title: The development of activating and inhibiting camelid VHH domains against human protein kinase C epsilon.
abstract: The 10 isozymes of the protein kinase C (PKC) family can have different roles on the same biological process, making isozyme specific analysis of function crucial. Currently, only few pharmacological compounds with moderate isozyme specific effects exist thus hampering research into individual PKC isozymes. The antigen binding regions of camelid single chain antibodies (VHHs) could provide a solution for obtaining PKC isozyme specific modulators. In the present study, we have successfully selected and characterized PKCɛ specific VHH antibodies from two immune VHH libraries using phage display. The VHHs were shown to exclusively bind to PKCɛ in ELISA and immunoprecipitation studies. Strikingly, five of the VHHs had an effect on PKCɛ kinase activity in vitro. VHHs A10, C1 and D1 increased PKCɛ kinase activity in a concentration-dependent manner (EC(50) values: 212-310nM), whereas E6 and G8 inhibited PKCɛ activity (IC(50) values: 103-233nM). None of these VHHs had an effect on the activity of the other novel PKC isozymes PKCδ and PKCθ. To our knowledge, these antibodies are the first described VHH activators and inhibitors for a protein kinase. Furthermore, the development of PKCɛ specific modulators is an important contribution to PKC research.
authors.affiliation: Cellular Dynamics, Department of Biology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands.
authors.forename: Milla M I
authors.initials: MM
authors.lastname: Paalanen
authors.forename: Elina
authors.initials: E
authors.lastname: Ekokoski
authors.forename: Mohamed
authors.initials: M
authors.lastname: El Khattabi
authors.forename: Raimo K
authors.initials: RK
authors.lastname: Tuominen
authors.forename: C Theo
authors.initials: CT
authors.lastname: Verrips
authors.forename: Johannes
authors.initials: J
authors.lastname: Boonstra
authors.forename: Christophe
authors.initials: C
authors.lastname: Blanchetot
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002161:Camelids, New World / Q000276:immunology; D022801:Complementarity Determining Regions; D006801:Humans; D007143:Immunoglobulin Heavy Chains; D020128:Inhibitory Concentration 50; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000276:immunology*; D019151:Peptide Library; D011485:Protein Binding; D051744:Protein Kinase C-epsilon / Q000037:antagonists & inhibitors / Q000276:immunology; D057127:Single-Chain Antibodies / Q000276:immunology* / Q000302:isolation & purification*
chemical_list: D022801:Complementarity Determining Regions; D007143:Immunoglobulin Heavy Chains; D007527:Isoenzymes; D019151:Peptide Library; D057127:Single-Chain Antibodies; C497255:PRKCE protein, human; D051744:Protein Kinase C-epsilon