Phosphoproteomic screening identifies Rab GTP ases as novel downstream targets of PINK 1
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 stimulat...
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| Vydáno v: | The EMBO journal Ročník 34; číslo 22; s. 2840 - 2861 |
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| Hlavní autoři: | , , , , , , , , , , , , , , |
| Médium: | Journal Article |
| Jazyk: | angličtina |
| Vydáno: |
12.11.2015
|
| ISSN: | 0261-4189, 1460-2075 |
| On-line přístup: | Získat plný text |
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| Shrnutí: | 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.
image
The Parkinson's disease‐mutated
PINK
1 kinase phosphorylates Parkin and ubiquitin. Phosphoproteomic screening reveals Rab8A, Rab8B and Rab13
GTP
ases as some of only few additional targets whose phosphorylation depends on
PINK
1 during mitophagy.
Activated PINK1 indirectly controls phosphorylation of serine 111 of Rab8A and closely related Rab GTPases.
Biochemical and cellular analysis imply an unknown intermediate PINK1‐dependent Rab8A Ser111 kinase or phosphatase.
PINK1‐directed activation of Parkin E3 ligase activity is independent of Rab8A Ser111 phosphorylation
Phosphorylation at Ser111 inhibits Rab8A activation by its guanine exchange factor, Rabin8. |
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| ISSN: | 0261-4189 1460-2075 |
| DOI: | 10.15252/embj.201591593 |