Phosphorylation of Parkin at Serine65 is essential for its activation in vivo

Thomas G. McWilliams (Lead / Corresponding author), Erica Barini, Risto Pohjolan-Pirhonen, Simon P. Brooks, Francois Singh, Sophie Burel, Kristin Balk, Atul Kumar, Lambert Montava Garriga, Alan Prescott, Sidi Mohamed Hassoun, François Mouton-Liger, Graeme Ball, Rachel Hills, Axel Knebel, Ayse Ulusoy, Donato A. Di Monte, Jevgenia Tamjar, Odetta Antico, Kyle Fears & 11 others Laura Smith, Riccardo Brambilla, Eino Palin, Miko Valori, Johanna Eerola-Rautio, Pentti Tienari, Olga Corti, Stephen B. Dunnett, Ian Ganley, Anu Suomalainen, Miratul Muqit

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Abstract

Mutations in PINK1 and Parkin result in autosomal recessive Parkinson's disease (PD). Cell culture and in vitro studies have elaborated the PINK1-dependent regulation of Parkin and defined how this dyad orchestrates the elimination of damaged mitochondria via mitophagy. PINK1 phosphorylates ubiquitin at serine 65 (Ser65) and Parkin at an equivalent Ser65 residue located within its N-terminal ubiquitin-like domain, resulting in activation; however, the physiological significance of Parkin Ser65 phosphorylation in vivo in mammals remains unknown. To address this, we generated a Parkin Ser65Ala (S65A) knock-in mouse model. We observe endogenous Parkin Ser65 phosphorylation and activation in mature primary neurons following mitochondrial depolarization and reveal this is disrupted in ParkinS65A/S65A neurons. Phenotypically, ParkinS65A/S65A mice exhibit selective motor dysfunction in the absence of any overt neurodegeneration or alterations in nigrostriatal mitophagy. The clinical relevance of our findings is substantiated by the discovery of homozygous PARKIN (PARK2) p.S65N mutations in two unrelated patients with PD. Moreover, biochemical and structural analysis demonstrates that the ParkinS65N/S65N mutant is pathogenic and cannot be activated by PINK1. Our findings highlight the central role of Parkin Ser65 phosphorylation in health and disease.

Original languageEnglish
Article number181108
Pages (from-to)1-18
Number of pages18
JournalOpen Biology
Volume8
Issue number11
DOIs
Publication statusPublished - 7 Nov 2018

Fingerprint

Phosphorylation
serine
Serine
phosphorylation
Chemical activation
Mitochondrial Degradation
Parkinson disease
ubiquitin
Ubiquitin
Neurons
Parkinson Disease
neurons
mutation
Mutation
Mitochondria
Mammals
Depolarization
Cell culture
Structural analysis
in vitro studies

Keywords

  • Parkinson's disease
  • Mitochondria
  • Mitophagy
  • Autophagy
  • mito-QC
  • Parkin
  • PINK1
  • Neurodegeneration

