Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1.

Yu‐Chiang Lai,Ronny Lehneck,Helen I Woodroof,Thomas J Macartney,Olga Corti,Brian D Dill,Matthias Trost,Chandana Kondapalli,Jean‐Christophe Corvol,James B Procter,Robert Gourlay,Mark Peggie,Aymelt Itzen,David G Campbell,Miratul Mk Muqit

doi:10.15252/embj.201591593

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 invitro 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 invivo. Our findings also suggest that disruption of Rab GTPase-mediated signalling may represent a major mechanism in the neurodegenerative cascade of Parkinson's disease.

Highlights

Human mutations in genes encoding the mitochondrial protein kinase, PTEN-induced kinase 1 (PINK1), and the ubiquitin E3 ligase, Parkin, are associated with autosomal recessive Parkinson’s disease (PD) (Kitada et al, 1998; Valente et al, 2004)
Using phospho-specific antibodies raised against phospho-serine 111 (Ser111) for each of the Rab GTPases, we demonstrate that Rab Ser111 phosphorylation is abolished in PINK1 knockout as well as PINK1 mutant patient-derived cells, indicating that this site is absolutely dependent on PINK1
We and other groups have previously reported that the Parkinson’s associated PINK1 kinase becomes activated in mammalian cells upon mitochondrial depolarisation that can be induced by mitochondrial uncouplers such as carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (Kondapalli et al, 2012; Okatsu et al, 2012)

Summary

Introduction

Human mutations in genes encoding the mitochondrial protein kinase, PTEN-induced kinase 1 (PINK1), and the ubiquitin E3 ligase, Parkin, are associated with autosomal recessive Parkinson’s disease (PD) (Kitada et al, 1998; Valente et al, 2004). The EMBO Journal the recruitment of Parkin, a cytosolic protein, to depolarised mitochondria where it ubiquitylates multiple mitochondrial substrates to trigger the removal of mitochondria by autophagy ( known as mitophagy; Narendra et al, 2008, 2010; Geisler et al, 2010; Matsuda et al, 2010; Vives-Bauza et al, 2010). We and other groups have found that upon activation, PINK1 directly phosphorylates Parkin at serine 65 (Ser65) within its ubiquitin-like (Ubl) domain (Kondapalli et al, 2012; Shiba-Fukushima et al, 2012) and ubiquitin at an equivalent Ser residue (Kane et al, 2014; Kazlauskaite et al, 2014b, 2015; Koyano et al, 2014), and together, phosphorylation of both residues leads to maximal recruitment and activation of Parkin at mitochondria (Kane et al, 2014; Kazlauskaite et al, 2014a,b, 2015; Koyano et al, 2014; Ordureau et al, 2014)

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