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Oxidative stress induced mitochondrial protein kinase A mediates cytochrome c oxidase dysfunction.

Srinivasan S, Spear J, Chandran K, Joseph J, Kalyanaraman B, Avadhani NG - PLoS ONE (2013)

Bottom Line: Instead, activation of hypoxia-induced PKA is dependent on reactive oxygen species (ROS).Substitution of wild type subunit Vb of CcO with phosphorylation resistant S40A mutant subunit attenuated the loss of CcO activity and reduced ROS production.The results also describe a novel mechanism of mitochondrial PKA activation which is independent of mitochondrial cAMP, but responsive to ROS.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Biology and the Mari Lowe Center for Comparative Oncology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Previously we showed that Protein kinase A (PKA) activated in hypoxia and myocardial ischemia/reperfusion mediates phosphorylation of subunits I, IVi1 and Vb of cytochrome c oxidase. However, the mechanism of activation of the kinase under hypoxia remains unclear. It is also unclear if hypoxic stress activated PKA is different from the cAMP dependent mitochondrial PKA activity reported under normal physiological conditions. In this study using RAW 264.7 macrophages and in vitro perfused mouse heart system we investigated the nature of PKA activated under hypoxia. Limited protease treatment and digitonin fractionation of intact mitochondria suggests that higher mitochondrial PKA activity under hypoxia is mainly due to increased sequestration of PKA Catalytic α (PKAα) subunit in the mitochondrial matrix compartment. The increase in PKA activity is independent of mitochondrial cAMP and is not inhibited by adenylate cyclase inhibitor, KH7. Instead, activation of hypoxia-induced PKA is dependent on reactive oxygen species (ROS). H89, an inhibitor of PKA activity and the antioxidant Mito-CP prevented loss of CcO activity in macrophages under hypoxia and in mouse heart under ischemia/reperfusion injury. Substitution of wild type subunit Vb of CcO with phosphorylation resistant S40A mutant subunit attenuated the loss of CcO activity and reduced ROS production. These results provide a compelling evidence for hypoxia induced phosphorylation as a signal for CcO dysfunction. The results also describe a novel mechanism of mitochondrial PKA activation which is independent of mitochondrial cAMP, but responsive to ROS.

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Hypoxia induces mitochondrial PKA activity in RAW 264.7 macrophages.A) PKA activity in isolated mitochondrial and cytosol of RAW 264.7 macrophages subjected to hypoxia for 0-12h. The activity of the corresponding normoxic fraction was taken as 100% activity. The inset shows immunoblot of representative mitochondrial and cytosolic fractions for cross contamination. Blot was probed with antibodies to Actin and TOM20 as markers of cytosol and mitochondria. 30µg of mitochondrial and cytosolic proteins were loaded in each well. B) PKA activity in mitochondria isolated from normoxic and hypoxic cells. H89 (1µM) and MPI (1µM) were added at the start of hypoxia (n=3) C) Effect of KH7 on hypoxia induced mitochondrial PKA activity. RAW 264.7 cells were treated with indicated concentrations of KH7 at the start of hypoxia (n=3) D) Immunoblots showing level of PKAα in mitochondria isolated from normoxic and hypoxic cells with or without KH7 (10µM) treatment. Values in parantheses underneath each blot are relative intensities of the bands. Blot is representative of two separate experiments. E) cAMP levels in 10µg of whole cell lysates and isolated mitochondria from normoxic and hypoxic cells. The difference in cAMP level between normoxic and hypoxic total lysates was not significant. *, p<0.01; **, p<0.005; ***,p<0.001.
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pone-0077129-g001: Hypoxia induces mitochondrial PKA activity in RAW 264.7 macrophages.A) PKA activity in isolated mitochondrial and cytosol of RAW 264.7 macrophages subjected to hypoxia for 0-12h. The activity of the corresponding normoxic fraction was taken as 100% activity. The inset shows immunoblot of representative mitochondrial and cytosolic fractions for cross contamination. Blot was probed with antibodies to Actin and TOM20 as markers of cytosol and mitochondria. 30µg of mitochondrial and cytosolic proteins were loaded in each well. B) PKA activity in mitochondria isolated from normoxic and hypoxic cells. H89 (1µM) and MPI (1µM) were added at the start of hypoxia (n=3) C) Effect of KH7 on hypoxia induced mitochondrial PKA activity. RAW 264.7 cells were treated with indicated concentrations of KH7 at the start of hypoxia (n=3) D) Immunoblots showing level of PKAα in mitochondria isolated from normoxic and hypoxic cells with or without KH7 (10µM) treatment. Values in parantheses underneath each blot are relative intensities of the bands. Blot is representative of two separate experiments. E) cAMP levels in 10µg of whole cell lysates and isolated mitochondria from normoxic and hypoxic cells. The difference in cAMP level between normoxic and hypoxic total lysates was not significant. *, p<0.01; **, p<0.005; ***,p<0.001.

