Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65

By Agne Kazlauskaite, Chandana Kondapalli, Robert Gourlay, D. Campbell, M. S. Ritorto, Kay Hofmann, D. Alessi, A. Knebel, M. Trost, M. Muqit. Biochemical Journal, 2014

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We have previously reported that the Parkinson's disease-associated kinase PINK1 (PTEN-induced putative kinase 1) is activated by mitochondrial depolarization and stimulates the Parkin E3 ligase by phosphorylating Ser65 within its Ubl (ubiquitin-like) domain. Using phosphoproteomic analysis, we identified a novel ubiquitin phosphopeptide phosphorylated at Ser65 that was enriched 14-fold in HEK (human embryonic kidney)-293 cells overexpressing wild-type PINK1 stimulated with the mitochondrial uncoupling agent CCCP (carbonyl cyanide m-chlorophenylhydrazone), to activate PINK1, compared with cells expressing kinase-inactive PINK1. Ser65 in ubiquitin lies in a similar motif to Ser65 in the Ubl domain of Parkin. Remarkably, PINK1 directly phosphorylates Ser65 of ubiquitin in vitro. We undertook a series of experiments that provide striking evidence that Ser65-phosphorylated ubiquitin (ubiquitinPhospho−Ser65) functions as a critical activator of Parkin. First, we demonstrate that a fragment of Parkin lacking the Ubl domain encompassing Ser65 (ΔUbl-Parkin) is robustly activated by ubiquitinPhospho−Ser65, but not by non-phosphorylated ubiquitin. Secondly, we find that the isolated Parkin Ubl domain phosphorylated at Ser65 (UblPhospho−Ser65) can also activate ΔUbl-Parkin similarly to ubiquitinPhospho−Ser65. Thirdly, we establish that ubiquitinPhospho−Ser65, but not non-phosphorylated ubiquitin or UblPhospho−Ser65, activates full-length wild-type Parkin as well as the non-phosphorylatable S65A Parkin mutant. Fourthly, we provide evidence that optimal activation of full-length Parkin E3 ligase is dependent on PINK1-mediated phosphorylation of both Parkin at Ser65 and ubiquitin at Ser65, since only mutation of both proteins at Ser65 completely abolishes Parkin activation. In conclusion, the findings of the present study reveal that PINK1 controls Parkin E3 ligase activity not only by phosphorylating Parkin at Ser65, but also by phosphorylating ubiquitin at Ser65. We propose that phosphorylation of Parkin at Ser65 serves to prime the E3 ligase enzyme for activation by ubiquitinPhospho−Ser65, suggesting that small molecules that mimic ubiquitinPhospho−Ser65 could hold promise as novel therapies for Parkinson's disease.

Subfields: Epidemiology, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/d71bf77993c507531d06f861accdc287714213f2

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Citations in the corpus

The Roles of PINK1, Parkin and Mitochondrial Fidelity in Parkinson's Disease (2015, methodology): “Parkin lacking the Ubl domain is still activated by PINK1, indicating that phospho-ubiquitin binding to Parkin doesmore than displace the Parkin Ubl domain (Kazlauskaite et al., 2014b).”
The PINK1-PARKIN mitochondrial ubiquitylation pathway drives a program of TBK1 activation and recruitment of OPTN and NDP52 to promote mitophagy (2015, background): “…on the mitochondrial outer membrane (MOM), where it promotes phosphorylation of PARKIN on S65 in its N-terminal UB-like domain, as well as the conserved S65 within UB on mitochondria (Kane et al., 2014; Kazlauskaite et al., 2014; Kondapalli et al., 2012; Koyano et al., 2014; Ordureau et al., 2014).”
A new pathway for mitochondrial quality control: mitochondrial‐derived vesicles (2014, background): “This exposes the kinase domain to the cytosol where it phosphorylates ubiquitin and Parkin, leading to stable Parkin recruitment and activation at the mitochondrial surface (Kim et al, 2008; ShibaFukushima et al, 2012; Iguchi et al, 2013; Kane et al, 2014; Kazlauskaite et al, 2014).”
Atg8 family LC3/GABARAP proteins are crucial for autophagosome–lysosome fusion but not autophagosome formation during PINK1/Parkin mitophagy and starvation (2016, background): “The selectivity is achieved by S65 phosphorylated ubiquitin chains placed on the surface of mitochondria by PINK1 and Parkin (Kane et al., 2014; Kazlauskaite et al., 2014; Koyano et al., 2014).”