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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 prevents damage to the mitochondrial network.(A) Representative examples of mitochondrial morphology, examined via transmission electron microscopy. Black arrows denote healthy, elongated mitochondria and white arrows denote rounded, swollen mitochondria. Scale bar represents 2 μm. (B) Quantification of electron microscopy, showing the average mitochondrial area per field. At least 15 fields were examined per cell type. (C) Percent elongated mitochondria per field, quantified from at least 15 microscope fields per cell type. (D) Mitochondrial mass of HEK 293 cells expressing each BNIP3 mutant, measured by flow cytometric analysis of the mean fluorescence intensity (MFI) of Mitotracker Green FM. (E) Mitochondrial protein levels in HEK 293 cells expressing each BNIP3 mutant, monitored by detection of mitochondrially-encoded cytochrome c oxidase subunit II (MT-CO2). For bar graphs, significant differences between control cells (without BNIP3) and cells expressing each BNIP3 mutant are denoted by * p<0.05, ** p<0.01, and *** p<0.001; significant differences between cells expressing WT BNIP3 and either control cells or cells expressing each BNIP3 mutant 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.
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pone.0129667.g002: C-terminal BNIP3 phosphorylation prevents damage to the mitochondrial network.(A) Representative examples of mitochondrial morphology, examined via transmission electron microscopy. Black arrows denote healthy, elongated mitochondria and white arrows denote rounded, swollen mitochondria. Scale bar represents 2 μm. (B) Quantification of electron microscopy, showing the average mitochondrial area per field. At least 15 fields were examined per cell type. (C) Percent elongated mitochondria per field, quantified from at least 15 microscope fields per cell type. (D) Mitochondrial mass of HEK 293 cells expressing each BNIP3 mutant, measured by flow cytometric analysis of the mean fluorescence intensity (MFI) of Mitotracker Green FM. (E) Mitochondrial protein levels in HEK 293 cells expressing each BNIP3 mutant, monitored by detection of mitochondrially-encoded cytochrome c oxidase subunit II (MT-CO2). For bar graphs, significant differences between control cells (without BNIP3) and cells expressing each BNIP3 mutant are denoted by * p<0.05, ** p<0.01, and *** p<0.001; significant differences between cells expressing WT BNIP3 and either control cells or cells expressing each BNIP3 mutant 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.

Mentions: Upon expression of each BNIP3 phosphomutant in HEK 293 cells, mitochondrial morphology and content were examined. Analysis of mitochondrial morphology using transmission electron microscopy revealed a disruption of the mitochondrial network, exemplified by rounding of mitochondria and mitochondrial swelling in cells expressing WT, ΔR, T188A, or 6N BNIP3 (Fig 2A, white arrows). This is consistent with previous studies of WT BNIP3 by electron microscopy [7, 24, 35]. Conversely, the mitochondria of cells expressing T188D or 6D BNIP3 maintained a healthy mitochondrial network, exemplified by the retention of elongated mitochondria (Fig 2A, black arrows). Importantly, comparison of the average mitochondrial area per field and the percent of elongated mitochondria per field revealed that expression of T188D or 6D BNIP3 did not significantly reduce mitochondrial area or increase mitochondrial fragmentation relative to control cells lacking BNIP3 (Fig 2B and 2C). Furthermore, expression of WT or nonphosphorylated ΔR, T188A, or 6N BNIP3 resulted in decreased mitochondrial mass, determined by Mitotracker Green FM fluorescence (Fig 2D). This is consistent with previous observations that WT BNIP3 induces a loss of mitochondrial mass [7]. In contrast, expression of the phosphomimetic T188D or 6D BNIP3 mutants did not significantly reduce mitochondrial mass (Fig 2D). These results were confirmed by Western blot analysis of MT-CO2 (mitochondrially encoded cytochrome c oxidase II) levels, where expression of WT or nonphosphorylated BNIP3 reduced MT-CO2 protein levels relative to control cells lacking BNIP3 (Fig 2E).


