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

TitlePhos-tag analysis of Rab10 phosphorylation by LRRK2: a powerful assay for assessing kinase function and inhibitors
Authors
Issue Date2016
PublisherPortland Press Ltd.. The Journal's web site is located at http://www.biochemj.org/bj/default.htm
Citation
Biochemical Journal, 2016, v. 473, p. 2671-2685 How to Cite?
AbstractAutosomal 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 Thr/Ser 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] knock-in 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) knock-in 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] knock-in 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 aide with evaluating the effect that inhibitors, mutations and other factors have on the LRRK2 signalling pathway.
Persistent Identifierhttp://hdl.handle.net/10722/232049
ISSN
2015 Impact Factor: 3.562
2015 SCImago Journal Rankings: 2.582
PubMed Central ID

 

DC FieldValueLanguage
dc.contributor.authorIto, G-
dc.contributor.authorKatsemonova, K-
dc.contributor.authorTonelli, F-
dc.contributor.authorLis, P-
dc.contributor.authorBaptista, MAS-
dc.contributor.authorShpiro, N-
dc.contributor.authorDuddy, G-
dc.contributor.authorWilson, S-
dc.contributor.authorHo, WL-
dc.contributor.authorHo, SL-
dc.contributor.authorReith, AD-
dc.contributor.authorAlessi, DR-
dc.date.accessioned2016-09-20T05:27:21Z-
dc.date.available2016-09-20T05:27:21Z-
dc.date.issued2016-
dc.identifier.citationBiochemical Journal, 2016, v. 473, p. 2671-2685-
dc.identifier.issn0264-6021-
dc.identifier.urihttp://hdl.handle.net/10722/232049-
dc.description.abstractAutosomal 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 Thr/Ser 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] knock-in 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) knock-in 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] knock-in 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 aide with evaluating the effect that inhibitors, mutations and other factors have on the LRRK2 signalling pathway.-
dc.languageeng-
dc.publisherPortland Press Ltd.. The Journal's web site is located at http://www.biochemj.org/bj/default.htm-
dc.relation.ispartofBiochemical Journal-
dc.rightsThe final version of record is available at [Journal URL].-
dc.titlePhos-tag analysis of Rab10 phosphorylation by LRRK2: a powerful assay for assessing kinase function and inhibitors-
dc.typeArticle-
dc.identifier.emailHo, WL: hwl2002@hku.hk-
dc.identifier.emailHo, SL: slho@hku.hk-
dc.identifier.authorityHo, WL=rp00259-
dc.identifier.authorityHo, SL=rp00240-
dc.identifier.doi10.1042/BCJ20160557-
dc.identifier.pmcidPMC5003698 -
dc.identifier.hkuros265530-
dc.identifier.volume473-
dc.identifier.spage2671-
dc.identifier.epage2685-
dc.publisher.placeUnited Kingdom-

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