Miro phosphorylation sites regulate Parkin recruitment and mitochondrial motility

Abstract

The PTEN-induced putative kinase 1 (PINK1)/Parkin pathway can tag damaged mitochondria and trigger their degradation by mitophagy. Before the onset of mitophagy, the pathway blocks mitochondrial motility by causing Miro degradation. PINK1 activates Parkin by phosphorylating both Parkin and ubiquitin. PINK1, however, has other mitochondrial substrates, including Miro (also called RhoT1 and -2), although the significance of those substrates is less clear. We show that mimicking PINK1 phosphorylation of Miro on S156 promoted the interaction of Parkin with Miro, stimulated Miro ubiquitination and degradation, recruited Parkin to the mitochondria, and via Parkin arrested axonal transport of mitochondria. Although Miro S156E promoted Parkin recruitment it was insufficient to trigger mitophagy in the absence of broader PINK1 action. In contrast, mimicking phosphorylation of Miro on T298/T299 inhibited PINK1-induced Miro ubiquitination, Parkin recruitment, and Parkin-dependent mitochondrial arrest. The effects of the T298E/T299E phosphomimetic were dominant over S156E substitution. We propose that the status of Miro phosphorylation influences the decision to undergo Parkin-dependent mitochondrial arrest, which, in the context of PINK1 action on other substrates, can restrict mitochondrial dynamics before mitophagy.is the second most common neurodegenerative disorder, and is closely linked to mitochondrial dysfunction (1, 2). Two hereditary forms of recessive PD are caused by mutations in PINK1 (PTEN-induced putative kinase 1), a Ser/Thr mitochondrial kinase, and Parkin, a cytosolic E3 ubiquitin ligase (3, 4). The realization that these proteins are in a single pathway, with PINK1 acting upstream of Parkin to influence mitochondrial properties, was a critical step in uncovering the underlying pathological mechanisms of PD (5-7). This pathway can trigger the selective autophagy of damaged mitochondria, termed mitophagy (8, 9), but additional cellular functions have also been indicated for PINK1 and Parkin (10-15). Much, however, remains unclear about how the PINK1/Parkin pathway is regulated.In current models of PINK1/Parkin mitophagy (reviewed in ref. 1), healthy mitochondria import a PINK1 precursor constitutively to the inner membrane, where it is cleaved (16-18). The cleaved form then returns to the cytoplasm and is degraded by the N-end rule pathway (19). Mitochondrial depolarization, or protein misfolding in the matrix of energized mitochondria (20), prevent the import and degradation of PINK1, resulting in the accumulation of PINK1 on the outer mitochondrial membrane (OMM) (9,21,22). Once on the OMM, PINK1 kinase activity recruits Parkin from the cytosol (8, 9). Although Parkin adopts a self-inhibited conformation in solution (23-25), it becomes fully activated in a PINK1-dependent manner on the mitochondria (9, 21). Parkin ubiquitinates numerous proteins of the OMM (26, 27), and thereby recruits autophagy-related proteins to the damaged mitochondrion for autophagosome assembly (28-30).How PINK1 recruits and activa...

Mitostasis in Neurons: Maintaining Mitochondria in an Extended Cellular Architecture

“…Early evidence for this model came from a study of the voltage sensitive dye JC-1 in axons, which found that those mitochondria moving retrograde had lower membrane potentials than those moving anterograde and that inhibition of the electron transport chain (ETC) with Antimycin A increased retrograde movement (Miller and Sheetz, 2004). A subsequent study, however, that used TMRM to assay mitochondrial membrane potential found no distinction between anterograde and retrograde populations (Verburg and Hollenbeck, 2008) and others have observed the arrest of mitochondrial movement in response to inhibitors of the ETC, an arrest mediated by the proteolytic degradation of Miro via the PINK1/Parkin pathway (Hsieh et al, 2016; Liu et al, 2012; Shlevkov et al, 2016; Wang et al, 2011). Most recently, a study employed exposures of dorsal root ganglion neurons for 6 hours or longer to very low levels of antimycin A to mimic a long-term mild stress, followed by an hour of recovery.…”

“…The events downstream of PINK1 stabilization include phosphorylation of Miro and Mitofusin (Figure 3), phosphorylation (and activation) of Parkin, an E3 ubiquitin ligase, and phosphorylation of ubiquitin. Parkin binds to both phospho-miro and phospho-ubiquitin and is thereby recruited to the mitochondrial surface (Heo et al, 2015; Kane et al, 2014; Lazarou et al, 2015; McWilliams and Muqit, 2017; Shlevkov et al, 2016; Wong and Holzbaur, 2014). The resulting positive feedback loop results in extensive addition of phospho-ubiquitin to the mitochondrial surface.…”

Mitochondrial-cytoskeletal interactions: dynamic associations that facilitate network function and remodeling

“…Multiple studies have linked changes in mitochondrial motility to mitochondrial quality control pathways such as mitophagy. In one pathway, damaged mitochondria are immobilized along the axon through the PINK1- and Parkin-dependent degradation of Miro [18], promoting localized mitophagy [19]. Parkinson’s disease-related mutations in PINK1, Parkin or LRRK2 slow the degradation of Miro, leading to the hypothesis that delayed arrest of damaged mitochondria along the axon also delays their degradation [20].…”

How to get rid of mitochondria: crosstalk and regulation of multiple mitophagy pathways

“…The substrates of Parkin are linked to important mitochondrial functions. The ubiquitylation of these substrates ensure the isolation of mitochondria by inhibiting fusion and promoting fission (substrates: Mfn1/2 and Drp1) (Gegg et al, 2010;Rakovic et al, 2013;Pryde et al, 2016), and by interfering with mitochondrial transport [substrate: MIRO (Mitochondrial Rho GTPase 1)] (Shlevkov et al, 2016). The formation of polyubiquitin chains has been shown to occur via K6-, K11-, K27-, K48-and K63-linkage (Geisler et al, 2010;Narendra et al, 2010;Geisler et al, 2014;Cunningham et al, 2015).…”