Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1 |
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| Authors: | Yu‐Chiang Lai Chandana Kondapalli Ronny Lehneck James B Procter Brian D Dill Helen I Woodroof Robert Gourlay Mark Peggie Thomas J Macartney Olga Corti Jean‐Christophe Corvol David G Campbell Aymelt Itzen Matthias Trost Miratul MK Muqit |
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| Affiliation: | 1. Centre for Integrated Protein Science Munich, Department Chemistry, Technische Universit?t München, Garching, Germany;2. Division of Computational Biology, College of Life Sciences, University of Dundee, Dundee, UK;3. MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee, UK;4. Division of Signal Transduction Therapy, College of Life Sciences, University of Dundee, Dundee, UK;5. Inserm U 1127, Paris, France;6. CNRS UMR 7225, Paris, France;7. Sorbonne Universités, UPMC Paris 06, UMR S 1127, Paris, France;8. Institut du Cerveau et de la Moelle épinière, ICM, Paris, France;9. Inserm, Centre d'Investigation Clinique (CIC), Paris, France;10. AP‐HP, Département des maladies du système nerveux, H?pital de la Pitié‐Salpêtrière, Paris, France;11. College of Medicine, Dentistry & Nursing, University of Dundee, Dundee, UK |
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| 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 (Ser65) 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 (Ser111) in response to PINK1 activation. Using phospho‐specific antibodies raised against Ser111 of each of the Rabs, we demonstrate that Rab Ser111 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 Ser111 phosphorylation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser111 phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser65. We further show mechanistically that phosphorylation at Ser111 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 Ser111 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. |
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| Keywords: | Parkinson''s disease phosphoproteomics PINK1 Rab GTPases |
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