Protein kinase C phosphorylates the inositol 1,4,5-trisphosphate receptor type 2 and decreases the mobilization of Ca2+in pancreatoma AR4-2J cells

Abstract

In non-excitable cells, the inositol 1,4,5-trisphosphate receptor channel, which plays a major (IP3R) is an intracellular Ca2+ role in Ca2+ signalling. Three isoforms of IP3R have been identified (IP3R-1, IP3R-2 and IP3R-3) and most cell types express different proportions of each isoform. The differences between the pharmacological and functional properties of the various isoforms of IP3R are poorly understood. AR4-2J cells, which express almost exclusively (~86%) the IP3R-2, represent an interesting model to study this particular isoform. Here, we investigated a regulatory mechanism by which protein kinase C (PKC) influences IP3R-2-mediated Ca2+ release. Using an immunoprecipitation approach, we confirmed that AR4-2J cells express almost exclusively the IP3R-2 isoform. Using an in vitro phosphorylation assay, we showed that the immunopurified IP3R-2 was efficiently phosphorylated by exogenous PKC. In intact AR4-2J cells metabolically labelled with 32Pi, we showed that phorbol-12-myristate-13-acetate (PMA) and Ca2+ mobilizing agonists cause the phosphorylation of IP3R-2. In saponin-permeabilized AR4-2J cells, IP3-induced Ca2+ release was reduced after a pre-treatment with PMA or with exogenous PKC. PMA also reduced the Ca2+ response of intact AR4-2J cells stimulated with carbachol and epidermal growth factor, two agonists that use different receptor types to activate phospholipase C. These results demonstrate that PKC decreases the Ca2+mobilizing activity of IP3R-2 and thus exerts a negative feedback on the agonists-induced Ca2+ response of AR4-2J cells.