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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2008;28:1803.)
© 2008 American Heart Association, Inc.

Cell Biology/Signaling

Protein Kinase G Phosphorylates Soluble Guanylyl Cyclase on Serine 64 and Inhibits Its Activity

Zongmin Zhou; Nazish Sayed; Anastasia Pyriochou; Charis Roussos; David Fulton; Annie Beuve; Andreas Papapetropoulos

From the 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.

Correspondence to Andreas Papapetropoulos, PhD, Laboratory of Molecular Pharmacology, Department of Pharmacy, University of Patras, Patras, Greece 26504. 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/β1 sGC was resistant to the PKG-induced reduction in activity. Using purified sGC we observed that the S64D 1 phosphomimetic /β1 dimer exhibited lower Vmax; moreover, the decrease in Km after NO stimulation was less pronounced in S64D 1/β1 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