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Abstract
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-mtFIS1101-152 ). 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 ~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.
Original language | English |
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Pages (from-to) | 6284-6301 |
Number of pages | 18 |
Journal | FASEB journal : official publication of the Federation of American Societies for Experimental Biology |
Volume | 34 |
Issue number | 5 |
Early online date | 22 Mar 2020 |
DOIs | |
Publication status | Published - 4 May 2020 |
Keywords
- endogenous
- mitophagy
- skeletal muscle
- tandem ubiquitin-binding entity (TUBE)
- ubiquitin
ASJC Scopus subject areas
- Biotechnology
- Biochemistry
- Molecular Biology
- Genetics
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- 1 Finished
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Role of AMPK in Nutrient Sensing and in Cancer (Investigator Award Renewal)
Hardie, G. (Investigator)
1/10/17 → 31/03/24
Project: Research