期刊
AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY
卷 296, 期 2, 页码 C306-C316出版社
AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajpcell.00216.2008
关键词
subcellular compartmentalization; signal transduction; cAMP; pancreatic beta-cells; insulin exocytosis; protein kinase A; A-kinase achoring protein
资金
- Centre National de la Recherche Scientifique
- University of Montpellier
- Association de Recherche sur le Diabete via the Institut National de la Sante et de la Recherche Me Medicale/Programme National de la Recherche sur le Diabete Program
- French Ministry of Foreign Affairs (through the Egide Foundation)
- European Foundation for the Study of Diabetes/European Association for the Study of Diabetes
Faruque OM, Le-Nguyen D, Lajoix A-D, Vives E, Petit P, Bataille D, Hani EH. Cell-permeable peptide-based disruption of endogenous PKA-AKAP complexes: a tool for studying the molecular roles of AKAP-mediated PKA subcellular anchoring. Am J Physiol Cell Physiol 296: C306-C316, 2009. First published December 10, 2008; doi:10.1152/ajpcell.00216.2008.-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 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.
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