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PKA and Calcineurin Scaffolding on AKAP79: A Small Angle Xray/Neutron Scattering Profile
Author(s) -
Hall James,
Copps Jeffrey,
Zhang Ping,
Heck Albert,
Heller William,
Blumenthal Don,
Taylor Susan
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.856.6
Subject(s) - calcineurin , protein subunit , gene isoform , protein kinase a , cytosol , calmodulin , biophysics , protein quaternary structure , chemistry , microbiology and biotechnology , docking (animal) , cav1.2 , scaffold protein , fkbp , kinase , biochemistry , biology , enzyme , signal transduction , medicine , transplantation , nursing , gene
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 (RIa, RIb, RIIa, and RIIb). 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 Ca v 1.2 L‐type calcium channels by AKAP79 and up‐regulates calcium influx by phosphorylating the channel's cytosolic C‐terminal tail. AKAP79 also binds the Ca 2+ /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, RIIb holoenzyme, and calcuneurin (“ARC” for short). We show a stoichiometry of 1:1:1 (AKAP79: RIIb 2 C 2 :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. Furthermore, we have identified a functional interaction between RIIb and CN using peptide arrays. It involves the flexible linker regions of RIIb including both of LxVP and RRxSV motifs. Altogether, we suggest beyond its function of localizing PKA and CN in close spatial proximity to the Ca v 1.2 L‐type calcium channel, the ARC signalosome fosters extended protein‐protein interactions and regulations that provide tight spatiotemporal control. Support or Funding Information Support for this work was provided through NIH GM034921