Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation

Agne Kazlauskaite, R Julio Martínez-Torres, Scott Wilkie, Atul Kumar, Julien Peltier, Alba Gonzalez, Clare Johnson, Jinwei Zhang, Anthony G Hope, Mark Peggie, Matthias Trost, Daan Mf van Aalten, Dario R Alessi, Alan R Prescott, Axel Knebel, Helen Walden, Miratul MK Muqit

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    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 (Ser65)-which lies within its ubiquitin-like domain (Ubl)-and indirectly through phosphorylation of ubiquitin at Ser65. How Ser65-phosphorylated ubiquitin (ubiquitinPhospho-Ser65) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitinPhospho-Ser65 binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser65 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 ubiquitinPhospho-Ser65, thereby promoting Parkin Ser65 phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser65 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 ubiquitinPhospho-Ser65 to Parkin disrupts the interaction between the Ubl domain and C-terminal region, thereby increasing the accessibility of Parkin Ser65. Finally, purified Parkin maximally phosphorylated at Ser65 in vitro cannot be further activated by the addition of ubiquitinPhospho-Ser65. Our results thus suggest that a major role of ubiquitinPhospho-Ser65 is to promote PINK1-mediated phosphorylation of Parkin at Ser65, leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho-Ser65-binding pocket on the surface of Parkin that is critical for the ubiquitinPhospho-Ser65 interaction. This study provides new mechanistic insights into Parkin activation by ubiquitinPhospho-Ser65, which could aid in the development of Parkin activators that mimic the effect of ubiquitinPhospho-Ser65.

    Original languageEnglish
    Pages (from-to)939-954
    Number of pages16
    JournalEMBO Reports
    Volume16
    Issue number8
    Early online date25 Jun 2015
    DOIs
    Publication statusPublished - 1 Aug 2015

    Fingerprint

    Phosphorylation
    Ubiquitin
    Serine
    Chemical activation
    Ubiquitin-Protein Ligases
    Mutagenesis
    Mutation
    Mitochondria

    Keywords

    • Parkin
    • Parkinson's disease
    • phosphorylation
    • PINK1
    • ubiquitin

    Cite this

    Kazlauskaite, Agne ; Martínez-Torres, R Julio ; Wilkie, Scott ; Kumar, Atul ; Peltier, Julien ; Gonzalez, Alba ; Johnson, Clare ; Zhang, Jinwei ; Hope, Anthony G ; Peggie, Mark ; Trost, Matthias ; van Aalten, Daan Mf ; Alessi, Dario R ; Prescott, Alan R ; Knebel, Axel ; Walden, Helen ; Muqit, Miratul MK. / Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation. In: EMBO Reports. 2015 ; Vol. 16, No. 8. pp. 939-954.
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    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 (Ser65)-which lies within its ubiquitin-like domain (Ubl)-and indirectly through phosphorylation of ubiquitin at Ser65. How Ser65-phosphorylated ubiquitin (ubiquitinPhospho-Ser65) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitinPhospho-Ser65 binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser65 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 ubiquitinPhospho-Ser65, thereby promoting Parkin Ser65 phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser65 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 ubiquitinPhospho-Ser65 to Parkin disrupts the interaction between the Ubl domain and C-terminal region, thereby increasing the accessibility of Parkin Ser65. Finally, purified Parkin maximally phosphorylated at Ser65 in vitro cannot be further activated by the addition of ubiquitinPhospho-Ser65. Our results thus suggest that a major role of ubiquitinPhospho-Ser65 is to promote PINK1-mediated phosphorylation of Parkin at Ser65, leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho-Ser65-binding pocket on the surface of Parkin that is critical for the ubiquitinPhospho-Ser65 interaction. This study provides new mechanistic insights into Parkin activation by ubiquitinPhospho-Ser65, which could aid in the development of Parkin activators that mimic the effect of ubiquitinPhospho-Ser65.",
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    author = "Agne Kazlauskaite and Mart{\'i}nez-Torres, {R Julio} and Scott Wilkie and Atul Kumar and Julien Peltier and Alba Gonzalez and Clare Johnson and Jinwei Zhang and Hope, {Anthony G} and Mark Peggie and Matthias Trost and {van Aalten}, {Daan Mf} and Alessi, {Dario R} and Prescott, {Alan R} and Axel Knebel and Helen Walden and Muqit, {Miratul MK}",
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    Kazlauskaite, A, Martínez-Torres, RJ, Wilkie, S, Kumar, A, Peltier, J, Gonzalez, A, Johnson, C, Zhang, J, Hope, AG, Peggie, M, Trost, M, van Aalten, DM, Alessi, DR, Prescott, AR, Knebel, A, Walden, H & Muqit, MMK 2015, 'Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation', EMBO Reports, vol. 16, no. 8, pp. 939-954. https://doi.org/10.15252/embr.201540352

    Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation. / Kazlauskaite, Agne; Martínez-Torres, R Julio; Wilkie, Scott; Kumar, Atul; Peltier, Julien; Gonzalez, Alba; Johnson, Clare; Zhang, Jinwei; Hope, Anthony G; Peggie, Mark; Trost, Matthias; van Aalten, Daan Mf; Alessi, Dario R; Prescott, Alan R; Knebel, Axel; Walden, Helen; Muqit, Miratul MK.

    In: EMBO Reports, Vol. 16, No. 8, 01.08.2015, p. 939-954.

    Research output: Contribution to journalArticle

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    AU - Kazlauskaite, Agne

    AU - Martínez-Torres, R Julio

    AU - Wilkie, Scott

    AU - Kumar, Atul

    AU - Peltier, Julien

    AU - Gonzalez, Alba

    AU - Johnson, Clare

    AU - Zhang, Jinwei

    AU - Hope, Anthony G

    AU - Peggie, Mark

    AU - Trost, Matthias

    AU - van Aalten, Daan Mf

    AU - Alessi, Dario R

    AU - Prescott, Alan R

    AU - Knebel, Axel

    AU - Walden, Helen

    AU - Muqit, Miratul MK

    N1 - © 2015 The Authors. Published under the terms of the CC BY 4.0 license.

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    N2 - 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 (Ser65)-which lies within its ubiquitin-like domain (Ubl)-and indirectly through phosphorylation of ubiquitin at Ser65. How Ser65-phosphorylated ubiquitin (ubiquitinPhospho-Ser65) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitinPhospho-Ser65 binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser65 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 ubiquitinPhospho-Ser65, thereby promoting Parkin Ser65 phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser65 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 ubiquitinPhospho-Ser65 to Parkin disrupts the interaction between the Ubl domain and C-terminal region, thereby increasing the accessibility of Parkin Ser65. Finally, purified Parkin maximally phosphorylated at Ser65 in vitro cannot be further activated by the addition of ubiquitinPhospho-Ser65. Our results thus suggest that a major role of ubiquitinPhospho-Ser65 is to promote PINK1-mediated phosphorylation of Parkin at Ser65, leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho-Ser65-binding pocket on the surface of Parkin that is critical for the ubiquitinPhospho-Ser65 interaction. This study provides new mechanistic insights into Parkin activation by ubiquitinPhospho-Ser65, which could aid in the development of Parkin activators that mimic the effect of ubiquitinPhospho-Ser65.

    AB - 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 (Ser65)-which lies within its ubiquitin-like domain (Ubl)-and indirectly through phosphorylation of ubiquitin at Ser65. How Ser65-phosphorylated ubiquitin (ubiquitinPhospho-Ser65) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitinPhospho-Ser65 binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser65 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 ubiquitinPhospho-Ser65, thereby promoting Parkin Ser65 phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser65 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 ubiquitinPhospho-Ser65 to Parkin disrupts the interaction between the Ubl domain and C-terminal region, thereby increasing the accessibility of Parkin Ser65. Finally, purified Parkin maximally phosphorylated at Ser65 in vitro cannot be further activated by the addition of ubiquitinPhospho-Ser65. Our results thus suggest that a major role of ubiquitinPhospho-Ser65 is to promote PINK1-mediated phosphorylation of Parkin at Ser65, leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho-Ser65-binding pocket on the surface of Parkin that is critical for the ubiquitinPhospho-Ser65 interaction. This study provides new mechanistic insights into Parkin activation by ubiquitinPhospho-Ser65, which could aid in the development of Parkin activators that mimic the effect of ubiquitinPhospho-Ser65.

    KW - Parkin

    KW - Parkinson's disease

    KW - phosphorylation

    KW - PINK1

    KW - ubiquitin

    U2 - 10.15252/embr.201540352

    DO - 10.15252/embr.201540352

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    Kazlauskaite A, Martínez-Torres RJ, Wilkie S, Kumar A, Peltier J, Gonzalez A et al. Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation. EMBO Reports. 2015 Aug 1;16(8):939-954. https://doi.org/10.15252/embr.201540352