title: Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1.
abstract: Mutations in the PTEN-induced kinase 1 (PINK1) are causative of autosomal recessive Parkinson's disease (PD). We have previously reported that PINK1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser(65)) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK1-dependent phosphorylation targets in HEK (human embryonic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub-family of Rab GTPases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser(111)) in response to PINK1 activation. Using phospho-specific antibodies raised against Ser(111) of each of the Rabs, we demonstrate that Rab Ser(111) phosphorylation occurs specifically in response to PINK1 activation and is abolished in HeLa PINK1 knockout cells and mutant PINK1 PD patient-derived fibroblasts stimulated by mitochondrial depolarisation. We provide evidence that Rab8A GTPase Ser(111) phosphorylation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser(111) phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser(65). We further show mechanistically that phosphorylation at Ser(111) significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor (GEF), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK1 is able to regulate the phosphorylation of Rab GTPases and indicate that monitoring phosphorylation of Rab8A/8B/13 at Ser(111) may represent novel biomarkers of PINK1 activity in vivo. Our findings also suggest that disruption of Rab GTPase-mediated signalling may represent a major mechanism in the neurodegenerative cascade of Parkinson's disease.
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences University of Dundee, Dundee, UK.
authors.forename: Yu-Chiang
authors.initials: YC
authors.lastname: Lai
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences University of Dundee, Dundee, UK.
authors.forename: Chandana
authors.initials: C
authors.lastname: Kondapalli
authors.affiliation: Centre for Integrated Protein Science Munich, Department Chemistry Technische Universität München, Garching, Germany.
authors.forename: Ronny
authors.initials: R
authors.lastname: Lehneck
authors.affiliation: Division of Computational Biology, College of Life Sciences University of Dundee, Dundee, UK.
authors.forename: James B
authors.initials: JB
authors.lastname: Procter
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences University of Dundee, Dundee, UK.
authors.forename: Brian D
authors.initials: BD
authors.lastname: Dill
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences University of Dundee, Dundee, UK.
authors.forename: Helen I
authors.initials: HI
authors.lastname: Woodroof
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences University of Dundee, Dundee, UK.
authors.forename: Robert
authors.initials: R
authors.lastname: Gourlay
authors.affiliation: Division of Signal Transduction Therapy, College of Life Sciences University of Dundee, Dundee, UK.
authors.forename: Mark
authors.initials: M
authors.lastname: Peggie
authors.affiliation: Division of Signal Transduction Therapy, College of Life Sciences University of Dundee, Dundee, UK.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Macartney
authors.affiliation: Inserm U 1127, Paris, France CNRS UMR 7225, Paris, France Sorbonne Universités UPMC Paris 06 UMR S 1127, Paris, France Institut du Cerveau et de la Moelle épinière ICM, Paris, France.
authors.forename: Olga
authors.initials: O
authors.lastname: Corti
authors.affiliation: Inserm U 1127, Paris, France CNRS UMR 7225, Paris, France Sorbonne Universités UPMC Paris 06 UMR S 1127, Paris, France Institut du Cerveau et de la Moelle épinière ICM, Paris, France Inserm Centre d'Investigation Clinique (CIC), Paris, France AP-HP, Département des maladies du système nerveux, Hôpital de la Pitié-Salpêtrière, Paris, France.
authors.forename: Jean-Christophe
authors.initials: JC
authors.lastname: Corvol
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences University of Dundee, Dundee, UK.
authors.forename: David G
authors.initials: DG
authors.lastname: Campbell
authors.affiliation: Centre for Integrated Protein Science Munich, Department Chemistry Technische Universität München, Garching, Germany.
authors.forename: Aymelt
authors.initials: A
authors.lastname: Itzen
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences University of Dundee, Dundee, UK m.trost@dundee.ac.uk m.muqit@dundee.ac.uk.
authors.forename: Matthias
authors.initials: M
authors.lastname: Trost
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences University of Dundee, Dundee, UK College of Medicine, Dentistry & Nursing, University of Dundee, Dundee, UK m.trost@dundee.ac.uk m.muqit@dundee.ac.uk.
authors.forename: Miratul Mk
authors.initials: MM
authors.lastname: Muqit
mesh_terms: D019943:Amino Acid Substitution; D004789:Enzyme Activation / Q000235:genetics; D000081410:Germinal Center Kinases; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D020125:Mutation, Missense; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D020734:Parkinsonian Disorders / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D010766:Phosphorylation / Q000235:genetics; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000099202:PTEN-Induced Putative Kinase
chemical_list: D000081410:Germinal Center Kinases; D015513:Oncogene Proteins; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D020691:rab GTP-Binding Proteins; D000099202:PTEN-Induced Putative Kinase; C529761:Rab8b protein, human; C085485:RAB13 protein, human; C082377:RAB8A protein, human
keywords: PINK1; Parkinson's disease; Rab GTPases; phosphoproteomics