Nitration of tyrosine 247 inhibits protein kinase G-1α activity by attenuating cyclic guanosine monophosphate binding

Abstract

The cGMP-dependent protein kinase G-1α (PKG-1α) is a downstream mediator of nitric oxide and natriuretic peptide signaling. Alterations in this pathway play a key role in the pathogenesis and progression of vascular diseases associated with increased vascular tone and thickness, such as pulmonary hypertension. Previous studies have shown that tyrosine nitration attenuates PKG-1α activity. However, little is known about the mechanisms involved in this event. Utilizing mass spectrometry, we found that PKG-1α is susceptible to nitration at tyrosine 247 and 425. Tyrosine to phenylalanine mutants, Y247F- and Y425F-PKG-1α, were both less susceptible to nitration than WT PKG-1α, but only Y247F-PKG-1α exhibited preserved activity, suggesting that the nitration of Tyr(247) is critical in attenuating PKG-1α activity. The overexpression of WT- or Y247F-PKG-1α decreased the proliferation of pulmonary artery smooth muscle cells (SMC), increased the expression of SMC contractile markers, and decreased the expression of proliferative markers. Nitrosative stress induced a switch from a contractile to a synthetic phenotype in cells expressing WT- but not Y247F-PKG-1α. An antibody generated against 3-NT-Y247 identified increased levels of nitrated PKG-1α in humans with pulmonary hypertension. Finally, to gain a more mechanistic understanding of how nitration attenuates PKG activity, we developed a homology model of PKG-1α. This model predicted that the nitration of Tyr(247) would decrease the affinity of PKG-1α for cGMP, which we confirmed using a [(3)H]cGMP binding assay. Our study shows that the nitration of Tyr(247) and the attenuation of cGMP binding is an important mechanism regulating in PKG-1α activity and SMC proliferation/differentiation.

Keywords: Cyclic GMP (cGMP); Enzyme Catalysis; Mass Spectrometry (MS); Molecular Modeling; Peroxynitrite; Protein Kinase G (PKG).

FIGURE 1.

Identification of the nitration sites on human PKG-1α. HEK-293T cells were transfected with an expression plasmid containing full-length WT-PKG-1α cDNA. After 48 h, the cells were exposed or not to SIN-1 (500 μm) for 30 min. The cells were lysed; PKG-1α was immunoprecipitated, and the protein was subjected to SDS-PAGE and staining by imperial protein stain. The band corresponding to PKG-1α was excised and trypsinized, and MS was performed on the extracted peptides. MS analysis of the human 3-NT modified PKG-1α sequence, LADVLEETHYENGEYIIR, corresponding to the peptide comprising the amino acids 233–250, and the sequence, QIMQGAHSDFIVRLYR, corresponding to the peptide comprising the amino acids 411–426, demonstrated the nitration of Tyr²⁴⁷ and Tyr⁴²⁵ (A). The peptide with m/z 2209.04 (parent peptide LADVLEETHYENGEYIIR with m/z 2164.04 + 45 Da of nitro group) was further fragmented, and MS/MS data were analyzed (B). Solid lines represent the predicted masses for the nitrated peptide found in the MS/MS with an error less than 0.5 ppm. The MS/MS spectrum of the 2209.04 m/z ion was obtained in positive reflector mode fitted with peptide ²³³LADVLEETHYENGEY(NO₂)IIR²⁵⁰ from the PKG-1α sequence.

FIGURE 2.

Nitration of Tyr²⁴⁷ attenuates PKG-1α activity. HEK-293T cells were transiently transfected with expression plasmids containing WT-, Y247F-, or Y425F-PKG-1α for 48 h. Immunoblot analysis verified increased expression of PKG-1α (A). Cells were also treated or not with SIN-1 (500 μm, 30 min). Protein extracts were immunoprecipitated using an antibody raised against PKG-1α, and the level of nitrated PKG-1α was determined by probing the membranes with an antiserum raised against 3-NT. The blots were then stripped and reprobed for PKG-1α to normalize for the efficiency of the immunoprecipitation. A representative blot is shown (B). The nitration of WT-PKG-1α was significantly increased in the presence of SIN-1 (B). However, there were no significant increases in the nitration levels of Y247F- or Y425F-PKG-1α in the presence of SIN-1 (B). Although SIN-1 did not alter cGMP-independent PKG activity (white bars), cGMP-dependent PKG activity was attenuated in cells expressing WT- and Y425F-PKG-1α, but not in cells expressing Y247F-PKG-1α (black bars, C). The data are means ± S.E., n = 3–7. *, p < 0.05 versus untreated WT-PKG-1α and Y425F-PKG-1α. IB, immunoblotting; IP, immunoprecipitation.

