Oxidation of cysteine 117 stimulates constitutive activation of the type I cGMP-dependent protein kinase

Jessica L. Sheehe, Adrian D. Bonev, Anna M. Schmoker, Bryan A. Ballif, Mark T. Nelson, Thomas M. Moon, Wolfgang R. Dostmann

Research output: Contribution to journalArticlepeer-review

Abstract

The type I cGMP-dependent protein kinase (PKG I) is an essential regulator of vascular tone. It has been demonstrated that the type I isoform can be constitutively activated by oxidizing conditions. However, the amino acid residues implicated in this phenomenon are not fully elucidated. To investigate the molecular basis for this mechanism, we studied the effects of oxidation using recombinant WT, truncated, and mutant constructs of PKG I. Using an in vitro assay, we observed that oxidation with hydrogen peroxide (H2O2) resulted in constitutive, cGMP-independent activation of PKG I. PKG I C42S and a truncation construct that does not contain Cys-42 (53) were both constitutively activated by H2O2. In contrast, oxidation of PKG I C117S maintained its cGMP-dependent activation characteristics, although oxidized PKG I C195S did not. To corroborate these results, we also tested the effects of our constructs on the PKG I-specific substrate, the large conductance potassium channel (KCa 1.1). Application of WT PKG I activated by either cGMP or H2O2 increased the open probabilities of the channel. Neither cGMP nor H2O2 activation of PKG I C42S significantly increased channel open probabilities. Moreover, cGMP-stimulated PKG I C117S increased KCa 1.1 activity, but this effect was not observed under oxidizing conditions. Finally, we observed that PKG I C42S caused channel flickers, indicating dramatically altered KCa 1.1 channel characteristics compared with channels exposed to WT PKG I. Cumulatively, these results indicate that constitutive activation of PKG I proceeds through oxidation of Cys-117 and further suggest that the formation of a sulfur acid is necessary for this phenotype.

Original languageEnglish
Pages (from-to)16791-16802
Number of pages12
JournalJournal of Biological Chemistry
Volume293
Issue number43
Early online date11 Sept 2018
DOIs
Publication statusPublished - 26 Oct 2018

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