Abstract 3379: cGMP Specific Phosphodiesterase Type 5A Activity is Regulated by S-nitrosylation at Cys 181
Introduction: cGMP specific phosphodiesterase type 5A (PDE5A) has a key role in limiting the effect of the second messenger cGMP as demonstrated by the use of the specific inhibitor, sildenafil. However, the mechanism by which PDE5 activity is regulated is not fully understood. Recently, it has been reported that PDE5A specifically degrades the pool of cGMP produced by the nitric oxide (NO) stimulated soluble guanylate cyclase. NO is known to regulate other proteins in this pathway through S-nitrosylation, a redox modification where an NO group is covalently added to the sulfur atom of a cysteine. Here we test the hypothesis that PDE5 activity is regulated by S-nitrosylation and determine the critical modified cysteine residues.
Methods: Rates of cGMP hydrolysis were measured by an in vitro activity assay using purified mouse flag-PDE5 treated with the NO-donor, S-Nitrosoglutathione (GSNO, 1–100μM) or control. The S-nitrosylation status of PDE5A was determined by biotin switch assay of cell lysates over expressing flag-PDE5A using the equivalent conditions. After NO modifications were replaced with biotin moieties, labeled proteins or trypsin digested peptides were captured with streptavidin agarose and evaluated by western blot or tandem mass spectrometry, respectively.
Results: PDE5A activity was found to be decreased in a dose dependent manner with NO donor inducing a 25.0 ± 3.8% (n=4, p<0.01) decrease with 100μM GSNO treatment compared to controls. PDE5A was S-nitrosylated by the NO donors in a similar dose dependent manner when analyzed using the biotin switch assay. Subsequent mapping studies identified 2 potential sites of S-nitrosylation at C181 and C210, both of which are located in the N-terminal regulatory domain. Independent mutation of candidate cysteines to serine residues revealed that C181 is more receptive to NO modification than C210. Additionally, the C181S PDE5A was found to be resistant to the NO-donor dependent decrease (0.55 ± 2.3%, p=0.9) in activity while C210S activity was found to be depressed in the presence of 100μM GSNO (28.9 ± 1.7%, p<0.01) (n=4).
Conclusions: These data indicates that PDE5A activity is negatively regulated by S-nitrosylaion at C181 which may contribute to the overall regulation of cGMP signaling in the cell.
This research has received full or partial funding support from the American Heart Association, AHA Mid-Atlantic Affiliate (Maryland, North Carolina, South Carolina, Virginia & Washington, DC).