Mentions: Previous studies from our laboratory showed that PKA activity associated with mitochondria increased in cells subjected to hypoxia and hearts subjected to ischemia-reperfusion [10]. In this study we investigated the sub-mitochondrial location as well as the nature of PKA activated under hypoxia. For this purpose RAW 264.7 macrophages were subjected to 0-12h of hypoxia (1% O2) and PKA activity was measured in both isolated mitochondria and cytosol. As seen in Figure 1A, there was a time dependent increase in PKA activity in mitochondria up to 12h of hypoxia. The activity in the cytosolic fraction, on the other hand, did not significantly change during this period. The inset in Figure 1A shows that mitochondria used were relatively free of cytosolic contamination. This is important since the cytoplasmic PKA activity is about 10 fold higher than mitochondrial PKA activity. Figure 1B shows the effect of inhibitors on mitochondrial PKA activity. Two PKA inhibitors; H89, an isoquinolinesulfonamide and MPI, a specific peptide inhibitor were used. Mitochondrial PKA activity in both normoxic and hypoxic cells were inhibited by H89, and MPI (Figure 1B). However, KH7, a specific inhibitor of soluble adenylate cyclase had no significant effect on hypoxia mediated increase in mitochondrial PKA activity (Figure 1C). In addition, KH7 did not have any effect on the mitochondrial level of PKAα subunit in hypoxic cells (Figure 1D). Since cAMP is the known activator of PKA, we measured the level of cAMP in both cytosol and mitochondria of cells subjected to hypoxia. As shown in Figure 1E, cAMP level decreased by about 15% in total lysate from hypoxic cells, but was unchanged in mitochondria. These results suggest that increase in mitochondrial PKA activity under hypoxia is independent of cAMP.


Oxidative stress induced mitochondrial protein kinase A mediates cytochrome c oxidase dysfunction.

Srinivasan S, Spear J, Chandran K, Joseph J, Kalyanaraman B, Avadhani NG - PLoS ONE (2013)

Hypoxia induces mitochondrial PKA activity in RAW 264.7 macrophages.A) PKA activity in isolated mitochondrial and cytosol of RAW 264.7 macrophages subjected to hypoxia for 0-12h. The activity of the corresponding normoxic fraction was taken as 100% activity. The inset shows immunoblot of representative mitochondrial and cytosolic fractions for cross contamination. Blot was probed with antibodies to Actin and TOM20 as markers of cytosol and mitochondria. 30µg of mitochondrial and cytosolic proteins were loaded in each well. B) PKA activity in mitochondria isolated from normoxic and hypoxic cells. H89 (1µM) and MPI (1µM) were added at the start of hypoxia (n=3) C) Effect of KH7 on hypoxia induced mitochondrial PKA activity. RAW 264.7 cells were treated with indicated concentrations of KH7 at the start of hypoxia (n=3) D) Immunoblots showing level of PKAα in mitochondria isolated from normoxic and hypoxic cells with or without KH7 (10µM) treatment. Values in parantheses underneath each blot are relative intensities of the bands. Blot is representative of two separate experiments. E) cAMP levels in 10µg of whole cell lysates and isolated mitochondria from normoxic and hypoxic cells. The difference in cAMP level between normoxic and hypoxic total lysates was not significant. *, p<0.01; **, p<0.005; ***,p<0.001.
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Related In: Results  -  Collection

