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Regulation of SERCA2 activity by PDE3A in human myocardium: Phosphorylation-dependent interaction of PDE3A1 with SERCA2.

Ahmad, Faiyaz; Shen, Weixing; Vandeput, Fabrice; Szabo-Fresnais, Nicolas; Krall, Judith; Degerman, Eva LU ; Goetz, Frank; Klussmann, Enno; Movsesian, Matthew and Manganiello, Vincent (2015) In Journal of Biological Chemistry 290(11). p.6763-6776
Abstract
PDE3 regulates cAMP-mediated signaling in the heart, and PDE3 inhibitors augment contractility in patients with heart failure. Studies in mice showed that PDE3A, not PDE3B, is the subfamily responsible for these inotropic effects, and that murine PDE3A1 associates with SERCA2, PLB and AKAP18 in a multi-protein signalosome in human SR. Immunohistochemical staining demonstrated that PDE3A co-localizes in Z-bands of human cardiac myocytes with desmin, SERCA2, PLB and AKAP18. In human SR fractions, cAMP increased PLB phosphorylation and SERCA2 activity; this was potentiated by PDE3 inhibition but not by PDE4 inhibition. During gel-filtration chromatography of solubilized SR membranes, PDE3 activity was recovered in distinct HMW and LMW peaks.... (More)
PDE3 regulates cAMP-mediated signaling in the heart, and PDE3 inhibitors augment contractility in patients with heart failure. Studies in mice showed that PDE3A, not PDE3B, is the subfamily responsible for these inotropic effects, and that murine PDE3A1 associates with SERCA2, PLB and AKAP18 in a multi-protein signalosome in human SR. Immunohistochemical staining demonstrated that PDE3A co-localizes in Z-bands of human cardiac myocytes with desmin, SERCA2, PLB and AKAP18. In human SR fractions, cAMP increased PLB phosphorylation and SERCA2 activity; this was potentiated by PDE3 inhibition but not by PDE4 inhibition. During gel-filtration chromatography of solubilized SR membranes, PDE3 activity was recovered in distinct HMW and LMW peaks. HMW peaks contained PDE3A1 and PDE3A2, while LMW peaks contained PDE3A1, PDE3A2 and PDE3A3. Western blotting showed that endogenous HMW PDE3A1 was the principal PKA-phosphorylated isoform. Phosphorylation of endogenous PDE3A by rPKAc increased cAMP-hydrolytic activity, correlated with shift of PDE3A from LMW to HMW peaks, and increased co-immumoprecipitation of SERCA2, cav3, PKARII, PP2A and AKAP18 with PDE3A. In experiments with recombinant proteins, phosphorylation of rhPDE3A isoforms by rPKAc increased co-immumoprecipitation with rSERCA2 and rAKAP18. Deletion of the rhPDE3A1/PDE3A2 N-terminus blocked interactions with rSERCA2. Serine-to-alanine substitutions identified S292/S293, a site unique to hPDE3A1, as the principal site regulating its interaction with SERCA2. These results indicate that phosphorylation of hPDE3A1 at a PKA site in its unique N-terminal extension promotes its incorporation into SERCA2/AKAP18 signalosomes, where it regulates a discrete cAMP pool that controls contractility by modulating phosphorylation-dependent protein-protein interactions, PLB phosphorylation and SERCA2 activity. (Less)
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Contribution to journal
publication status
published
subject
in
Journal of Biological Chemistry
volume
290
issue
11
pages
6763 - 6776
publisher
ASBMB
external identifiers
  • pmid:25593322
  • wos:000350991500013
  • scopus:84925012261
ISSN
1083-351X
DOI
10.1074/jbc.M115.638585
language
English
LU publication?
yes
id
378379f9-2886-4c4d-8cd5-337b34f7499e (old id 5040324)
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http://www.ncbi.nlm.nih.gov/pubmed/25593322?dopt=Abstract
date added to LUP
2015-02-03 17:58:14
date last changed
2017-07-02 03:06:33
@article{378379f9-2886-4c4d-8cd5-337b34f7499e,
  abstract     = {PDE3 regulates cAMP-mediated signaling in the heart, and PDE3 inhibitors augment contractility in patients with heart failure. Studies in mice showed that PDE3A, not PDE3B, is the subfamily responsible for these inotropic effects, and that murine PDE3A1 associates with SERCA2, PLB and AKAP18 in a multi-protein signalosome in human SR. Immunohistochemical staining demonstrated that PDE3A co-localizes in Z-bands of human cardiac myocytes with desmin, SERCA2, PLB and AKAP18. In human SR fractions, cAMP increased PLB phosphorylation and SERCA2 activity; this was potentiated by PDE3 inhibition but not by PDE4 inhibition. During gel-filtration chromatography of solubilized SR membranes, PDE3 activity was recovered in distinct HMW and LMW peaks. HMW peaks contained PDE3A1 and PDE3A2, while LMW peaks contained PDE3A1, PDE3A2 and PDE3A3. Western blotting showed that endogenous HMW PDE3A1 was the principal PKA-phosphorylated isoform. Phosphorylation of endogenous PDE3A by rPKAc increased cAMP-hydrolytic activity, correlated with shift of PDE3A from LMW to HMW peaks, and increased co-immumoprecipitation of SERCA2, cav3, PKARII, PP2A and AKAP18 with PDE3A. In experiments with recombinant proteins, phosphorylation of rhPDE3A isoforms by rPKAc increased co-immumoprecipitation with rSERCA2 and rAKAP18. Deletion of the rhPDE3A1/PDE3A2 N-terminus blocked interactions with rSERCA2. Serine-to-alanine substitutions identified S292/S293, a site unique to hPDE3A1, as the principal site regulating its interaction with SERCA2. These results indicate that phosphorylation of hPDE3A1 at a PKA site in its unique N-terminal extension promotes its incorporation into SERCA2/AKAP18 signalosomes, where it regulates a discrete cAMP pool that controls contractility by modulating phosphorylation-dependent protein-protein interactions, PLB phosphorylation and SERCA2 activity.},
  author       = {Ahmad, Faiyaz and Shen, Weixing and Vandeput, Fabrice and Szabo-Fresnais, Nicolas and Krall, Judith and Degerman, Eva and Goetz, Frank and Klussmann, Enno and Movsesian, Matthew and Manganiello, Vincent},
  issn         = {1083-351X},
  language     = {eng},
  number       = {11},
  pages        = {6763--6776},
  publisher    = {ASBMB},
  series       = {Journal of Biological Chemistry},
  title        = {Regulation of SERCA2 activity by PDE3A in human myocardium: Phosphorylation-dependent interaction of PDE3A1 with SERCA2.},
  url          = {http://dx.doi.org/10.1074/jbc.M115.638585},
  volume       = {290},
  year         = {2015},
}