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Phosphoproteomic screening identifies Rab GTP ases as novel downstream targets of PINK 1
Author(s) -
Lai YuChiang,
Kondapalli Chandana,
Lehneck Ronny,
Procter James B,
Dill Brian D,
Woodroof Helen I,
Gourlay Robert,
Peggie Mark,
Macartney Thomas J,
Corti Olga,
Corvol JeanChristophe,
Campbell David G,
Itzen Aymelt,
Trost Matthias,
Muqit Miratul MK
Publication year - 2015
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.15252/embj.201591593
Subject(s) - phosphorylation , biology , rab , hek 293 cells , phosphoproteomics , microbiology and biotechnology , ubiquitin ligase , gtpase , parkin , ubiquitin , serine , biochemistry , protein phosphorylation , protein kinase a , gene , medicine , disease , pathology , parkinson's disease
Mutations in the PTEN ‐induced kinase 1 ( PINK 1) are causative of autosomal recessive Parkinson's disease ( PD ). We have previously reported that PINK 1 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 PINK 1‐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 GTP ases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser 111 ) in response to PINK 1 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 PINK 1 activation and is abolished in HeLa PINK 1 knockout cells and mutant PINK 1 PD patient‐derived fibroblasts stimulated by mitochondrial depolarisation. We provide evidence that Rab8A GTP ase Ser 111 phosphorylation is not directly regulated by PINK 1 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 PINK 1 is able to regulate the phosphorylation of Rab GTP ases and indicate that monitoring phosphorylation of Rab8A/8B/13 at Ser 111 may represent novel biomarkers of PINK 1 activity in vivo . Our findings also suggest that disruption of Rab GTP ase‐mediated signalling may represent a major mechanism in the neurodegenerative cascade of Parkinson's disease.