Modulation of serines 17 and 24 in the LC3-interacting region of Bnip3 determines pro-survival mitophagy versus apoptosis

Abstract

BH3-only proteins integrate apoptosis and autophagy pathways, yet regulation and functional consequences of pathway cross-talk are not fully resolved. The BH3-only protein Bnip3 is an autophagy receptor that signals autophagic degradation of mitochondria (mitophagy) via interaction of its LC3-interacting region (LIR) with Atg8 proteins. Here we report that phosphorylation of serine residues 17 and 24 flanking the Bnip3 LIR promotes binding to specific Atg8 members LC3B and GATE-16. Using quantitative multispectral image-based flow cytometry, we demonstrate that enhancing Bnip3-Atg8 interactions via phosphorylation-mimicked LIR mutations increased mitochondrial sequestration, lysosomal delivery, and degradation. Importantly, mitochondria were targeted by mitophagy prior to cytochrome c release, resulting in reduced cellular cytochrome c release capacity. Intriguingly, pro-survival Bcl-x(L) positively regulated Bnip3 binding to LC3B, sequestration, and mitochondrial autophagy, further supporting an anti-apoptotic role for Bnip3-induced mitophagy. The ensemble of these results demonstrates that the phosphorylation state of the Bnip3 LIR signals either the induction of apoptosis or pro-survival mitophagy.

FIGURE 1.

Bnip3 up-regulation correlates with mitochondrial autophagy in cardiac HL-1 cells. A, hypoxia/reoxygenation increased Bnip3 expression and decreased mitochondrial content. HL-1 cells were submitted to 18 h of hypoxia, followed by 2 h of reoxygenation Western blot detection of mitochondrial markers Tim23, COXIV, Bnip3, and RhoGDI as a loading control. Shown is densitometric quantification of Tim23 and COXIV, normalized to RhoGDI. B, immunodetection of endogenous Bnip3 targeted to mitochondria, where it colocalized with GFP-LC3B following hypoxia/reoxygenation. Bi, HL-1 cells expressing GFP-LC3B were submitted to 18 h of hypoxia, followed by 2 h of reoxygenation. GFP-LC3B-labeled autophagosomes (pseudocolored light blue) and antibody labeled Tom20 (red) and Bnip3 (purple) were imaged by high resolution microscopy. Bnip3-targeted mitochondria that colocalized with autophagosomes appear dark blue. Bii, the region of interest from Bi is magnified, and the ImageJ plot profile function was used to illustrate colocalizations within the bounded region between autophagosomes (blue), Bnip3 (purple), and Tom20 (red). Error bars, S.E.

FIGURE 2.

The Bnip3 LIR determines the interaction with LC3B, in a manner dependent on serine/threonine phosphorylation. A, sequence alignment demonstrating that Bnip3 contains a core tetrapeptide LIR, identical to Nix. B and C, Bnip3 WVEL is a functioning LIR and is responsible for interactions with LC3B. B, RFP-tagged Bnip3 WT and LIR mutant W18A/L21A were expressed in MCF-7 cells stably expressing GFP-LC3B. At 48 h of expression, immunoprecipitations were performed with α-GFP. Shown is Western blot detection of RFP. C, CCCP-enhanced mitophagy is dependent on Bnip3 LIR. Ci, RFP-tagged Bnip3 WT and Bnip3 W18A/L21A co-expressed with GFP-LC3B in HeLa cells for 24 h, treated with CCCP (10 μm; 6 h), and imaged by high resolution microscopy. Bnip3 is pseudocolored light blue, and LC3B is pseudocolored purple. Dark blue indicates colocalization due to sequestration of mitochondria. Cii, plot profiles of Bnip3 (light blue) and LC3B (purple) intensities demonstrating localizations of Bnip3-targeted mitochondria and autophagosomes. Ciii, colocalization of Bnip3 WT and Bnip3 W18A/L21A with GFP-LC3B was quantified using the ImageJ colocalization color map plugin. Dot plots indicate the data set distribution and mean correlation index (I_corr) ± S.E. of at least 10 cells (Z-stacks) per condition. D, the serine/threonine phosphatase inhibitor okadaic acid (OA) increases binding of Bnip3 to LC3B. RFP-Bnip3 WT was expressed in MCF-7 cells stably expressing GFP-LC3B. At 42 h of expression, cells were treated with increasing concentrations of okadaic acid (1, 10, 50, and 100 nm) for 6 h, and immunoprecipitations (IP) were then performed with α-GFP. Western blot (WB) detection of RFP and GFP. *, both bands correspond to RFP-Bnip3. E, binding of Bnip3 to LC3B is dependent on serine/threonine kinase activities. RFP-Bnip3 WT was expressed in MCF-7 cells stably expressing GFP-LC3B. At 42 h of expression, cells were treated with concentrations of K252c (staurosporine aglycone; 16, 160, and 1600 nm) for 6 h, and immunoprecipitations were then performed with α-GFP. Shown is Western blot detection of RFP and GFP. *, both bands correspond to RFP-Bnip3.

