Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1

Research output: Contribution to journal › Article › peer-review

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Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1. / Lai, Yu Chiang; Kondapalli, Chandana; Lehneck, Ronny; Procter, James B.; Dill, Brian D.; Woodroof, Helen I.; Gourlay, Robert; Peggie, Mark; Macartney, Thomas J.; Corti, Olga; Corvol, Jean Christophe; Campbell, David G.; Itzen, Aymelt; Trost, Matthias; Muqit, Miratul M.K.

In: EMBO Journal, Vol. 34, No. 22, 12.11.2015, p. 2840-2861.

Research output: Contribution to journal › Article › peer-review

Harvard

Lai, YC, Kondapalli, C, Lehneck, R, Procter, JB, Dill, BD, Woodroof, HI, Gourlay, R, Peggie, M, Macartney, TJ, Corti, O, Corvol, JC, Campbell, DG, Itzen, A, Trost, M & Muqit, MMK 2015, 'Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1', EMBO Journal, vol. 34, no. 22, pp. 2840-2861. https://doi.org/10.15252/embj.201591593

APA

Lai, Y. C., Kondapalli, C., Lehneck, R., Procter, J. B., Dill, B. D., Woodroof, H. I., Gourlay, R., Peggie, M., Macartney, T. J., Corti, O., Corvol, J. C., Campbell, D. G., Itzen, A., Trost, M., & Muqit, M. M. K. (2015). Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1. EMBO Journal, 34(22), 2840-2861. https://doi.org/10.15252/embj.201591593

Vancouver

Lai YC, Kondapalli C, Lehneck R, Procter JB, Dill BD, Woodroof HI et al. Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1. EMBO Journal. 2015 Nov 12;34(22):2840-2861. https://doi.org/10.15252/embj.201591593

Author

Lai, Yu Chiang ; Kondapalli, Chandana ; Lehneck, Ronny ; Procter, James B. ; Dill, Brian D. ; Woodroof, Helen I. ; Gourlay, Robert ; Peggie, Mark ; Macartney, Thomas J. ; Corti, Olga ; Corvol, Jean Christophe ; Campbell, David G. ; Itzen, Aymelt ; Trost, Matthias ; Muqit, Miratul M.K. / Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1. In: EMBO Journal. 2015 ; Vol. 34, No. 22. pp. 2840-2861.

Bibtex

@article{c6f2813204184a7e8b40011bd727f83d,
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 (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.",
keywords = "Parkinson's disease, phosphoproteomics, PINK1, Rab GTPases, Membrane and intracellular transport, Methods and resources, Post-translational modifications, Proteolysis and proteomics",
author = "Lai, {Yu Chiang} and Chandana Kondapalli and Ronny Lehneck and Procter, {James B.} and Dill, {Brian D.} and Woodroof, {Helen I.} and Robert Gourlay and Mark Peggie and Macartney, {Thomas J.} and Olga Corti and Corvol, {Jean Christophe} and Campbell, {David G.} and Aymelt Itzen and Matthias Trost and Muqit, {Miratul M.K.}",
year = "2015",
month = nov,
day = "12",
doi = "10.15252/embj.201591593",
language = "English",
volume = "34",
pages = "2840--2861",
journal = "The EMBO journal",
issn = "0261-4189",
publisher = "EMBO Press",
number = "22",

}

RIS

TY - JOUR

T1 - Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1

AU - Lai, Yu Chiang

AU - Kondapalli, Chandana

AU - Lehneck, Ronny

AU - Procter, James B.

AU - Dill, Brian D.

AU - Woodroof, Helen I.

AU - Gourlay, Robert

AU - Peggie, Mark

AU - Macartney, Thomas J.

AU - Corti, Olga

AU - Corvol, Jean Christophe

AU - Campbell, David G.

AU - Itzen, Aymelt

AU - Trost, Matthias

AU - Muqit, Miratul M.K.

PY - 2015/11/12

Y1 - 2015/11/12

N2 - 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.

AB - 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.

KW - Parkinson's disease

KW - phosphoproteomics

KW - PINK1

KW - Rab GTPases

KW - Membrane and intracellular transport

KW - Methods and resources

KW - Post-translational modifications

KW - Proteolysis and proteomics

UR - http://www.scopus.com/inward/record.url?scp=84949520315&partnerID=8YFLogxK

U2 - 10.15252/embj.201591593

DO - 10.15252/embj.201591593

M3 - Article

C2 - 26471730

AN - SCOPUS:84949520315

VL - 34

SP - 2840

EP - 2861

JO - The EMBO journal

JF - The EMBO journal

SN - 0261-4189

IS - 22

ER -