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pone-0077129-g001: Hypoxia induces mitochondrial PKA activity in RAW 264.7 macrophages.A) PKA activity in isolated mitochondrial and cytosol of RAW 264.7 macrophages subjected to hypoxia for 0-12h. The activity of the corresponding normoxic fraction was taken as 100% activity. The inset shows immunoblot of representative mitochondrial and cytosolic fractions for cross contamination. Blot was probed with antibodies to Actin and TOM20 as markers of cytosol and mitochondria. 30µg of mitochondrial and cytosolic proteins were loaded in each well. B) PKA activity in mitochondria isolated from normoxic and hypoxic cells. H89 (1µM) and MPI (1µM) were added at the start of hypoxia (n=3) C) Effect of KH7 on hypoxia induced mitochondrial PKA activity. RAW 264.7 cells were treated with indicated concentrations of KH7 at the start of hypoxia (n=3) D) Immunoblots showing level of PKAα in mitochondria isolated from normoxic and hypoxic cells with or without KH7 (10µM) treatment. Values in parantheses underneath each blot are relative intensities of the bands. Blot is representative of two separate experiments. E) cAMP levels in 10µg of whole cell lysates and isolated mitochondria from normoxic and hypoxic cells. The difference in cAMP level between normoxic and hypoxic total lysates was not significant. *, p<0.01; **, p<0.005; ***,p<0.001.
Mentions: Previous studies from our laboratory showed that PKA activity associated with mitochondria increased in cells subjected to hypoxia and hearts subjected to ischemia-reperfusion [10]. In this study we investigated the sub-mitochondrial location as well as the nature of PKA activated under hypoxia. For this purpose RAW 264.7 macrophages were subjected to 0-12h of hypoxia (1% O2) and PKA activity was measured in both isolated mitochondria and cytosol. As seen in Figure 1A, there was a time dependent increase in PKA activity in mitochondria up to 12h of hypoxia. The activity in the cytosolic fraction, on the other hand, did not significantly change during this period. The inset in Figure 1A shows that mitochondria used were relatively free of cytosolic contamination. This is important since the cytoplasmic PKA activity is about 10 fold higher than mitochondrial PKA activity. Figure 1B shows the effect of inhibitors on mitochondrial PKA activity. Two PKA inhibitors; H89, an isoquinolinesulfonamide and MPI, a specific peptide inhibitor were used. Mitochondrial PKA activity in both normoxic and hypoxic cells were inhibited by H89, and MPI (Figure 1B). However, KH7, a specific inhibitor of soluble adenylate cyclase had no significant effect on hypoxia mediated increase in mitochondrial PKA activity (Figure 1C). In addition, KH7 did not have any effect on the mitochondrial level of PKAα subunit in hypoxic cells (Figure 1D). Since cAMP is the known activator of PKA, we measured the level of cAMP in both cytosol and mitochondria of cells subjected to hypoxia. As shown in Figure 1E, cAMP level decreased by about 15% in total lysate from hypoxic cells, but was unchanged in mitochondria. These results suggest that increase in mitochondrial PKA activity under hypoxia is independent of cAMP.

Bottom Line: Instead, activation of hypoxia-induced PKA is dependent on reactive oxygen species (ROS).Substitution of wild type subunit Vb of CcO with phosphorylation resistant S40A mutant subunit attenuated the loss of CcO activity and reduced ROS production.The results also describe a novel mechanism of mitochondrial PKA activation which is independent of mitochondrial cAMP, but responsive to ROS.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Biology and the Mari Lowe Center for Comparative Oncology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Previously we showed that Protein kinase A (PKA) activated in hypoxia and myocardial ischemia/reperfusion mediates phosphorylation of subunits I, IVi1 and Vb of cytochrome c oxidase. However, the mechanism of activation of the kinase under hypoxia remains unclear. It is also unclear if hypoxic stress activated PKA is different from the cAMP dependent mitochondrial PKA activity reported under normal physiological conditions. In this study using RAW 264.7 macrophages and in vitro perfused mouse heart system we investigated the nature of PKA activated under hypoxia. Limited protease treatment and digitonin fractionation of intact mitochondria suggests that higher mitochondrial PKA activity under hypoxia is mainly due to increased sequestration of PKA Catalytic α (PKAα) subunit in the mitochondrial matrix compartment. The increase in PKA activity is independent of mitochondrial cAMP and is not inhibited by adenylate cyclase inhibitor, KH7. Instead, activation of hypoxia-induced PKA is dependent on reactive oxygen species (ROS). H89, an inhibitor of PKA activity and the antioxidant Mito-CP prevented loss of CcO activity in macrophages under hypoxia and in mouse heart under ischemia/reperfusion injury. Substitution of wild type subunit Vb of CcO with phosphorylation resistant S40A mutant subunit attenuated the loss of CcO activity and reduced ROS production. These results provide a compelling evidence for hypoxia induced phosphorylation as a signal for CcO dysfunction. The results also describe a novel mechanism of mitochondrial PKA activation which is independent of mitochondrial cAMP, but responsive to ROS.

Show MeSH
Related in: MedlinePlus