dc.contributor.author | de Los Heros, P | |
dc.contributor.author | Alessi, DR | |
dc.contributor.author | Gourlay, R | |
dc.contributor.author | Campbell, DG | |
dc.contributor.author | Deak, M | |
dc.contributor.author | Macartney, TJ | |
dc.contributor.author | Kahle, KT | |
dc.contributor.author | Zhang, J | |
dc.date.accessioned | 2018-04-04T10:33:28Z | |
dc.date.accessioned | 2018-07-09T15:04:25Z | |
dc.date.issued | 2014-03-15 | |
dc.description.abstract | Precise 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.sponsorship | This 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.citation | Vol. 458 (3), pp. 559 - 573 | en_GB |
dc.identifier.doi | 10.1042/BJ20131478 | |
dc.identifier.uri | http://hdl.handle.net/10871/33420 | |
dc.language.iso | en | en_GB |
dc.publisher | Portland Press for Biochemical Society | en_GB |
dc.relation.url | https://www.ncbi.nlm.nih.gov/pubmed/24393035 | en_GB |
dc.relation.url | http://hdl.handle.net/10871/32310 | en_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.subject | Amino Acid Sequence | en_GB |
dc.subject | Cell Line | en_GB |
dc.subject | Chlorides | en_GB |
dc.subject | Humans | en_GB |
dc.subject | Molecular Sequence Data | en_GB |
dc.subject | Phosphopeptides | en_GB |
dc.subject | Phosphorylation | en_GB |
dc.subject | Potassium | en_GB |
dc.subject | Protein Isoforms | en_GB |
dc.subject | Protein-Serine-Threonine Kinases | en_GB |
dc.subject | Signal Transduction | en_GB |
dc.subject | Sodium-Potassium-Chloride Symporters | en_GB |
dc.subject | Symporters | en_GB |
dc.subject | Transcription Factors | en_GB |
dc.title | The WNK-regulated SPAK/OSR1 kinases directly phosphorylate and inhibit the K+-Cl- co-transporters | en_GB |
dc.type | Article | en_GB |
dc.date.available | 2018-04-04T10:33:28Z | |
dc.date.available | 2018-07-09T15:04:25Z | |
exeter.place-of-publication | England | en_GB |
dc.description | This is the final version of the article. Available from Portland Press via the DOI in this record. | en_GB |
dc.description | There is another ORE record for this publication: http://hdl.handle.net/10871/32310 | en_GB |
dc.identifier.journal | Biochemical Journal | en_GB |