FIGURE 3.

The effect of nitration on pulmonary arterial smooth muscle cell growth and metabolism. PASMC were transiently transfected with expression plasmids containing WT-PKG-1α, Y247F-PKG-1α, or pDEST40 (as a control) for 20 h. Cells were then exposed or not to SIN-1 (500 μm, 48 h), and the effect on PKG protein levels (A) and activity was determined (B). SIN-1 had no effect on PKG-1α protein levels but attenuated the cGMP-dependent increase in PKG activity in the cells transfected with WT-PKG-1α, but not those expressing the Y247F PKG-1α mutant (A). The effects of SIN-1 on cellular proliferation (C) and cellular metabolic activity (D) were also determined. PASMC expressing either WT- or Y247F-PKG-1α were less proliferative and metabolically active than the pDEST40 transfected control cells. SIN-1 exposure stimulated proliferation and metabolism in WT-, but not Y247F-PKG-1α-transfected PASMC. The transfection efficiency in the PASMC was ∼20%. The data are means ± S.E., n = 4. *, p < 0.05 versus pDEST40; †, p < 0.05 versus WT-PKG-1α; ‡, p < 0.05 versus WT-PKG-1α + SIN-1.

FIGURE 4.

The effect of nitration on pulmonary arterial smooth muscle cell phenotype. PASMC were transiently transfected with expression plasmids containing WT-PKG-1α, Y247F-PKG-1α, or PDEST40 (as a control) for 20 h. Cells were then exposed or not to SIN-1 (500 μm, 48 h), and the effect on synthetic and contractile markers was determined. The levels of MYH (A), calponin-1 (B), and vimentin (C) were determined. The blots were then stripped and reprobed for β-actin to normalize for protein loading. A representative blot is shown for each. Under basal conditions, PASMC transfected with the WT- and the Y247F-PKG-1α exhibited increased expression of the contractile markers MYH and calponin-1 and decreased expression of the proliferative marker vimentin, indicative of a contractile phenotype. SIN-1 decreased the expression of the contractile markers MYH and calponin-1 and increased the expression of the proliferative marker, vimentin, in the WT-PKG-1α-transfected cells, indicative of a proliferative phenotype. The Y247F PKG-1α-expressing cells were resistant to this phenotypic conversion. PASMC were also subjected to immunohistochemistry using antibodies to SM22-α (5 μg/ml) and PCNA (1 μg/ml). Relevant secondary antibodies linked to Alexa Fluor 488 were then applied (green). DAPI was also used to stain the cell nuclei (blue). PASMC expressing WT- or Y247F-PKG-1α acquired a contractile phenotype with the increased filamentous binding of the SM22-α protein on the actin stress fibers (D and E). The nuclear localization of PCNA was also reduced in these cells (F and G). However, when PASMC were treated with SIN-1, the WT PKG-1α-expressing cells exhibited decreased filamentous SM22-α expression and increased nuclear staining of PCNA, whereas the Y247F-PKG-1α-expressing cells were unaffected. The data are means ± S.E., n = 4–7. *, p < 0.05 versus pDEST40; †, p < 0.05 versus WT-PKG-1α; ‡, p < 0.05 versus WT-PKG-1α + SIN-1.

FIGURE 5.

