Oxidant sensor in the cGMP-binding pocket of PKGIα regulates nitroxyl-mediated kinase activity

Figures

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  Figure 1. Identification of HNO-modified cysteines in PKGIα by NOxICAT. (A) Exposure of recombinant human PKGIα to HNO donors induced oxidation of Cys42, Cys117 and Cys195 in PKGIα. The table summarises the mass charge (m/z) in molecular ions (mi) of the detected peptides containing the cysteines in the reduced (blue) and oxidised (pink) state. (B) Fourier Transform Mass Spectrometry spectra are expressed as the relative abundance of the detected peptides in m/z containing Cys42 (left), Cys117 (middle) and Cys195 (right), vehicle-treated (top), after exposure to 1-nitrosocyclohexyl acetate (NCA; 25 mmol/L, 15 min) (middle) or Angeli’s salt (AS; 25 µmol/L, 15 min) (bottom). (C) Quantification of the detected oxidised (pink) versus reduced (blue) peptide fraction is shown as percent of the total detected peptides.

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  Figure 2. Effect of HNO-mediated PKGIα oxidation on kinase activity. HEK-293 cells were transfected to express PKGIα WT or various mutants Cys42Ser, Cys117/195Ser, Cys42/117/195Ser, exposed to (A) NCA (100 µmol/L, 30 min) or (B) AS (500 µmol/L, 15 min). In vitro kinase (IVK) assays were performed in cell lysates by addition of recombinant His6-tagged C1-M-C2 domain of cardiac myosin-binding protein C as a substrate in the presence of γ32P-ATP. Substrate phosphorylation was detected by autoradiography (figure shows cropped version, full representation in online supplement). Bar charts represent the results of 5 independent experiments. Data are expressed in comparison to the WT response after HNO treatment. *P < 0.05, **P < 0.01, ***P < 0.001 by comparison against each respective unstimulated control or WT after HNO-exposure. Western immunoblot analysis for PKGIα under non-reducing (NR) or reducing (R) conditions was performed in the same samples (figure shows cropped reducing blots, full representation in online supplement). Representative immunoblots show PKGIα migrating at 75 kDa (monomer) and 150 kDa (dimer). Data are representative of 5 independent experiments. (C) Analysis of…

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  Figure 3. Assessment of intradisulfide-induced changes in the cGMP-binding domain of PKGIα. (A) β-Escin-permeabilised primary mouse VSMCs stably expressing the FRET sensor cGi500 were superfused with intracellular-like medium (ICM) containing increasing concentrations of cGMP (0.1, 1, 10 µmol/L). This was followed by incubation with DMSO (0.02%; upper panel) or NCA (100 µmol/L; bottom panel) and by another incubation with ICM supplemented with increasing concentrations of cGMP (0.1, 1, 10 µmol/L). Changes of the FRET signals were recorded by epifluorescence microscopy. Representative FRET traces are shown on the left. The bar charts on the right summarise the FRET results from 10 cells per group as the ΔR/ΔR0 (amplitude relative to the signal induced by the first application of 10 µmol/L cGMP) induced by cGMP incubation before (white bars) or after exposure to DMSO (black; upper panel) or NCA (red; bottom panel). **P < 0.01, ***P < 0.001, comparing cGMP-induced changes in FRET ratio before and after DMSO or NCA by two-way ANOVA. (B) In vitro cGMP binding to PKGIα after exposure to DTT (100 mmol/L, 10 min) (black squares) or NCA (100 µmol/L, 30 min) (red dots) was investigated by measuring…

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  Figure 4. Correlation of intradisulfide formation in endogenous PKGIα with HNO-mediated vasorelaxation in vitro. The effect of NCA (A) or AS (B) on vasorelaxation was assessed in mesenteric arteries from wildtype (WT; black) mice. U46619 (100 nmol/L) was used to preconstrict vessels and then increasing concentrations of NCA (0.1, 0.3, 1, 3, 10, 30, 100, 300 µmol/L) or AS (0.1, 0.3, 1, 3, 10, 30, 100 µmol/L) were administered. Experiments were performed in 1-2 vessels derived from at least 4 animals for each HNO donor. Western immunoblot analysis for PKGIα was performed under reducing (R) or non-reducing (NR) conditions in vessel homogenates exposed to 30 µmol/L NCA or 3 µmol/L AS.

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  Figure 5. Correlation of intradisulfide formation in endogenous PKGIα with HNO-mediated vasorelaxation in vivo. Both HNO donors induced concentration-dependent dilations in arterioles in vivo. (A) The effect of NCA was studied in 68 vessels from 6 mice. (B) The effect of AS was studied in 73 vessels from 6 mice. Data are given as mean ± SEM. *** indicates P < 0.001 for paired comparisons (t-test). Western immunoblot analysis for PKGIα was performed under reducing (R) and non-reducing (NR) conditions in isolated cremaster muscles exposed to 50 µmol/L NCA or AS. Black arrows indicate the positions of monomeric (75 kDa), inter- (150 kDa) and intradisulfide (below 150 kDa) PKGIα.

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  Figure 6. Illustration of the domain structure of the PKGIα homodimer and the localisation of cysteines that are modified by exposure to HNO donors to form inter- and intradisulfide bonds. DD: docking and dimerisation domain; AI: autoinhibitory domain; cGMP-A: high affinity cGMP-binding domain; cGMP-B: low affinity cGMP-binding domain; SW: switch helix; ATP: ATP-binding domain; sGC: soluble guanylate cyclase.