AKAP79/150 Interacts with AC8 and Regulates Ca2+-dependent cAMP Synthesis in Pancreatic and Neuronal Systems

Debbie Willoughby,Nanako Masada,Sebastian Wachten,Mario Pagano,Michelle L Halls,Katy L Everett,Antonio Ciruela,Dermot M.F Cooper

doi:10.1074/jbc.m110.120725

Debbie Willoughby, Nanako Masada + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m110.120725

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Abstract

Protein kinase A anchoring proteins (AKAPs) provide the backbone for targeted multimolecular signaling complexes that serve to localize the activities of cAMP. Evidence is accumulating of direct associations between AKAPs and specific adenylyl cyclase (AC) isoforms to facilitate the actions of protein kinase A on cAMP production. It happens that some of the AC isoforms (AC1 and AC5/6) that bind specific AKAPs are regulated by submicromolar shifts in intracellular Ca2+. However, whether AKAPs play a role in the control of AC activity by Ca2+ is unknown. Using a combination of co-immunoprecipitation and high resolution live cell imaging techniques, we reveal an association of the Ca2+-stimulable AC8 with AKAP79/150 that limits the sensitivity of AC8 to intracellular Ca2+ events. This functional interaction between AKAP79/150 and AC8 was observed in HEK293 cells overexpressing the two signaling molecules. Similar findings were made in pancreatic insulin-secreting cells and cultured hippocampal neurons that endogenously express AKAP79/150 and AC8, which suggests important physiological implications for this protein-protein interaction with respect to Ca2+-stimulated cAMP production.

Highlights

Associate with a range of other signaling proteins, including protein kinase C, Epac, phosphatases (e.g. PP2B), and cAMP-dependent phosphodiesterases (e.g. PDE4) as well as a wide range of specific ion channel and receptor subtypes [1, 4]
AKAP79/150 Associates with the N Terminus of AC8—To probe for potential interactions between the Ca2ϩ-stimulated AC8 and AKAPs, a number of HA, Myc, or FLAG-tagged AKAPs were transiently overexpressed in HEK293 cells and screened by GST pull-down assays (Fig. 1)
Comparisons were made for the interaction of each AKAP with GST alone or with GST fused to the N terminus of AC8, the cytosolic C1b domain of AC8, and the C terminus of AC8

Summary

Introduction

Associate with a range of other signaling proteins, including protein kinase C, Epac (exchange protein directly activated by cAMP), phosphatases (e.g. PP2B), and cAMP-dependent phosphodiesterases (e.g. PDE4) as well as a wide range of specific ion channel and receptor subtypes [1, 4]. Measurements of intracellular Ca2ϩ changes showed comparable Ca2ϩ entry upon the addition of 2 mM extracellular Ca2ϩ to store-depleted cells under all four transfection conditions (Fig. 4B), indicating more direct effects of the AKAPs on Ca2ϩ-regulated AC8 activity.

Results

Conclusion