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

• Published in: The Journal of biological chemistry 2014-03, Vol.289 (11), p.7948-7961
• Main Authors: Aggarwal, Saurabh, Gross, Christine M., Rafikov, Ruslan, Kumar, Sanjiv, Fineman, Jeffrey R., Ludewig, Britta, Jonigk, Danny, Black, Stephen M.
• Format: Article
• Language: English
• Subjects: Adult; Animals; Aorta - cytology; Cardiovascular Diseases - metabolism; Catalytic Domain; Cell Differentiation; Cell Proliferation; Cells, Cultured; Cyclic GMP (cGMP); Cyclic GMP - chemistry; Cyclic GMP-Dependent Protein Kinase Type I - antagonists & inhibitors; Cyclic GMP-Dependent Protein Kinase Type I - metabolism; Enzyme Catalysis; Female; HEK293 Cells; Humans; Male; Mass Spectrometry; Mass Spectrometry (MS); Middle Aged; Models, Molecular; Molecular Modeling; Myocytes, Smooth Muscle - metabolism; Nitrogen - chemistry; Peroxynitrite; Peroxynitrous Acid - chemistry; Protein Binding; Protein Kinase G (PKG); Protein Structure and Folding; Sheep; Tyrosine - chemistry; Young Adult
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.534313

The cGMP-dependent protein kinase G-1α (PKG-1α) 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-1α activity. However, little is known about the mechanisms involved in this event. Utilizing mass spectrometry, we found that PKG-1α is susceptible to nitration at tyrosine 247 and 425. Tyrosine to phenylalanine mutants, Y247F- and Y425F-PKG-1α, were both less susceptible to nitration than WT PKG-1α, but only Y247F-PKG-1α exhibited preserved activity, suggesting that the nitration of Tyr247 is critical in attenuating PKG-1α activity. The overexpression of WT- or Y247F-PKG-1α 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-1α. An antibody generated against 3-NT-Y247 identified increased levels of nitrated PKG-1α 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-1α. This model predicted that the nitration of Tyr247 would decrease the affinity of PKG-1α for cGMP, which we confirmed using a [3H]cGMP binding assay. Our study shows that the nitration of Tyr247 and the attenuation of cGMP binding is an important mechanism regulating in PKG-1α activity and SMC proliferation/differentiation. Background: PKG-1α nitration plays an important role in the development of pulmonary hypertension. Results: We identified Tyr247 as the key residue susceptible to nitration and inhibition of PKG-1α. Conclusion: Nitration attenuates PKG activity by reducing its affinity for cGMP. Significance: Preventing the nitration of PKG-1α could prevent the phenotypic remodeling in the blood vessels during the development of a number of cardiovascular diseases.