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This Article Full Text Full Text (PDF) All Versions of this Article: 296/2/C306    most recent 00216.2008v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Faruque, O. M. Articles by Hani, E.-H. Search for Related Content PubMed PubMed Citation Articles by Faruque, O. M. Articles by Hani, E.-H.

METHODS IN CELL PHYSIOLOGY

Cell-permeable peptide-based disruption of endogenous PKA-AKAP complexes: a tool for studying the molecular roles of AKAP-mediated PKA subcellular anchoring

¹Centre National de la Recherche Scientifique (CNRS) UMR5232, Centre de Pharmacologie et d'Innovation dans le Diabète, Faculté de Pharmacie, Université de Montpellier 1; ²Institut National de la Santé et de la Recherche Médicale (INSERM) U376, Centre Hospitalier Universitaire Arnaud-de-Villeneuve; ³CNRS FRE-3009; and ⁴INSERM U896, Centre Régional de Lutte Contre le Cancer, Val d'Aurelle-Paul Lamarque, Montpellier, France

Submitted 21 April 2008 ; accepted in final form 3 December 2008

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 β-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 β-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.

subcellular compartmentalization; signal transduction; cAMP; pancreatic β-cells; insulin exocytosis; protein kinase A; A-kinase achoring protein