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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 decreases the BNIP3-OPA1 interaction at the mitochondrial membrane.(A) Detection of OPA1 by Western blot following immunoprecipitation of BNIP3 with an α-His tag antibody. (B) Reverse co-immunoprecipitation in which OPA1 was immunoprecipitated (α-OPA1 antibody), and co-immunoprecipitated BNIP3 was detected by Western blot. Blots are representative of 3 experiments. WCL = whole cell lysate. (C) Colocalization of BNIP3 and OPA1 at the mitochondria, examined by confocal microscopy following transient OPA1 overexpression. A minimum of 30 cells expressing each BNIP3 mutant were examined in 3 independent experiments. Scale bar represents 10 μm. (D) Quantification of BNIP3-OPA1 colocalization, expressed as the number of colocalized BNIP3-OPA1 pixels per cell. Significance of each cell type vs control cells (no BNIP3) is denoted by *** p<0.001; significance of each cell type vs cells expressing WT BNIP3 is denoted by # p<0.05, ## p<0.01, and ### p<0.001. (E) Percent Annexin V positive cells expressing BNIP3 with either normal levels of OPA1 (transfected with empty vector, EV) or high levels of OPA1 (transfected with OPA1) for 24 hr. Significance of each cell type vs control cells (no BNIP3) with normal levels of OPA1 is denoted by * p<0.05, ** p<0.01, and *** p<0.001; significant differences between each cell type overexpressing OPA1 vs control cells (no BNIP3) overexpressing OPA1 is denoted by $ p<0.05. Significant differences between cells expressing WT BNIP3 with cells not expressing BNIP3 (None) or expressing each BNIP3 mutant, all with normal levels of OPA1 is denoted by # p<0.05, ## p<0.01, and ### p<0.001; significant differences between each cell type overexpressing OPA1 vs cells expressing WT BNIP3 with exogenous OPA1 are denoted by ++ p<0.01; significant differences between conditions (normal levels of OPA1 vs OPA1 overexpression) for each cell type are denoted in brackets.
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pone.0129667.g006: C-terminal BNIP3 phosphorylation decreases the BNIP3-OPA1 interaction at the mitochondrial membrane.(A) Detection of OPA1 by Western blot following immunoprecipitation of BNIP3 with an α-His tag antibody. (B) Reverse co-immunoprecipitation in which OPA1 was immunoprecipitated (α-OPA1 antibody), and co-immunoprecipitated BNIP3 was detected by Western blot. Blots are representative of 3 experiments. WCL = whole cell lysate. (C) Colocalization of BNIP3 and OPA1 at the mitochondria, examined by confocal microscopy following transient OPA1 overexpression. A minimum of 30 cells expressing each BNIP3 mutant were examined in 3 independent experiments. Scale bar represents 10 μm. (D) Quantification of BNIP3-OPA1 colocalization, expressed as the number of colocalized BNIP3-OPA1 pixels per cell. Significance of each cell type vs control cells (no BNIP3) is denoted by *** p<0.001; significance of each cell type vs cells expressing WT BNIP3 is denoted by # p<0.05, ## p<0.01, and ### p<0.001. (E) Percent Annexin V positive cells expressing BNIP3 with either normal levels of OPA1 (transfected with empty vector, EV) or high levels of OPA1 (transfected with OPA1) for 24 hr. Significance of each cell type vs control cells (no BNIP3) with normal levels of OPA1 is denoted by * p<0.05, ** p<0.01, and *** p<0.001; significant differences between each cell type overexpressing OPA1 vs control cells (no BNIP3) overexpressing OPA1 is denoted by $ p<0.05. Significant differences between cells expressing WT BNIP3 with cells not expressing BNIP3 (None) or expressing each BNIP3 mutant, all with normal levels of OPA1 is denoted by # p<0.05, ## p<0.01, and ### p<0.001; significant differences between each cell type overexpressing OPA1 vs cells expressing WT BNIP3 with exogenous OPA1 are denoted by ++ p<0.01; significant differences between conditions (normal levels of OPA1 vs OPA1 overexpression) for each cell type are denoted in brackets.

