Membrane-surface anchoring of charged diacylglycerol-lactones correlates with biological activities

Abstract

Synthetic diacylglycerol-lactones (DAG-lactones) are effective modulators of critical cellular signaling pathways, downstream of the lipophilic second messenger diacylglycerol, that activate a host of protein kinase C (PKC) isozymes and other nonkinase proteins that share similar C1 membrane-targeting domains with PKC. A fundamental determinant of the biological activity of these amphiphilic molecules is the nature of their interactions with cellular membranes. This study examines the biological properties of charged DAG-lactones exhibiting different alkyl groups attached to the heterocyclic nitrogen of an α-pyridylalkylidene chain, and particularly the relationship between membrane interactions of the substituted DAG-lactones and their respective biological activities. Our results suggest that bilayer interface localization of the N-alkyl chain in the R(2) position of the DAG-lactones inhibits translocation of PKC isoenzymes onto the cellular membrane. However, the orientation of a branched alkyl chain at the bilayer surface facilitates PKC binding and translocation. This investigation emphasizes that bilayer localization of the aromatic side residues of positively charged DAG-lactone derivatives play a central role in determining biological activity, and that this factor contributes to the diversity of biological actions of these synthetic biomimetic ligands.

Figure 1

(I) Structure of a DAG-lactone prototype with the embedded DAG backbone shown with heavier lines; (II) Structure of a prototype DAG-lactone with an α-arylalkylidene chain. Structures of compounds 1–5.

Figure 2

Effect of DAG-lactone 1 on the plasma membrane localization of PKC isoenzymes. Confocal images of MCF-7 cells expressing PKCα-EGFP (A), PKCε-EGFP (C) or PKCδ-ECFP (E) stimulated with 40 μM DAG-lactone 1 (upper panels) or 40 μM PMA (lower panels). Time in seconds after stimulation is indicated in each micrograph. The protein localization was measured by a line profile (pixel density) traced in each frame as indicated in the materials and methods section. The resulting net change in PKCα-EGFP (B), PKCε-EGFP (D) or PKCδ-ECFP (F) plasma membrane localization upon DAG-lactone (●) and PMA (□) stimulations is expressed as the (Imb-Icyt)/Imb ratio (%) and is represented versus time. The red arrow indicates the stimulation time; Rmax and t1/2 parameters were calculated graphically [15]. The profiles are representative of the results obtained in the cells analyzed (n=16 for both DAG-lactone and PMA for PKCα-EGFP; n=12 and 10 for DAG-lactone and PMA, respectively for PKCε-EGFP; n=13 and 12 for DAG-lactone and PMA, respectively for PKCδ-ECFP). The scale bars indicated in the micrographs correspond to 12 μm.

Figure 3

Effect of DAG-lactone 5 on the plasma membrane localization of PKC isoenzymes. Confocal images of MCF-7 cells expressing PKCε-EGFP (A) or PKCδ-ECFP (B) stimulated with 40 μM DAG-lactone 5. Time in seconds after stimulation is indicated in each micrograph. The scale bars indicated correspond to 12 μm. (C, D) The percentage of protein localization was measured by a line profile (pixel density) traced in each frame as indicated in the materials and methods section. The resulting net change in PKCε-EGFP or PKCδ-ECFP plasma membrane localization upon DAG-lactone (●) stimulation is expressed as the (Imb-Icyt)/Imb ratio (%) and is represented versus time. The red arrow indicates the stimulation time; Rmax and t1/2 parameters were calculated graphically [15]. The profiles are representative of the results obtained in the cells analyzed (n=12 and 10 for PKCε-EGFP or PKCδ-ECFP, respectively).

Figure 4

Fluorescence quenching. Fluorescence intensities of NBD-PE dye embedded within DMPC vesicles following pre-incubation with DAG-lactones 1–5. Initial fluorescence (at time 0, upon addition of sodium thionite) is defined as 100%.

Figure 5

DSC thermograms. Spectra were acquired following incubation of DAG-lactones 1–5 with DMPC multilamellar vesicles.

Figure 6

Solvent accessible surface of the C1B domain of PKCα(A) and PKCδ(B) in the absence of ligands. They were calculated by using DSVisualizer 2.0 using a probe radius of 1.4 Å. Positively and negatively charged regions are shown in blue and red respectively, while the hydrophobic surface is represented in grey. Amino acidic residues of reference have been labeled to help orientate the molecules. The molecules on the left correspond to a front view with critical W or Y residues labeled. The molecules on the centre correspond to the back view and those in the right correspond to a top view showing the DAG-lactone binding cleft.