Abstract
Diacylglycerol (DAG) is a key lipid second messenger downstream of cellular receptors that binds to the C1 domain in many regulatory proteins. Protein kinase C (PKC) isoforms constitute the most prominent family of signaling proteins with DAG-responsive C1 domains, but six other families of proteins, including the chimaerins, Ras-guanyl nucleotide-releasing proteins (RasGRPs), and Munc13 isoforms, also play important roles. Their significant involvement in cancer, immunology, and neurobiology has driven intense interest in the C1 domain as a therapeutic target. As with other classes of targets, however, a key issue is the establishment of selectivity. Here, using [3H]phorbol 12,13-dibutyrate ([3H]PDBu) competition binding assays, we found that a synthetic DAG-lactone, AJH-836, preferentially binds to the novel PKC isoforms PKCδ and PKCϵ relative to classical PKCα and PKCβII. Assessment of intracellular translocation, a hallmark for PKC activation, revealed that AJH-836 treatment stimulated a striking preferential redistribution of PKCϵ to the plasma membrane relative to PKCα. Moreover, unlike with the prototypical phorbol ester phorbol 12-myristate 13-acetate (PMA), prolonged exposure of cells to AJH-836 selectively down-regulated PKCδ and PKCϵ without affecting PKCα expression levels. Biologically, AJH-836 induced major changes in cytoskeletal reorganization in lung cancer cells, as determined by the formation of membrane ruffles, via activation of novel PKCs. We conclude that AJH-836 represents a C1 domain ligand with PKC-activating properties distinct from those of natural DAGs and phorbol esters. Our study supports the feasibility of generating selective C1 domain ligands that promote novel biological response patterns.
Highlights
Diacylglycerol (DAG) is a key lipid second messenger downstream of cellular receptors that binds to the C1 domain in many regulatory proteins
The C1 domains from Protein kinase C (PKC) and other proteins with phorbol ester/DAG– binding capabilities possess an overall similar globular structure, stabilized by twin zinc fingers, with the phorbol ester/DAG– binding site formed from a hydrophilic cavity bounded by a hydrophobic rim
Our initial objective was to examine the ability of AJH836 to bind to recombinant PKC␣, PKCII, PKC␦, and PKC⑀ under parallel conditions using a competition [203H]phorbol 12,13-dibutyrate ([3H]PDBu) binding assay with phosphatidylserine vesicles as cofactor [5]
Summary
Extensive synthetic efforts have generated DAG-lactones substituted with a diversity of saturated and unsaturated alkyl and aryl chains at the sn-1 and sn-2 positions on the DAGlactone [21, 26]. Our initial objective was to examine the ability of AJH836 to bind to recombinant PKC␣, PKCII, PKC␦, and PKC⑀ (four of the main phorbol ester–responsive PKCs expressed in cancer cells) under parallel conditions using a competition [203H]phorbol 12,13-dibutyrate ([3H]PDBu) binding assay with phosphatidylserine vesicles as cofactor [5]. In vitro binding assays using reconstituted phospholipid vesicles will necessarily fail to capture the full range of differences in C1 domain ligand recognition occurring in a cellular context To tackle this issue, we examined subcellular translocation of the individual PKC isoforms expressed in cells as GFP-fused proteins.
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