title: Cell-permeable peptide-based disruption of endogenous PKA-AKAP complexes: a tool for studying the molecular roles of AKAP-mediated PKA subcellular anchoring.
abstract: Stimulation of numerous G protein-coupled receptors leads to the elevation of intracellular concentrations of cAMP, which subsequently activates the PKA pathway. Specificity of the PKA signaling module is determined by a sophisticated subcellular targeting network that directs the spatiotemporal activation of the kinase. This specific compartmentalization mechanism occurs through high-affinity interactions of PKA with A-kinase anchoring proteins (AKAPs), the role of which is to target the kinase to discrete subcellular microdomains. Recently, a peptide designated "AKAPis" has been proposed to competitively inhibit PKA-AKAP interactions in vitro. We therefore sought to characterize a cell-permeable construct of the AKAPis inhibitor and use it as a tool to characterize the impact of PKA compartmentalization by AKAPs. Using insulin-secreting pancreatic beta-cells (INS-1 cells), we showed that TAT-AKAPis (at a micromolar range) dose dependently disrupted a significant fraction of endogenous PKA-AKAP interactions. Immunoflurescent analysis also indicated that TAT-AKAPis significantly affected PKA subcellular localization. Furthermore, TAT-AKAPis markedly attenuated glucagon-induced phosphorylations of p44/p42 MAPKs and cAMP response element binding protein, which are downstream effectors of PKA. In parallel, TAT-AKAPis dose dependently inhibited the glucagon-induced potentiation of insulin release. Therefore, AKAP-mediated subcellular compartmentalization of PKA represents a key mechanism for PKA-dependent phosphorylation events and potentiation of insulin secretion in intact pancreatic beta-cells. More interestingly, our data highlight the effectiveness of the cell-permeable peptide-mediated approach to monitoring in cellulo PKA-AKAP interactions and delineating PKA-dependent phosphorylation events underlying specific cellular responses.
authors.affiliation: CNRS UMR-5232, Faculté de Pharmacie, Bat. D, 15 Ave. Charles Flahaut, Montpellier 34093 Cedex 5, France.
authors.forename: Omar M
authors.initials: OM
authors.lastname: Faruque
authors.forename: Dung
authors.initials: D
authors.lastname: Le-Nguyen
authors.forename: Anne-Dominique
authors.initials: AD
authors.lastname: Lajoix
authors.forename: Eric
authors.initials: E
authors.lastname: Vives
authors.forename: Pierre
authors.initials: P
authors.lastname: Petit
authors.forename: Dominique
authors.initials: D
authors.lastname: Bataille
authors.forename: El-Habib
authors.initials: EH
authors.lastname: Hani
mesh_terms: D054758:A Kinase Anchor Proteins / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002463:Cell Membrane Permeability; D000242:Cyclic AMP / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D054754:Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005934:Glucagon / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D047468:Immunoprecipitation; D007328:Insulin / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000201:enzymology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D021381:Protein Transport; D051381:Rats; D015398:Signal Transduction; D013997:Time Factors; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000378:metabolism
chemical_list: D054758:A Kinase Anchor Proteins; C086194:Akap8 protein, rat; D017362:Cyclic AMP Response Element-Binding Protein; D054754:Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit; D007328:Insulin; D009687:Nuclear Proteins; D010446:Peptide Fragments; D054322:tat Gene Products, Human Immunodeficiency Virus; D005934:Glucagon; D000242:Cyclic AMP; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D005947:Glucose