FIGURE 3.

Serine phosphorylation of the Bnip3 LIR motif determines its affinity for LC3B. A, putative serine phosphorylation sites of interest and generated mutants. Ai, alignment of the region containing the Bnip3 LIR across mammalian species. Aii, table describing Bnip3 mutants used in this study. B, phosphorylation state of the LIR determines autophagosomal sequestration of mitochondria. Bi, representative high resolution images of GFP-LC3B colocalization with RFP-Bnip3 WT and the indicated multisite LIR mutants at mitochondria in HeLa cells at 24 h. Plot profiles illustrate colocalization between Bnip3-targeted mitochondria and autophagosomes. Bii, MIFC analysis of Bnip3 WT, 2SA, and 2SE induction of mitophagy. The indicated Bnip3 constructs were expressed with GFP-LC3B in cardiac HL-1 cells for 24 h and imaged using the ImageStream X flow cytometer. Representative images and plot profiles are shown. Biii, using MIFC, quantification of the subpopulation of Bnip3 WT-targeted mitochondria with high GFP-LC3B colocalization was achieved using the colocalization function and gating on the fraction of cells with high colocalization. The identical analysis was applied to Bnip3 mutant populations. Population fractions are indicated as well as the percentage change of the two mutants compared with the wild type. C, localization of serine residues, which control binding affinity with LC3B. Ci, the indicated RFP-Bnip3 WT and multisite and single site mutant constructs were expressed in MCF-7 cells stably expressing GFP-LC3B. At 48 h of expression, immunoprecipitations (IP) were performed with α-GFP. Shown is Western blot detection of RFP, GFP, and actin. Cii, double mutant S17A/S24E and S17E/S24A Bnip3 constructs were expressed in MCF-7 stably expressing GFP-LC3B. At 48 h of expression, immunoprecipitations were performed with α-GFP. Shown are Western blot detection of RFP and GFP. Ciii, in vitro analysis of serine 17 phosphorylation during LC3B binding to Bnip3. Bnip3 S24E and 2SE mutants were expressed for 48 h in MCF-7 cells stably expressing GFP-LC3B. Cell lysates were incubated with λ-phosphatase (800 units) prior to immunoprecipitation with α-GFP. Shown is Western blot detection of RFP and GFP. PI, phosphatase inhibitor.

FIGURE 4.

Serine phosphorylation of the Bnip3 LIR motif determines its affinity for GATE-16. A, LIR serine residues control binding affinity for GATE-16. The indicated RFP-Bnip3 WT and multisite mutant constructs were expressed in MCF-7 stably expressing GFP-GATE-16. At 48 h of expression, immunoprecipitations (IP) were performed with α-GFP. Shown is Western blot (WB) detection of Bnip3. *, this band of RFP-Bnip3 overlaps with IgG heavy chain. B, phosphorylation at serine 17 is required for, whereas phosphorylation at serine 24 promotes, Bnip3 binding to GATE-16. The indicated RFP-Bnip3 WT and multisite and single site mutant constructs were expressed in MCF-7 stably expressing GFP-GATE-16. At 48 h of expression, immunoprecipitations were performed with α-GFP. Shown is Western blot detection of RFP, GFP, and actin. C, LIR phosphorylation enhances GATE-16 sequestration of Bnip3-targeted mitochondria. Shown is high resolution imaging of GFP-GATE-16 colocalization with WT and multisite LIR mutant Bnip3 at mitochondria in HeLa cells at 24 h. Plot profiles identify colocalizations between Bnip3-targeted mitochondria and autophagosomes. D, LIR phosphorylation does not enhance binding of GABARAP and GABARAP-L1. Strongly binding RFP-Bnip3 S24E and non-binding RFP-Bnip3 S24A were co-expressed with GFP-fused GABARAP, GABARAP-L1, or GABARAP-L2/GATE-16 in HeLa cells. At 24 h of expression, immunoprecipitations were performed with α-GFP antibody. Shown is Western blot detection of RFP and GFP. E, dual phosphorylation at serine 17 and serine 24 enhances GATE-16 binding. Double mutant S17A/S24E and S17E/S24A Bnip3 constructs were expressed in MCF-7 cells stably expressing GFP-GATE-16. At 48 h of expression, immunoprecipitations were performed with α-GFP. Shown is Western blot detection of RFP and GFP. F, okadaic acid treatment increases binding of Bnip3 to GATE-16. RFP-Bnip3 WT was expressed in MCF-7 cells stably expressing GATE-16. At 42 h of expression, cells were treated with increasing concentrations of okadaic acid (1, 10, 50, and 100 nm) for 6 h, and immunoprecipitations were then performed with α-GFP. Shown is Western blot detection of RFP and GFP.

