Modulation of Serines 17 and 24 in the LC3-interacting Region of Bnip3 Determines Pro-survival Mitophagy versus Apoptosis

• Published in: The Journal of biological chemistry Vol. 288; no. 2; pp. 1099 - 1113
• Main Authors: Zhu, Yanyan, Massen, Stefan, Terenzio, Marco, Lang, Verena, Chen-Lindner, Silu, Eils, Roland, Novak, Ivana, Dikic, Ivan, Hamacher-Brady, Anne, Brady, Nathan R.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 11.01.2013; American Society for Biochemistry and Molecular Biology
• Subjects: Apoptosis; Autophagy; Base Sequence; Bcl-2 Family; Bcl-xL; Bnip3; Cell Biology; Cell Line; Cell Survival; DNA Primers; Flow Cytometry; Humans; Immunoprecipitation; LC3; LIR Domain; Membrane Proteins - chemistry; Membrane Proteins - metabolism; Membrane Proteins - physiology; Microscopy, Fluorescence; Microtubule-Associated Proteins - metabolism; Mitochondria; Mitochondrial Apoptosis; Mitophagy; Phosphorylation; Proto-Oncogene Proteins - chemistry; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins - physiology; Serine - metabolism; Mitochondria; Bcl-xL; Bcl-2 Family; Mitochondrial Apoptosis; Bnip3; Mitophagy; Autophagy; LC3; LIR Domain; Apoptosis
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.399345

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-xL 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. Background: Bnip3 is both a pro-apoptotic BH3-only protein and a mitochondrial autophagy receptor. Results: Serine phosphorylation of the Bnip3 LC3-interacting region (LIR) increased binding to Atg8 members and consequently mitophagy, in a manner positively regulated by Bcl-xL. Conclusion: The Bnip3 LIR activity state determines either pro-survival mitophagy or mitochondrial apoptosis. Significance: Bnip3-induced mitophagy is serine kinase-regulated and thus a targetable pathway.