The PKC-ζ pseudosubstrate peptide induces glutamate release from retinal pigment epithelium cells through kinase- independent activation of Best1

Abstract

Aims: The retinal pigment epithelium (RPE) is a highly specialized cell monolayer, that plays a key role in the maintenance of photoreceptor function and the blood-retina barrier (BRB). In this study, we found that a myristoylated pseudosubstrate of PKC-ζ (PKCζ PS), considered as a PKC-ζ inhibitor, plays a distinct role in RPE.

Main methods: We demonstrated that PKCζ PS stimulates the release of Glutamate (Glu) using in vitro³H-Glutamate release experiments. By western blot, kinase assays, and Fluoresence Ca⁺² Concentration Measurements, we determined the cellular mechanisms involved in such release.

Key findings: Surprisingly, PKCζ PS has no effect on either phosphorylation of T560, essential for catalytic activity, nor it has an effect on kinase activity. It induces the dose-dependent release of Glu by increasing intracellular Ca⁺² levels. Interestingly, this release was not observed upon stimulation by other non-competitive PKC-ζ inhibitors. We here demonstrated that the PKCζ PS stimulates the release of Glutamate from RPE by activating the Ca²⁺-dependent Cl channel Bestrophin 1 (Best1).

Significance: These results question PKCζ PS specificity as an inhibitor of this enzyme. Furthermore, the present results underline the relevance of clarifying the molecular mechanisms involved in glutamate release from the retina under conditions derived from excitotoxic stimuli.

Keywords: 1-transpyrrolidine-2,-4-dicarboxylic acid (trans-PDC); 5-Nitro-2-(3-phenylpropylamino) benzoate (NPPB); Amino-oxyacetic acid; Calcium; Cell signaling; Chloride channels; Fluo-4 AM; Ionomycin; Isopropyl 2-amino-4-(3,4-dichlorophenyl)-3-thiophenecarboxylate (dichloro); Isopropyl 2-amino-4-(3,4-dimethoxyphenyl)-3-thiophenecarboxylate (dimethoxy); Methionine sulfoximine; Nifedipine; Niflumic acid; Peptide inhibitors; Retina.