Persistent Activation of cGMP-Dependent Protein Kinase by a Nitrated Cyclic Nucleotide via Site Specific Protein S-Guanylation

Abstract

8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) is a nitrated derivative of guanosine 3',5'-cyclic monophosphate (cGMP) formed endogenously under conditions associated with production of both reactive oxygen species and nitric oxide. It acts as an electrophilic second messenger in the regulation of cellular signaling by inducing a post-translational modification of redox-sensitive protein thiols via covalent adduction of cGMP moieties to protein thiols (protein S-guanylation). Here, we demonstrate that 8-nitro-cGMP potentially S-guanylates thiol groups of cGMP-dependent protein kinase (PKG), the enzyme that serves as one of the major receptor proteins for intracellular cGMP and controls a variety of cellular responses. S-Guanylation of PKG was found to occur in a site specific manner; Cys42 and Cys195 were the susceptible residues among 11 Cys residues. Importantly, S-guanylation at Cys195, which is located in the high-affinity cGMP binding domain of PKG, causes persistent enzyme activation as determined by in vitro kinase assay as well as by an organ bath assay. In vivo, S-guanylation of PKG was demonstrated to occur in mice without any specific treatment and was significantly enhanced by lipopolysaccharide administration. These findings warrant further investigation in terms of the physiological and pathophysiological roles of S-guanylation-dependent persistent PKG activation.

Synthesis and Characterization of 8-Nitroguanosine 3′,5′-Cyclic Monophosphorothioate Rp-Isomer as a Potent Inhibitor of Protein Kinase G1α

“…4,5) We previously determined the lipophilicity of 8-nitro-cGMP and 8-bromocGMP on the basis of octanol/water partition coefficient measurements, and we found 8-nitro-cGMP to be less lipophilic than 8-bromo-cGMP. 12) Consistent with this result, we found that tissue levels of Rp-8-nitro-cGMPS were lower than those of Rp-8-bromo-cGMPS after mouse aortic rings were incubated with those inhibitors (Fig. 7).…”

“…12) In brief, 1 µg/mL PKG1α (total 10 ng) was incubated with Rp-8-bromo-cGMP or Rp-8-nitro-cGMP at concentrations of 1, 10, or 100 µM for 90 min before the kinase experiments. PKG1α reaction mixtures with inhibitors were then used as kinase mixes during incubation with cGMP at different concentrations (0, 1, 10, or 100 µM) in 20 mM TrisHCl (pH 7.4) containing 16 mM MgCl 2 , 10 mM dithiothreitol, 0.1 mM ATP, and 60 µM Glasstide (Merck Millipore) at 37°C for 30 min.…”

“…Accumulating evidence suggested that PKG inhibition may be a potential target in the treatment of certain diseases such as bacterial toxin-induced diarrhea and cancers of the lung, colon, breast, and ovary. 4,5) We discovered endogenous formation of the nitrated cGMP derivative 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP) under conditions associated with excess production of both nitric oxide (NO) and reactive oxygen species in mammalian cells [7][8][9][10][11][12][13][14][15][16][17] as well as in plants. 18,19) Biochemical analyses revealed that 8-nitro-cGMP can activate PKG1α to a similar extent as native cGMP can.…”

“…18,19) Biochemical analyses revealed that 8-nitro-cGMP can activate PKG1α to a similar extent as native cGMP can. 9,12) Although native cGMP is degraded by phosphodiesterase to form guanosine 5′-mono-phosphate, 8-nitro-cGMP was resistant to phosphodiesterasemediated degradation. 9) Furthermore, 8-nitro-cGMP covalently bound to cysteine residues in PKG1α through the formation of cysteinyl-cGMP adducts in the enzyme (i.e., the process called protein S-guanylation).…”

“…9) Furthermore, 8-nitro-cGMP covalently bound to cysteine residues in PKG1α through the formation of cysteinyl-cGMP adducts in the enzyme (i.e., the process called protein S-guanylation). 12) Two S-guanylation sites were identified in PKG1α: Cys42, near the autoinhibitory domain, and Cys195, located at the high-affinity cGMP-binding site.…”