Phosphorylation of Parkin at Serine65 is essential for activation: elaboration of a Miro1 substrate-based assay of Parkin E3 ligase activity

Agne Kazlauskaite, Van Kelly, Clare Johnson, Carla Baillie, C. James Hastie, Mark Peggie, Thomas Macartney, Helen I. Woodroof, Dario R Alessi, Patrick G. A. Pedrioli, Miratul M. K. Muqit

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    Abstract

    Mutations in PINK1 and Parkin are associated with early-onset Parkinson's disease. We recently discovered that PINK1 phosphorylates Parkin at serine65 (Ser65) within its Ubl domain, leading to its activation in a substrate-free activity assay. We now demonstrate the critical requirement of Ser65 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 Ser65 by wild-type, but not kinase-inactive PINK1 or a Parkin Ser65Ala mutant, suggesting a possible mechanism of how Ser65 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 Ser65 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.

    Original languageEnglish
    Article number130213
    Number of pages14
    JournalOpen Biology
    Volume4
    DOIs
    Publication statusPublished - 19 Mar 2014

    Fingerprint

    Phosphorylation
    Ubiquitin-Protein Ligases
    ligases
    Assays
    phosphorylation
    ubiquitin
    Ubiquitin
    Chemical activation
    assays
    Substrates
    mutation
    Mutation
    Parkinson disease
    Ubiquitination
    Parkinson Disease
    development aid
    GTP Phosphohydrolases
    guanosinetriphosphatase
    Ligases
    Lysine

    Cite this

    Kazlauskaite, Agne ; Kelly, Van ; Johnson, Clare ; Baillie, Carla ; Hastie, C. James ; Peggie, Mark ; Macartney, Thomas ; Woodroof, Helen I. ; Alessi, Dario R ; Pedrioli, Patrick G. A. ; Muqit, Miratul M. K. / Phosphorylation of Parkin at Serine65 is essential for activation : elaboration of a Miro1 substrate-based assay of Parkin E3 ligase activity. In: Open Biology. 2014 ; Vol. 4.
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    abstract = "Mutations in PINK1 and Parkin are associated with early-onset Parkinson's disease. We recently discovered that PINK1 phosphorylates Parkin at serine65 (Ser65) within its Ubl domain, leading to its activation in a substrate-free activity assay. We now demonstrate the critical requirement of Ser65 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 Ser65 by wild-type, but not kinase-inactive PINK1 or a Parkin Ser65Ala mutant, suggesting a possible mechanism of how Ser65 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 Ser65 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.",
    author = "Agne Kazlauskaite and Van Kelly and Clare Johnson and Carla Baillie and Hastie, {C. James} and Mark Peggie and Thomas Macartney and Woodroof, {Helen I.} and Alessi, {Dario R} and Pedrioli, {Patrick G. A.} and Muqit, {Miratul M. K.}",
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    Phosphorylation of Parkin at Serine65 is essential for activation : elaboration of a Miro1 substrate-based assay of Parkin E3 ligase activity. / Kazlauskaite, Agne; Kelly, Van; Johnson, Clare; Baillie, Carla; Hastie, C. James; Peggie, Mark; Macartney, Thomas; Woodroof, Helen I.; Alessi, Dario R; Pedrioli, Patrick G. A.; Muqit, Miratul M. K.

    In: Open Biology, Vol. 4, 130213, 19.03.2014.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Phosphorylation of Parkin at Serine65 is essential for activation

    T2 - elaboration of a Miro1 substrate-based assay of Parkin E3 ligase activity

    AU - Kazlauskaite, Agne

    AU - Kelly, Van

    AU - Johnson, Clare

    AU - Baillie, Carla

    AU - Hastie, C. James

    AU - Peggie, Mark

    AU - Macartney, Thomas

    AU - Woodroof, Helen I.

    AU - Alessi, Dario R

    AU - Pedrioli, Patrick G. A.

    AU - Muqit, Miratul M. K.

    PY - 2014/3/19

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    N2 - Mutations in PINK1 and Parkin are associated with early-onset Parkinson's disease. We recently discovered that PINK1 phosphorylates Parkin at serine65 (Ser65) within its Ubl domain, leading to its activation in a substrate-free activity assay. We now demonstrate the critical requirement of Ser65 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 Ser65 by wild-type, but not kinase-inactive PINK1 or a Parkin Ser65Ala mutant, suggesting a possible mechanism of how Ser65 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 Ser65 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.

    AB - Mutations in PINK1 and Parkin are associated with early-onset Parkinson's disease. We recently discovered that PINK1 phosphorylates Parkin at serine65 (Ser65) within its Ubl domain, leading to its activation in a substrate-free activity assay. We now demonstrate the critical requirement of Ser65 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 Ser65 by wild-type, but not kinase-inactive PINK1 or a Parkin Ser65Ala mutant, suggesting a possible mechanism of how Ser65 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 Ser65 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.

    U2 - 10.1098/rsob.130213

    DO - 10.1098/rsob.130213

    M3 - Article

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