Journal of Biological Chemistry
Volume 293, Issue 43, 26 October 2018, Pages 16791-16802
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Enzymology
Oxidation of cysteine 117 stimulates constitutive activation of the type Iα cGMP-dependent protein kinase

https://doi.org/10.1074/jbc.RA118.004363Get rights and content
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The type I cGMP-dependent protein kinase (PKG I) is an essential regulator of vascular tone. It has been demonstrated that the type Iα 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 Iα. PKG Iα C42S and a truncation construct that does not contain Cys-42 (Δ53) were both constitutively activated by H2O2. In contrast, oxidation of PKG Iα C117S maintained its cGMP-dependent activation characteristics, although oxidized PKG Iα C195S did not. To corroborate these results, we also tested the effects of our constructs on the PKG Iα–specific substrate, the large conductance potassium channel (KCa 1.1). Application of WT PKG Iα activated by either cGMP or H2O2 increased the open probabilities of the channel. Neither cGMP nor H2O2 activation of PKG Iα C42S significantly increased channel open probabilities. Moreover, cGMP-stimulated PKG Iα C117S increased KCa 1.1 activity, but this effect was not observed under oxidizing conditions. Finally, we observed that PKG Iα C42S caused channel flickers, indicating dramatically altered KCa 1.1 channel characteristics compared with channels exposed to WT PKG Iα. Cumulatively, these results indicate that constitutive activation of PKG Iα proceeds through oxidation of Cys-117 and further suggest that the formation of a sulfur acid is necessary for this phenotype.

cyclic GMP (cGMP)
oxidation-reduction (redox)
protein kinase G (PKG)
potassium channel
signal transduction
AGC kinases
cGMP-dependent protein kinase
allostery
constitutive activation

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This work was supported by National Institutes of Health Grants 5T32 HL007594-30 (to J. L. S.) and R01 HL121706 and R01 HL131181 (to M. T. N.) and additional support from the Totman Trust for Biomedical Research. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

This article contains Figs. S1–S5.

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The abbreviations used are:

    PKG I

    cGMP-dependent protein kinase I

    TCEP

    tris(2-carboxyethyl)phosphine

    PDB

    Protein Data Bank

    TEV

    tobacco etch virus

    MWCO

    molecular weight cut-off

    MeCN

    acetonitrile

    Xcorr

    cross-correlation.