Phosphorylation of Mitochondrial Polyubiquitin by PINK1 Promotes Parkin Mitochondrial Tethering

Abstract

The kinase PINK1 and the E3 ubiquitin (Ub) ligase Parkin participate in mitochondrial quality control. The phosphorylation of Ser65 in Parkin's ubiquitin-like (UBl) domain by PINK1 stimulates Parkin activation and translocation to damaged mitochondria, which induces mitophagy generating polyUb chain. However, Parkin Ser65 phosphorylation is insufficient for Parkin mitochondrial translocation. Here we report that Ser65 in polyUb chain is also phosphorylated by PINK1, and that phosphorylated polyUb chain on mitochondria tethers Parkin at mitochondria. The expression of Tom70MTS-4xUb SE, which mimics phospho-Ser65 polyUb chains on the mitochondria, activated Parkin E3 activity and its mitochondrial translocation. An E3-dead form of Parkin translocated to mitochondria with reduced membrane potential in the presence of Tom70MTS-4xUb SE, whereas non-phospho-polyUb mutant Tom70MTS-4xUb SA abrogated Parkin translocation. Parkin binds to the phospho-polyUb chain through its RING1-In-Between-RING (IBR) domains, but its RING0-linker is also required for mitochondrial translocation. Moreover, the expression of Tom70MTS-4xUb SE improved mitochondrial degeneration in PINK1-deficient, but not Parkin-deficient, Drosophila. Our study suggests that the phosphorylation of mitochondrial polyUb by PINK1 is implicated in both Parkin activation and mitochondrial translocation, predicting a chain reaction mechanism of mitochondrial phospho-polyUb production by which rapid translocation of Parkin is achieved.

Methylene Blue Reduces Acute Cerebral Ischemic Injury via the Induction of Mitophagy

“…The MMP is an indicator of the mitochondrial functional status, and deregulation of MMP leads to a large spectrum of major human diseases, including diabetes, neurodegeneration, cancer and others (52). It has been reported that MB maintains the MMP when mitochondria are dysfunctional (2), and the MMP is a regulator of mitophagy (27). Accordingly, we investigated the effect of MB on the MMP and the relationship between the MMP and the induction of mitophagy.…”

“…Thus, mitophagy plays a crucial role in controlling the quality of mitochondria by preventing the generation of ROS by dysfunctional mitochondria (24,25). Deficiency in mitophagy is associated with neurodegenerative diseases such as Parkinson's disease (26,27), Alzheimer's disease (28,29) and Huntington's disease (30). Stroke is another type of neurodegenerative disease.…”

“…We confirmed this effect of MB using an oxygen-glucose deprivation (OGD) model in vitro and identified that the protection conferred by MB during OGD was abolished due to the inhibition of mitophagy. As mitophagy is directly induced by the loss of mitochondrial membrane potential (MMP) (27,(31)(32)(33), we further explored whether the effect of MB on mitophagy was mediated by the regulation of MMP. We found that the MMP dramatically declined after cells were exposed to OGD, whereas MB attenuated this decline.…”

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

“…In addition to the differential effects of various types of Ub chains on mitophagy, a further layer of complexity is added by the potential for these chains to be phosphorylated by PINK1 (Shiba-Fukushima et al 2014a;Okatsu et al 2015b;Wauer et al 2015). The functional consequences of PINK1-mediated Ub chain phosphorylation are currently unknown.…”

Flies with Parkinson's disease

“…Hence, the work conducted both in mammalian neurons and in flies, suggests that dysfunctional mitochondria fail to be transported in the cell. Stabilized Pink1 also targets other proteins: it recruits and phosphorylates Parkin (Shiba-Fukushima et al, 2012) and ubiquitin (Kane et al, 2014;Kazlauskaite et al, 2014;Koyano et al, 2014;Shiba-Fukushima et al, 2014), and Parkin then ubiquitinates mitochondrial targets that tag the organelle for degradation. The evidence indicates this pathway is implicated in mitochondrial quality control, but the targets ubiquitinated by Parkin including Mitofusin (Poole et al, 2010;Ziviani et al, 2010) and VDAC (Geisler et al, 2010;D.…”