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WNK1 phosphorylation sites in TBC1D1 and TBC1D4 modulate cell surface expression of GLUT1

2019-12-06Journal article Restricted access
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dc.contributor.authorHenriques, Andreia F.A.
dc.contributor.authorMatos, Paulo
dc.contributor.authorCarvalho, Ana Sofia
dc.contributor.authorAzkargorta, Mikel
dc.contributor.authorElortza, Felix
dc.contributor.authorMatthiesen, Rune
dc.contributor.authorJordan, Peter
dc.date.accessioned2020-05-05T22:17:08Z
dc.date.available2020-05-05T22:17:08Z
dc.date.issued2019-12-06
dc.description.abstractGlucose uptake by mammalian cells is a key mechanism to maintain cell and tissue homeostasis and relies mostly on plasma membrane-localized glucose transporter proteins (GLUTs). Two main cellular mechanisms regulate GLUT proteins in the cell: first, expression of GLUT genes is under dynamic transcriptional control and is used by cancer cells to increase glucose availability. Second, GLUT proteins are regulated by membrane traffic from storage vesicles to the plasma membrane (PM). This latter process is triggered by signaling mechanisms and well-studied in the case of insulin-responsive cells, which activate protein kinase AKT to phosphorylate TBC1D4, a RAB-GTPase activating protein involved in membrane traffic regulation. Previously, we identified protein kinase WNK1 as another kinase able to phosphorylate TBC1D4 and regulate the surface expression of the constitutive glucose transporter GLUT1. Here we describe that downregulation of WNK1 through RNA interference in HEK293 cells led to a 2-fold decrease in PM GLUT1 expression, concomitant with a 60% decrease in glucose uptake. By mass spectrometry, we identified serine (S) 704 in TBC1D4 as a WNK1-regulated phosphorylation site, and also S565 in the paralogue TBC1D1. Transfection of the respective phosphomimetic or unphosphorylatable TBC1D mutants into cells revealed that both affected the cell surface abundance of GLUT1. The results reinforce a regulatory role for WNK1 in cell metabolism and have potential impact for the understanding of cancer cell metabolism and therapeutic options in type 2 diabetes.pt_PT
dc.description.abstractHighlights: Expression levels of protein kinase WNK1 modulate cellular glucose uptake; WNK1 phosphorylates the RAB-GAP proteins TBC1D4 and TBC1D1 in vitro; WNK1-specific phosphorylation sites were identified in TBC1D4 and TBC1D1; Phosphomimetic TBC1D mutants modulate plasma membrane expression of GLUT1 in cells.pt_PT
dc.description.sponsorshipThis work was supported by Fundação para a Ciência e Tecnologia(FCT) [grants PTDC/SAU-MET/117236/2010 to PJ, grant UID/MULTI/04046/2019 to the research unit BioISI, and fellowship SFRH/BD/106080/2015 from the BioSYS PhD programme PD65-2012 to AFAH].The authors acknowledge José Ferrão for regular mycoplasma testing incultured cells and Patrícia Barros for critical reading of the manuscript.Anthony Albiston (Melbourne, Australia) and Florian Lang (Universityof Tubingen, Germany) kindly provided constucts pCR3.1/AS160-2mycor pIRES2-EGFP-SGK1, respectively, for this studypt_PT
dc.description.versioninfo:eu-repo/semantics/publishedVersionpt_PT
dc.identifier.citationArch Biochem Biophys. 2020 Jan 15;679:108223. doi: 10.1016/j.abb.2019.108223. Epub 2019 Dec 6pt_PT
dc.identifier.doi10.1016/j.abb.2019.108223pt_PT
dc.identifier.issn0003-9861
dc.identifier.urihttp://hdl.handle.net/10400.18/6595
dc.language.isoengpt_PT
dc.peerreviewedyespt_PT
dc.publisherElsevierpt_PT
dc.relationNew insights into the regulation of insulin-sensitive glucose transporters by protein kinases
dc.relation.publisherversionhttps://www.sciencedirect.com/science/article/pii/S0003986119306447?via%3Dihubpt_PT
dc.subjectArabidopsis Proteinspt_PT
dc.subjectBinding Sitespt_PT
dc.subjectBiological Transportpt_PT
dc.subjectGTPase-Activating Proteinspt_PT
dc.subjectGlucosept_PT
dc.subjectGlucose Transporter Type 1pt_PT
dc.subjectHEK293 Cellspt_PT
dc.subjectHumanspt_PT
dc.subjectImmediate-Early Proteinspt_PT
dc.subjectInsulinpt_PT
dc.subjectPhosphorylationpt_PT
dc.subjectProtein-Serine-Threonine Kinasespt_PT
dc.subjectProto-Oncogene Proteins c-aktpt_PT
dc.subjectWNK Lysine-Deficient Protein Kinase 1pt_PT
dc.subjectGene Expression Regulationpt_PT
dc.subjectVias de Transdução de Sinal e Patologias Associadaspt_PT
dc.titleWNK1 phosphorylation sites in TBC1D1 and TBC1D4 modulate cell surface expression of GLUT1pt_PT
dc.typejournal article
dspace.entity.typePublication
oaire.awardTitleNew insights into the regulation of insulin-sensitive glucose transporters by protein kinases
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/3599-PPCDT/PTDC%2FSAU-MET%2F117236%2F2010/PT
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/5876/UID%2FMulti%2F04046%2F2013/PT
oaire.citation.startPage108223pt_PT
oaire.citation.titleArchives of Biochemistry and Biophysicspt_PT
oaire.citation.volume679pt_PT
oaire.fundingStream3599-PPCDT
oaire.fundingStream5876
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.nameFundação para a Ciência e a Tecnologia
project.funder.nameFundação para a Ciência e a Tecnologia
rcaap.embargofctDe acordo com política editorial da revista.pt_PT
rcaap.rightsembargoedAccesspt_PT
rcaap.typearticlept_PT
relation.isProjectOfPublication16266ac0-ff70-4b67-acf4-c75a288de616
relation.isProjectOfPublicationdc84f768-e6f2-4eea-b294-6c8ebbd1a156
relation.isProjectOfPublication.latestForDiscoverydc84f768-e6f2-4eea-b294-6c8ebbd1a156

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