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A2263 - Tyrosine Nitration of Protein Kinase G-1a Disrupts Cyclic Nucleotide Cross-Talk and Is Involved in the Development of Acute Lung Injury
Author Block: E. Zemskov1, S. Aggarwal2, C. Gross3, R. Rafikov1, Q. Lu1, S. M. Black1; 1Department of Medicine, University of Arizona, Tucson, AZ, United States, 2Department of Anesthesiology and Perioperative Medicine, University of Alabama, Birmingham, AL, United States, 3Vascular Biology Center, Augusta University, Augusta, GA, United States.
RATIONALE: Protein tyrosine nitration is intimately associated with the development of acute lung injury (ALI). However, the key protein targets have not been identified. cGMP-dependent protein kinase 1α (PKG-1α) has been shown to regulate cAMP/cGMP signaling cross-talk and is important for maintenance of the endothelial barrier. Further, we have recently shown that PKG-1α can be inhibited by nitration at Tyr247. Taking into account the dramatic decrease in cAMP and impaired cAMP-dependent signaling associated with ALI, our studies focused on the potential role of PKG-1α nitration in disrupting cAMP signaling. Since cyclic nucleotide-specific phosphodiesterases (PDEs) can be modulated by phosphorylation, we explored the functional link between nitration-mediated PKG-1α inhibition and PDE(s) activation. METHODS: Mice challenged with i.t. E. coli LPS (6.75×104 EU/g, 12h) were utilized as an ALI model. A subset of mice were also pretreated with the peroxynitrite scavenger, MnTMPyP (5 mg/kg) to prevent protein nitration. Lung tissues were used to compare cyclic nucleotide levels, PKA/PKG/PDEs activities and PKG nitration. IP/immunoblotting were performed to detect changes in protein expression/modifications. Mass-spectrometry (MS) was used to analyze PDE3A post-translation modifications. RESULTS: LPS altered cyclic nucleotide levels in the mouse lung such that cAMP levels were decreased and cGMP levels were increased. These changes were abrogated by peroxynitrite scavenging. LPS also decreased the activities of PKA and PKG in a peroxynitrite-dependent manner. We identified an increase in PKG-1α nitration at Tyr247 and an increase in the activity of PDE3. NO-dependent activation of PKG in vitro induced an increase in the serine phosphorylation of PDE3A that correlated with a decrease in its activity. MS-analysis identified a novel PKG-1α-dependent phosphorylation site, Ser654, in PDE3A. Expression of PKG-1α in PKG-deficient cells confirmed an interaction between PKG-1α and PDE3A, as well as PKG-dependent PDE3A phosphorylation. This phosphorylation suppressed PDE3A activity which led to an elevation of cellular cAMP. However, the overexpression of a Ser654Ala PDE3A mutant abolished the inhibitory effect of PKG-1α and cAMP levels did not increase. CONCLUSIONS: We have identified a novel mechanism of cyclic nucleotide cross-talk that is regulated by a PKG-dependent inhibitory phosphorylation of PDE3A. In ALI, PKG-1α nitration at Tyr247 abolishes PDE3A phosphorylation leading to a loss of cAMP signaling and endothelial barrier dysfunction. These data suggest a therapeutic approach targeted at maintaining PKG activity could have efficacy in the treatment of ALI.