Dynamic recruitment and activation of ALS-associated TBK1 with its target optineurin are required for efficient mitophagy

Abstract

Mitochondria play an essential role in maintaining cellular homeostasis. The removal of damaged or depolarized mitochondria occurs via mitophagy, in which damaged mitochondria are targeted for degradation via ubiquitination induced by PTEN-induced putative kinase 1 (PINK1) and Parkin. Mitophagy receptors, including optineurin (OPTN), nuclear dot 52 kDa protein (NDP52), and Tax1-binding protein 1 (TAX1BP1), are recruited to mitochondria via ubiquitin binding and mediate autophagic engulfment through their association with microtubule-associated protein light chain 3 (LC3). Here, we use livecell imaging to demonstrate that OPTN, NDP52, and TAX1BP1 are recruited to mitochondria with similar kinetics following either mitochondrial depolarization or localized generation of reactive oxygen species, leading to sequestration by the autophagosome within ∼45 min after insult. Despite this corecruitment, we find that depletion of OPTN, but not NDP52, significantly slows the efficiency of sequestration. OPTN is phosphorylated by the kinase TANK-binding kinase 1 (TBK1) at serine 177; we find that TBK1 is corecruited with OPTN to depolarized mitochondria. Inhibition or depletion of TBK1, or expression of amyotrophic lateral sclerosis (ALS)-associated OPTN or TBK1 mutant blocks efficient autophagosome formation. Together, these results indicate that although there is some functional redundancy among mitophagy receptors, efficient sequestration of damaged mitochondria in response to mitochondrial stress requires both TBK1 and OPTN. Notably, ALSlinked mutations in OPTN and TBK1 can interfere with mitophagy, suggesting that inefficient turnover of damaged mitochondria may represent a key pathophysiological mechanism contributing to neurodegenerative disease.itochondria form interconnected networks that continuously remodel in response to shifting cellular needs (1). These dynamic networks serve as hubs for diverse cellular functions, including aerobic metabolism, calcium homeostasis (2), and redox signaling (3). Several key mitochondrial functions rely on the potential across the mitochondrial inner membrane. Loss of membrane potential is associated with mitochondrial fragmentation and impaired trafficking (4), and can potentially activate cell death pathways (5). To safeguard against these deleterious outcomes, eukaryotic cells have developed quality control mechanisms to monitor the membrane potential of resident mitochondria and selectively eliminate depolarized organelles through mitophagy.In mitophagy, damaged mitochondria are recognized and then sequestered by a double-membrane autophagosome, leading to selective degradation. Regulation of this process involves the ubiquitin kinase PTEN-induced putative kinase 1 (PINK1) (6) and the E3-ubiquitin ligase Parkin (7). Specifically, PINK1 accumulates on the surface of depolarized mitochondria, where it phosphorylates ubiquitin on local outer membrane proteins, resulting in the recruitment of Parkin (7-11). Parkin, in turn, modifies additional mitochondrial outer membrane proteins...

Expanding perspectives on the significance of mitophagy in cancer

“…Furthermore, p62/SQSTM1 is a multi-functional protein with roles in cell signaling and inflammation beyond its function as an autophagy adaptor, including control of NRF2 and mTORC1 activity [64,65]. It has also been questioned to what extent p62/SQSTM1 is actually involved in mitophagy [50,58] with the role of the OPTN, NDP52 and TAX1BP1 cargo adaptors emerging recently as being more critical for mitophagy [50,59,61,62]. OPTN in particular appears to be essential for Parkin/PINK1-dependent mitophagy induced by depolarization or excess ROS [59,61,62].…”

“…It has also been questioned to what extent p62/SQSTM1 is actually involved in mitophagy [50,58] with the role of the OPTN, NDP52 and TAX1BP1 cargo adaptors emerging recently as being more critical for mitophagy [50,59,61,62]. OPTN in particular appears to be essential for Parkin/PINK1-dependent mitophagy induced by depolarization or excess ROS [59,61,62]. OPTN is inactivated in amyotrophic lateral sclerosis (ALS) and ALS-linked mutations prevent recruitment of OPTN to mitochondria with Parkin [59].…”

“…TANK-binding kinase (TBK1), a Parkin/PINK1 target, phosphorylates OPTN, promoting the co-recruitment of OPTN and TBK1 to depolarized mitochondria. At depolarized mitochondria, OPTN binds to both phosphorylated ubiquitin chains and processed LC3 at growing phagophores [51,61,62]. Similar to OPTN, mutations in TBK1 that are associated with ALS, block its ability to promote mitophagy [61].…”

“…At depolarized mitochondria, OPTN binds to both phosphorylated ubiquitin chains and processed LC3 at growing phagophores [51,61,62]. Similar to OPTN, mutations in TBK1 that are associated with ALS, block its ability to promote mitophagy [61]. Interestingly, TBK1 has been linked to tumor dormancy in prostate cancer [66] and TBK1 emerged from an shRNA screen for synthetic lethality with oncogenic K-Ras in human NSCLC cell lines [67] where it was suggested that TBK1 was selectively required in K-Ras transformed tumor cells to prevent apoptosis mediated via NF-κB induced transcription of Bcl-X L [67].…”

“…Both NDP52 and TAX1BP1 are also recruited to depolarized mitochondria but it not yet clear if these cargo adaptors play as extensive a role as OPTN in mitophagy [61,62] although, it has been suggested that NDP52 could synergize with OPTN to promote mitophagy [51]. Several of these studies suggested that p62/SQSTM1 was not required for PINK1/Parkin-dependent mitophagy [50,51,61] but further work is needed to determine the specific contexts in which one autophagy adaptor molecule is utilized preferentially in PINK1/Parkin-dependent mitophagy over another.…”