학술논문

Nitration of Tyrosine 247 Inhibits Protein Kinase G-1α Activity by Attenuating Cyclic Guanosine Monophosphate Binding*
Document Type
article
Source
Journal of Biological Chemistry. 289(11)
Subject
Medical Physiology
Biomedical and Clinical Sciences
Aetiology
2.1 Biological and endogenous factors
Cardiovascular
Adult
Animals
Aorta
Cardiovascular Diseases
Catalytic Domain
Cell Differentiation
Cell Proliferation
Cells
Cultured
Cyclic GMP
Cyclic GMP-Dependent Protein Kinase Type I
Female
HEK293 Cells
Humans
Male
Mass Spectrometry
Middle Aged
Models
Molecular
Myocytes
Smooth Muscle
Nitrogen
Peroxynitrous Acid
Protein Binding
Sheep
Tyrosine
Young Adult
Enzyme Catalysis
Molecular Modeling
Protein Kinase G
Peroxynitrite
Chemical Sciences
Biological Sciences
Medical and Health Sciences
Biochemistry & Molecular Biology
Biological sciences
Biomedical and clinical sciences
Chemical sciences
Language
Abstract
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 Tyr(247) 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 Tyr(247) would decrease the affinity of PKG-1α for cGMP, which we confirmed using a [(3)H]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-1α activity and SMC proliferation/differentiation.