title: Type II PKAs are anchored to mature insulin secretory granules in INS-1 β-cells and required for cAMP-dependent potentiation of exocytosis.
abstract: Specificity of the cAMP-dependent protein kinase (PKA) pathway relies on an extremely sophisticated compartmentalization mechanism of the kinase within a given cell, based on high-affinity binding of PKA tetramer pools to different A-Kinase Anchoring Proteins (AKAPs). We and others have previously shown that AKAPs-dependent PKA subcellular targeting is a requisite for optimal cAMP-dependent potentiation of insulin exocytosis. We thus hypothesized that a PKA pool may directly anchor to the secretory compartment to potentiate insulin exocytosis. Here, using immunofluorescence analyses combined to subcellular fractionations and purification of insulin secretory granules (ISGs), we identified discrete subpools of type II PKAs, RIIα and RIIβ PKAs, along with the catalytic subunit, physically associated with ISGs within pancreatic insulin-secreting β-cells. Ultrastructural analysis of native rodent β-cells confirmed in vivo the occurrence of PKA on dense-core ISGs. Isoform-selective disruption of binding of PKAs to AKAPs reinforced the requirement of type II PKA isoforms for cAMP potentiation of insulin exocytosis. This granular localization of PKA was of critical importance since siRNA-mediated depletion of either RIIα or RIIβ PKAs resulted in a significant reduction of cAMP-dependent potentiation of insulin release. The present work provides evidence for a previously unrecognized pool of type II PKAs physically anchored to the β-cell ISGs compartment and supports a non-redundant function for type II PKAs during cAMP potentiation of exocytosis.
authors.affiliation: Mammalian Cell Biology Group, Institute of Human Genetics, CNRS-UPR1142, 141 Rue de La Cardonille, 34396, Montpellier, France; University of Montpellier-Faculty of Pharmacy, 15 Avenue Charles Flahaut, 34093, Montpellier, France.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Villalpando
authors.affiliation: Centre Régional d'Imagerie Cellulaire (CRIC), 641 Avenue du Doyen Gaston Giraud, 34093, Montpellier, France.
authors.forename: Chantal
authors.initials: C
authors.lastname: Cazevieille
authors.affiliation: Mammalian Cell Biology Group, Institute of Human Genetics, CNRS-UPR1142, 141 Rue de La Cardonille, 34396, Montpellier, France.
authors.forename: Anne
authors.initials: A
authors.lastname: Fernandez
authors.affiliation: Mammalian Cell Biology Group, Institute of Human Genetics, CNRS-UPR1142, 141 Rue de La Cardonille, 34396, Montpellier, France.
authors.forename: Ned J
authors.initials: NJ
authors.lastname: Lamb
authors.affiliation: Mammalian Cell Biology Group, Institute of Human Genetics, CNRS-UPR1142, 141 Rue de La Cardonille, 34396, Montpellier, France. Electronic address: el-habib.hani@igh.cnrs.fr.
authors.forename: El-Habib
authors.initials: EH
authors.lastname: Hani
mesh_terms: D054758:A Kinase Anchor Proteins / Q000378:metabolism*; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D005089:Exocytosis; D006801:Humans; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism*
chemical_list: D054758:A Kinase Anchor Proteins; D007527:Isoenzymes; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases
keywords: Compartmentalization; Endocrine; Exocytosis; Insulin; Pancreatic; Potentiation; Protein kinase-A; cAMP