CoChat
Sign Up
Cited by 183

The DOI matches its Crossref record.

Phos-tag analysis of Rab10 phosphorylation by LRRK2: a powerful assay for assessing kinase function and inhibitors

Ito, Genta, Katsemonova, Kristina, Tonelli, Francesca, Lis, Pawel, Baptista, Marco A.S., Shpiro, Natalia, Duddy, Graham, Wilson, Steve, Ho, Philip Wing-Lok, Ho, Shu-Leong, Reith, Alastair D., Alessi, Dario R.·2016·Biochemical Journal
DOI 10.1042/bcj20160557PMC PMC5003698
Open this paper in CoChat — free account

Abstract

Autosomal dominant mutations that activate the leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson's disease. Recent work has revealed that LRRK2 directly phosphorylates a conserved threonine/serine residue in the effector-binding switch-II motif of a number of Rab GTPase proteins, including Rab10. Here we describe a facile and robust method to assess phosphorylation of endogenous Rab10 in mouse embryonic fibroblasts (MEFs), lung and spleen-derived B-cells, based on the ability of the Phos-tag reagent to retard the electrophoretic mobility of LRRK2-phosphorylated Rab10. We exploit this assay to show that phosphorylation of Rab10 is ablated in kinase-inactive LRRK2[D2017A] knockin MEFs and mouse lung, demonstrating that LRRK2 is the major Rab10 kinase in these cells/tissue. We also establish that the Phos-tag assay can be deployed to monitor the impact that activating LRRK2 pathogenic (G2019S and R1441G) knockin mutations have on stimulating Rab10 phosphorylation. We show that upon addition of LRRK2 inhibitors, Rab10 is dephosphorylated within 1–2 min, markedly more rapidly than the Ser935 and Ser1292 biomarker sites that require 40–80 min. Furthermore, we find that phosphorylation of Rab10 is suppressed in LRRK2[S910A+S935A] knockin MEFs indicating that phosphorylation of Ser910 and Ser935 and potentially 14-3-3 binding play a role in facilitating the phosphorylation of Rab10 by LRRK2 in vivo. The Rab Phos-tag assay has the potential to significantly aid with evaluating the effect that inhibitors, mutations and other factors have on the LRRK2 signalling pathway.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1042/bcj20160557

Cites

Statistics

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Several RABs identified as being controlled by Retromer are substrates for LRRK2-mediated phosphorylation in response to disrupted lysosomal homoeostasis.

    “several RABs identified as being controlled by Retromer are substrates for LRKK2-mediated phosphorylation in response to disrupted lysosomal homoeostasis127–129.”

    From Mapping of endosomal proximity proteomes reveals Retromer as a hub for RAB GTPase regulation (2025) · cites this paper as [127]

    The Introduction (page unavailable) identifies Rab8A and Rab10 as direct physiological substrates of LRRK2, and the Results demonstrate LRRK2-mediated Rab10 phosphorylation. Our copy does not establish that these Rabs are controlled by Retromer or that their phosphorylation occurs in response to disrupted lysosomal homoeostasis. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • Rab10 was identified as phosphorylated by LRRK2 combining two independent genetic and chemical phosphoproteomic screens and has been validated in subsequent studies.

    “Rab10 specifically was identified as phosphorylated by LRRK2 combining two independent genetic and chemical phosphoproteomic screens and has been validated in subsequent studies [19, 28, 32, 34, 35, 41, 79].”

    From Cellular and subcellular localization of Rab10 and phospho-T73 Rab10 in the mouse and human brain (2023) · cites this paper as [28]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

No references could be resolved yet.

