Spatial Organization of a cAMP/Ca2+‐Regulated Signaling Complex: A Solution Structural Small‐Angle X‐ray and Neutron Scattering Study of an AKAP79‐Scaffolded Complex Containing Type IIβ PKA and Calcineurin

Abstract

cAMP‐dependent protein kinase (PKA) regulates many cell processes including development, differentiation, and memory. In its resting state, PKA is a tetrameric holoenzyme consisting of a regulatory (R) subunit dimer and two catalytic (C) subunits. Four isoforms of the R‐subunit exist (RIα, RIβ, RIIα, and RIIβ). Although each isoform varies in sequence and quaternary structure, all share a common domain organization. The strength, duration, and specificity of PKA activity within the cell are mostly dependent upon isoform specific subcellular localization. The D/D domain interface of the R‐subunits serves as a docking site for A Kinase Anchoring Proteins (AKAPs) that target PKA to specific cellular locations. In cardiac myocytes and neurons, PKA is localized to Cav1.2 L‐type calcium channels by AKAP79 and up‐regulates calcium influx by phosphorylating the channel's cytosolic C‐terminal tail. AKAP79 also binds the Ca2+/calmodulin‐dependent protein phosphatase type 2B (PP2B, or calcineurin) at a site very close to that of PKA binding. Given the close proximity of the calcineurin and PKA binding sites on AKAP79, calcineurin has been suggested to play roles in regulation of calcium channels, along with regulation of PKA activity itself. Using a variety of biochemical and biophysical techniques, we have isolated and characterized a stable complex of the C‐terminal tail of AKAP79, RIIβ holoenzyme, and calcuneurin (“ARC” for short). We show a stoichiometry of 1:1:1 (AKAP79: RIIβ2C2:CN) using native mass spectroscopy, and SAXS/SANS techniques confirm that this is a compact complex, suggesting a close interaction between PKA and calcineurin when bound to AKAP79. Altogether, we suggest beyond its function of localizing PKA and CN in close spatial proximity to the Cav1.2 L‐type calcium channel, the ARC signalosome fosters extended protein‐protein interactions and regulations that suggest tight spatiotemporal control.Support or Funding InformationJames Hall is a San Diego IRACDA Fellow supported by National Institutes of Health (NIH)/NIGMS K12 GM06852 Award. Support for this work was provided through NIH GM034921.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.