on July 17, 2008
Published online before print July 17, 2008, doi: 10.1161/ATVBAHA.108.165043
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Submitted on February 26, 2008
Accepted on July 2, 2008
Protein Kinase G Phosphorylates Soluble Guanylyl Cyclase on Serine 64 and Inhibits Its Activity
Zongmin Zhou ;
From the G.P. Livanos and M. Simou Laboratories, Evangelismos Hospital, Critical Care Department (Z.Z., C.R., A.P.), University of Athens School of Medicine, Greece; the Department of Pharmacology (N.S., A.B.), New Jersey Medical School, UDMNJ, Newark; the Laboratory of Molecular Pharmacology, Department of Pharmacy (A.P.), University of Patras, Greece; and the Vascular Biology Center (D.F.), Medical College of Georgia, Augusta.
* To whom correspondence should be addressed. E-mail: apapapet{at}upatras.gr.
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.
Methods and Results—Overexpression of a constitutively active form of PKG (
PKG) stimulated 32P incorporation into the 
1 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 PKG exhibited lower basal and NO-stimulated cGMP accumulation, whereas the S64A 1/
1 sGC was resistant to the PKG-induced reduction in activity. Using purified sGC we observed that the S64D 1 phosphomimetic /1 dimer exhibited lower Vmax; moreover, the decrease in Km after NO stimulation was less pronounced in S64D 1/1 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 1 subunit and that phosphorylation inhibits sGC activity, establishing a negative feedback loop.
Key words: cGMP • soluble guanylyl cyclase • PKG • nitric oxide • phosphorylation
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