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.
Highlights
Mitochondria play an important role in maintaining skeletal muscle function.[1]
The important questions in the field of skeletal muscle mitophagy are: (a) whether the PTEN-induced kinase 1 (PINK1)-Parkin signaling pathway is functionally active; (b) how adenosine monophosphate activated protein kinase (AMPK) is involved in the regulation of mitophagy; and (c) whether it cooperates with PINK1-Parkin signaling
These data suggest that AMPK regulates the activation of TANK-binding kinase 1 (TBK1) in skeletal muscle cells, and that this occurs in a PINK1-Parkin independent manner
Summary
Mitochondria play an important role in maintaining skeletal muscle function.[1]. The removal of defective mitochondria (known as mitophagy) has been implicated in the sarcopenia of aging muscle.[1]. Allosteric activation of Parkin through phospho-Ub binding,[11] along with PINK1 mediated Parkin phosphorylation,[12] maximally activates Parkin,[13] allowing Parkin to ubiquitylate OMM proteins such as mitofusin-1/2 (MFN-1/2) and CDGSH iron sulfur domain 1 (CISD1).[14] ubiquitylation of OMM proteins provides a docking site for activated autophagy receptors, such as optineurin (OPTN), nuclear dot protein 52 (NDP52), and sequestosome-1 (SQSTM1/p62), to bind, linking ubiquitylated cargo to autophagic membranes.[15] TANK-binding kinase 1 (TBK1) is thought to be an important signaling node in this process, activating autophagy receptors that link ubiquitylated cargo to the autophagosome.[16,17,18,19,20,21] TBK1 is known to be activated following phosphorylation at Ser 17222 and recent work has suggested that its activation upon mitochondrial depolarization requires both PINK1 and Parkin.[16,17,23] Despite these advances, much of the research that underpins our current understanding of PINK1-Parkin mediated mitophagy has been conducted in mammalian cells that stably express non-native PINK1 and/ or Parkin.[6,7,9,14,16,19,23,24,25,26] murine models have shown that basal mitophagy occurs in skeletal muscle even in the absence of PINK1.27,28 These studies highlight the importance of studying mitophagy and its signaling events using endogenous proteins. Our results suggest that in skeletal muscle AMPK drives mitophagy by enhancing mitochondrial fission (via MFF phosphorylation) and mitochondrial autophagosome engulfment (via TBK1 phosphorylation), in a PINK1Parkin independent manner
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