Cite this

McWilliams, T. G., Barini, E., Pohjolan-Pirhonen, R., Brooks, S. P., Singh, F., Burel, S., ... Muqit, M. (2018). Phosphorylation of Parkin at Serine65 is essential for its activation in vivo. Open Biology, 8(11), 1-18. [181108]. https://doi.org/10.1098/rsob.180108
McWilliams, Thomas G. ; Barini, Erica ; Pohjolan-Pirhonen, Risto ; Brooks, Simon P. ; Singh, Francois ; Burel, Sophie ; Balk, Kristin ; Kumar, Atul ; Montava Garriga, Lambert ; Prescott, Alan ; Hassoun, Sidi Mohamed ; Mouton-Liger, François ; Ball, Graeme ; Hills, Rachel ; Knebel, Axel ; Ulusoy, Ayse ; Di Monte, Donato A. ; Tamjar, Jevgenia ; Antico, Odetta ; Fears, Kyle ; Smith, Laura ; Brambilla, Riccardo ; Palin, Eino ; Valori, Miko ; Eerola-Rautio, Johanna ; Tienari, Pentti ; Corti, Olga ; Dunnett, Stephen B. ; Ganley, Ian ; Suomalainen, Anu ; Muqit, Miratul. / Phosphorylation of Parkin at Serine65 is essential for its activation in vivo. In: Open Biology. 2018 ; Vol. 8, No. 11. pp. 1-18.
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abstract = "Mutations in PINK1 and Parkin result in autosomal recessive Parkinson's disease (PD). Cell culture and in vitro studies have elaborated the PINK1-dependent regulation of Parkin and defined how this dyad orchestrates the elimination of damaged mitochondria via mitophagy. PINK1 phosphorylates ubiquitin at serine 65 (Ser65) and Parkin at an equivalent Ser65 residue located within its N-terminal ubiquitin-like domain, resulting in activation; however, the physiological significance of Parkin Ser65 phosphorylation in vivo in mammals remains unknown. To address this, we generated a Parkin Ser65Ala (S65A) knock-in mouse model. We observe endogenous Parkin Ser65 phosphorylation and activation in mature primary neurons following mitochondrial depolarization and reveal this is disrupted in ParkinS65A/S65A neurons. Phenotypically, ParkinS65A/S65A mice exhibit selective motor dysfunction in the absence of any overt neurodegeneration or alterations in nigrostriatal mitophagy. The clinical relevance of our findings is substantiated by the discovery of homozygous PARKIN (PARK2) p.S65N mutations in two unrelated patients with PD. Moreover, biochemical and structural analysis demonstrates that the ParkinS65N/S65N mutant is pathogenic and cannot be activated by PINK1. Our findings highlight the central role of Parkin Ser65 phosphorylation in health and disease.",
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author = "McWilliams, {Thomas G.} and Erica Barini and Risto Pohjolan-Pirhonen and Brooks, {Simon P.} and Francois Singh and Sophie Burel and Kristin Balk and Atul Kumar and {Montava Garriga}, Lambert and Alan Prescott and Hassoun, {Sidi Mohamed} and Fran{\cc}ois Mouton-Liger and Graeme Ball and Rachel Hills and Axel Knebel and Ayse Ulusoy and {Di Monte}, {Donato A.} and Jevgenia Tamjar and Odetta Antico and Kyle Fears and Laura Smith and Riccardo Brambilla and Eino Palin and Miko Valori and Johanna Eerola-Rautio and Pentti Tienari and Olga Corti and Dunnett, {Stephen B.} and Ian Ganley and Anu Suomalainen and Miratul Muqit",
note = "We thank our colleagues from the MRC PPU support teams for assistance with tissue culture and genotyping and our colleagues in Biological Services at both the University of Dundee and Cardiff University for excellent animal husbandry and care. M.M.K.M. is funded by a Wellcome Trust Senior Research Fellowship in Clinical Science (101022/Z/13/Z). This work was supported by the Wellcome Trust; Medical Research Council; Parkinson’s UK; the Michael J. Fox Foundation for Parkinson’s disease research; and a EMBO Young Investigator Programme (YIP) award. A. J. Macdonald Menzies Charitable Trust Prize Studentship funds K.B. O.A. is funded by a Rosetrees Trust PhD Studentship. G.B. is supported by an MRC Next Generation Optical Microscopy Award (MR/K015869/1) and Dundee Imaging Facility is supported by the ‘Wellcome Trust Technology Platform’ award (097945/B/11/Z). We are grateful to the sequencing service (School of Life Sciences, University of Dundee); and the MRC PPU Reagents and Services antibody purification teams (coordinated by James Hastie and Hilary McLauchlan) for excellent technical support. O.C. and colleagues (France) are supported by Fondation Institut du Cerveau et de la Moelle Epinie`re and Agence Nationale pour la Recherche (‘Investissements d’avenir’, grant ANR-10-IAIHU-06) and Innovative Medicines Initiative Joint Undertaking under grant agreement no.115568, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies’ in kind contribution. AS and colleagues (Finland) would like to thank the following funding sources: Sigrid Juselius Foundation; Academy of Finland, Helsinki University Central Hospital, and University of Helsinki (to A.S.), Biomedicum Helsinki Foundation, The Finnish Cultural Foundation and The Finnish Medical Foundation (to R.P-P.). Work by S.P.B. and colleagues (Cardiff ) was supported by a Ser Cymru PhD studentship, Life Sciences Research Network Wales (K.F.) and a Wellcome Trust Studentship (L.S.).",
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McWilliams, TG, Barini, E, Pohjolan-Pirhonen, R, Brooks, SP, Singh, F, Burel, S, Balk, K, Kumar, A, Montava Garriga, L, Prescott, A, Hassoun, SM, Mouton-Liger, F, Ball, G, Hills, R, Knebel, A, Ulusoy, A, Di Monte, DA, Tamjar, J, Antico, O, Fears, K, Smith, L, Brambilla, R, Palin, E, Valori, M, Eerola-Rautio, J, Tienari, P, Corti, O, Dunnett, SB, Ganley, I, Suomalainen, A & Muqit, M 2018, 'Phosphorylation of Parkin at Serine65 is essential for its activation in vivo', Open Biology, vol. 8, no. 11, 181108, pp. 1-18. https://doi.org/10.1098/rsob.180108

Phosphorylation of Parkin at Serine65 is essential for its activation in vivo. / McWilliams, Thomas G. (Lead / Corresponding author); Barini, Erica; Pohjolan-Pirhonen, Risto; Brooks, Simon P. ; Singh, Francois; Burel, Sophie; Balk, Kristin; Kumar, Atul; Montava Garriga, Lambert; Prescott, Alan; Hassoun, Sidi Mohamed; Mouton-Liger, François ; Ball, Graeme; Hills, Rachel; Knebel, Axel; Ulusoy, Ayse; Di Monte, Donato A.; Tamjar, Jevgenia; Antico, Odetta; Fears, Kyle; Smith, Laura; Brambilla, Riccardo; Palin, Eino; Valori, Miko; Eerola-Rautio, Johanna; Tienari, Pentti; Corti, Olga; Dunnett, Stephen B.; Ganley, Ian; Suomalainen, Anu; Muqit, Miratul (Lead / Corresponding author).

In: Open Biology, Vol. 8, No. 11, 181108, 07.11.2018, p. 1-18.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Phosphorylation of Parkin at Serine65 is essential for its activation in vivo

AU - McWilliams, Thomas G.

AU - Barini, Erica

AU - Pohjolan-Pirhonen, Risto

AU - Brooks, Simon P.

AU - Singh, Francois

AU - Burel, Sophie

AU - Balk, Kristin

AU - Kumar, Atul

AU - Montava Garriga, Lambert

AU - Prescott, Alan

AU - Hassoun, Sidi Mohamed

AU - Mouton-Liger, François

AU - Ball, Graeme

AU - Hills, Rachel

AU - Knebel, Axel

AU - Ulusoy, Ayse

AU - Di Monte, Donato A.

AU - Tamjar, Jevgenia

AU - Antico, Odetta

AU - Fears, Kyle

AU - Smith, Laura

AU - Brambilla, Riccardo

AU - Palin, Eino

AU - Valori, Miko

AU - Eerola-Rautio, Johanna

AU - Tienari, Pentti

AU - Corti, Olga

AU - Dunnett, Stephen B.

AU - Ganley, Ian

AU - Suomalainen, Anu

AU - Muqit, Miratul

N1 - We thank our colleagues from the MRC PPU support teams for assistance with tissue culture and genotyping and our colleagues in Biological Services at both the University of Dundee and Cardiff University for excellent animal husbandry and care. M.M.K.M. is funded by a Wellcome Trust Senior Research Fellowship in Clinical Science (101022/Z/13/Z). This work was supported by the Wellcome Trust; Medical Research Council; Parkinson’s UK; the Michael J. Fox Foundation for Parkinson’s disease research; and a EMBO Young Investigator Programme (YIP) award. A. J. Macdonald Menzies Charitable Trust Prize Studentship funds K.B. O.A. is funded by a Rosetrees Trust PhD Studentship. G.B. is supported by an MRC Next Generation Optical Microscopy Award (MR/K015869/1) and Dundee Imaging Facility is supported by the ‘Wellcome Trust Technology Platform’ award (097945/B/11/Z). We are grateful to the sequencing service (School of Life Sciences, University of Dundee); and the MRC PPU Reagents and Services antibody purification teams (coordinated by James Hastie and Hilary McLauchlan) for excellent technical support. O.C. and colleagues (France) are supported by Fondation Institut du Cerveau et de la Moelle Epinie`re and Agence Nationale pour la Recherche (‘Investissements d’avenir’, grant ANR-10-IAIHU-06) and Innovative Medicines Initiative Joint Undertaking under grant agreement no.115568, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies’ in kind contribution. AS and colleagues (Finland) would like to thank the following funding sources: Sigrid Juselius Foundation; Academy of Finland, Helsinki University Central Hospital, and University of Helsinki (to A.S.), Biomedicum Helsinki Foundation, The Finnish Cultural Foundation and The Finnish Medical Foundation (to R.P-P.). Work by S.P.B. and colleagues (Cardiff ) was supported by a Ser Cymru PhD studentship, Life Sciences Research Network Wales (K.F.) and a Wellcome Trust Studentship (L.S.).

PY - 2018/11/7

Y1 - 2018/11/7

N2 - Mutations in PINK1 and Parkin result in autosomal recessive Parkinson's disease (PD). Cell culture and in vitro studies have elaborated the PINK1-dependent regulation of Parkin and defined how this dyad orchestrates the elimination of damaged mitochondria via mitophagy. PINK1 phosphorylates ubiquitin at serine 65 (Ser65) and Parkin at an equivalent Ser65 residue located within its N-terminal ubiquitin-like domain, resulting in activation; however, the physiological significance of Parkin Ser65 phosphorylation in vivo in mammals remains unknown. To address this, we generated a Parkin Ser65Ala (S65A) knock-in mouse model. We observe endogenous Parkin Ser65 phosphorylation and activation in mature primary neurons following mitochondrial depolarization and reveal this is disrupted in ParkinS65A/S65A neurons. Phenotypically, ParkinS65A/S65A mice exhibit selective motor dysfunction in the absence of any overt neurodegeneration or alterations in nigrostriatal mitophagy. The clinical relevance of our findings is substantiated by the discovery of homozygous PARKIN (PARK2) p.S65N mutations in two unrelated patients with PD. Moreover, biochemical and structural analysis demonstrates that the ParkinS65N/S65N mutant is pathogenic and cannot be activated by PINK1. Our findings highlight the central role of Parkin Ser65 phosphorylation in health and disease.

AB - Mutations in PINK1 and Parkin result in autosomal recessive Parkinson's disease (PD). Cell culture and in vitro studies have elaborated the PINK1-dependent regulation of Parkin and defined how this dyad orchestrates the elimination of damaged mitochondria via mitophagy. PINK1 phosphorylates ubiquitin at serine 65 (Ser65) and Parkin at an equivalent Ser65 residue located within its N-terminal ubiquitin-like domain, resulting in activation; however, the physiological significance of Parkin Ser65 phosphorylation in vivo in mammals remains unknown. To address this, we generated a Parkin Ser65Ala (S65A) knock-in mouse model. We observe endogenous Parkin Ser65 phosphorylation and activation in mature primary neurons following mitochondrial depolarization and reveal this is disrupted in ParkinS65A/S65A neurons. Phenotypically, ParkinS65A/S65A mice exhibit selective motor dysfunction in the absence of any overt neurodegeneration or alterations in nigrostriatal mitophagy. The clinical relevance of our findings is substantiated by the discovery of homozygous PARKIN (PARK2) p.S65N mutations in two unrelated patients with PD. Moreover, biochemical and structural analysis demonstrates that the ParkinS65N/S65N mutant is pathogenic and cannot be activated by PINK1. Our findings highlight the central role of Parkin Ser65 phosphorylation in health and disease.

KW - Parkinson's disease

KW - Mitochondria

KW - Mitophagy

KW - Autophagy

KW - mito-QC

KW - Parkin

KW - PINK1

KW - Neurodegeneration

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U2 - 10.1098/rsob.180108

DO - 10.1098/rsob.180108

M3 - Article

VL - 8

SP - 1

EP - 18

JO - Open Biology

JF - Open Biology

SN - 2046-2441

IS - 11

M1 - 181108

ER -

McWilliams TG, Barini E, Pohjolan-Pirhonen R, Brooks SP, Singh F, Burel S et al. Phosphorylation of Parkin at Serine65 is essential for its activation in vivo. Open Biology. 2018 Nov 7;8(11):1-18. 181108. https://doi.org/10.1098/rsob.180108