Systematic proteomic analysis of LRRK2-mediated Rab GTPase phosphorylation establishes a connection to ciliogenesis

Martin Steger, Federico Diez, Herschel S. Dhekne, Pawel Lis, Raja S. Nirujogi, Ozge Karayel, Francesca Tonelli, Terina N. Martinez, Esben Lorentzen, Suzanne R. Pfeffer, Dario R. Alessi (Lead / Corresponding author), Matthias Mann

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Abstract

We previously reported that Parkinson’s disease (PD) kinase LRRK2 phosphorylates a subset of Rab GTPases on a conserved residue in their switch-II domains (Steger, Tonelli et al., 2016) (PMID: 26824392). Here, we systematically analyzed the Rab protein family and found 14 of them (Rab3A/B/C/D, Rab5A/B/C, Rab8A/B, Rab10, Rab12, Rab29, Rab35 and Rab43) to be specifically phosphorylated by LRRK2, with evidence for endogenous phosphorylation for ten of them (Rab3A/B/C/D, Rab8A/B, Rab10, Rab12, Rab35 and Rab43). Affinity enrichment mass spectrometry revealed that the primary ciliogenesis regulator, RILPL1 specifically interacts with the LRRK2-phosphorylated forms of Rab8A and Rab10, whereas RILPL2 binds to phosphorylated Rab8A, Rab10, and Rab12. Induction of primary cilia formation by serum starvation led to a two-fold reduction in ciliogenesis in fibroblasts derived from pathogenic LRRK2-R1441G knock-in mice. These results implicate LRRK2 in primary ciliogenesis and suggest that Rab-mediated protein transport and/or signaling defects at cilia may contribute to LRRK2-dependent
pathologies.
Original languageEnglish
Article numbere31012
Pages (from-to)1-22
Number of pages22
JournaleLife
Volume6
DOIs
Publication statusPublished - 10 Nov 2017

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rab GTP-Binding Proteins
Phosphorylation
Cilia
Proteomics
Pathology
Protein Transport
Fibroblasts
Starvation
Mass spectrometry
Parkinson Disease
Mass Spectrometry
Proteins
Phosphotransferases
Switches
Defects
Serum

Cite this

Steger, Martin ; Diez, Federico ; Dhekne, Herschel S. ; Lis, Pawel ; Nirujogi, Raja S. ; Karayel, Ozge ; Tonelli, Francesca ; Martinez, Terina N. ; Lorentzen, Esben ; Pfeffer, Suzanne R. ; Alessi, Dario R. ; Mann, Matthias. / Systematic proteomic analysis of LRRK2-mediated Rab GTPase phosphorylation establishes a connection to ciliogenesis. In: eLife. 2017 ; Vol. 6. pp. 1-22.
@article{e9ef5c9ac721440fbecbbcb17ed7400c,
title = "Systematic proteomic analysis of LRRK2-mediated Rab GTPase phosphorylation establishes a connection to ciliogenesis",
abstract = "We previously reported that Parkinson’s disease (PD) kinase LRRK2 phosphorylates a subset of Rab GTPases on a conserved residue in their switch-II domains (Steger, Tonelli et al., 2016) (PMID: 26824392). Here, we systematically analyzed the Rab protein family and found 14 of them (Rab3A/B/C/D, Rab5A/B/C, Rab8A/B, Rab10, Rab12, Rab29, Rab35 and Rab43) to be specifically phosphorylated by LRRK2, with evidence for endogenous phosphorylation for ten of them (Rab3A/B/C/D, Rab8A/B, Rab10, Rab12, Rab35 and Rab43). Affinity enrichment mass spectrometry revealed that the primary ciliogenesis regulator, RILPL1 specifically interacts with the LRRK2-phosphorylated forms of Rab8A and Rab10, whereas RILPL2 binds to phosphorylated Rab8A, Rab10, and Rab12. Induction of primary cilia formation by serum starvation led to a two-fold reduction in ciliogenesis in fibroblasts derived from pathogenic LRRK2-R1441G knock-in mice. These results implicate LRRK2 in primary ciliogenesis and suggest that Rab-mediated protein transport and/or signaling defects at cilia may contribute to LRRK2-dependent pathologies.",
author = "Martin Steger and Federico Diez and Dhekne, {Herschel S.} and Pawel Lis and Nirujogi, {Raja S.} and Ozge Karayel and Francesca Tonelli and Martinez, {Terina N.} and Esben Lorentzen and Pfeffer, {Suzanne R.} and Alessi, {Dario R.} and Matthias Mann",
note = "This work was supported The Michael J. Fox Foundation for Parkinson’s Research (grant ID 6986.05), the Max-Planck Society for the Advancement of Science and the Medical Research Council (grant numbers MC_UU_12016/2 (to D.R.A.). We thank S. Uebel, M. Spitaler and S. Pettera from the MPIB Biochemistry Core Facility and S. Kroiss, G. Sowa, K. Mayr and I. Paron from the department of Proteomics and Signal Transduction for technical assistance. Members of the Dundee MRC-PPU Cloning, DNA Sequencing, antibody, protein production, tissue culture and mass spectrometry teams for technical assistance. Sandy Chou at Abcam for generation of the phospho-Rab8 polyclonal antibody. LRRK2 R1441G MEFs were kindly provided by Dr ShuLeong Ho (Division of Neurology, Department of Medicine, University of Hong Kong) and have been described previously (Ito et al., 2016).",
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Systematic proteomic analysis of LRRK2-mediated Rab GTPase phosphorylation establishes a connection to ciliogenesis. / Steger, Martin; Diez, Federico; Dhekne, Herschel S. ; Lis, Pawel; Nirujogi, Raja S.; Karayel, Ozge ; Tonelli, Francesca; Martinez, Terina N.; Lorentzen, Esben; Pfeffer, Suzanne R.; Alessi, Dario R. (Lead / Corresponding author); Mann, Matthias.

