Analysis by Fluorescence Resonance Energy Transfer of the Interaction between Ligands and Protein Kinase Cδ in the Intact Cell

Abstract

The role of the protein kinase C (PKC) family of serine/ threonine kinases in cellular differentiation, proliferation, apoptosis, and other responses makes them attractive therapeutic targets. The activation of PKCs by ligands in vivo varies depending upon cell type; therefore, methods are needed to screen the potency of PKCs in this context. Here we describe a genetically encoded chimera of native PKC␦ fused to yellow-and cyanshifted green fluorescent protein, which can be expressed in mammalian cells. This chimeric protein kinase, CY-PKC␦, retains native or near-native activity in the several biological and biochemical parameters that we tested. Binding assays showed that CY-PKC␦ and native human PKC␦ have similar binding affinity for phorbol 12,13-dibutyrate. Analysis of translocation by Western blotting and confocal microscopy showed that CY-PKC␦ translocates from the cytosol to the membrane upon treatment with ligand, that the translocation has similar dose dependence as that of endogenous PKC␦, and that the pattern of translocation is indistinguishable from that of the green fluorescent protein-PKC␦ fusion well characterized from earlier studies. Treatment with phorbol ester of cells expressing CY-PKC␦ resulted in a dose-dependent increase in FRET that could be visualized in situ by confocal microscopy or measured fluorometrically. By using this construct, we were able to measure the kinetics and potencies of 12 known PKC ligands, with respect to CY-PKC␦, in the intact cell. The CY-PKC␦ chimera and the in vivo assays described here therefore show potential for high throughput screening of prospective PKC␦ ligands within the context of cell type.The members of the protein kinase C (PKC) 1 family of serine/ threonine kinases represent critical signaling molecules in the cell. PKCs are activated by the second messenger sn-1,2-diacylglycerol (DAG) or by their ultrapotent analogues, the phorbol esters. Because PKCs activate downstream cellular pathways that regulate cell proliferation, apoptosis, differentiation, and other responses, they are important therapeutic targets. Indeed, a number of compounds targeting protein kinase C are currently at different stages of drug development. Examples include LY333531, being evaluated for diabetic retinopathy and anti-angiogenesis (1, 2), prostratin and dPP, being evaluated for AIDS chemotherapy (3, 4), and bryostatin 1 and ingenol 3-angelate, being evaluated as cancer chemotherapeutic agents (5-7).The known isoforms of protein kinase C include the classical PKCs (␣, ␤ I , ␤ II , and ␥), which are calcium-dependent; the novel PKCs (␦, ⑀, , and ), which are calcium-independent, and the atypical PKCs ( and ), which are not activated by DAG or the phorbol esters (8, 9). The classical and novel PKCs each contain one or more highly conserved C1 domains, zinc finger motifs that act as the binding site for DAG and the phorbol esters (10, 11).Biochemical assays of PKC activation have limited predictive value for the understanding of structure-activity relationships because of ex...

PKCγ mutations in spinocerebellar ataxia type 14 affect C1 domain accessibility and kinase activity leading to aberrant MAPK signaling

“…When we monitored the changes in protein conformation upon activation of PKC by PMA, we consistently observed a FRET increase for all constructs (data not shown), whereas a decrease was expected, as this would correspond to the open active conformation. Similar observations have been reported for the PKCδ isoform (Braun et al, 2005). This can be explained by accumulation of PKC at the plasma membrane induced by PMA.…”

Kinetics of Penetration Influence the Apparent Potency of Vanilloids on TRPV1

“…The first compound was olvanil, which has been described as a potent nonpungent agonist of TRPV1. Olvanil has a calculated log P of 8.00, and we know that phorbol esters with a log P in this range enter cells slowly (Braun et al, 2005a) and only slowly induce responses of their intracellular target, protein kinase C (Wang et al, 2000;Braun et al, 2005b). In our standard 45 Ca 2ϩ uptake assay with a 5-min incubation time, the EC 50 for olvanil was 115 Ϯ 32 nM; the EC 50 for 45 Ca 2ϩ uptake determined with a 30-min assay was only slightly lower at 81 Ϯ 12 nM (mean Ϯ S.E.M., n ϭ 3 experiments).…”

“…Its predicted log P value is 8.03. We know that fluorescent phorbol esters with such a hydrophobicity penetrate slowly (Braun et al, 2005a) and that nonfluorescent phorbol esters with similar lipophilicity activate protein kinase C slowly (Braun et al, 2005b) and cause slow membrane translocation of protein kinase C (a measure of its interaction) (Wang et al, 2000). Furthermore, […”

Conserved Modular Domains Team up to Latch-open Active Protein Kinase Cα

“…Vitro-We engineered an initial unimolecular FRET sensor (mCer-PKC␣-mCit-FLAG; each element is listed from the N to the C termini and dashes consist of a (GSG) 2-4 linker) capitalizing on the design scheme of a previously reported PKC␦ sensor that elicited changes in FRET upon activation both in vitro and in cells (23). This PKC␣ flanking sensor (50 nM) showed enhanced FRET upon activation with Ca 2ϩ and liposomes containing DAG and PS ( Fig.…”