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

Saurabh Aggarwal, Christine M. Gross, Ruslan Rafikov, Sanjiv Kumar, Jeffrey R. Fineman, Britta Ludewig, Danny Jonigk, Stephen Matthew Black

Research output: Contribution to journalArticle

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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 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.

Original languageEnglish (US)
Pages (from-to)7948-7961
Number of pages14
JournalJournal of Biological Chemistry
Volume289
Issue number11
DOIs
StatePublished - Mar 14 2014

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Nitration
Cyclic GMP-Dependent Protein Kinases
Cyclic GMP
Tyrosine
Smooth Muscle Myocytes
Muscle
Cells
Pulmonary Hypertension
Natriuretic Peptides
Cell proliferation
Phenylalanine
Vascular Diseases

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Nitration of tyrosine 247 inhibits protein kinase G-1α activity by attenuating cyclic guanosine monophosphate binding. / Aggarwal, Saurabh; Gross, Christine M.; Rafikov, Ruslan; Kumar, Sanjiv; Fineman, Jeffrey R.; Ludewig, Britta; Jonigk, Danny; Black, Stephen Matthew.

In: Journal of Biological Chemistry, Vol. 289, No. 11, 14.03.2014, p. 7948-7961.

Research output: Contribution to journalArticle

Aggarwal, Saurabh ; Gross, Christine M. ; Rafikov, Ruslan ; Kumar, Sanjiv ; Fineman, Jeffrey R. ; Ludewig, Britta ; Jonigk, Danny ; Black, Stephen Matthew. / Nitration of tyrosine 247 inhibits protein kinase G-1α activity by attenuating cyclic guanosine monophosphate binding. In: Journal of Biological Chemistry. 2014 ; Vol. 289, No. 11. pp. 7948-7961.
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