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Spatial Organization of a cAMP/Ca 2+ ‐Regulated Signaling Complex: A Structural Study of an AKAP79‐Scaffolded Complex Containing Type IIβ PKA and Calcineurin
Author(s) -
Hall James,
Copps Jeff,
Barry Rachael,
Zhang Ping,
Blumenthal Donald K.,
Taylor Susan
Publication year - 2019
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.2019.33.1_supplement.478.7
Subject(s) - calcineurin , protein subunit , protein kinase a , chemistry , calmodulin , cytosol , gene isoform , biophysics , protein quaternary structure , cav1.2 , microbiology and biotechnology , kinase , biochemistry , enzyme , biology , medicine , transplantation , 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 (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 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 the 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 calciuneurin (“ARC” for short). We show a stoichiometry of 1:1:1 (AKAP79ct: RIIβ 2 ‐C 2 :CNt) 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 Ca v 1.2 L‐type calcium channel, the ARC signalosome fosters extended protein‐protein interactions and regulations that suggest tight spatiotemporal control. Support for this work was provided through NIH GM034921. Support or Funding Information James Hall is an IRACDA fellow and was supported by NIGMS/NIH award K12GM068524. This work was also supported by a grant from the National Institutes of Health GM034921 to S.S. Taylor. This Research used resources at the High Flux Isotope Reactor and Spallation Neutron Source, which are DOE Office of Science User Facilities operated by Oak Ridge National Laboratory. This work utilized SasView software, originally developed by the DANSE project under NSF award DMR‐0520547. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .