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Phosphorylation of the BNIP3 C-Terminus Inhibits Mitochondrial Damage and Cell Death without Blocking Autophagy.

Liu KE, Frazier WA - PLoS ONE (2015)

Bottom Line: However, some highly respiring cells and cancer cells tolerate BNIP3 expression, suggesting that a yet unknown mechanism exists to restrain the lethal effects of BNIP3 on mitochondria.These findings establish phosphorylation as a switch to determine the pro-survival and pro-death effects of the protein.Our findings also suggest a novel target for the regulation of these activities in transformed cells where BNIP3 is often highly expressed.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, United States of America.

ABSTRACT
BNIP3 is a dual function protein, able to activate autophagy and induce cell death. Upon expression of BNIP3, which is upregulated by hypoxia, the protein induces mitochondrial dysfunction, often leading to cell death. However, some highly respiring cells and cancer cells tolerate BNIP3 expression, suggesting that a yet unknown mechanism exists to restrain the lethal effects of BNIP3 on mitochondria. Here we present evidence that BNIP3 undergoes several phosphorylation events at its C-terminus, adjacent to the transmembrane domain. Phosphorylation at these residues inhibits BNIP3-induced mitochondrial damage, preventing a loss of mitochondrial mass and mitochondrial membrane potential, as well as preventing an increase in reactive oxygen species. This decrease in mitochondrial damage, as well as the reduction of cell death upon C-terminal BNIP3 phosphorylation, can be explained by a diminished interaction between BNIP3 and OPA1, a key regulator of mitochondrial fusion and mitochondrial inner membrane structure. Importantly, phosphorylation of these C-terminal BNIP3 residues blocks cell death without preventing autophagy, providing evidence that the two functional roles of BNIP3 can be regulated independently. These findings establish phosphorylation as a switch to determine the pro-survival and pro-death effects of the protein. Our findings also suggest a novel target for the regulation of these activities in transformed cells where BNIP3 is often highly expressed.

No MeSH data available.


