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dc.contributor.authorde Los Heros, P
dc.contributor.authorAlessi, DR
dc.contributor.authorGourlay, R
dc.contributor.authorCampbell, DG
dc.contributor.authorDeak, M
dc.contributor.authorMacartney, TJ
dc.contributor.authorKahle, KT
dc.contributor.authorZhang, J
dc.date.accessioned2018-04-04T10:33:28Z
dc.date.accessioned2018-07-09T15:04:25Z
dc.date.issued2014-03-15
dc.description.abstractPrecise homoeostasis of the intracellular concentration of Cl- is achieved via the co-ordinated activities of the Cl- influx and efflux. We demonstrate that the WNK (WNK lysine-deficient protein kinase)-activated SPAK (SPS1-related proline/alanine-rich kinase)/OSR1 (oxidative stress-responsive kinase 1) known to directly phosphorylate and stimulate the N[K]CCs (Na+-K+ ion co-transporters), also promote inhibition of the KCCs (K+-Cl- co-transporters) by directly phosphorylating a recently described C-terminal threonine residue conserved in all KCC isoforms [Site-2 (Thr1048)]. First, we demonstrate that SPAK and OSR1, in the presence of the MO25 regulatory subunit, robustly phosphorylates all KCC isoforms at Site-2 in vitro. Secondly, STOCK1S-50699, a WNK pathway inhibitor, suppresses SPAK/OSR1 activation and KCC3A Site-2 phosphorylation with similar efficiency. Thirdly, in ES (embryonic stem) cells lacking SPAK/OSR1 activity, endogenous phosphorylation of KCC isoforms at Site-2 is abolished and these cells display elevated basal activity of 86Rb+ uptake that was not markedly stimulated further by hypotonic high K+ conditions, consistent with KCC3A activation. Fourthly, a tight correlation exists between SPAK/OSR1 activity and the magnitude of KCC3A Site-2 phosphorylation. Lastly, a Site-2 alanine KCC3A mutant preventing SPAK/OSR1 phosphorylation exhibits increased activity. We also observe that KCCs are directly phosphorylated by SPAK/OSR1, at a novel Site-3 (Thr5 in KCC1/KCC3 and Thr6 in KCC2/KCC4), and a previously recognized KCC3-specific residue, Site-4 (Ser96). These data demonstrate that the WNK-regulated SPAK/OSR1 kinases directly phosphorylate the N[K]CCs and KCCs, promoting their stimulation and inhibition respectively. Given these reciprocal actions with anticipated net effects of increasing Cl- influx, we propose that the targeting of WNK-SPAK/OSR1 with kinase inhibitors might be a novel potent strategy to enhance cellular Cl- extrusion, with potential implications for the therapeutic modulation of epithelial and neuronal ion transport in human disease states.en_GB
dc.description.sponsorshipThis work was supported by the Medical Research Council and the Wellcome Trust [grant number 091415] as well as the pharmaceutical companies supporting the Division of Signal Transduction Therapy Unit (AstraZeneca, Boehringer-Ingelheim, GlaxoSmithKline, Merck KgaA, Janssen Pharmaceutica and Pfizer). K.T.K. is supported by the Manton Center for Orphan Diseases at Children's Hospital Boston at Harvard Medical School, and the Harvard/MIT Joint Research Grants Program in Basic Neuroscience.en_GB
dc.identifier.citationVol. 458 (3), pp. 559 - 573en_GB
dc.identifier.doi10.1042/BJ20131478
dc.identifier.urihttp://hdl.handle.net/10871/33420
dc.language.isoenen_GB
dc.publisherPortland Press for Biochemical Societyen_GB
dc.relation.urlhttps://www.ncbi.nlm.nih.gov/pubmed/24393035en_GB
dc.relation.urlhttp://hdl.handle.net/10871/32310en_GB
dc.rights© 2014 The author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC-BY) (http://creativecommons.org/licenses/by/3.0/) which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.en_GB
dc.subjectAmino Acid Sequenceen_GB
dc.subjectCell Lineen_GB
dc.subjectChloridesen_GB
dc.subjectHumansen_GB
dc.subjectMolecular Sequence Dataen_GB
dc.subjectPhosphopeptidesen_GB
dc.subjectPhosphorylationen_GB
dc.subjectPotassiumen_GB
dc.subjectProtein Isoformsen_GB
dc.subjectProtein-Serine-Threonine Kinasesen_GB
dc.subjectSignal Transductionen_GB
dc.subjectSodium-Potassium-Chloride Symportersen_GB
dc.subjectSymportersen_GB
dc.subjectTranscription Factorsen_GB
dc.titleThe WNK-regulated SPAK/OSR1 kinases directly phosphorylate and inhibit the K+-Cl- co-transportersen_GB
dc.typeArticleen_GB
dc.date.available2018-04-04T10:33:28Z
dc.date.available2018-07-09T15:04:25Z
exeter.place-of-publicationEnglanden_GB
dc.descriptionThis is the final version of the article. Available from Portland Press via the DOI in this record.en_GB
dc.descriptionThere is another ORE record for this publication: http://hdl.handle.net/10871/32310en_GB
dc.identifier.journalBiochemical Journalen_GB


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