Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65
| Autor: | Agne, Kazlauskaite, Chandana, Kondapalli, Robert, Gourlay, David G, Campbell, Maria Stella, Ritorto, Kay, Hofmann, Dario R, Alessi, Axel, Knebel, Matthias, Trost, Miratul M K, Muqit |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2014 |
| Předmět: |
CCCP
carbonyl cyanide m-chlorophenylhydrazone HOIL1 haem-oxidized IRP2 (iron-regulatory protein 2) ubiquitin ligase 1 IKK IκB (inhibitor of nuclear factor κB) kinase Ubiquitin-Protein Ligases Molecular Sequence Data MBP maltose-binding protein ISG15 interferon-induced 17 kDa protein Accelerated Publication Nedd8 neural-precursor-cell-expressed developmentally down-regulated 8 HEK human embryonic kidney PINK1 PTEN (phosphatase and tensin homologue deleted on chromosome 10)-induced putative kinase 1 NUAK1 NUAK family SNF1-like kinase 1 ubiquitin Serine Humans Amino Acid Sequence TcPINK1 Tribolium castaneum PINK1 Parkin PD Parkinson’s disease TCEP tris-(2-carboxyethyl)phosphine phosphorylation SUMO small ubiquitin-related modifier HRP horseradish peroxidase GSK3β glycogen synthase kinase-3β PTEN (phosphatase and tensin homologue deleted on chromosome 10)-induced putative kinase 1 (PINK1) nervous system diseases HEK293 Cells CDK2 cyclin-dependent kinase 2 PLK1 Polo-like kinase 1 Ni-NTA Ni2+-nitrilotriacetate Parkinson’s disease MLK1 mixed lineage kinase 1 SILAC stable isotope labelling by amino acids in cell culture Ubl ubiquitin-like OTU1 OTU (ovarian tumour) domain-containing protein 1 Protein Kinases |
| Zdroj: | Biochemical Journal |
| ISSN: | 1470-8728 0264-6021 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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