In: eLife, Vol. 6, e31012, 10.11.2017, p. 1-22.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Systematic proteomic analysis of LRRK2-mediated Rab GTPase phosphorylation establishes a connection to ciliogenesis

AU - Steger, Martin

AU - Diez, Federico

AU - Dhekne, Herschel S.

AU - Lis, Pawel

AU - Nirujogi, Raja S.

AU - Karayel, Ozge

AU - Tonelli, Francesca

AU - Martinez, Terina N.

AU - Lorentzen, Esben

AU - Pfeffer, Suzanne R.

AU - Alessi, Dario R.

AU - Mann, Matthias

N1 - This work was supported The Michael J. Fox Foundation for Parkinson’s Research (grant ID 6986.05), the Max-Planck Society for the Advancement of Science and the Medical Research Council (grant numbers MC_UU_12016/2 (to D.R.A.). We thank S. Uebel, M. Spitaler and S. Pettera from the MPIB Biochemistry Core Facility and S. Kroiss, G. Sowa, K. Mayr and I. Paron from the department of Proteomics and Signal Transduction for technical assistance. Members of the Dundee MRC-PPU Cloning, DNA Sequencing, antibody, protein production, tissue culture and mass spectrometry teams for technical assistance. Sandy Chou at Abcam for generation of the phospho-Rab8 polyclonal antibody. LRRK2 R1441G MEFs were kindly provided by Dr ShuLeong Ho (Division of Neurology, Department of Medicine, University of Hong Kong) and have been described previously (Ito et al., 2016).

PY - 2017/11/10

Y1 - 2017/11/10

N2 - We previously reported that Parkinson’s disease (PD) kinase LRRK2 phosphorylates a subset of Rab GTPases on a conserved residue in their switch-II domains (Steger, Tonelli et al., 2016) (PMID: 26824392). Here, we systematically analyzed the Rab protein family and found 14 of them (Rab3A/B/C/D, Rab5A/B/C, Rab8A/B, Rab10, Rab12, Rab29, Rab35 and Rab43) to be specifically phosphorylated by LRRK2, with evidence for endogenous phosphorylation for ten of them (Rab3A/B/C/D, Rab8A/B, Rab10, Rab12, Rab35 and Rab43). Affinity enrichment mass spectrometry revealed that the primary ciliogenesis regulator, RILPL1 specifically interacts with the LRRK2-phosphorylated forms of Rab8A and Rab10, whereas RILPL2 binds to phosphorylated Rab8A, Rab10, and Rab12. Induction of primary cilia formation by serum starvation led to a two-fold reduction in ciliogenesis in fibroblasts derived from pathogenic LRRK2-R1441G knock-in mice. These results implicate LRRK2 in primary ciliogenesis and suggest that Rab-mediated protein transport and/or signaling defects at cilia may contribute to LRRK2-dependent pathologies.

AB - We previously reported that Parkinson’s disease (PD) kinase LRRK2 phosphorylates a subset of Rab GTPases on a conserved residue in their switch-II domains (Steger, Tonelli et al., 2016) (PMID: 26824392). Here, we systematically analyzed the Rab protein family and found 14 of them (Rab3A/B/C/D, Rab5A/B/C, Rab8A/B, Rab10, Rab12, Rab29, Rab35 and Rab43) to be specifically phosphorylated by LRRK2, with evidence for endogenous phosphorylation for ten of them (Rab3A/B/C/D, Rab8A/B, Rab10, Rab12, Rab35 and Rab43). Affinity enrichment mass spectrometry revealed that the primary ciliogenesis regulator, RILPL1 specifically interacts with the LRRK2-phosphorylated forms of Rab8A and Rab10, whereas RILPL2 binds to phosphorylated Rab8A, Rab10, and Rab12. Induction of primary cilia formation by serum starvation led to a two-fold reduction in ciliogenesis in fibroblasts derived from pathogenic LRRK2-R1441G knock-in mice. These results implicate LRRK2 in primary ciliogenesis and suggest that Rab-mediated protein transport and/or signaling defects at cilia may contribute to LRRK2-dependent pathologies.

U2 - 10.7554/eLife.31012

DO - 10.7554/eLife.31012

M3 - Article

C2 - 29125462

VL - 6

SP - 1

EP - 22

JO - eLife

JF - eLife

SN - 2050-084X

M1 - e31012

ER -