Identification of Y247-PKG-1α nitration in vitro and in vivo. Recombinant PKG-1α protein (A) was incubated in the presence or absence of SIN-1 (500 μm, 30 min). The protein was immunoblotted and probed with an antibody raised against 3-NT-Y247-PKG-1α and then normalized with total PKG-1α antibody. Our results demonstrate that 3-NT-Y247-PKG-1α antibody preferentially binds to the nitrated PKG-1α. In addition, HEK-293T cells (B) or PASMC (C) were transiently transfected with expression plasmids containing WT- or Y247F-PKG-1α for 48 h. Cells were then treated or not with SIN-1 (500 μm, 30 min). Protein extracts were immunoblotted and probed with anti-3-NT-Y247-PKG-1α antibody. The blots were then stripped and reprobed for total PKG-1α and β-actin to normalize loading. WT-PKG-1α nitration was significantly increased in the presence of SIN-1. However, there were no significant increases in the nitration levels of Y247F-PKG-1α in the presence of SIN-1. The 3-NT-Y247-PKG-1α antibody also detected higher PKG-1α nitration levels in peripheral lung tissues of lambs with pulmonary hypertension secondary to increased pulmonary blood flow (D). Finally, immunohistochemical analysis was performed on lung sections prepared from humans with pulmonary hypertension (PH). The antibodies used were goat anti-PKG-1α (red), 3-NT-Y247-PKG-1α (red), and anti-caldesmon (green). The fluorescently stained sections were then analyzed using confocal microscopy and a representative image is shown (E). The 3-NT-Y247-PKG-1α antibody identified significantly higher levels of nitrated PKG-1α in the lungs of patients with pulmonary hypertensive (F), and this was predominant in the smooth muscle layer (E). The data are means ± S.E., n = 3–6. *, p < 0.05 versus untreated WT-PKG-1α for B and C, control lambs for D, and normal human reference lungs for F. †, p < 0.05 versus WT-PKG-1α + SIN-1 (B and C).

FIGURE 6.

Generation of a homology model of human PKG-1α. The YASARA homology modeling software was used to build a homology model of the PKG-1α regulatory domain using the known crystal structure of the PKA regulatory domain (PDB code 1NE4), as a template. The known crystal structure of the catalytic domain of PKA (PDB code 2CPK) was used to construct the corresponding homology model of the catalytic domain of PKG-1α. Using the homology models of these two domains of PKG-1α, a complete three-dimensional model of the protein was generated. The AutoDock program was then used to dock two cGMP molecules to the cGMP-binding sites: A and B and an ATP molecule to the ATP-binding site. The analysis of the PKG-1α structure indicates that Tyr²⁴⁷ lies in close proximity to the cGMP-binding site B (A). Further, the comparison of the recently crystallized structure of PKG-1α and our homology model demonstrated high similarity within the cGMP-binding site B, even though this crystal structure was not used to build our homology model (B). The YASARA homology modeling software was also used to predict the affinity of cGMP for the cGMP-binding site B in the PKG-1α homology model under control (C) and nitrosative stress conditions (D). The addition of a NO₂ group to Tyr²⁴⁷ is predicted to decrease the total hydrogen bonding energy between cGMP and PKG-1α from 91.93 to 54.02 kJ/mol (C and D).

FIGURE 7.

The cGMP binding and dissociation characteristics of PKG-1α under nitrosative stress. PASMC were transiently transfected with expression plasmids containing WT- or Y247F-PKG-1α for 48 h. The cells were then serum-starved for 4 h and exposed or not to SIN-1 (500 μm, 30 min), and PKG-1α was immunoprecipitated. Immunoprecipitated WT- and Y247F-PKG-1α protein (100 ng) was then analyzed in a [³H]cGMP binding assay (A) and a [³H]cGMP dissociation assay (B). SIN-1 treatment significantly attenuated maximal [³H]cGMP binding to WT-PKG-1α, but not to the Y247F-PKG-1α mutant (A). In the dissociation assay, a 100-fold excess of unlabeled cGMP was added at time 0 s (B_o) to initiate the dissociation (exchange) of bound [³H]cGMP. The reaction was stopped with cold aqueous saturated (NH₄)₂SO₄ at various time points. The results were plotted as ln(B/B_o) with B_o as the initial [³H]cGMP bound] and B as the [³H]cGMP remaining bound at various time points. SIN-1 treatment enhanced the dissociation/exchange of [³H]cGMP from WT-PKG-1α, but not from Y247F-PKG-1α (B). Enzyme kinetics were also determined using varying concentrations of cGMP (0–10 μm). The change in the enzyme activity for each concentration of cGMP was plotted in pmol/min/μg protein using nonlinear regression (curve fit) analysis. The maximum velocity (V_max) of the phosphotransferase reaction of WT-PKG-1α, but not Y247F-PKG-1α, was significantly decreased with SIN-1 exposure (C). Each value represents the mean of three separate experiments. The data are means ± S.E., n = 3. *, p < 0.05 versus untreated WT-PKG-1α.