Related in: MedlinePlus

C-terminal BNIP3 phosphorylation inhibits BNIP3-induced cell death.(A) Quantification of the percent of Annexin V positive cells 24 hr after induction of BNIP3 expression, analyzed by flow cytometry. Data is expressed as the average of 4 independent experiments. Significant differences between control cells (no BNIP3) and cells expressing each form of BNIP3 are denoted by * p<0.05, ** p<0.01, and *** p<0.001; significant differences between complementary pairs of BNIP3 mutants are shown in brackets. (B), (C), and (D): Percent Annexin V positive cells expressing WT or phosphomutant BNIP3 with or without the following treatments: (B) 150μM H2O2 for 120 min, (C) 10μM FCCP for 120 min, and (D) 48 hr hypoxia. In (B) and (C), significant differences between control cells (without BNIP3) and cells expressing each BNIP3 mutant in normal conditions are denoted by * p<0.05, ** p<0.01, and *** p<0.001; significant differences between control cells and cells expressing each BNIP3 mutant, all undergoing additional cellular stress (H2O2, or FCCP), are denoted by $ p<0.05, $ $ p<0.01, and $ $ $ p<0.001. Significant differences between cells expressing WT BNIP3 and cells either lacking BNIP3 (None) or expressing each BNIP3 mutant in normal conditions are denoted by # p<0.05, ## p<0.01, and ### p<0.001; significant differences between cells expressing WT BNIP3 and cells either lacking BNIP3 (None) or expressing each BNIP3 mutant in the presence of H2O2 or FCCP are denoted by + p<0.05, ++ p<0.01, and +++ p<0.001; significant differences between complementary pairs of BNIP3 mutants treated with H2O2 or FCCP are shown in brackets. In (D), significant differences between control cells (without BNIP3) and cells expressing each BNIP3 mutant in normoxia are denoted by * p<0.05, ** p<0.01, and *** p<0.001; significant differences between cells expressing ΔTM BNIP3 and cells expressing each other form of BNIP3, all cultured in hypoxia, are denoted by & p<0.05, && p<0.01, and &&& p<0.001. Significant differences between cells expressing WT BNIP3 and cells either lacking BNIP3 (None) or expressing each BNIP3 mutant in normal conditions are denoted by # p<0.05, ## p<0.01, and ### p<0.001; significant differences between cells expressing WT BNIP3 and cells either lacking BNIP3 (None) or expressing each BNIP3 mutant in hypoxic conditions are denoted by + p<0.05, ++ p<0.01, and +++ p<0.001; significant differences between treatment conditions in cells expressing the same BNIP3 mutant are shown in brackets.
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pone.0129667.g005: C-terminal BNIP3 phosphorylation inhibits BNIP3-induced cell death.(A) Quantification of the percent of Annexin V positive cells 24 hr after induction of BNIP3 expression, analyzed by flow cytometry. Data is expressed as the average of 4 independent experiments. Significant differences between control cells (no BNIP3) and cells expressing each form of BNIP3 are denoted by * p<0.05, ** p<0.01, and *** p<0.001; significant differences between complementary pairs of BNIP3 mutants are shown in brackets. (B), (C), and (D): Percent Annexin V positive cells expressing WT or phosphomutant BNIP3 with or without the following treatments: (B) 150μM H2O2 for 120 min, (C) 10μM FCCP for 120 min, and (D) 48 hr hypoxia. In (B) and (C), significant differences between control cells (without BNIP3) and cells expressing each BNIP3 mutant in normal conditions are denoted by * p<0.05, ** p<0.01, and *** p<0.001; significant differences between control cells and cells expressing each BNIP3 mutant, all undergoing additional cellular stress (H2O2, or FCCP), are denoted by $ p<0.05, $ $ p<0.01, and $ $ $ p<0.001. Significant differences between cells expressing WT BNIP3 and cells either lacking BNIP3 (None) or expressing each BNIP3 mutant in normal conditions are denoted by # p<0.05, ## p<0.01, and ### p<0.001; significant differences between cells expressing WT BNIP3 and cells either lacking BNIP3 (None) or expressing each BNIP3 mutant in the presence of H2O2 or FCCP are denoted by + p<0.05, ++ p<0.01, and +++ p<0.001; significant differences between complementary pairs of BNIP3 mutants treated with H2O2 or FCCP are shown in brackets. In (D), significant differences between control cells (without BNIP3) and cells expressing each BNIP3 mutant in normoxia are denoted by * p<0.05, ** p<0.01, and *** p<0.001; significant differences between cells expressing ΔTM BNIP3 and cells expressing each other form of BNIP3, all cultured in hypoxia, are denoted by & p<0.05, && p<0.01, and &&& p<0.001. Significant differences between cells expressing WT BNIP3 and cells either lacking BNIP3 (None) or expressing each BNIP3 mutant in normal conditions are denoted by # p<0.05, ## p<0.01, and ### p<0.001; significant differences between cells expressing WT BNIP3 and cells either lacking BNIP3 (None) or expressing each BNIP3 mutant in hypoxic conditions are denoted by + p<0.05, ++ p<0.01, and +++ p<0.001; significant differences between treatment conditions in cells expressing the same BNIP3 mutant are shown in brackets.

Mentions: Given the differential ability of phosphomimetic and nonphosphorylated C-terminal BNIP3 mutants to damage mitochondria, levels of cell death in HEK 293 cells expressing each BNIP3 phosphomutant were measured by Annexin V fluorescence. Consistent with previous reports of increased cell death upon expression of WT or ΔR BNIP3, the percent of Annexin V positive cells was highest in HEK 293 cells expressing WT or nonphosphorylated BNIP3 (Fig 5A) [9, 32]. Conversely, expression of ΔTM, T188D, or 6D BNIP3 did not significantly increase cell death (Fig 5A). Thus, under normal conditions, C-terminal BNIP3 phosphorylation prevents cell death while allowing autophagy to proceed. Levels of HEK 293 cell death were also monitored upon exposure to stress conditions. The addition of H2O2 or FCCP to cells was chosen to increase cellular oxidative stress and mitochondrial stress, respectively. Upon addition of H2O2, cells expressing WT or nonphosphorylated BNIP3 exhibited significant increases in Annexin V fluorescence, whereas cells expressing 6D BNIP3 exhibited full protection from H2O2 toxicity (Fig 5B). In contrast, cells expressing T188D BNIP3 exhibited an intermediate level of cell death during H2O2-induced cellular stress, with cell death being significantly increased compared to H2O2-stressed control cells (not expressing BNIP3), but still significantly lower compared to the level of cell death observed in the H2O2-treated cells expressing the complementary T188A BNIP3 mutant (Fig 5B). Similar results were observed upon mitochondrial stress using FCCP (Fig 5C).


