72 - The tyrosine nitration of PKG-1α inhibits its regulatory function in the pulmonary endothelium

Abstract

Protein tyrosine nitration secondary to peroxynitrite generation is involved in the pathogenesis of vascular diseases. We have previously shown that tyrosine nitration is associated with the development of acute lung injury (ALI). Protein kinase PKG-1a, which integrates cAMP/cGMP signaling and is important for the endothelial barrier maintenance, can be inhibited by nitration at Tyr247. We studied a tyrosine nitration effect on PKG-1a regulatory function in the pulmonary endothelium. Since cyclic nucleotide-specific phosphodiesterases (PDEs) can be modulated by phosphorylation, we explored the functional link between nitration-mediated PKG-1a inhibition and PDE(s) activation. LPS-challenged mice were utilized as ALI model. The peroxynitrite scavenger, MnTMPyP was used to attenuate protein nitration. Cyclic nucleotide levels, PKA/PKG/PDEs activities and PKG nitration were evaluated. PKG-1a-dependent phosphorylation sites on PDE3A were elucidated by mass-spectrometry. LPS-treatment decreased cAMP and increased cGMP levels in mouse lungs. PKA/PKG activities were also decreased. Nitrosative stress played a pivotal role in these changes. We identified an increase in PKG-1a nitration at Tyr247 and PDE3A activity increase. PKG activation in vitro induced an increase in PDE3A serine phosphorylation that correlated with a decrease in its activity. Mass-spectrometry identified a novel PKG-1a-dependent phosphorylation site, Ser654, in PDE3A. We confirmed PKG-1a-PDE3A interaction, and PKG-dependent PDE3A phosphorylation. This phosphorylation suppressed PDE3A activity and elevated intracellular cAMP levels. Ser654Ala-PDE3A mutant overexpression abolished PKG-1a-dependent inhibitory effect and prevented cAMP increase. We designed a peptide aimed to protect PKG-1a from Tyr247 nitration. This peptide attenuated the endothelial barrier disruption in LPS-challenged mice. We have identified a novel mechanism of cyclic nucleotide cross-talk regulated by PKG-dependent inhibitory phosphorylation of PDE3A. In ALI, PKG-1a Tyr247 nitration abolishes PDE3A phosphorylation leading to cAMP signaling impairment and endothelial dysfunction. Prevention of PKG-1a nitration significantly improved the barrier-protective mechanisms in ALI model. These data suggest a therapeutic approach of PKG activity protection in ALI treatment.