Journal
FASEB JOURNAL
Volume 34, Issue 5, Pages 6284-6301Publisher
WILEY
DOI: 10.1096/fj.201903051R
Keywords
endogenous; mitophagy; skeletal muscle; tandem ubiquitin-binding entity (TUBE); ubiquitin
Categories
Funding
- BBSRC
- MRC Versus Arthritis Centre for Musculoskeletal Ageing Research
- Wellcome Trust [204766/Z/16/Z, 104612/Z/14/Z]
- MRC [MR/N00275X/1, MR/S025618/1]
- Diabetes UK [17/0005681]
- European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program [715884]
- MRC [1854365, MR/N00275X/1, MR/S025618/1] Funding Source: UKRI
- Wellcome Trust [104612/Z/14/Z] Funding Source: researchfish
- Wellcome Trust [104612/Z/14/Z] Funding Source: Wellcome Trust
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Mitophagy is a key process regulating mitochondrial quality control. Several mechanisms have been proposed to regulate mitophagy, but these have mostly been studied using stably expressed non-native proteins in immortalized cell lines. In skeletal muscle, mitophagy and its molecular mechanisms require more thorough investigation. To measure mitophagy directly, we generated a stable skeletal muscle C2C12 cell line, expressing a mitophagy reporter construct (mCherry-green fluorescence protein-mtFIS1(101-152)). Here, we report that both carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment and adenosine monophosphate activated protein kinase (AMPK) activation by 991 promote mitochondrial fission via phosphorylation of MFF and induce mitophagy by similar to 20%. Upon CCCP treatment, but not 991, ubiquitin phosphorylation, a read-out of PTEN-induced kinase 1 (PINK1) activity, and Parkin E3 ligase activity toward CDGSH iron sulfur domain 1 (CISD1) were increased. Although the PINK1-Parkin signaling pathway is active in response to CCCP treatment, we observed no change in markers of mitochondrial protein content. Interestingly, our data shows that TANK-binding kinase 1 (TBK1) phosphorylation is increased after both CCCP and 991 treatments, suggesting TBK1 activation to be independent of both PINK1 and Parkin. Finally, we confirmed in non-muscle cell lines that TBK1 phosphorylation occurs in the absence of PINK1 and is regulated by AMPK-dependent signaling. Thus, AMPK activation promotes mitophagy by enhancing mitochondrial fission (via MFF phosphorylation) and autophagosomal engulfment (via TBK1 activation) in a PINK1-Parkin independent manner.
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