Universität zu Köln

Kazlauskaite, Agne, Kondapalli, Chandana, Gourlay, Robert, Campbell, David G., Ritorto, Maria Stella ORCID: 0000-0001-9861-6863, Hofmann, Kay ORCID: 0000-0002-2289-9083, Alessi, Dario R., Knebel, Axel, Trost, Matthias ORCID: 0000-0002-5732-700X and Muqit, Miratul M. K. (2014). Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser(65). Biochem. J., 460. S. 127 - 140. LONDON: PORTLAND PRESS LTD. ISSN 1470-8728

Full text not available from this repository.

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, PlNK1 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) (Delta Ubl-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 Delta Ubl-Parkin similarly to ubiquitin(PhosPh-Ser65). Thirdly, we establish that ubiquitin(PhosPh-Ser65), but not non-phosphorylated ubiquitin or Ubl(PhosPh-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(PhosPh-Ser65), suggesting that small molecules that mimic ubiquitin(PhosPh-Ser65) could hold promise as novel therapies for Parkinson's disease.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Kazlauskaite, Agne UNSPECIFIED UNSPECIFIED UNSPECIFIED Kondapalli, Chandana UNSPECIFIED UNSPECIFIED UNSPECIFIED Gourlay, Robert UNSPECIFIED UNSPECIFIED UNSPECIFIED Campbell, David G. UNSPECIFIED UNSPECIFIED UNSPECIFIED Ritorto, Maria Stella UNSPECIFIED orcid.org/0000-0001-9861-6863 UNSPECIFIED Hofmann, Kay UNSPECIFIED orcid.org/0000-0002-2289-9083 UNSPECIFIED Alessi, Dario R. UNSPECIFIED UNSPECIFIED UNSPECIFIED Knebel, Axel UNSPECIFIED UNSPECIFIED UNSPECIFIED Trost, Matthias UNSPECIFIED orcid.org/0000-0002-5732-700X UNSPECIFIED Muqit, Miratul M. K. UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-438315
DOI:	10.1042/BJ20140334
Journal or Publication Title:	Biochem. J.
Volume:	460
Page Range:	S. 127 - 140
Date:	2014
Publisher:	PORTLAND PRESS LTD
Place of Publication:	LONDON
ISSN:	1470-8728
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Mathematics and Natural Sciences > Department of Biology > Institute for Genetics
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language CRYSTAL-STRUCTURE; DISEASE; DOMAIN; MUTATIONS; PROTEOME; PINK1; RING; DROSOPHILA-PINK1; DYSFUNCTION; MECHANISMS Multiple languages Biochemistry & Molecular Biology Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/43831