Nitration of tyrosine 247 inhibits protein kinase G-1α activity by attenuating cyclic guanosine monophosphate binding.

Academic Article

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.
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    Keywords

  • Cyclic GMP (cGMP), Enzyme Catalysis, Mass Spectrometry (MS), Molecular Modeling, Peroxynitrite, Protein Kinase G (PKG), 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
  • Digital Object Identifier (doi)

    Author List

  • Aggarwal S; Gross CM; Rafikov R; Kumar S; Fineman JR; Ludewig B; Jonigk D; Black SM
  • Start Page

  • 7948
  • End Page

  • 7961
  • Volume

  • 289
  • Issue

  • 11