The three 'P's of mitophagy: PARKIN, PINK1, and post-translational modifications

Abstract

Two Parkinson's disease (PD)-associated proteins, the mitochondrial kinase PINK1 and the E3-ubiquitin (Ub) ligase PARKIN, are central to mitochondrial quality control. In this pathway, PINK1 accumulates on defective mitochondria, eliciting the translocation of PARKIN from the cytosol to mediate the clearance of damaged mitochondria via autophagy (mitophagy). Throughout the different stages of mitophagy, post-translational modifications (PTMs) are critical for the regulation of PINK1 and PARKIN activity and function. Indeed, activation and recruitment of PARKIN onto damaged mitochondria involves PINK1-mediated phosphorylation of both PARKIN and Ub. Through a stepwise cascade, PARKIN is converted from an autoinhibited enzyme into an active phospho-Ub-dependent E3 ligase. Upon activation, PARKIN ubiquitinates itself in concert with many different mitochondrial substrates. The Ub conjugates attached to these substrates can in turn be phosphorylated by PINK1, which triggers further cycles of PARKIN recruitment and activation. This feed-forward amplification loop regulates both PARKIN activity and mitophagy. However, the precise steps and sequence of PTMs in this cascade are only now being uncovered. For instance, the Ub conjugates assembled by PARKIN consist predominantly of noncanonical K6-linked Ub chains. Moreover, these modifications are reversible and can be disassembled by deubiquitinating enzymes (DUBs), including Ub-specific protease 8 (USP8), USP15, and USP30. However, PINK1-mediated phosphorylation of Ub can impede the activity of these DUBs, adding a new layer of complexity to the regulation of PARKIN-mediated mitophagy by PTMs. It is therefore evident that further insight into how PTMs regulate the PINK1-PARKIN pathway will be critical for our understanding of mitochondrial quality control.

Keywords: PARKIN; PINK1; deubiquitination; mitophagy; phosphorylation; ubiquitin.

Figure 1.

Schematic representation of the functional domains and PD-associated mutations in PINK1 and PARKIN. (A) Schematic of the domain structure and PD-associated mutations in PINK1. (B) Schematic highlighting sites of PARKIN autoinhibition. PARKIN activity depends on two functional sites: a binding site for the E2 on RING1 and a catalytic site at C431 in the RING2 domain. Both sites are occluded in the autoinhibited structure. (C) An outline of the domains in PARKIN and its PD-associated mutations. (Ubl) Ub-like domain; (RING) really interesting new gene; (IBR) in-between RINGs; (REP) repressor element of PARKIN domain. S65 in the Ubl is the site of PINK1-mediated phosphorylation. C431 is the PARKIN active site cysteine. W403 is a residue in the REP domain that, when mutated to an alanine (W403A), abolishes this autoinhibition and causes PARKIN to be hyperactive (Trempe et al. 2013). All indicated mutations in both PINK1 and PARKIN represent homozygous or compound heterozygous mutations associated with PD.

Figure 2.

Model for PARKIN activation and recruitment onto damaged mitochondria. Following a loss in mitochondrial membrane potential, PINK1 accumulates on depolarized mitochondria. (1) When present on the OMM, PINK1 phosphorylates both S65 in the N-terminal Ubl of PARKIN and S65 within the Ub moieties in Ub conjugates that are attached to mitochondrial substrates on the outer mitochondrial membrane. (2) These phosphorylation events activate PARKIN and elicit its mitochondrial recruitment via direct interaction with the phospho-Ub conjugates on the mitochondria. (3) Active PARKIN promotes the ubiquitination of multiple mitochondrial substrates. PINK1 phosphorylates these Ub conjugates, which in turn acts as a feed-forward mechanism to further promote PARKIN activation and recruitment. (IMM) Inner mitochondrial membrane; (OMM) outer mitochondrial membrane.

Figure 3.

Model for the types of linkages in Ub conjugates formed by PARKIN. PARKIN forms Ub conjugates comprised of four distinct linkages: K6, K11, K48, and K63. (Left) In one potential scenario, PARKIN forms homotypic chains on its substrates comprised of one linkage only, albeit to varying degrees between chains. (Right) Alternatively, PARKIN can form chains that are mixed in nature, consisting of varying levels of each of the four linkages.