Ubiquitin phosphorylation in Parkinson’s disease: Implications for pathogenesis and treatment

PINK1 phosphorylates ubiquitin at Ser65 in response to mitochondrial depolarization

Mitochondria are double membrane-bound organelles with four distinct submitochondrial compartments: the outer mitochondrial membrane (OMM), the inner mitochondrial membrane (IMM), the intermembrane space (IMS), and the matrix. The compartmentalization is crucial to mitochondria-mediated processes, including energy production, metabolism, redox control, calcium homeostasis, and programmed cell death [19, 20]. Mitochondrial dysfunction is implicated as a key factor in PD pathogenesis [21–23]. Human genetic studies revealed that homozygous mutations in mitochondrial kinase PINK1 cause autosomal recessive, early-onset PD [7–9, 24], whereas heterozygous mutations in PINK1 increase the risk for developing late-onset PD [25–27], highlighting the importance of knowing the sites and mechanisms of PINK1 action.

PINK1 is an ubiquitously expressed, 581-amino-acid protein Ser/Thr kinase with an N-terminal mitochondrial targeting sequence [28]. Under normal physiological conditions, PINK1 is imported into healthy mitochondria through the translocase of outer membrane (TOM) and translocase of inner membrane (TIM) complexes [29], where the 64-kDa full-length PINK1 can undergo sequential proteolytic cleavages by the matrix-localized mitochondrial processing peptidase (MPP) and the IMM-localized protease PARL to generate a 52-kDa processed form of PINK1 [30–33]. According to one model, the 52-kDa processed form of PINK1 is retrotranslocated to the cytosol for rapid degradation by the proteasome through the N-end rule pathway [34] and consequently, PINK1 protein levels are virtually undetectable under normal conditions, thus arguing against a function of PINK1 in healthy mitochondria [30, 34, 35]. In contrast, other studies reported significant levels of PINK1 protein under normal conditions, but localized PINK1 to either the OMM with its kinase domain facing the cytoplasm [36–38] or to the IMM/IMS with its kinase domain facing the IMS [39–43]. Recently, super-resolution imaging analyses using three-dimensional structured illumination microscopy (3D-SIM) [44] or a combination of tracking and localization microscopy (TALM) and fluorescence photoactivation localization microscopy (F-PALM) [45] clearly showed that, under normal conditions, PINK1 is not present on the OMM of healthy mitochondria, but rather PINK1 resides in the IMM/IMS where it is mainly localized to the cristae membrane and intracristae space. Furthermore, PINK1 was found to colocalize with the mitochondrial chaperone TRAP1, a previously identified PINK1 substrate [40, 46], in these submitochondrial compartments [44]. Together, these results support a model (Fig. 1) that PINK1 plays an intramitochondrial signaling role by phosphorylating TRAP1 [40] and potentially also other IMM/IMS-localized proteins, such as the complex I subunit NdufA10 [47] and the mitochondrial serine protease HtrA2 [48], to regulate activities of polarized mitochondria.

PINK1-mediated intramitochondrial signaling in healthy mitochondria. PINK1 is imported into healthy mitochondria through the TOM and TIM complexes and is then cleaved sequentially by mitochondrial processing peptidase (MPP) and PARL to generate a processed form of PINK1 that resides in the intermembrane space. There, PINK1 can phosphorylate mitochondrial chaperone TRAP1 and perhaps also other substrates to regulate the activities of polarized mitochondria, such as respiration and redox control

Super-resolution imaging analyses showed that, in response to mitochondrial depolarization, PINK1 changes its submitochondrial localization from the IMM/IMS to the OMM of depolarized mitochondria [44, 45], whereas TRAP remains in the IMM/IMS [44]. The PINK1 localization on the OMM of depolarized mitochondria is likely due to the blockade of PINK1 mitochondrial import through the IMM by the loss of mitochondrial membrane potential [29]. As expected, the mitochondrial import blockade prevents the cleavage of PINK1 by PARL, leading to accumulation of full-length PINK1 on damage mitochondria [29, 30, 33]. Recently, accumulation of misfolded proteins in the mitochondrial matrix was reported to cause PINK1 localization on the OMM without mitochondrial depolarization [49], which might be explained by the possibility that misfolded proteins may somehow inhibit PINK1 mitochondrial import through the TIM complex. Increasing evidence supports that mitochondrial dysfunction-triggered PINK1 localization on the OMM serves as a damage-sensing, quality-control mechanism to mark damaged mitochondria for clearance by mitophagy [29, 35, 44, 49, 50].

Mitochondrial depolarization not only causes PINK1 localization on the OMM of damaged mitochondria but also induces PINK1 dimerization and autophosphorylation at its Ser228 and Ser402 residues [51, 52], which could be an activation mechanism for enhancing PINK1 kinase activity [53]. Intriguingly, PINK1 was recently found to phosphorylate the residue Ser65 of either ubiquitin [54–57] or ubiquitin chains conjugated to mitochondrial proteins in response to mitochondrial depolarization [57, 58], indicating a function of PINK1 as a ubiquitin kinase. This finding is very exciting because previous phosphoproteomic analyses revealed that ubiquitin can be phosphorylated at multiple sites, including Ser65 [16, 17, 59], but the identity of the kinases for phosphorylating ubiquitin was unknown. Quantitative proteomic analysis showed that, under normal conditions, the Ser65-phosphorylated form of ubiquitin (phospho-Ser65-ubiquitin) is essentially undetectable on healthy mitochondria, but upon mitochondrial depolarization, the level of phospho-Ser65-ubiquitin increases to ~20 % of the total ubiquitin level on damaged mitochondria [57], indicating ubiquitin phosphorylation at Ser65 is a stress-responsive signal that can be induced by mitochondrial dysfunction. Interestingly, a study in yeast [18] demonstrated that ubiquitin phosphorylation at Ser65 can also be induced by oxidative stress, although another kinase must be involved because no PINK1 orthologue exists in yeast. Emerging data indicate that ubiquitin phosphorylation at Ser65 causes significant changes in the structures of ubiquitin and ubiquitin chains and affects ubiquitination and deubiquitination cascades catalyzed by a number of E2 ubiquitin-conjugating enzymes, E3 ligases, and deubiquitinating enzymes [18, 60]. Thus, ubiquitin phosphorylation can have a profound impact on the ubiquitin signalling system. Below, we will focus on the role of PINK1-mediated ubiquitin phosphorylation in activating parkin to promote mitophagy.