Abstract 11361: S-nitrosation of cGMP Specific Phosphodiesterase Type 5A at C181 Inhibits cGMP Binding in the Regulatory GAF A Domain Affecting Enzyme Activity
Introduction: cGMP specific phosphodiesterase type 5A (PDE5A) plays a key role in the negative feedback of the second messenger cGMP. PDE5A is activated by Protein Kinase G (PKG) phosphorylation (S92) and cGMP binding to GAF regulatory domains in the N-terminus and normally targets the cGMP pool generated by nitric oxide (NO)-stimulated soluble guanylate cyclase. NO can also directly modulate proteins via S-nitrosation (SNO), the covalent attachment of an NO to a cysteine thiol. Here we tested the hypothesis that PDE5 is also regulated by SNO, identified the targeted residues, and tested the impact of their modification.
Methods: SNO-modified residues were detected and identified on PDE5A using the biotin switch assay coupled with mass spectrometry. Activity and binding studies were performed using purified mouse flag-PDE5A treated with the NO-donor, S-nitrosoglutathione (GSNO, 1-100 µM) or control compounds.
Results: Treatment of purified flag-PDE5A with 100 µM GSNO produced a 25% decrease in cGMP hydrolysis compared to control (1.04 ± 0.024 vs. 1.38 ± 0.031 nmoles Pi/µg/min, p<0.01, n=4). PDE5A was determined to be SNO-modified in vitro and in vivo and mapped to residues C181 and C210 in the regulatory GAF A domain. Allosteric binding of cGMP at the GAF A domain is known to dramatically increase hydrolytic activity. Independent mutation of these residues to serine revealed C181 is more reactive to SNO than C210. C181S PDE5A was resistant to the NO-donor dependent decrease in activity (1.37 ± 0.03 nmoles Pi/µg/min) while C210S PDE5A activity showed an equivalent inhibition to wild type (0.96 ± 0.02 nmoles Pi/µg/min). S-nitrosation at C181 was found to inhibit allosteric cGMP binding at the GAF A domain producing a 4 fold increase in KD (60 ± 9.9 nM vs. 245 ± 28 nM) and a 25% decrease in Bmax (9.2 ± 0.24 vs. 6.9 ± 0.16 pmoles/µg) while the C181S mutant was unaffected (KD, 79 ± 9.2 nM; Bmax, 8.7 ± 0.20 pmoles/µg) (n=3).
Conclusions: Based on these data PDE5 is inhibited directly by SNO-modification at C181. We propose this modification simultaneously reduces catabolic activity and releases cGMP from the GAF A domain increasing the available concentration. These effects favor activation of PKG and may contribute to the overall regulation of cGMP signaling in the cell.
- © 2011 by American Heart Association, Inc.