title: Phosphorylation of Parkin at Serine65 is essential for activation: elaboration of a Miro1 substrate-based assay of Parkin E3 ligase activity.
abstract: Mutations in PINK1 and Parkin are associated with early-onset Parkinson's disease. We recently discovered that PINK1 phosphorylates Parkin at serine65 (Ser(65)) within its Ubl domain, leading to its activation in a substrate-free activity assay. We now demonstrate the critical requirement of Ser(65) phosphorylation for substrate ubiquitylation through elaboration of a novel in vitro E3 ligase activity assay using full-length untagged Parkin and its putative substrate, the mitochondrial GTPase Miro1. We observe that Parkin efficiently ubiquitylates Miro1 at highly conserved lysine residues, 153, 230, 235, 330 and 572, upon phosphorylation by PINK1. We have further established an E2-ubiquitin discharge assay to assess Parkin activity and observe robust discharge of ubiquitin-loaded UbcH7 E2 ligase upon phosphorylation of Parkin at Ser(65) by wild-type, but not kinase-inactive PINK1 or a Parkin Ser65Ala mutant, suggesting a possible mechanism of how Ser(65) phosphorylation may activate Parkin E3 ligase activity. For the first time, to the best of our knowledge, we report the effect of Parkin disease-associated mutations in substrate-based assays using full-length untagged recombinant Parkin. Our mutation analysis indicates an essential role for the catalytic cysteine Cys431 and reveals fundamental new knowledge on how mutations may confer pathogenicity via disruption of Miro1 ubiquitylation, free ubiquitin chain formation or by impacting Parkin's ability to discharge ubiquitin from a loaded E2. This study provides further evidence that phosphorylation of Parkin at Ser(65) is critical for its activation. It also provides evidence that Miro1 is a direct Parkin substrate. The assays and reagents developed in this study will be important to uncover new insights into Parkin biology as well as aid in the development of screens to identify small molecule Parkin activators for the treatment of Parkinson's disease.
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK.
authors.forename: Agne
authors.initials: A
authors.lastname: Kazlauskaite
authors.forename: Van
authors.initials: V
authors.lastname: Kelly
authors.forename: Clare
authors.initials: C
authors.lastname: Johnson
authors.forename: Carla
authors.initials: C
authors.lastname: Baillie
authors.forename: C James
authors.initials: CJ
authors.lastname: Hastie
authors.forename: Mark
authors.initials: M
authors.lastname: Peggie
authors.forename: Thomas
authors.initials: T
authors.lastname: Macartney
authors.forename: Helen I
authors.initials: HI
authors.lastname: Woodroof
authors.forename: Dario R
authors.initials: DR
authors.lastname: Alessi
authors.forename: Patrick G A
authors.initials: PG
authors.lastname: Pedrioli
authors.forename: Miratul M K
authors.initials: MM
authors.lastname: Muqit
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D006801:Humans; D024101:Mitochondrial Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010300:Parkinson Disease / Q000378:metabolism / Q000473:pathology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D000099202:PTEN-Induced Putative Kinase
chemical_list: D024101:Mitochondrial Proteins; D011494:Protein Kinases; D011994:Recombinant Proteins; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D020741:rho GTP-Binding Proteins; D000099202:PTEN-Induced Putative Kinase; C079437:UBE2L3 protein, human; C111567:parkin protein; C473124:RHOT1 protein, human
keywords: Miro1; PINK1; Parkin; Parkinson's disease; phosphorylation; ubiquitin