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 prevents damage to the mitochondrial network.(A) Representative examples of mitochondrial morphology, examined via transmission electron microscopy. Black arrows denote healthy, elongated mitochondria and white arrows denote rounded, swollen mitochondria. Scale bar represents 2 μm. (B) Quantification of electron microscopy, showing the average mitochondrial area per field. At least 15 fields were examined per cell type. (C) Percent elongated mitochondria per field, quantified from at least 15 microscope fields per cell type. (D) Mitochondrial mass of HEK 293 cells expressing each BNIP3 mutant, measured by flow cytometric analysis of the mean fluorescence intensity (MFI) of Mitotracker Green FM. (E) Mitochondrial protein levels in HEK 293 cells expressing each BNIP3 mutant, monitored by detection of mitochondrially-encoded cytochrome c oxidase subunit II (MT-CO2). For bar graphs, significant differences between control cells (without BNIP3) and cells expressing each BNIP3 mutant are denoted by * p<0.05, ** p<0.01, and *** p<0.001; significant differences between cells expressing WT BNIP3 and either control cells or cells expressing each BNIP3 mutant 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.
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Related In: Results  -  Collection

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pone.0129667.g002: C-terminal BNIP3 phosphorylation prevents damage to the mitochondrial network.(A) Representative examples of mitochondrial morphology, examined via transmission electron microscopy. Black arrows denote healthy, elongated mitochondria and white arrows denote rounded, swollen mitochondria. Scale bar represents 2 μm. (B) Quantification of electron microscopy, showing the average mitochondrial area per field. At least 15 fields were examined per cell type. (C) Percent elongated mitochondria per field, quantified from at least 15 microscope fields per cell type. (D) Mitochondrial mass of HEK 293 cells expressing each BNIP3 mutant, measured by flow cytometric analysis of the mean fluorescence intensity (MFI) of Mitotracker Green FM. (E) Mitochondrial protein levels in HEK 293 cells expressing each BNIP3 mutant, monitored by detection of mitochondrially-encoded cytochrome c oxidase subunit II (MT-CO2). For bar graphs, significant differences between control cells (without BNIP3) and cells expressing each BNIP3 mutant are denoted by * p<0.05, ** p<0.01, and *** p<0.001; significant differences between cells expressing WT BNIP3 and either control cells or cells expressing each BNIP3 mutant 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.
Mentions: Upon expression of each BNIP3 phosphomutant in HEK 293 cells, mitochondrial morphology and content were examined. Analysis of mitochondrial morphology using transmission electron microscopy revealed a disruption of the mitochondrial network, exemplified by rounding of mitochondria and mitochondrial swelling in cells expressing WT, ΔR, T188A, or 6N BNIP3 (Fig 2A, white arrows). This is consistent with previous studies of WT BNIP3 by electron microscopy [7, 24, 35]. Conversely, the mitochondria of cells expressing T188D or 6D BNIP3 maintained a healthy mitochondrial network, exemplified by the retention of elongated mitochondria (Fig 2A, black arrows). Importantly, comparison of the average mitochondrial area per field and the percent of elongated mitochondria per field revealed that expression of T188D or 6D BNIP3 did not significantly reduce mitochondrial area or increase mitochondrial fragmentation relative to control cells lacking BNIP3 (Fig 2B and 2C). Furthermore, expression of WT or nonphosphorylated ΔR, T188A, or 6N BNIP3 resulted in decreased mitochondrial mass, determined by Mitotracker Green FM fluorescence (Fig 2D). This is consistent with previous observations that WT BNIP3 induces a loss of mitochondrial mass [7]. In contrast, expression of the phosphomimetic T188D or 6D BNIP3 mutants did not significantly reduce mitochondrial mass (Fig 2D). These results were confirmed by Western blot analysis of MT-CO2 (mitochondrially encoded cytochrome c oxidase II) levels, where expression of WT or nonphosphorylated BNIP3 reduced MT-CO2 protein levels relative to control cells lacking BNIP3 (Fig 2E).

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