Nitration of Tyrosine 247 Inhibits Protein Kinase G-1α Activity by Attenuating Cyclic Guanosine Monophosphate Binding

Background: PKG-1 nitration plays an important role in the development of pulmonary hypertension. Results: We identified Tyr(247) as the key residue susceptible to nitration and inhibition of PKG-1. Conclusion: Nitration attenuates PKG activity by reducing its affinity for cGMP. Significance: Preventing the nitration of PKG-1 could prevent the phenotypic remodeling in the blood vessels during the development of a number of cardiovascular diseases. The cGMP-dependent protein kinase G-1 (PKG-1) is a downstream mediator of nitric oxide and natriuretic peptide signaling. Alterations in this pathway play a key role in the pathogenesis and progression of vascular diseases associated with increased vascular tone and thickness, such as pulmonary hypertension. Previous studies have shown that tyrosine nitration attenuates PKG-1 activity. However, little is known about the mechanisms involved in this event. Utilizing mass spectrometry, we found that PKG-1 is susceptible to nitration at tyrosine 247 and 425. Tyrosine to phenylalanine mutants, Y247F- and Y425F-PKG-1, were both less susceptible to nitration than WT PKG-1, but only Y247F-PKG-1 exhibited preserved activity, suggesting that the nitration of Tyr(247) is critical in attenuating PKG-1 activity. The overexpression of WT- or Y247F-PKG-1 decreased the proliferation of pulmonary artery smooth muscle cells (SMC), increased the expression of SMC contractile markers, and decreased the expression of proliferative markers. Nitrosative stress induced a switch from a contractile to a synthetic phenotype in cells expressing WT- but not Y247F-PKG-1. An antibody generated against 3-NT-Y247 identified increased levels of nitrated PKG-1 in humans with pulmonary hypertension. Finally, to gain a more mechanistic understanding of how nitration attenuates PKG activity, we developed a homology model of PKG-1. This model predicted that the nitration of Tyr(247) would decrease the affinity of PKG-1 for cGMP, which we confirmed using a [H-3]cGMP binding assay. Our study shows that the nitration of Tyr(247) and the attenuation of cGMP binding is an important mechanism regulating in PKG-1 activity and SMC proliferation/differentiation.