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

Agne Kazlauskaite,Miratul M K Muqit,Matthias Trost,David G Campbell,Dario R Alessi,Maria Stella Ritorto,Chandana Kondapalli,Kay Hofmann,Robert Gourlay,Axel Knebel

doi:10.1042/bj20140334

Abstract

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.

Highlights

Mutations in PINK1 [PTEN-induced putative kinase 1] and Parkin are associated with early-onset autosomal-recessive PD (Parkinson’s disease) [1,2]
We have previously reported that PINK1 is activated in response to mitochondrial depolarization and that this leads to autophosphorylation of PINK1 at Thr257 as well as phosphorylation of Parkin at Ser65 in vivo [13]
We have previously reported that PINK1-dependent phosphorylation of Parkin Ser65 that lies within the Ubl domain stimulates Parkin E3 ligase activity [13]

Summary

Introduction

Mutations in PINK1 [PTEN (phosphatase and tensin homologue deleted on chromosome 10)-induced putative kinase 1] and Parkin are associated with early-onset autosomal-recessive PD (Parkinson’s disease) [1,2]. PINK1 has been reported to be required for Parkin recruitment to mitochondria upon mitochondrial membrane depolarization in mammalian cell lines [9,10,11,12]. We recently found that PINK1 can phosphorylate Parkin directly at a highly conserved residue, Ser, that lies within the Ubl (ubiquitin-like) domain of Parkin and demonstrated that phosphorylation stimulates Parkin E3 ligase activity [13]. Recent high-resolution crystal structures of Parkin lacking the Ubl domain suggest that Parkin is autoinhibited; the studies do not shed any light on the mechanism of how Ser phosphorylation triggers conformational change and activation of Parkin [14,15,16]

Methods

Results

Conclusion