FIGURE 5.

Autophagy induction is required for Bnip3 binding to LC3B. A, Bnip3 binding to LC3B depends on LC3B lipidation. GFP-LC3B and GFP-LC3B (G120A) were co-expressed with either RFP-Bnip3 WT or Bnip3 2SE. At 18 h of expression, high resolution imaging was performed. Plot profiles illustrate colocalizations between Bnip3-targeted mitochondria and autophagosomes. B, Beclin-1 knockdown inhibits the interaction between Bnip3 and LC3B. Bi, stable control and Beclin-1 shRNA knockdown were performed in MCF7 cells stably expressing GFP-LC3B. Bii, Bnip3 WT, Bnip3 2A, and Bnip3 2E were expressed in MCF-7/GFP-LC3B with stable Beclin-1 knockdown or control knockdown. At 48 h of expression, immunoprecipitations (IP) were performed with α-GFP. Shown is Western blot (WB) detection of RFP and GFP. *, this band of RFP-Bnip3 overlaps with IgG heavy chain. C, CCCP drives mitochondrial sequestration in a LIR-dependent manner. Ci, Bnip3 2SA was co-expressed with GFP-LC3B in HeLa cells for 24 h, treated with CCCP (10 μm, 6 h), and imaged by high resolution microscopy. Cii, colocalization of Bnip3 WT, Bnip3 2A, and Bnip3 2E with GFP-LC3B in HeLa cells treated with CCCP (10 μm, 6 h) was quantified using the ImageJ colocalization color map plugin. Dot plots indicate the data set distribution and mean correlation index (I_corr) ± S.E. of at least 10 cells (Z-stacks) per condition. Ciii, RFP-tagged Bnip3 WT was expressed in MCF-7 cells stably expressing GFP-LC3B for 48 h. Cells were treated with CCCP as indicated, and immunoprecipitations were performed with α-GFP. Shown is Western blot detection of RFP and GFP.

FIGURE 6.

Bnip3 LIR phosphorylation regulates maturation and autophagic degradation of mitochondria. A, mitophagy is enhanced by Bnip3 LIR phosphorylation. RFP-Bnip3 WT and the indicated multisite and single site mutant constructs were expressed in HeLa cells for 48 h. Ai, high resolution imaging of RFP-Bnip3-targeted mitochondria immunolabeled for Tom20 (green). Aii, Western blot analysis of mitochondrial protein levels. HeLa cells expressing RFP or the indicated RFP-Bnip3 construct were harvested 48 h after transfection and analyzed by Western blot. The levels of Tim23 and VDAC were quantified by densitometric analysis and normalized to RhoGDI. Aiii, use of MIFC to quantify subpopulations of cells with high mitophagy (low mitochondrial mass) through detection of mitochondrial Tom20 content in cell populations. Example phenotypes of high and low Tom20-labeled cells are shown below the graph. B, LIR phosphorylation enhances Bnip3 targeting to lysosomes. Bi, RFP-Bnip3 WT and indicated multisite and single site mutant constructs were co-expressed with the endolysosomal marker GFP-Rab7 in HeLa cells for 24 h. Shown are plot profiles of Bnip3 (light blue) and Rab7 (purple) demonstrating localizations of Bnip3-targeted mitochondria and autolysosomes. Bii, MIFC analysis of Bnip3 WT, 2SA, and 2SE colocalization with the endo-/lysosomal marker Rab7. The indicated Bnip3 constructs were expressed with GFP-LC3B in cardiac HL-1 cells for 24 h and imaged using the ImageStream X flow cytometer. Representative images and plot profiles are shown. Biii, using MIFC, the subpopulation of Bnip3 WT-targeted mitochondria with high GFP-Rab7 colocalization was quantified, gating on the fraction of cells with high colocalization. Population fractions are indicated as well as the percentage change of the two Bnip3 mutants compared with Bnip3 WT. Error bars, S.E.

FIGURE 7.