Research works printed but not linked: 43

  1. Bras, 2015, SnapShot: genetics of Parkinson's disease, Cell, 160, 570, 10.1016/j.cell.2015.01.019 · DOI
  2. Singleton, 2013, The genetics of Parkinson's disease: progress and therapeutic implications, Mov. Disord., 28, 14, 10.1002/mds.25249 · DOI
  3. Zimprich, 2004, Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology, Neuron, 44, 601, 10.1016/j.neuron.2004.11.005 · DOI
  4. Paisan-Ruiz, 2004, Cloning of the gene containing mutations that cause PARK8-linked Parkinson's disease, Neuron, 44, 595, 10.1016/j.neuron.2004.10.023 · DOI
  5. Thaler, 2009, The LRRK2 G2019S mutation as the cause of Parkinson's disease in Ashkenazi Jews, J. Neural. Transm. (Vienna), 116, 1473, 10.1007/s00702-009-0303-0 · DOI
  6. West, 2005, Parkinson's disease-associated mutations in leucine-rich repeat kinase 2 augment kinase activity, Proc. Natl. Acad. Sci. U.S.A., 102, 16842, 10.1073/pnas.0507360102 · DOI · Paper page
  7. Jaleel, 2007, LRRK2 phosphorylates moesin at threonine-558: characterization of how Parkinson's disease mutants affect kinase activity, Biochem. J., 405, 307, 10.1042/BJ20070209 · DOI · Paper page
  8. Mata, 2006, LRRK2 in Parkinson's disease: protein domains and functional insights, Trends Neurosci, 29, 286, 10.1016/j.tins.2006.03.006 · DOI
  9. Li, 2009, Mutant LRRK2(R1441G) BAC transgenic mice recapitulate cardinal features of Parkinson's disease, Nat. Neurosci., 12, 826, 10.1038/nn.2349 · DOI
  10. Tong, 2009, R1441C mutation in LRRK2 impairs dopaminergic neurotransmission in mice, Proc. Natl. Acad. Sci. U.S.A., 106, 14622, 10.1073/pnas.0906334106 · DOI
  11. Daniels, 2011, Insight into the mode of action of the LRRK2 Y1699C pathogenic mutant, J. Neurochem., 116, 304, 10.1111/j.1471-4159.2010.07105.x · DOI
  12. Nichols, 2010, 14-3-3 binding to LRRK2 is disrupted by multiple Parkinson's disease associated mutations and regulates cytoplasmic localisation, Biochem. J., 430, 393, 10.1042/BJ20100483 · DOI
  13. Steger, 2016, Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases, eLife, 5, e12813, 10.7554/eLife.12813 · DOI · Paper page
  14. Pereira-Leal, 2000, The mammalian Rab family of small GTPases: definition of family and subfamily sequence motifs suggests a mechanism for functional specificity in the Ras superfamily, J. Mol. Biol., 301, 1077, 10.1006/jmbi.2000.4010 · DOI
  15. Cherfils, 2013, Regulation of small GTPases by GEFs, GAPs, and GDIs, Physiol. Rev., 93, 269, 10.1152/physrev.00003.2012 · DOI · Paper page
  16. Pfeffer, 2001, Rab GTPases: specifying and deciphering organelle identity and function, Trends Cell Biol., 11, 487, 10.1016/S0962-8924(01)02147-X · DOI
  17. Tucci, 2010, Genetic variability at the PARK16 locus, Eur. J. Hum. Genet., 18, 1356, 10.1038/ejhg.2010.125 · DOI · Paper page
  18. Pihlstrom, 2015, Fine mapping and resequencing of the PARK16 locus in Parkinson's disease, J. Hum. Genet., 60, 357, 10.1038/jhg.2015.34 · DOI · Paper page
  19. MacLeod, 2013, RAB7L1 interacts with LRRK2 to modify intraneuronal protein sorting and Parkinson's disease risk, Neuron, 77, 425, 10.1016/j.neuron.2012.11.033 · DOI · Paper page
  20. Wilson, 2014, Mutations in RAB39B cause X-linked intellectual disability and early-onset Parkinson disease with alpha-synuclein pathology, Am. J. Hum. Genet., 95, 729, 10.1016/j.ajhg.2014.10.015 · DOI
  21. Mata, 2015, The RAB39B p.G192R mutation causes X-linked dominant Parkinson's disease, Mol. Neurodegener., 10, 50, 10.1186/s13024-015-0045-4 · DOI
  22. Cooper, 2006, Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson's models, Science, 313, 324, 10.1126/science.1129462 · DOI
  23. Gitler, 2008, The Parkinson's disease protein alpha-synuclein disrupts cellular Rab homeostasis, Proc. Natl. Acad. Sci. U.S.A., 105, 145, 10.1073/pnas.0710685105 · DOI
  24. Lai, 2015, Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1, EMBO J., 34, 2840, 10.15252/embj.201591593 · DOI
