Nitration of protein kinase G-Iα modulates cyclic nucleotide crosstalk via phosphodiesterase 3A: Implications for acute lung injury

Figures

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  Figure 1. Altered phosphodiesterase (PDE) activities and cyclic nucleotide levels in LPS-induced animal model of ALI. Lungs were isolated from mice treated with LPS and/or MnTMPyP as described in the Experimental procedures section. The tissue extracts were used to evaluate the protein levels of PDE2, PDE3, and PDE5 (A–C) and respective activities (D–F). LPS increases PDE3 activity and a decrease in PDE5 activity, despite no changes in protein levels. PDE activity changes were sensitive to MnTMPyP pretreatment indicative of a peroxynitrite-dependent mechanism. LPS decreased cAMP levels (G), whereas cGMP levels were increased (H). MnTMPyP pretreatment prevented these changes (G and H). Data represent mean ± SEM, n = 3 to 5. ∗p < 0.05 versus control lungs; †p < 0.05 versus LPS alone. ALI, acute lung injury; LPS, lipopolysaccharide; MnTMPyP, manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin.

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  Figure 2. LPS treatment impairs PKA and PKG activities in vivo via peroxynitrite-dependent mechanism. The activity of PKA and PKG was tested in lung extracts obtained from mice treated with LPS and/or MnTMPyP as described in the Experimental procedures section. The protein levels of respective enzymes were evaluated by immunoblotting. LPS treatment did not affect the protein levels of either PKG-Iα (A) or PKA (D). However, LPS attenuated both PKG-Iα (B and C) and PKA (E) activity. Again, MnTMPyP pretreatment preserved the activity of PKA and PKG (B, C, and E). Data are mean ± SEM, n = 3 to 5. ∗p < 0.05 versus control lungs; †p < 0.05 versus LPS alone. LPS, lipopolysaccharide; MnTMPyP, manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin; PKG, protein kinase G.

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  Figure 3. Nitrosative stress induced by LPS in vivo leads to PKG-Iα tyrosine nitration.A, peroxynitrite levels, estimated using DHR oxidation, were significantly increased in LPS-treated mice. Specificity of signal was demonstrated by MnTMPyP pretreatment attenuating DHR123 oxidation. B, immunoprecipitation analysis using an antibody specific to nitrotyrosine (3-NT) demonstrated an increase in the tyrosine nitration of PKG-Iα immunoprecipitated from LPS-exposed tissues. MnTMPyP pretreatment prevented the nitration of PKG-Iα. IgG (HC) is the immunoglobulin heavy chain. C, immunoblotting using an antibody specific to nitro-Y247 in PKG-Iα confirmed the increase in PKG-Iα nitration in HEK293T cells transfected with a PKG-Iα expression plasmid and treated with 3-morpholinosydnonimine. D, an increase in PKG-Iα nitration in mouse lungs exposed to LPS was also shown to occur at Y247. MnTMPyP reduced Y247 nitration in PKG-Iα in response to LPS. Data represent mean ± SEM, n = 3 to 5. ∗p < 0.05 versus control lungs; †p < 0.05 versus LPS alone. DHR, dihydrorhodamine; HEK293T, human embryonic kidney 293T cells; LPS, lipopolysaccharide; MnTMPyP, manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin;…

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  Figure 4. Expression of PKG-Iα downregulates PDE3A activity and increases cAMP levels in vitro. PKG-Iα was transiently expressed in PKG-Iα–deficient HEK293T cells; as a negative control, the cells were transfected with an empty vector. A, immunoprecipitation analysis identified an association of PDE3A with PKG-Iα. PKG-Iα–expressing cells also exhibit a significant decrease in PDE3 activity (B) and a significant increase in cAMP levels (C). Data represent mean ± SEM, n = 3. ∗p < 0.05 versus empty vector. D, comparative analysis of human and mouse PDE3A amino acid sequences using NetPhos 3.1 revealed conserved PKG-specific phosphorylation sites (shown in red). HEK293T, human embryonic kidney 293T cells; PDE3A, phosphodiesterase 3A; PKG-Iα, protein kinase G-Iα.

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  Figure 5. PKG-Iα phosphorylates PDE3A at S654. HEK293T cells were transfected with an expression plasmid for PDE3A either alone or cotransfected with a PKG-Iα expression plasmid. Then cell lysates were subjected to PDE3A immunoprecipitation followed by SDS-PAGE and MALDI-TOF MS analysis as described in the Experimental procedures section. A, transfection and expression of PDE3A and PKG-Iα in HEK293T cells were confirmed by immunoblotting using antibodies against FLAG-tag, PDE3A, and PKG-Iα. B and C, MS/MS identified a PDE3A-specific peptide containing a serine located at position 654 (predicted PKG phosphorylation site) that was only phosphorylated when cells were transfected with PDE3A and PKG-Iα. D, the antibody generated against PKG-specific phosphorylation site, phospho-S654 in PDE3A, specifically recognized this modification when PDE3A expressed with PKG-Iα in HEK293T cells and did not react with PDE3A expressed alone. E, the antibody specific to phospho-S654-PDE3A demonstrated dramatic changes of this PDE3A modification in mouse model of ALI. Data represent mean ± SEM, n = 7. ∗p < 0.05 control versus lipopolysaccharide (LPS)-treated mice. HEK293T, human embryonic kidney 293T cells;…

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  Figure 6. PKG-Iα–dependent phosphorylation of PDE3A at S654inhibits PDE3A activity and protects intracellular cAMP from degradation.A, in HEK293T cells, overexpressed PKG-Iα associates with both wtPDE3A and mutant S654A-PDE3A. B, however, only wtPDE3A is phosphorylated at S654 when cells were treated with 8-bromo-cGMP (4 h). C, the activity of wtPDE3A was also downregulated by PKG-Iα overexpression, whereas the S654A-mutant PDE3A was unaffected. D, similarly, cAMP levels were increased by PKG-Iα overexpression in cells expressing wtPDE3A but not in cells expressing the S654A-PDE3A mutant. Data in B represent mean ± SEM, n = 3. ∗p < 0.05 wtPDE3A alone versus wtPDE3A + PKG-Iα; †p < 0.05 mutPDE3A + PKG-Iα versus wtPDE3A + PKG-Iα. Data in C and D represent mean ± SEM, n = 4. ∗p < 0.05 wtPDE3A alone versus wtPDE3A + PKG-Iα; †p < 0.05 wtPDE3A + PKG-Iα versus mutPDE3A + PKG-Iα. HEK293T, human embryonic kidney 293T cells; PDE3A, phosphodiesterase 3A; PKG-Iα, protein kinase G-Iα; S654, serine 654.

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  Figure 7. Schematic representation of PKG-Iα–dependent cAMP/cGMP crosstalk in pulmonary endothelial cells.A, under normal physiological conditions, NO-mediated cGMP generation activates PKG-Iα leading to inhibitory phosphorylation of PDE3A at S654. This results in cAMP elevation and stimulation of barrier-enhancing effectors. B, LPS-mediated generation of peroxynitrite inhibits PKG-Iα via protein tyrosine nitration. Although cGMP levels are high because of LPS-dependent inhibition of PDE5, nitrated PKG-Iα cannot phosphorylate and inhibit PDE3A. This leads to PDE3A-dependent cAMP degradation and impaired barrier function. LPS, lipopolysaccharide; PDE3A, phosphodiesterase 3A; PKG-Iα, protein kinase G-Iα; NO, nitric oxide; S654, serine 654.