Nitration of tyrosine 247 inhibits protein kinase G-1α activity by attenuating cyclic guanosine monophosphate binding.
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.
From the Pulmonary Disease Program, Vascular Biology Center, Georgia Regents University, Augusta, Georgia 30912.
Saurabh
S
Aggarwal
Christine M
CM
Gross
Ruslan
R
Rafikov
Sanjiv
S
Kumar
Jeffrey R
JR
Fineman
Britta
B
Ludewig
Danny
D
Jonigk
Stephen M
SM
Black
D000328:Adult; D000818:Animals; D001011:Aorta / Q000166:cytology; D002318:Cardiovascular Diseases / Q000378:metabolism; D020134:Catalytic Domain; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000737:chemistry*; D062965:Cyclic GMP-Dependent Protein Kinase Type I / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D008875:Middle Aged; D008958:Models, Molecular; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D009584:Nitrogen / Q000737:chemistry*; D030421:Peroxynitrous Acid / Q000737:chemistry; D011485:Protein Binding; D012756:Sheep; D014443:Tyrosine / Q000737:chemistry*; D055815:Young Adult
D030421:Peroxynitrous Acid; D014443:Tyrosine; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D006152:Cyclic GMP; D009584:Nitrogen
Cyclic GMP (cGMP); Enzyme Catalysis; Mass Spectrometry (MS); Molecular Modeling; Peroxynitrite; Protein Kinase G (PKG)