title: Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation.
abstract: Mutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser(65))--which lies within its ubiquitin-like domain (Ubl)--and indirectly through phosphorylation of ubiquitin at Ser(65). How Ser(65)-phosphorylated ubiquitin (ubiquitin(Phospho-Ser65)) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitin(Phospho-Ser65) binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser(65) by PINK1 in vitro. Analysis of the Parkin structure, corroborated by site-directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitin(Phospho-Ser65), thereby promoting Parkin Ser(65) phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser(65) phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitin(Phospho-Ser65) to Parkin disrupts the interaction between the Ubl domain and C-terminal region, thereby increasing the accessibility of Parkin Ser(65). Finally, purified Parkin maximally phosphorylated at Ser(65) in vitro cannot be further activated by the addition of ubiquitin(Phospho-Ser65). Our results thus suggest that a major role of ubiquitin(Phospho-Ser65) is to promote PINK1-mediated phosphorylation of Parkin at Ser(65), leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho-Ser(65)-binding pocket on the surface of Parkin that is critical for the ubiquitin(Phospho-Ser65) interaction. This study provides new mechanistic insights into Parkin activation by ubiquitin(Phospho-Ser65), which could aid in the development of Parkin activators that mimic the effect of ubiquitin(Phospho-Ser65).
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences University of Dundee, Dundee, UK.
authors.forename: Agne
authors.initials: A
authors.lastname: Kazlauskaite
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences University of Dundee, Dundee, UK.
authors.forename: R Julio
authors.initials: RJ
authors.lastname: Martínez-Torres
authors.affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences University of Dundee, Dundee, UK.
authors.forename: Scott
authors.initials: S
authors.lastname: Wilkie
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences University of Dundee, Dundee, UK.
authors.forename: Atul
authors.initials: A
authors.lastname: Kumar
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences University of Dundee, Dundee, UK.
authors.forename: Julien
authors.initials: J
authors.lastname: Peltier
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences University of Dundee, Dundee, UK.
authors.forename: Alba
authors.initials: A
authors.lastname: Gonzalez
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences University of Dundee, Dundee, UK.
authors.forename: Clare
authors.initials: C
authors.lastname: Johnson
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences University of Dundee, Dundee, UK.
authors.forename: Jinwei
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences University of Dundee, Dundee, UK.
authors.forename: Anthony G
authors.initials: AG
authors.lastname: Hope
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences University of Dundee, Dundee, UK.
authors.forename: Mark
authors.initials: M
authors.lastname: Peggie
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences University of Dundee, Dundee, UK.
authors.forename: Matthias
authors.initials: M
authors.lastname: Trost
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences University of Dundee, Dundee, UK.
authors.forename: Daan M F
authors.initials: DM
authors.lastname: van Aalten
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences University of Dundee, Dundee, UK.
authors.forename: Dario R
authors.initials: DR
authors.lastname: Alessi
authors.affiliation: Division of Cell Signalling and Immunology, College of Life Sciences University of Dundee, Dundee, UK.
authors.forename: Alan R
authors.initials: AR
authors.lastname: Prescott
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences University of Dundee, Dundee, UK.
authors.forename: Axel
authors.initials: A
authors.lastname: Knebel
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences University of Dundee, Dundee, UK.
authors.forename: Helen
authors.initials: H
authors.lastname: Walden
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences University of Dundee, Dundee, UK College of Medicine, Dentistry & Nursing University of Dundee, Dundee, UK m.muqit@dundee.ac.uk.
authors.forename: Miratul M K
authors.initials: MM
authors.lastname: Muqit
mesh_terms: D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D000099202:PTEN-Induced Putative Kinase
chemical_list: D011494:Protein Kinases; D012694:Serine; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D000099202:PTEN-Induced Putative Kinase; C111567:parkin protein
keywords: PINK1; Parkin; Parkinson's disease; phosphorylation; ubiquitin