Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser ⁶⁵

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 Ser ⁶⁵ within its Ubl (ubiquitin-like) domain. Using phosphoproteomic analysis, we identified a novel ubiquitin phosphopeptide phosphorylated at Ser ⁶⁵ 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. Ser ⁶⁵ in ubiquitin lies in a similar motif to Ser ⁶⁵ in the Ubl domain of Parkin. Remarkably, PINK1 directly phosphorylates Ser ⁶⁵ of ubiquitin in vitro. We undertook a series of experiments that provide striking evidence that Ser ⁶⁵-phosphorylated ubiquitin (ubiquitin ^{Phospho−Ser65}) functions as a critical activator of Parkin. First, we demonstrate that a fragment of Parkin lacking the Ubl domain encompassing Ser ⁶⁵ (ΔUbl-Parkin) is robustly activated by ubiquitin ^{Phospho−Ser65}, but not by non-phosphorylated ubiquitin. Secondly, we find that the isolated Parkin Ubl domain phosphorylated at Ser ⁶⁵ (Ubl ^{Phospho−Ser65}) can also activate ΔUbl-Parkin similarly to ubiquitin ^{Phospho−Ser65}. Thirdly, we establish that ubiquitin ^{Phospho−Ser65}, but not non-phosphorylated ubiquitin or Ubl ^{Phospho−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 Ser ⁶⁵ and ubiquitin at Ser ⁶⁵, since only mutation of both proteins at Ser ⁶⁵ 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 Ser ⁶⁵, but also by phosphorylating ubiquitin at Ser ⁶⁵. We propose that phosphorylation of Parkin at Ser ⁶⁵ serves to prime the E3 ligase enzyme for activation by ubiquitin ^{Phospho−Ser65}, suggesting that small molecules that mimic ubiquitin ^{Phospho−Ser65} could hold promise as novel therapies for Parkinson's disease.

Abstract

We describe a novel and unexpected mechanism by which PINK1 protein kinase activates Parkin E3 ligase. We show that PINK1 phosphorylates ubiquitin at Ser ⁶⁵ and that phosphorylated ubiquitin acts as a direct activator of Parkin.