Phosphorylation of Parkin at serine 65 is essential for its activation in vivo.

Olga Corti,Miratul M. K. Muqit,Erica Barini,Stephen B. Dunnett,Rachel Hills,Graeme Ball,Lambert Montava-Garriga,Atul Kumar,Laura A. Smith ,Anu Suomalainen,Eino Palin,Ian G. Ganley,Sidi Mohamed Hassoun,Risto Pohjolan-Pirhonen,Simon Philip Brooks ,Miko Valori,Johanna Eerola‐Rautio ,François Mouton-Liger,Axel Knebel,Kyle Fears,Pentti J. Tienari ,Alan R. Prescott,J. Tamjar ,Ayşe Ulusoy ,Odetta Antico,François Singh,Kristin Balk,Sophie Burel,Thomas G. McWilliams,Riccardo Brambilla,Donato A. Di Monte

doi:10.1098/rsob.180108

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.

Highlights

Mutations in genes encoding PTEN-induced kinase 1 (PINK1) and the ubiquitin E3 ligase Parkin are causal for early onset& 2018 The Authors
To test the hypothesis that Parkin serine 65 (Ser65) phosphorylation is central to Parkin activation and neuronal homeostasis in vivo, we generated a knock-in mouse model in which the codon encoding Parkin Ser65 was altered by homologous recombination to alanine to prevent Parkin protein phosphorylation by PINK1
Homozygous ParkinS65A/S65A mice were born at expected Mendelian frequencies and wild-type and mutant mice were indistinguishable in terms of gross measures of growth, weight and development

Summary

Introduction

Mutations in genes encoding PTEN-induced kinase 1 (PINK1) (human PARK6) and the ubiquitin E3 ligase Parkin (human PARK2) are causal for early onset& 2018 The Authors. PINK1 is activated upon mitochondrial depolarization that can be induced using mitochondrial uncoupling agents, e.g. antimycin A/oligomycin (A/O) This results in the recruitment and activation of Parkin at the outer mitochondrial membrane (OMM) [4,5,6]. Parkin ubiquitylates multiple substrates at the OMM leading to both de novo assembly and elongation of existing ubiquitin chains that are, in turn, phosphorylated by PINK1. Together, this generates a feed-forward enhancement of Parkin activation and mitochondrial ubiquitylation that heralds the recruitment of selective autophagy adaptors necessary for the completion of mitophagy [12 – 15]

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Results

Conclusion