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

1 Centre National de la Recherche Scientifique (CNRS) UMR5232, Centre de Pharmacologie et d'Innovation dans le Diabète, Faculté de Pharmacie, Université de Montpellier 1; 2 Institut National de la Santé et de la Recherche Médicale (INSERM) U376, Centre Hospitalier Universitaire Arnaud-de-Villeneuve; 3 CNRS FRE-3009; and 4 INSERM 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 A