title: Activation of the thiazide-sensitive Na+-Cl- cotransporter by the WNK-regulated kinases SPAK and OSR1.
abstract: Mutations increasing WNK1 kinase expression in humans cause the pseudohypoaldosteronism type II hypertension syndrome. This condition is treated effectively by thiazide diuretics, which exert their effects by inhibiting the Na+-Cl(-) cotransporter (NCC), suggesting a link between WNK1 and NCC. Here, we demonstrate that the SPAK and OSR1 kinases that are activated by WNK1 phosphorylate human NCC at three conserved residues (Thr46, Thr55 and Thr60). Activation of the WNK1-SPAK/OSR1 signalling pathway by treatment of HEK293 or mpkDCT kidney distal-convoluted-tubule-derived cells with hypotonic low-chloride conditions induced phosphorylation of NCC at residues phosphorylated by SPAK/OSR1. Efficient phosphorylation of NCC was dependent upon a docking interaction between an RFXI motif in NCC and SPAK/OSR1. Mutation of Thr60 to Ala in NCC markedly inhibited phosphorylation of Thr46 and Thr55 as well as NCC activation induced by hypotonic low-chloride treatment of HEK293 cells. Our results establish that the WNK1-SPAK/OSR1 signalling pathway plays a key role in controlling the phosphorylation and activity of NCC. They also suggest a mechanism by which increased WNK1 overexpression could lead to hypertension and that inhibitors of SPAK/OSR1 might be of use in reducing blood pressure by suppressing phosphorylation and hence activity of NCC.
authors.affiliation: MRC Protein Phosphorylation Unit, MSI/WTB complex, University of Dundee, Dow Street, Dundee, DD1 5EH, UK. c.j.z.richardson@dundee.ac.uk
authors.forename: Ciaran
authors.initials: C
authors.lastname: Richardson
authors.forename: Fatema H
authors.initials: FH
authors.lastname: Rafiqi
authors.forename: Håkan K R
authors.initials: HK
authors.lastname: Karlsson
authors.forename: Ntsane
authors.initials: N
authors.lastname: Moleleki
authors.forename: Alain
authors.initials: A
authors.lastname: Vandewalle
authors.forename: David G
authors.initials: DG
authors.lastname: Campbell
authors.forename: Nick A
authors.initials: NA
authors.lastname: Morrice
authors.forename: Dario R
authors.initials: DR
authors.lastname: Alessi
mesh_terms: D020816:Amino Acid Motifs / Q000502:physiology; D000595:Amino Acid Sequence / Q000502:physiology; D002460:Cell Line; D004789:Enzyme Activation / Q000502:physiology; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D006973:Hypertension / Q000188:drug therapy / Q000378:metabolism / Q000503:physiopathology; D007038:Hypotonic Solutions / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D007684:Kidney Tubules / Q000166:cytology / Q000378:metabolism*; D015778:Minor Histocompatibility Antigens; D010766:Phosphorylation; D017354:Point Mutation / Q000502:physiology; D011485:Protein Binding / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D049993:Sodium Chloride Symporter Inhibitors / Q000494:pharmacology; D049992:Sodium Chloride Symporters / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D000076204:WNK Lysine-Deficient Protein Kinase 1
chemical_list: D007038:Hypotonic Solutions; D047908:Intracellular Signaling Peptides and Proteins; D015778:Minor Histocompatibility Antigens; D049993:Sodium Chloride Symporter Inhibitors; D049992:Sodium Chloride Symporters; C118135:OXSR1 protein, human; D017346:Protein Serine-Threonine Kinases; C486409:STK39 protein, human; D000076204:WNK Lysine-Deficient Protein Kinase 1; C498743:WNK1 protein, human