Mentions: BNIP3 and OPA1 have been shown to interact in the intermembrane space of mitochondria, in a manner dependent on the extreme C-terminus of BNIP3 [16]. To determine whether C-terminal BNIP3 phosphorylation regulates the interaction of BNIP3 with OPA1, co-immunoprecipitation assays were performed. Due to the reduced level of OPA1 present in HEK 293 cells expressing WT or nonphosphorylated BNIP3, the co-immunoprecipitation assays were performed using HEK 293 cells expressing each BNIP3 mutant with simultaneous OPA1 overexpression, thus minimizing differences in OPA1 availability between cell types (S6A Fig). Following immunoprecipitation of BNIP3 using an α-His tag antibody, OPA1 was detected by Western blot. The highest amounts of co-immunoprecipitated OPA1 were detected in cells expressing WT, T188A, or 6N BNIP3 (Fig 6A). Consistent with previous reports, a markedly decreased amount of OPA1 co-immunoprecipitated with ΔR BNIP3, which lacks the extreme C-terminus (Fig 1D) [16]. Importantly, the lowest amount of OPA1 was co-immunoprecipitated from cells expressing T188D or 6D BNIP3, suggesting that C-terminal BNIP3 phosphorylation decreases the BNIP3-OPA1 interaction (Fig 6A). The decreased interaction between ΔR, T188D, or 6D BNIP3 with OPA1 was confirmed using the reverse co-immunoprecipitation assay, in which an α-OPA1 antibody co-immunoprecipitated low levels of ΔR, T188D, and 6D BNIP3 and higher levels of WT, T188A, and 6N BNIP3 (Fig 6B). In both co-immunoprecipitation experiments, ΔTM BNIP3 did not interact with OPA1, as expected (Fig 6A and 6B). Additionally, the colocalization of BNIP3 and OPA1 was observed via confocal microscopy, where WT, T188A and 6N BNIP3 exhibited strong colocalization with OPA1, and ΔR, T188D, and 6D BNIP3 had decreased colocalization with OPA1 (Fig 6C). Quantification of the number of colocalized BNIP3-OPA1 pixels per cell confirmed that ΔR, T188D, and 6D BNIP3 exhibited significantly decreased colocalization with OPA1 relative to WT BNIP3 (Fig 6D). To ensure the accuracy of these observations, the colocalization of WT or mutant BNIP3 with OPA1 at mitochondria was also monitored using simultaneous detection of MT-CO2, BNIP3, and OPA1 by confocal microscopy (S7 Fig).


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 decreases the BNIP3-OPA1 interaction at the mitochondrial membrane.(A) Detection of OPA1 by Western blot following immunoprecipitation of BNIP3 with an α-His tag antibody. (B) Reverse co-immunoprecipitation in which OPA1 was immunoprecipitated (α-OPA1 antibody), and co-immunoprecipitated BNIP3 was detected by Western blot. Blots are representative of 3 experiments. WCL = whole cell lysate. (C) Colocalization of BNIP3 and OPA1 at the mitochondria, examined by confocal microscopy following transient OPA1 overexpression. A minimum of 30 cells expressing each BNIP3 mutant were examined in 3 independent experiments. Scale bar represents 10 μm. (D) Quantification of BNIP3-OPA1 colocalization, expressed as the number of colocalized BNIP3-OPA1 pixels per cell. Significance of each cell type vs control cells (no BNIP3) is denoted by *** p<0.001; significance of each cell type vs cells expressing WT BNIP3 is denoted by # p<0.05, ## p<0.01, and ### p<0.001. (E) Percent Annexin V positive cells expressing BNIP3 with either normal levels of OPA1 (transfected with empty vector, EV) or high levels of OPA1 (transfected with OPA1) for 24 hr. Significance of each cell type vs control cells (no BNIP3) with normal levels of OPA1 is denoted by * p<0.05, ** p<0.01, and *** p<0.001; significant differences between each cell type overexpressing OPA1 vs control cells (no BNIP3) overexpressing OPA1 is denoted by $ p<0.05. Significant differences between cells expressing WT BNIP3 with cells not expressing BNIP3 (None) or expressing each BNIP3 mutant, all with normal levels of OPA1 is denoted by # p<0.05, ## p<0.01, and ### p<0.001; significant differences between each cell type overexpressing OPA1 vs cells expressing WT BNIP3 with exogenous OPA1 are denoted by ++ p<0.01; significant differences between conditions (normal levels of OPA1 vs OPA1 overexpression) for each cell type are denoted in brackets.
