Protein kinase G phosphorylates soluble guanylyl cyclase on serine 64 and inhibits its activity

Abstract

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 (ΔPKG) stimulated 32P incorporation into the α1 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 ΔPKG exhibited lower basal and NO-stimulated cGMP accumulation, whereas the S64A α1/β1 sGC was resistant to the PKG-induced reduction in activity. Using purified sGC we observed that the S64D α1 phosphomimetic /β1 dimer exhibited lower Vmax; moreover, the decrease in Km after NO stimulation was less pronounced in S64D α1/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 α1 subunit and that phosphorylation inhibits sGC activity, establishing a negative feedback loop. © 2008 American Heart Association, Inc.