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

ABSTRACT: The cGMP-dependent protein kinase G-1? (PKG-1?) 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-1? activity. However, little is known about the mechanisms involved in this event. Utilizing mass spectrometry, we found that PKG-1? is susceptible to nitration at tyrosine 247 and 425. Tyrosine to phenylalanine mutants, Y247F- and Y425F-PKG-1?, were both less susceptible to nitration than WT PKG-1?, but only Y247F-PKG-1? exhibited preserved activity, suggesting that the nitration of Tyr(247) is critical in attenuating PKG-1? activity. The overexpression of WT- or Y247F-PKG-1? 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-1?. An antibody generated against 3-NT-Y247 identified increased levels of nitrated PKG-1? 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-1?. This model predicted that the nitration of Tyr(247) would decrease the affinity of PKG-1? 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-1? activity and SMC proliferation/differentiation.

SUBMITTER: Aggarwal S 

PROVIDER: S-EPMC3953305 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature