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Functional interactions of the SPAK/OSR1 kinases with their upstream activator WNK1 and downstream substrate NKCC1

Abstract : The STE20/SPS1-related Proline-Alanine-rich Kinase (SPAK) and Oxidative Stress Response kinase-1 (OSR1) kinases interact and phosphorylate the Na-K-Cl cotransporter-1 (NKCC1), leading to its activation. Recent studies indicated that SPAK and OSR1 are phosphorylated and activated by the WNK1 (With-No-K(Lys) protein kinase-1) and WNK4, genes mutated in humans affected by Gordon‘s hypertension syndrome. Here we identify 3 residues in NKCC1 (Thr175/Thr179/Thr184-shark or Thr203/Thr207/Thr212-human), that are phosphorylated by SPAK and OSR1, and develop a peptide substrate (CATCHtide), to assess SPAK and OSR1 activity. Exposure of 293 cells to osmotic stress, which leads to phosphorylation and activation of NKCC1, increased phosphorylation of NKCC1 at the sites targetted by SPAK/OSR1. The residues on NKCC1, phosphorylated by SPAK/OSR1, are conserved in other cation cotransporters, such as the NaCl cotransporter, the target of thiazide drugs that lower blood pressure in humans with Gordon‘s syndrome. Furthermore, we characterise the properties of a 92 residue Conserved C-Terminal (CCT) domain on SPAK and OSR1 that interacts with an RFXV motif present in the substrate NKCC1 and its activators WNK1/WNK4. A peptide containing the RFXV motif interacts with nanomolar affinity with the CCT domains of SPAK/OSR1, and can be utilised to affinity purify SPAK and OSR1 from cell extracts. Mutation of the Arg, Phe or Val within this peptide abolishes binding to SPAK/OSR1. We identify specific residues within the CCT required for interaction with the RFXV motif and demonstrate that mutation of these in OSR1 inhibited phosphorylation of NKCC1, but not of the CATCHtide peptide that does not possess an RFXV motif. We establish that an intact CCT domain is required for WNK1 to efficiently phosphorylate and activate OSR1. These data establish that the CCT domain functions as a multi-purpose docking-site, enabling SPAK/OSR1 to interact with substrates (NKCC1) and activators (WNK1/WNK4).
Mots-clés : Life Sciences
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Submitted on : Friday, April 30, 2010 - 3:43:20 PM
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Alberto C Vitari, Jacob Thastrup, Fatema Rafiqi, Maria Deak, Nick A Morrice, et al.. Functional interactions of the SPAK/OSR1 kinases with their upstream activator WNK1 and downstream substrate NKCC1. Biochemical Journal, Portland Press, 2006, 397 (1), pp.223-231. ⟨10.1042/BJ20060220⟩. ⟨hal-00478531⟩



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