Phosphorylation of the BNIP3 C-Terminus Inhibits Mitochondrial Damage and Cell Death without Blocking Autophagy.

Liu KE, Frazier WA - PLoS ONE (2015)

C-terminal BNIP3 phosphorylation inhibits BNIP3-induced cell death.(A) Quantification of the percent of Annexin V positive cells 24 hr after induction of BNIP3 expression, analyzed by flow cytometry. Data is expressed as the average of 4 independent experiments. Significant differences between control cells (no BNIP3) and cells expressing each form of BNIP3 are denoted by * p<0.05, ** p<0.01, and *** p<0.001; significant differences between complementary pairs of BNIP3 mutants are shown in brackets. (B), (C), and (D): Percent Annexin V positive cells expressing WT or phosphomutant BNIP3 with or without the following treatments: (B) 150μM H2O2 for 120 min, (C) 10μM FCCP for 120 min, and (D) 48 hr hypoxia. In (B) and (C), significant differences between control cells (without BNIP3) and cells expressing each BNIP3 mutant in normal conditions are denoted by * p<0.05, ** p<0.01, and *** p<0.001; significant differences between control cells and cells expressing each BNIP3 mutant, all undergoing additional cellular stress (H2O2, or FCCP), are denoted by $ p<0.05, $ $ p<0.01, and $ $ $ p<0.001. Significant differences between cells expressing WT BNIP3 and cells either lacking BNIP3 (None) or expressing each BNIP3 mutant in normal conditions are denoted by # p<0.05, ## p<0.01, and ### p<0.001; significant differences between cells expressing WT BNIP3 and cells either lacking BNIP3 (None) or expressing each BNIP3 mutant in the presence of H2O2 or FCCP are denoted by + p<0.05, ++ p<0.01, and +++ p<0.001; significant differences between complementary pairs of BNIP3 mutants treated with H2O2 or FCCP are shown in brackets. In (D), significant differences between control cells (without BNIP3) and cells expressing each BNIP3 mutant in normoxia are denoted by * p<0.05, ** p<0.01, and *** p<0.001; significant differences between cells expressing ΔTM BNIP3 and cells expressing each other form of BNIP3, all cultured in hypoxia, are denoted by & p<0.05, && p<0.01, and &&& p<0.001. Significant differences between cells expressing WT BNIP3 and cells either lacking BNIP3 (None) or expressing each BNIP3 mutant in normal conditions are denoted by # p<0.05, ## p<0.01, and ### p<0.001; significant differences between cells expressing WT BNIP3 and cells either lacking BNIP3 (None) or expressing each BNIP3 mutant in hypoxic conditions are denoted by + p<0.05, ++ p<0.01, and +++ p<0.001; significant differences between treatment conditions in cells expressing the same BNIP3 mutant are shown in brackets.
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pone.0129667.g005: C-terminal BNIP3 phosphorylation inhibits BNIP3-induced cell death.(A) Quantification of the percent of Annexin V positive cells 24 hr after induction of BNIP3 expression, analyzed by flow cytometry. Data is expressed as the average of 4 independent experiments. Significant differences between control cells (no BNIP3) and cells expressing each form of BNIP3 are denoted by * p<0.05, ** p<0.01, and *** p<0.001; significant differences between complementary pairs of BNIP3 mutants are shown in brackets. (B), (C), and (D): Percent Annexin V positive cells expressing WT or phosphomutant BNIP3 with or without the following treatments: (B) 150μM H2O2 for 120 min, (C) 10μM FCCP for 120 min, and (D) 48 hr hypoxia. In (B) and (C), significant differences between control cells (without BNIP3) and cells expressing each BNIP3 mutant in normal conditions are denoted by * p<0.05, ** p<0.01, and *** p<0.001; significant differences between control cells and cells expressing each BNIP3 mutant, all undergoing additional cellular stress (H2O2, or FCCP), are denoted by $ p<0.05, $ $ p<0.01, and $ $ $ p<0.001. Significant differences between cells expressing WT BNIP3 and cells either lacking BNIP3 (None) or expressing each BNIP3 mutant in normal conditions are denoted by # p<0.05, ## p<0.01, and ### p<0.001; significant differences between cells expressing WT BNIP3 and cells either lacking BNIP3 (None) or expressing each BNIP3 mutant in the presence of H2O2 or FCCP are denoted by + p<0.05, ++ p<0.01, and +++ p<0.001; significant differences between complementary pairs of BNIP3 mutants treated with H2O2 or FCCP are shown in brackets. In (D), significant differences between control cells (without BNIP3) and cells expressing each BNIP3 mutant in normoxia are denoted by * p<0.05, ** p<0.01, and *** p<0.001; significant differences between cells expressing ΔTM BNIP3 and cells expressing each other form of BNIP3, all cultured in hypoxia, are denoted by & p<0.05, && p<0.01, and &&& p<0.001. Significant differences between cells expressing WT BNIP3 and cells either lacking BNIP3 (None) or expressing each BNIP3 mutant in normal conditions are denoted by # p<0.05, ## p<0.01, and ### p<0.001; significant differences between cells expressing WT BNIP3 and cells either lacking BNIP3 (None) or expressing each BNIP3 mutant in hypoxic conditions are denoted by + p<0.05, ++ p<0.01, and +++ p<0.001; significant differences between treatment conditions in cells expressing the same BNIP3 mutant are shown in brackets.
Mentions: Given the differential ability of phosphomimetic and nonphosphorylated C-terminal BNIP3 mutants to damage mitochondria, levels of cell death in HEK 293 cells expressing each BNIP3 phosphomutant were measured by Annexin V fluorescence. Consistent with previous reports of increased cell death upon expression of WT or ΔR BNIP3, the percent of Annexin V positive cells was highest in HEK 293 cells expressing WT or nonphosphorylated BNIP3 (Fig 5A) [9, 32]. Conversely, expression of ΔTM, T188D, or 6D BNIP3 did not significantly increase cell death (Fig 5A). Thus, under normal conditions, C-terminal BNIP3 phosphorylation prevents cell death while allowing autophagy to proceed. Levels of HEK 293 cell death were also monitored upon exposure to stress conditions. The addition of H2O2 or FCCP to cells was chosen to increase cellular oxidative stress and mitochondrial stress, respectively. Upon addition of H2O2, cells expressing WT or nonphosphorylated BNIP3 exhibited significant increases in Annexin V fluorescence, whereas cells expressing 6D BNIP3 exhibited full protection from H2O2 toxicity (Fig 5B). In contrast, cells expressing T188D BNIP3 exhibited an intermediate level of cell death during H2O2-induced cellular stress, with cell death being significantly increased compared to H2O2-stressed control cells (not expressing BNIP3), but still significantly lower compared to the level of cell death observed in the H2O2-treated cells expressing the complementary T188A BNIP3 mutant (Fig 5B). Similar results were observed upon mitochondrial stress using FCCP (Fig 5C).