  25. Kinoshita, 2004, Recognition of phosphate monoester dianion by an alkoxide-bridged dinuclear zinc(II) complex, Dalton Trans., 1189, 10.1039/b400269e · DOI
  26. Kinoshita, 2005, Novel immobilized zinc(II) affinity chromatography for phosphopeptides and phosphorylated proteins, J. Sep. Sci., 28, 155, 10.1002/jssc.200401833 · DOI
  27. Kinoshita, 2006, Phosphate-binding tag, a new tool to visualize phosphorylated proteins, Mol. Cell. Proteomics, 5, 749, 10.1074/mcp.T500024-MCP200 · DOI
  28. Dzamko, 2010, Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser(910)/Ser(935), disruption of 14-3-3 binding and altered cytoplasmic localization, Biochem. J., 430, 405, 10.1042/BJ20100784 · DOI
  29. Reith, 2012, GSK2578215A; a potent and highly selective 2-arylmethyloxy-5-substitutent-N-arylbenzamide LRRK2 kinase inhibitor, Bioorg. Med. Chem. Lett., 22, 5625, 10.1016/j.bmcl.2012.06.104 · DOI
  30. Choi, 2012, Brain penetrant LRRK2 inhibitor, ACS Med. Chem. Lett., 3, 658, 10.1021/ml300123a · DOI
  31. Miller, 2014, Compounds inhibiting leucine-rich repeat kinase enzyme activity, Int. Pat.
  32. Koike, 2007, Polyacrylamide gel for electrophoresis, polyacrylamide gel electrophoresis method using the same, method of producing the same, and acrylamide compound, Int. Pat.
  33. Liu, 2014, LRRK2 R1441G mice are more liable to dopamine depletion and locomotor inactivity, Ann. Clin. Transl. Neurol., 1, 199, 10.1002/acn3.45 · DOI
  34. Parisiadou, 2009, Phosphorylation of ezrin/radixin/moesin proteins by LRRK2 promotes the rearrangement of actin cytoskeleton in neuronal morphogenesis, J. Neurosci., 29, 13971, 10.1523/JNEUROSCI.3799-09.2009 · DOI
  35. Wiggin, 2002, MSK1 and MSK2 are required for the mitogen- and stress-induced phosphorylation of CREB and ATF1 in fibroblasts, Mol. Cell. Biol., 22, 2871, 10.1128/MCB.22.8.2871-2881.2002 · DOI
  36. Davies, 2013, Comprehensive characterization and optimization of anti-LRRK2 (leucine-rich repeat kinase 2) monoclonal antibodies, Biochem. J., 453, 101, 10.1042/BJ20121742 · DOI
  37. Fell, 2015, MLi-2, a potent, selective, and centrally active compound for exploring the therapeutic potential and safety of LRRK2 kinase inhibition, J. Pharmacol. Exp. Ther., 355, 397, 10.1124/jpet.115.227587 · DOI
  38. Sheng, 2012, Ser1292 autophosphorylation is an indicator of LRRK2 kinase activity and contributes to the cellular effects of PD mutations, Sci. Transl. Med., 4, 164ra161, 10.1126/scitranslmed.3004485 · DOI
  39. Fraser, 2013, LRRK2 secretion in exosomes is regulated by 14-3-3, Hum. Mol. Genet., 22, 4988, 10.1093/hmg/ddt346 · DOI
  40. Fraser, 2016, Urinary LRRK2 phosphorylation predicts parkinsonian phenotypes in G2019S LRRK2 carriers, Neurology, 86, 994, 10.1212/WNL.0000000000002436 · DOI
  41. Chia, 2014, Phosphorylation of LRRK2 by casein kinase 1alpha regulates trans-Golgi clustering via differential interaction with ARHGEF7, Nat. Commun., 5, 5827, 10.1038/ncomms6827 · DOI
  42. Dzamko, 2012, The IkappaB kinase family phosphorylates the Parkinson's disease kinase LRRK2 at Ser935 and Ser910 during Toll-like receptor signaling, PLos One, 7, e39132, 10.1371/journal.pone.0039132 · DOI
  43. Li, 2011, Phosphorylation-dependent 14-3-3 binding to LRRK2 is impaired by common mutations of familial Parkinson's disease, PLos One, 6, e17153, 10.1371/journal.pone.0017153 · DOI

Cited by

Sorted by citations

Metadata

Authors
Ito, G., Katsemonova, K., Tonelli, F., Lis, P., Baptista, M. A. S., Shpiro, N., Duddy, G., Wilson, S., Ho, P. W.-L., Ho, S.-L., Reith, A. D., & Alessi, D. R.
Venue
Biochemical Journal
Publisher
Portland Press Ltd.
Published
2016-08-30
Volume
473
Issue
17
Pages
2671-2685
ISSN
  • 0264-6021
  • 1470-8728
DOI
10.1042/bcj20160557
PMC
PMC5003698
Cited by
183
References
0
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (vor, 1059 d)

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review