title: 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 (IP(3)R) is an intracellular Ca(2+) role in Ca(2+) signalling. Three isoforms of IP(3)R have been identified (IP(3)R-1, IP(3)R-2 and IP(3)R-3) and most cell types express different proportions of each isoform. The differences between the pharmacological and functional properties of the various isoforms of IP(3)R are poorly understood. AR4-2J cells, which express almost exclusively (~86%) the IP(3)R-2, represent an interesting model to study this particular isoform. Here, we investigated a regulatory mechanism by which protein kinase C (PKC) influences IP(3)R-2-mediated Ca(2+) release. Using an immunoprecipitation approach, we confirmed that AR4-2J cells express almost exclusively the IP(3)R-2 isoform. Using an in vitro phosphorylation assay, we showed that the immunopurified IP(3)R-2 was efficiently phosphorylated by exogenous PKC. In intact AR4-2J cells metabolically labelled with (32)Pi, we showed that phorbol-12-myristate-13-acetate (PMA) and Ca(2+) mobilizing agonists cause the phosphorylation of IP(3)R-2. In saponin-permeabilized AR4-2J cells, IP(3)-induced Ca(2+) release was reduced after a pre-treatment with PMA or with exogenous PKC. PMA also reduced the Ca(2+) 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 Ca(2+)mobilizing activity of IP(3)R-2 and thus exerts a negative feedback on the agonists-induced Ca(2+) response of AR4-2J cells.
authors.affiliation: Department of Pharmacology, Faculty of Medicine and Health Sciences, University of Sherbrooke, 3001 12th Avenue North, Sherbrooke, Quebec, Canada J1H 5N4.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Arguin
authors.forename: Yannik
authors.initials: Y
authors.lastname: Regimbald-Dumas
authors.forename: Marc-Olivier
authors.initials: MO
authors.lastname: Fregeau
authors.forename: Annabelle Z
authors.initials: AZ
authors.lastname: Caron
authors.forename: Gaetan
authors.initials: G
authors.lastname: Guillemette
mesh_terms: D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000032:analysis / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism*; D045744:Cell Line, Tumor; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism / Q000494:pharmacology; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism*; D010179:Pancreas / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D013050:Spectrometry, Fluorescence
chemical_list: D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D015544:Inositol 1,4,5-Trisphosphate; D011493:Protein Kinase C; D002118:Calcium