Bcl-x_L overexpression enhances autophagic sequestration of Bnip3-targeted mitochondria. A, Bcl-x_L enhances Bnip3-induced mitochondrial sequestration. RFP-Bnip3 WT and the indicated multisite mutants were co-expressed with GFP-LC3B for 24 h and imaged by high resolution microscopy. As a control, MCF-7 cells were stably transfected with pcDNA3.1 (Ai), and MCF-7 cells were generated to stably overexpress Bcl-x_L (Aii), shown in Bi. B, Bcl-x_L enhances LIR-dependent Bnip3 binding to LC3B. RFP, RFP-Bnip3 WT, and 2SE were expressed in MCF-7 cells stably expressing GFP-LC3B, together with either Bcl-x_L or vector control pcDNA3.1. Bi, Western blot detection of Bcl-x_L expression in control pcDNA- and pcDNA-Bcl-x_L-expressing MCF-7 cells. Bii, at 48 h of expression immunoprecipitations (IP) were performed with α-GFP. Shown is Western blot (WB) detection of RFP and GFP. Biii, immunoprecipitated Bnip3 was quantified by densitometric analysis and normalized to input. C, Bnip3 promotes Bcl-x_L localization to autophagosomes. Ci, GFP-LC3B and RFP-Bcl-x_L were co-expressed with either pcDNA3.1 or pcDNA-Bnip3 in HeLa cells for 24 h and imaged by high resolution microscopy. Plot profiles illustrate colocalizations between Bcl-x_L-targeted mitochondria and autophagosomes. Cii, autophagosome sequestration of Bcl-x_L-targeted mitochondria in the absence and presence of Bnip3 was quantified using the ImageJ colocalization color map plugin. Dot plots indicate the data set distribution and mean correlation index (I_corr) ± S.E. of at least 10 cells (Z-stacks) per condition. D, the Bnip3 BH3 domain is required for LC3B binding. RFP-fused WT Bnip3 and Bnip3 deleted for its BH3 domain (Bnip3ΔBH3) were expressed in MCF-7 cells stably expressing GFP-LC3B. Di, at 48 h of expression, immunoprecipitations were performed with α-GFP. Shown is Western blot detection of RFP and GFP. Dii, RFP-Bnip3ΔBH3 and GFP-LC3B were expressed in HeLa cells, in the presence and absence of pcDNA3.1-Bcl-x_L. High resolution imaging was performed at 24 h. Plot profiles illustrate colocalizations between Bnip3ΔBH3-targeted mitochondria and autophagosomes. E, shRNA depletion of Bcl-x_L reduced Bnip3 binding to LC3B. Ei, Western blot detection of Bcl-x_L expression in control shRNA and Bcl-x_L knock-down shRNA HeLa cells. Eii, at 24 h of expression, immunoprecipitations were performed with α-GFP. Shown is Western blot detection of RFP and GFP. Error bars, S.E.

FIGURE 8.

Mimicked phosphorylation of Bnip3 LIR promotes mitochondrial cytochrome c degradation. A, quantification of cellular cytochrome c levels by Western blot. HeLa cells expressing RFP or the indicated Bnip3 construct were harvested 48 h after transfection and analyzed via Western blot. The levels of cytochrome c were quantified by densitometric analysis and normalized to RhoGDI. Cellular cytochrome c content was found decreased in RFP-Bnip3 WT and RFP-Bnip3 2SE-expressing cells, compared with cells expressing RFP. B, cellular cytochrome c content regulation by LIR phosphorylation. RFP-Bnip3 WT and indicated multisite and single site mutant constructs were expressed in HeLa cells for 48 h. Subpopulations of cells with low mitochondrial cytochrome c content were quantified by MIFC detection of mitochondrial cytochrome c. Example phenotypes of high and low cytochrome c-positive cells are shown below the graph. C, MCF-7 cells expressing RFP-Bnip3 2SE and BFP-LC3B for 24 h were stained with rhodamine 123 (Rhod123; 50 nm) to assess mitochondrial polarization state. D, Bnip3-induced mitophagy of cytochrome c-containing mitochondria is enhanced by Bcl-x_L. Di, RFP-LC3B and Bcl-x_L alone or in combination with Bnip3 were expressed in MCF-7 cells stably expressing cytochrome c-GFP. At 24 h, high resolution imaging was performed. Dii, enlarged region of Bnip3/Bcl-x_L cell in i. The plot profile illustrates colocalizations between Bnip3-targeted mitochondria and autophagosomes in the enlarged region. iii, autophagosome sequestration of cytochrome c-containing mitochondria, in the presence of Bnip3 and Bnip3/Bcl-x_L expression, was quantified using the ImageJ colocalization color map plugin. Dot plots indicate the data set distribution and mean correlation index (I_corr) ± S.E. of at least 10 cells (Z-stacks) per condition. E, effect of Bnip3-induced mitophagy on caspase 3 activation. Following 24 h of expression of RFP, RFP-Bnip3, RFP-Bnip3 2SA, or RFP-Bnip3 2SE, HeLa cells were treated with TNFα for 6 h and analyzed by Western blot. The levels of active caspase-3 were quantified by densitometric analysis and normalized to actin. F, schematic for Bnip3 regulation and interactions that form the cell fate decision of apoptosis versus mitophagy. Error bars, S.E.