The WNK-regulated SPAK/OSR1 kinases directly phosphorylate and inhibit the K+–Cl− co-transporters

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The WNK-regulated SPAK/OSR1 kinases directly phosphorylate and inhibit the K+–Cl− co-transporters

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Title: The WNK-regulated SPAK/OSR1 kinases directly phosphorylate and inhibit the K+–Cl− co-transporters
Author: delos Heros, Paola; Alessi, Dario R.; Gourlay, Robert; Campbell, David G.; Deak, Maria; Macartney, Thomas J.; Kahle, Kristopher T.; Zhang, Jinwei

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Citation: delos Heros, Paola, Dario R. Alessi, Robert Gourlay, David G. Campbell, Maria Deak, Thomas J. Macartney, Kristopher T. Kahle, and Jinwei Zhang. 2014. “The WNK-regulated SPAK/OSR1 kinases directly phosphorylate and inhibit the K+–Cl− co-transporters.” Biochemical Journal 458 (Pt 3): 559-573. doi:10.1042/BJ20131478. http://dx.doi.org/10.1042/BJ20131478.
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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.
Published Version: doi:10.1042/BJ20131478
Other Sources: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3940040/pdf/
Terms of Use: This article is made available under the terms and conditions applicable to Other Posted Material, as set forth at http://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA
Citable link to this page: http://nrs.harvard.edu/urn-3:HUL.InstRepos:12064547
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