PKCε hold promise as future therapeutics, in addition to being useful in research into PKCε 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 PKCε. Here we report a thorough kinetic analysis of these VHHs. The inhibiting VHHs act as non-competitive inhibitors of PKCε activity, whereas the activating VHHs have several different modes of action, either increasing Vmax and/or decreasing Km values. We also show that the binding of the VHHs to PKCε 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 PKCε nor can they bind the rat form of the protein in immunoprecipitation studies despite the 98% identity between the human and rat PKCε proteins. Finally, we show for the first time that the VHHs can influence PKCε function also in cells, since an activating VHH increases the rate of PKCε translocation in response to PMA in HeLa cells, whereas an inhibiting VHH slows down the translocation. These results give insight into the mechanisms of PKCε activity modulation and highlight the importance of protein conformation on VHH binding.
Highlights
Protein kinase C (PKC) is a family of serine/threonine kinases that regulate several signaling pathways in cells
Affinity measurements using Surface Plasmon Resonance We have already shown by immunoprecipitation (IP) and kinase activity assays that the VHH activators and inhibitors of PKCe bind the human PKCe protein [23]
These results were in strong contrast to the enzyme-linked immunosorbent assay (ELISA) and IP results described previously [23]
Summary
Protein kinase C (PKC) is a family of serine/threonine kinases that regulate several signaling pathways in cells. The ten PKC isozymes have distinct biological functions and are divided into three groups based on cofactor requirements [1]. Conventional PKCs (a, bI, bII and c) are activated by Ca2+ and diacylglycerol (DAG), novel PKCs (d, e, g and h) require only DAG for activation, and atypical PKCs (f and i/l) are insensitive to both DAG and Ca2+ [2]. Conventional and novel PKC isozymes translocate to the plasma membrane when DAG or its surrogate, phorbol 12-myristate 13-acetate (PMA), which is often used as a PKC activator in cellular assays, become available [3]. In addition to cofactor binding, PKC activity is regulated by priming phosphorylations of three conserved phosphorylation motifs [1] and protein-protein interactions such as binding to receptors for activated C kinase (RACKs) [4]
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