Open AccessAKAP6 and phospholamban colocalize and interact in HEK‐293T cells and primary murine cardiomyocytes
Author(s) -
Hakem Zadeh Farigol,
Teng Allen C. T.,
Kuzmanov Uros,
Chambers Paige J.,
Tupling Allan R.,
Gramolini Anthony O.
Publication year - 2019
Publication title -
physiological reports
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.918
H-Index - 39
ISSN - 2051-817X
DOI - 10.14814/phy2.14144
Subject(s) - hek 293 cells , phospholamban , colocalization , immunoprecipitation , microbiology and biotechnology , phosphorylation , transfection , endoplasmic reticulum , chemistry , biology , biochemistry , receptor , gene
Phospholamban ( PLN ) is an important Ca 2+ modulator at the sarcoplasmic reticulum ( SR ) of striated muscles. It physically interacts and inhibits sarcoplasmic reticulum Ca 2+ ATP ase ( SERCA 2) function, whereas a protein kinase A ( PKA )‐dependent phosphorylation at its serine 16 reverses the inhibition. The underlying mechanism of this post‐translational modification, however, remains not fully understood. Using publicly available databases, we identified A‐kinase anchoring protein 6 ( AKAP 6) as a candidate that might play some roles in PLN phosphorylation. Immunofluorescence showed colocalization between GFP ‐ AKAP 6 and PLN in transfected HEK ‐293T cells and cultured mouse neonatal cardiomyocytes ( CMNC s). Co‐immunoprecipitation confirmed the functional interaction between AKAP 6 and PLN in HEK ‐293T and isolated adult rat cardiomyocytes in response to isoproterenol stimulation. Functionally, AKAP 6 promoted Ca 2+ uptake activity of SERCA 1 in cotransfected HEK ‐293T cells despite the presence of PLN . These results were further confirmed in adult rat cardiomyocytes. Immunofluorescence showed colocalization of both proteins around the perinuclear region, while protein–protein interaction was corroborated by immunoprecipitation of the nucleus‐enriched fraction of rat hearts. Our findings suggest AKAP 6 as a novel interacting partner to PLN in HEK ‐293T and murine cardiomyocytes.