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

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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 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.

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Most cited references 35

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Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS.

S Gerber, J. Rush, O. Stemman … (2003)

A need exists for technologies that permit the direct quantification of differences in protein and posttranslationally modified protein expression levels. Here we present a strategy for the absolute quantification (termed AQUA) of proteins and their modification states. Peptides are synthesized with incorporated stable isotopes as ideal internal standards to mimic native peptides formed by proteolysis. These synthetic peptides can also be prepared with covalent modifications (e.g., phosphorylation, methylation, acetylation, etc.) that are chemically identical to naturally occurring posttranslational modifications. Such AQUA internal standard peptides are then used to precisely and quantitatively measure the absolute levels of proteins and posttranslationally modified proteins after proteolysis by using a selected reaction monitoring analysis in a tandem mass spectrometer. In the present work, the AQUA strategy was used to (i) quantify low abundance yeast proteins involved in gene silencing, (ii) quantitatively determine the cell cycle-dependent phosphorylation of Ser-1126 of human separase protein, and (iii) identify kinases capable of phosphorylating Ser-1501 of separase in an in vitro kinase assay. The methods described here represent focused, alternative approaches for studying the dynamically changing proteome.

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The Rab7 effector protein RILP controls lysosomal transport by inducing the recruitment of dynein-dynactin motors.

Ingrid Jordens, Mar Fernandez-Borja, Marije Marsman … (2001)

Many intracellular compartments, including MHC class II-containing lysosomes, melanosomes, and phagosomes, move along microtubules in a bidirectional manner and in a stop-and-go fashion due to the alternating activities of a plus-end directed kinesin motor and a minus-end directed dynein-dynactin motor. It is largely unclear how motor proteins are targeted specifically to different compartments. Rab GTPases recruit and/or activate several proteins involved in membrane fusion and vesicular transport. They associate with specific compartments after activation, which makes Rab GTPases ideal candidates for controlling motor protein binding to specific membranes. We and others [7] have identified a protein, called RILP (for Rab7-interacting lysosomal protein), that interacts with active Rab7 on late endosomes and lysosomes. Here we show that RILP prevents further cycling of Rab7. RILP expression induces the recruitment of functional dynein-dynactin motor complexes to Rab7-containing late endosomes and lysosomes. Consequently, these compartments are transported by these motors toward the minus end of microtubules, effectively inhibiting their transport toward the cell periphery. This signaling cascade may be responsible for timed and selective dynein motor recruitment onto late endosomes and lysosomes.

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Coordination of Rab8 and Rab11 in primary ciliogenesis.

Amlan Das, Wei-Guo Chai, Shanshan Feng … (2010)

Primary cilia are microtubule-based membrane projections located at the surface of many cells. Defects in primary cilia formation have been implicated in a number of genetic disorders, such as Bardet-Biedl Syndrome and Polycystic Kidney Disease. Recent studies have demonstrated that polarized vesicular transport involving Rab8 and its guanine nucleotide-exchange factor Rabin8 is essential for primary ciliogenesis. Here we report that Rabin8 is a direct downstream effector of Rab11, which functions in membrane trafficking from the trans-Golgi network and recycling endosomes. Rab11, in its GTP-bound form, interacts with Rabin8 and kinetically stimulates the guanine nucleotide-exchange activity of Rabin8 toward Rab8. Rab11 is enriched at the base of the primary cilia and inhibition of Rab11 function by a dominant-negative mutant or RNA interference blocks primary ciliogenesis. Our results suggest that Rab GTPases coordinate with each other in the regulation of vesicular trafficking during primary ciliogenesis.

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Author and article information

Contributors

Herschel S Dhekne

Pawel Lis

Raja S Nirujogi

Ozge Karayel

Francesca Tonelli

Terina N Martinez

Esben Lorentzen:

ORCID: http://orcid.org/0000-0001-6493-7220

Suzanne R Pfeffer:

ORCID: http://orcid.org/0000-0002-6462-984X

Dario R Alessi:

ORCID: http://orcid.org/0000-0002-2140-9185

Matthias Mann:

ORCID: http://orcid.org/0000-0003-1292-4799

Ivan Dikic: Role: Reviewing Editor

Journal

Journal ID (nlm-ta): eLife

Journal ID (iso-abbrev): Elife

Journal ID (publisher-id): eLife

Title: eLife

Publisher: eLife Sciences Publications, Ltd

ISSN (Electronic): 2050-084X

Publication date (Electronic, pub): 10 November 2017

Publication date Collection: 2017

Volume: 6

Electronic Location Identifier: e31012

Affiliations

[1 ]deptDepartment of Proteomics and Signal Transduction Max-Planck-Institute of Biochemistry MartinsriedGermany

[2 ]deptMedical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences University of Dundee DundeeUnited Kingdom

[3 ]deptDepartment of Biochemistry Stanford University School of Medicine StanfordUnited States

[4 ]The Michael J. Fox Foundation for Parkinson’s Research New YorkUnited States

[5 ]deptDepartment of Molecular Biology and Genetics Aarhus University AarhusDenmark

Goethe University Frankfurt Germany

Author notes

[†]

These authors contributed equally to this work.

Author information

Martin Steger http://orcid.org/0000-0003-1637-8190

Esben Lorentzen http://orcid.org/0000-0001-6493-7220

Suzanne R Pfeffer http://orcid.org/0000-0002-6462-984X

Dario R Alessi http://orcid.org/0000-0002-2140-9185

Matthias Mann http://orcid.org/0000-0003-1292-4799

Article

Publisher ID: 31012

DOI: 10.7554/eLife.31012

PMC ID: 5695910

PubMed ID: 29125462

SO-VID: 661aeb03-1b32-465b-b0df-9e93dff65c9b

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This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

History

Date received : 04 August 2017

Date accepted : 09 November 2017

Funding

Funded by: FundRef http://dx.doi.org/10.13039/501100004189, Max-Planck-Gesellschaft;

Award Recipient : Martin Steger Ozge Karayel Matthias Mann

Funded by: FundRef http://dx.doi.org/10.13039/100000864, Michael J. Fox Foundation for Parkinson's Research;

Award ID: 6986.05

Award Recipient : Martin Steger Federico Diez Herschel S Dhekne Pawel Lis Raja S Nirujogi Ozge Karayel Francesca Tonelli Terina N Martinez Suzanne R Pfeffer Dario R Alessi Matthias Mann

Funded by: FundRef http://dx.doi.org/10.13039/501100000265, Medical Research Council;

Award ID: 357811350 R60

Award Recipient : Federico Diez Pawel Lis Raja S Nirujogi Francesca Tonelli Dario R Alessi

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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Author impact statement Parkinson's kinase LRRK2 phosphorylates a distinct subset of Rabs, and LRRK2-dependent phosphorylation links LRKK2 to ciliogenesis.

ScienceOpen disciplines: Life sciences

Keywords: rab gtpases,lrrk2,mass spectrometry,parkinson's disease,ciliogenesis,proteomics,human,mouse

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ScienceOpen disciplines: Life sciences

Keywords: rab gtpases, lrrk2, mass spectrometry, parkinson's disease, ciliogenesis, proteomics, human, mouse

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

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Most cited references 35

Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS.

The Rab7 effector protein RILP controls lysosomal transport by inducing the recruitment of dynein-dynactin motors.

Coordination of Rab8 and Rab11 in primary ciliogenesis.

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