Volume 7 Supplement 1

3rd International Conference on cGMP Generators, Effectors and Therapeutic Implications

Open Access

Protein kinase G phosphorylates soluble guanylyl cyclase and inhibits its activity

  • Andreas Papapetropoulos1, 3Email author,
  • Zongmin Zhou1,
  • Nazish Sayed2,
  • Anastasia Pyriochou3,
  • Charis Roussos1,
  • David Fulton4 and
  • Annie Beuve2
BMC Pharmacology20077(Suppl 1):P45

DOI: 10.1186/1471-2210-7-S1-P45

Published: 25 July 2007

Soluble guanylyl cyclase (sGC) is a receptor for the signaling molecule nitric oxide (NO). Binding of NO to the sGC heme moiety causes up to 400-fold stimulation of its activity, leading to increased cGMP levels and cGMP-dependent protein kinase (PKG) activation. As sGC subunits contain putative phosphorylation sites for PKG, we tested the hypothesis that sGC activity is regulated by PKG. In vitro kinase assays revealed that sGC is a PKG substrate. In vivo, a constitutively active form of PKG stimulated incorporation of 32P into sGC. We then proceeded to map the exact phosphorylation site by generating serine to alanine mutations of putative PKG sites. Wild-type (wt) sGC co-expressed with a constitutively active form of PKG exhibited lower basal and NO-stimulated cGMP accumulation, while the serine to alanine sGC was resistant to the PKG-induced reduction in activity. In line with these observations, a phosphomimetic serine to aspartate sGC mutant showed reduced ability to synthesize cGMP. Using purified sGC and mutants, we observed that the phosphomimetic sGC mutant exhibited lower Vmax both under basal and NO-stimulated conditions and that the decrease in Km after NO stimulation was less pronounced than that for the wt. Moreover, the phosphorylation deficient sGC exhibited reduced desensitization to acute NO exposure and facilitated greater VASP phosphorylation. We conclude that PKG phosphorylates sGC on serine and phosphorylation inhibits sGC activity, establishing a negative feedback loop.

Declarations

Acknowledgements

Supported by the Greek Ministry of Education and the Thorax Foundation.

Authors’ Affiliations

(1)
G.P. Livanos and M. Simou Laboratories, Evangelismos Hospital, Critical Care Department, University of Athens School of Medicine
(2)
Department of Pharmacology, New Jersey Medical School,UDMNJ
(3)
Laboratory of Molecular Pharmacology, Department of Pharmacy, University of Patras
(4)
Vascular Biology Center, Medical College of Georgia

Copyright

© Papapetropoulos et al; licensee BioMed Central Ltd. 2007

This article is published under license to BioMed Central Ltd.

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