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. 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.