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pone.0129667.g006: C-terminal BNIP3 phosphorylation decreases the BNIP3-OPA1 interaction at the mitochondrial membrane.(A) Detection of OPA1 by Western blot following immunoprecipitation of BNIP3 with an α-His tag antibody. (B) Reverse co-immunoprecipitation in which OPA1 was immunoprecipitated (α-OPA1 antibody), and co-immunoprecipitated BNIP3 was detected by Western blot. Blots are representative of 3 experiments. WCL = whole cell lysate. (C) Colocalization of BNIP3 and OPA1 at the mitochondria, examined by confocal microscopy following transient OPA1 overexpression. A minimum of 30 cells expressing each BNIP3 mutant were examined in 3 independent experiments. Scale bar represents 10 μm. (D) Quantification of BNIP3-OPA1 colocalization, expressed as the number of colocalized BNIP3-OPA1 pixels per cell. Significance of each cell type vs control cells (no BNIP3) is denoted by *** p<0.001; significance of each cell type vs cells expressing WT BNIP3 is denoted by # p<0.05, ## p<0.01, and ### p<0.001. (E) Percent Annexin V positive cells expressing BNIP3 with either normal levels of OPA1 (transfected with empty vector, EV) or high levels of OPA1 (transfected with OPA1) for 24 hr. Significance of each cell type vs control cells (no BNIP3) with normal levels of OPA1 is denoted by * p<0.05, ** p<0.01, and *** p<0.001; significant differences between each cell type overexpressing OPA1 vs control cells (no BNIP3) overexpressing OPA1 is denoted by $ p<0.05. Significant differences between cells expressing WT BNIP3 with cells not expressing BNIP3 (None) or expressing each BNIP3 mutant, all with normal levels of OPA1 is denoted by # p<0.05, ## p<0.01, and ### p<0.001; significant differences between each cell type overexpressing OPA1 vs cells expressing WT BNIP3 with exogenous OPA1 are denoted by ++ p<0.01; significant differences between conditions (normal levels of OPA1 vs OPA1 overexpression) for each cell type are denoted in brackets.
Mentions: BNIP3 and OPA1 have been shown to interact in the intermembrane space of mitochondria, in a manner dependent on the extreme C-terminus of BNIP3 [16]. To determine whether C-terminal BNIP3 phosphorylation regulates the interaction of BNIP3 with OPA1, co-immunoprecipitation assays were performed. Due to the reduced level of OPA1 present in HEK 293 cells expressing WT or nonphosphorylated BNIP3, the co-immunoprecipitation assays were performed using HEK 293 cells expressing each BNIP3 mutant with simultaneous OPA1 overexpression, thus minimizing differences in OPA1 availability between cell types (S6A Fig). Following immunoprecipitation of BNIP3 using an α-His tag antibody, OPA1 was detected by Western blot. The highest amounts of co-immunoprecipitated OPA1 were detected in cells expressing WT, T188A, or 6N BNIP3 (Fig 6A). Consistent with previous reports, a markedly decreased amount of OPA1 co-immunoprecipitated with ΔR BNIP3, which lacks the extreme C-terminus (Fig 1D) [16]. Importantly, the lowest amount of OPA1 was co-immunoprecipitated from cells expressing T188D or 6D BNIP3, suggesting that C-terminal BNIP3 phosphorylation decreases the BNIP3-OPA1 interaction (Fig 6A). The decreased interaction between ΔR, T188D, or 6D BNIP3 with OPA1 was confirmed using the reverse co-immunoprecipitation assay, in which an α-OPA1 antibody co-immunoprecipitated low levels of ΔR, T188D, and 6D BNIP3 and higher levels of WT, T188A, and 6N BNIP3 (Fig 6B). In both co-immunoprecipitation experiments, ΔTM BNIP3 did not interact with OPA1, as expected (Fig 6A and 6B). Additionally, the colocalization of BNIP3 and OPA1 was observed via confocal microscopy, where WT, T188A and 6N BNIP3 exhibited strong colocalization with OPA1, and ΔR, T188D, and 6D BNIP3 had decreased colocalization with OPA1 (Fig 6C). Quantification of the number of colocalized BNIP3-OPA1 pixels per cell confirmed that ΔR, T188D, and 6D BNIP3 exhibited significantly decreased colocalization with OPA1 relative to WT BNIP3 (Fig 6D). To ensure the accuracy of these observations, the colocalization of WT or mutant BNIP3 with OPA1 at mitochondria was also monitored using simultaneous detection of MT-CO2, BNIP3, and OPA1 by confocal microscopy (S7 Fig).

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