Cyclic GMP-dependent Protein Kinase*

Cyclic GMP (cGMP) inhibits intracellular calcium ([Ca2+Ii) mobilization in vascular smooth muscle cells by a mechanism that is not well understood. Because several studies suggest that cGMP inhibits inositol 1,4,5trisphosphate (IP,) action, we examined the effects of cGMP-dependent protein kinase on IP, receptor phosphorylation. The purified IP, receptor was phosphorylated using either the cGMPor CAMP-dependent protein kinase in vitro. Phosphorylation was time-dependent and stoichiometric using both kinases. Two-dimensional phosphopeptide mapping, high performance liquid chromatography analysis, and amino acid analysis showed that identical sites were phosphorylated using either kinase, and identified serine 1755 as the site of phosphorylation. The synthetic peptide corresponding to serine 1755 (GRRESLTSFG) was phosphorylated with a K , in the range of 30-40 PM by both kinases. The kinetic analysis revealed that this peptide substrate is the best substrate described for cGMP kinase to date. Vascular smooth muscle cells prelabeled with [32Plorthophosphate and treated with atrial natriuretic peptide or sodium nitroprusside to elevate cGMP also resulted in increased labeling of the IP, receptor. Phosphorylation of IP, receptor by cGMP kinase may regulate the function of IP, receptor in vascular smooth muscle cells and contribute to the effect of cGMP to regulate intracellular calcium levels.