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

Abstract

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 assay, we observed that oxidation with hydrogen peroxide (HO) 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 HO 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 (K 1.1). Application of WT PKG Iα activated by either cGMP or HO increased the open probabilities of the channel. Neither cGMP nor HO activation of PKG Iα C42S significantly increased channel open probabilities. Moreover, cGMP-stimulated PKG Iα C117S increased K 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 K 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.

Long-lasting blood pressure lowering effects of nitrite are NO-independent and mediated by hydrogen peroxide, persulfides, and oxidation of protein kinase G1α redox signalling

“…C195 was also shown to adduct the electrophile 8-nitro-cGMP, with this S-guanylation modification leading to persistent activation of the kinase 35 . Hyperoxidation of C117 has been implicated as a mechanism of PKG1α activation by H 2 O 2 , 36 whilst activation by this oxidant was not observed by others 32 . In summary, the redox control of PKG1α is complex, with our understanding continuing to evolve as contemporary studies on the subject emerge.…”

cGMP: a unique 2nd messenger molecule – recent developments in cGMP research and development

“…Data from an in vitro assay suggest that constitutive activation of PKG-Iα is not mediated via Cys42, but rather through oxyacid formation at Cys117. This mechanism could be particularly relevant to PKG anchored near hydrogen peroxide sources or under pathophysiological conditions associated with high intracellular concentrations of hydrogen peroxide (Sheehe et al 2018).…”

Fresh evidence overturns the identification of a factor involved in blood-vessel dilation

“…Vasodilation by cis-WOOH ultimately derives from the oxidation of Cys 42 in PKG1α. However, Cys 42 might not be the only cysteine residue that can be oxidized to activate the enzyme; Cys 117 seems to be another 12 . The effects of the Cys 42 mutation that Stanley et al used to prevent PKG1α dimerization are also controversial.…”

“…The effects of the Cys 42 mutation that Stanley et al used to prevent PKG1α dimerization are also controversial. Some studies 12,13 have found that it might depress cGMP-stimulated PKG1α activity. Furthermore, PKG1α dimerization triggered by Cys 42 oxidation occurs in the hearts of mice stressed by high blood pressure (pressure overload), yet its prevention in mice with the Cys 42 mutation enhances PKG1αmediated protection against such stress 14 -not by altering PKG1α activation, but by changing the intracellular localization of that enzyme and therefore its interactions with other proteins.…”

“…Besides full error correction and large-scale computation, which are probably still some distance away, the techniques developed in these studies could have other applications. For example, GKP-encoded qubits can be used for high-resolution displacement sensing 11 , whereas exponential-SWAP gates integrated in a circuit might enable the demonstration of specific instances in which quantum computation outperforms its classical counterpart 12 . More generally, the exquisite level of control attained in these two very different architectures shows that infinite-dimensional systems, despite their analog character, are a cuttingedge contender in the race for advanced quantum information processing.…”