Bottom Line: However, some highly respiring cells and cancer cells tolerate BNIP3 expression, suggesting that a yet unknown mechanism exists to restrain the lethal effects of BNIP3 on mitochondria.These findings establish phosphorylation as a switch to determine the pro-survival and pro-death effects of the protein.Our findings also suggest a novel target for the regulation of these activities in transformed cells where BNIP3 is often highly expressed.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, United States of America.

ABSTRACT
BNIP3 is a dual function protein, able to activate autophagy and induce cell death. Upon expression of BNIP3, which is upregulated by hypoxia, the protein induces mitochondrial dysfunction, often leading to cell death. However, some highly respiring cells and cancer cells tolerate BNIP3 expression, suggesting that a yet unknown mechanism exists to restrain the lethal effects of BNIP3 on mitochondria. Here we present evidence that BNIP3 undergoes several phosphorylation events at its C-terminus, adjacent to the transmembrane domain. Phosphorylation at these residues inhibits BNIP3-induced mitochondrial damage, preventing a loss of mitochondrial mass and mitochondrial membrane potential, as well as preventing an increase in reactive oxygen species. This decrease in mitochondrial damage, as well as the reduction of cell death upon C-terminal BNIP3 phosphorylation, can be explained by a diminished interaction between BNIP3 and OPA1, a key regulator of mitochondrial fusion and mitochondrial inner membrane structure. Importantly, phosphorylation of these C-terminal BNIP3 residues blocks cell death without preventing autophagy, providing evidence that the two functional roles of BNIP3 can be regulated independently. These findings establish phosphorylation as a switch to determine the pro-survival and pro-death effects of the protein. Our findings also suggest a novel target for the regulation of these activities in transformed cells where BNIP3 is often highly expressed.

No MeSH data available.


Related in: MedlinePlus