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 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.
Highlights
Blood pressure is maintained in part through constriction and relaxation of smooth muscle cells in the peripheral vasculature
Previous analyses of protein kinase I (PKG I) isoforms determined that PKG I␣, but not PKG I, exhibits sensitivity to oxidation [10, 12]
It was concluded that Cys-42, the only cysteine residue unique to PKG I␣, must be the primary regulator of oxidation-dependent activation [10]
Summary
There are two confirmed sites where disulfide bonds form in the PKG I␣ holoenzyme, which are an interprotomer disulfide bond between Cys-42 and Cys42Ј and an intraprotomer disulfide bond between Cys-117 and Cys-195 (Fig. 1A) [11]. After 30 min of exposure, we observed the greatest effect on the cGMP-dependent activation of the kinase, wherein the -fold activation was reduced by ϳ90% to 2.5 (Table 1). These conditions were used for all subsequent measurements of the oxidation phenotype. Oxidation resulted in a 5– 6-fold increase in basal activity in the absence of cGMP (1.13 mol/min ϫ mg; p Ͻ 0.001 compared with WTred; Fig. 2A). In agreement with previous studies, analysis of WT PKG I␣ by nonreducing SDS-PAGE showed that increasing concentrations of H2O2 resulted in formation of an interprotomer disulfide bond (Fig. 2D). For C42Sox, we observed a significant increase in basal activity
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