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

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

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 previo...

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Main Authors: Summanen, Milla, Granqvist, Niko, Tuominen, Raimo K., Yliperttula, Marjo, Verrips, C. Theo, Boonstra, Johannes, Blanchetot, Christophe, Ekokoski, Elina
Format: Online
Language:English
Published: Public Library of Science 2012
Online Access:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3334965/
id pubmed-3334965
recordtype oai_dc
spelling pubmed-33349652012-04-25 Kinetics of PKCε Activating and Inhibiting Llama Single Chain Antibodies and Their Effect on PKCε Translocation in HeLa Cells Summanen, Milla Granqvist, Niko Tuominen, Raimo K. Yliperttula, Marjo Verrips, C. Theo Boonstra, Johannes Blanchetot, Christophe Ekokoski, Elina Research Article 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. Public Library of Science 2012-04-20 /pmc/articles/PMC3334965/ /pubmed/22536418 http://dx.doi.org/10.1371/journal.pone.0035630 Text en Summanen et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
repository_type Open Access Journal
institution_category Foreign Institution
institution US National Center for Biotechnology Information
building NCBI PubMed
collection Online Access
language English
format Online
author Summanen, Milla
Granqvist, Niko
Tuominen, Raimo K.
Yliperttula, Marjo
Verrips, C. Theo
Boonstra, Johannes
Blanchetot, Christophe
Ekokoski, Elina
spellingShingle Summanen, Milla
Granqvist, Niko
Tuominen, Raimo K.
Yliperttula, Marjo
Verrips, C. Theo
Boonstra, Johannes
Blanchetot, Christophe
Ekokoski, Elina
Kinetics of PKCε Activating and Inhibiting Llama Single Chain Antibodies and Their Effect on PKCε Translocation in HeLa Cells
author_facet Summanen, Milla
Granqvist, Niko
Tuominen, Raimo K.
Yliperttula, Marjo
Verrips, C. Theo
Boonstra, Johannes
Blanchetot, Christophe
Ekokoski, Elina
author_sort Summanen, Milla
title Kinetics of PKCε Activating and Inhibiting Llama Single Chain Antibodies and Their Effect on PKCε Translocation in HeLa Cells
title_short Kinetics of PKCε Activating and Inhibiting Llama Single Chain Antibodies and Their Effect on PKCε Translocation in HeLa Cells
title_full Kinetics of PKCε Activating and Inhibiting Llama Single Chain Antibodies and Their Effect on PKCε Translocation in HeLa Cells
title_fullStr Kinetics of PKCε Activating and Inhibiting Llama Single Chain Antibodies and Their Effect on PKCε Translocation in HeLa Cells
title_full_unstemmed Kinetics of PKCε Activating and Inhibiting Llama Single Chain Antibodies and Their Effect on PKCε Translocation in HeLa Cells
title_sort kinetics of pkcε activating and inhibiting llama single chain antibodies and their effect on pkcε translocation in hela cells
description 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.
publisher Public Library of Science
publishDate 2012
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3334965/
_version_ 1611524105173991424

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