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1.  Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65 
Biochemical Journal  2014;460(Pt 1):127-139.
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.
We describe a novel and unexpected mechanism by which PINK1 protein kinase activates Parkin E3 ligase. We show that PINK1 phosphorylates ubiquitin at Ser65 and that phosphorylated ubiquitin acts as a direct activator of Parkin.
doi:10.1042/BJ20140334
PMCID: PMC4000136  PMID: 24660806
Parkin; Parkinson’s disease; phosphorylation; PTEN (phosphatase and tensin homologue deleted on chromosome 10)-induced putative kinase 1 (PINK1); ubiquitin; CCCP, carbonyl cyanide m-chlorophenylhydrazone; CDK2, cyclin-dependent kinase 2; GSK3β, glycogen synthase kinase-3β; HEK, human embryonic kidney; HOIL1, haem-oxidized IRP2 (iron-regulatory protein 2) ubiquitin ligase 1; HRP, horseradish peroxidase; IKK, IκB (inhibitor of nuclear factor κB) kinase; ISG15, interferon-induced 17 kDa protein; MBP, maltose-binding protein; MLK1, mixed lineage kinase 1; Nedd8, neural-precursor-cell-expressed developmentally down-regulated 8; Ni-NTA, Ni2+-nitrilotriacetate; NUAK1, NUAK family SNF1-like kinase 1; OTU1, OTU (ovarian tumour) domain-containing protein 1; PD, Parkinson’s disease; PINK1, PTEN (phosphatase and tensin homologue deleted on chromosome 10)-induced putative kinase 1; PLK1, Polo-like kinase 1; SILAC, stable isotope labelling by amino acids in cell culture; SUMO, small ubiquitin-related modifier; TCEP, tris-(2-carboxyethyl)phosphine; TcPINK1, Tribolium castaneum PINK1; Ubl, ubiquitin-like
2.  PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65 
Open Biology  2012;2(5):120080.
Summary
Missense mutations in PTEN-induced kinase 1 (PINK1) cause autosomal-recessive inherited Parkinson's disease (PD). We have exploited our recent discovery that recombinant insect PINK1 is catalytically active to test whether PINK1 directly phosphorylates 15 proteins encoded by PD-associated genes as well as proteins reported to bind PINK1. We have discovered that insect PINK1 efficiently phosphorylates only one of these proteins, namely the E3 ligase Parkin. We have mapped the phosphorylation site to a highly conserved residue within the Ubl domain of Parkin at Ser65. We show that human PINK1 is specifically activated by mitochondrial membrane potential (Δψm) depolarization, enabling it to phosphorylate Parkin at Ser65. We further show that phosphorylation of Parkin at Ser65 leads to marked activation of its E3 ligase activity that is prevented by mutation of Ser65 or inactivation of PINK1. We provide evidence that once activated, PINK1 autophosphorylates at several residues, including Thr257, which is accompanied by an electrophoretic mobility band-shift. These results provide the first evidence that PINK1 is activated following Δψm depolarization and suggest that PINK1 directly phosphorylates and activates Parkin. Our findings indicate that monitoring phosphorylation of Parkin at Ser65 and/or PINK1 at Thr257 represent the first biomarkers for examining activity of the PINK1-Parkin signalling pathway in vivo. Our findings also suggest that small molecule activators of Parkin that mimic the effect of PINK1 phosphorylation may confer therapeutic benefit for PD.
doi:10.1098/rsob.120080
PMCID: PMC3376738  PMID: 22724072
PINK1; Parkin; Parkinson's disease

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