Protein kinase G phosphorylates soluble guanylyl cyclase on serine 64 and inhibits its activity.
OBJECTIVE
Binding of nitric oxide (NO) to soluble guanylyl cyclase (sGC) leads to increased cGMP synthesis that activates cGMP-dependent protein kinase (PKG). Herein, we tested whether sGC activity is regulated by PKG.


RESULTS
Overexpression of a constitutively active form of PKG (DeltaPKG) stimulated (32)P incorporation into the alpha1 subunit. Serine to alanine mutation of putative sites revealed that Ser64 is the main phosphorylation site for PKG. Using a phospho-specific antibody we observed that endogenous sGC phosphorylation on Ser 64 increases in cells and tissues exposed to NO, in a PKG-inhibitable manner. Wild-type (wt) sGC coexpressed with DeltaPKG exhibited lower basal and NO-stimulated cGMP accumulation, whereas the S64A alpha1/beta1 sGC was resistant to the PKG-induced reduction in activity. Using purified sGC we observed that the S64D alpha1 phosphomimetic /beta1 dimer exhibited lower Vmax; moreover, the decrease in Km after NO stimulation was less pronounced in S64D alpha1/beta1 compared to wild-type sGC. Expression of a phosphorylation-deficient sGC showed enhanced responsiveness to endothelium-derived NO, reduced desensitization to acute NO exposure, and allowed for greater VASP phosphorylation.


CONCLUSIONS
We conclude that PKG phosphorylates sGC on Ser64 of the alpha1 subunit and that phosphorylation inhibits sGC activity, establishing a negative feedback loop.
Zhou
Zongmin
Z
Critical Care Department, Evangelismos Hospital, University of Athens School of Medicine, Greece.
Sayed
Nazish
N
Pyriochou
Anastasia
A
Roussos
Charis
C
Fulton
David
D
Beuve
Annie
A
Papapetropoulos
Andreas
A
D000818:Animals; D019556:COS Cells; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002522:Chlorocebus aethiops; D006152:Cyclic GMP / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D025461:Feedback, Physiological; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008840:Microfilament Proteins / Q000378:metabolism; D009154:Mutation; D009569:Nitric Oxide / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine; D015398:Signal Transduction; D000071756:Soluble Guanylyl Cyclase; D013379:Substrate Specificity; D014162:Transfection; D000098662:Vasodilator-Stimulated Phosphoprotein
D015815:Cell Adhesion Molecules; D008840:Microfilament Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D000098662:Vasodilator-Stimulated Phosphoprotein; D009569:Nitric Oxide; D012694:Serine; D017869:Cyclic GMP-Dependent Protein Kinases; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase; D006152:Cyclic GMP