Activation of the thiazide-sensitive Na-Cl cotransporter by the WNK-regulated kinases SPAK and OSR1

MRC Protein Phosphorylation Unit, MSI/WTB complex, University of Dundee, Dow Street, Dundee, DD1 5EH, UK.
Journal of Cell Science (Impact Factor: 5.43). 04/2008; 121(Pt 5):675-84. DOI: 10.1242/jcs.025312
Source: PubMed


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.

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Available from: Nick Morrice, Jan 24, 2014
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    • "Multiple laboratories, including our own, have provided evidence that the angiotensin II regulates distal convoluted tubule NCC acutely by increasing apical distribution via trafficking to the apical membrane (Sandberg et al. 2007; Ko et al. 2015), and chronically by increasing its abundance and phosphorylation (Gamba, 2012; Nguyen et al. 2013). NCC is phosphorylated at multiple sites and phosphorylation is associated with its activation (Richardson et al. 2008). Additionally, NCC-P is located exclusively in the apical membrane (Pedersen et al. 2010; Lee et al. 2013). "
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    ABSTRACT: In Sprague Dawley rats, 2-week angiotensin II (AngII) infusion increases Na(+) transporter abundance and activation from cortical thick ascending loop of Henle (TALH) to medullary collecting duct (CD) and raises blood pressure associated with a pressure natriuresis, accompanied by depressed Na(+) transporter abundance and activation from proximal tubule (PT) through medullary TALH. This study tests the hypothesis that early during AngII infusion, before blood pressure raises, Na(+) transporters' abundance and activation increase all along the nephron. Male Sprague Dawley rats were infused via osmotic minipumps with a subpressor dose of AngII (200 ng/kg/min) or vehicle for 3 days. Overnight urine was collected in metabolic cages and sodium transporters' abundance and phosphorylation were determined by immunoblotting homogenates of renal cortex and medulla. There were no significant differences in body weight gain, overnight urine volume, urinary Na(+) and K(+) excretion, or rate of excretion of a saline challenge between AngII and vehicle infused rats. The 3-day nonpressor AngII infusion significantly increased the abundance of PT Na(+)/H(+) exchanger 3 (NHE3), cortical TALH Na-K-2Cl cotransporter 2 (NKCC2), distal convoluted tubule (DCT) Na-Cl cotransporter (NCC), and cortical CD ENaC subunits. Additionally, phosphorylation of cortical NKCC2, NCC, and STE20/SPS1-related proline-alanine-rich kinase (SPAK) were increased; medullary NKCC2 and SPAK were not altered. In conclusion, 3-day AngII infusion provokes PT NHE3 accumulation as well as NKCC2, NCC, and SPAK accumulation and activation in a prehypertensive phase before evidence for intrarenal angiotensinogen accumulation.
    Full-text · Article · Sep 2015
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    • "Overexpression of WNK1 causes hypertension and hyperkalemia in humans by altering renal Na+ and K+ transport [11]. WNK1 activates the downstream protein kinases STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) and oxidative stress responsive 1 (OSR1) through phosphorylation of the t-loop in the catalytic domain [12], [13], [14], [15]. Activated SPAK and OSR1 associate and then phosphorylate and activate other ion co-transporters, including Na+/K+/2Cl− co-transporter 1 (NKCC1) [16], [17]. "
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    ABSTRACT: The WNK1 (WNK lysine deficient protein kinase 1) protein is a serine/threonine protein kinase with emerging roles in cancer. WNK1 causes hypertension and hyperkalemia when overexpressed and cardiovascular defects when ablated in mice. In this study, the role of Wnk1 in angiogenesis was explored using the zebrafish model. There are two zebrafish wnk1 isoforms, wnk1a and wnk1b, and both contain all the functional domains found in the human WNK1 protein. Both isoforms are expressed in the embryo at the initiation of angiogenesis and in the posterior cardinal vein (PCV), similar to fms-related tyrosine kinase 4 (flt4). Using morpholino antisense oligonucleotides against wnk1a and wnk1b, we observed that wnk1 morphants have defects in angiogenesis in the head and trunk, similar to flk1/vegfr2 morphants. Furthermore, both wnk1a and wnk1b mRNA can partially rescue the defects in vascular formation caused by flk1/vegfr2 knockdown. Mutation of the kinase domain or the Akt/PI3K phosphorylation site within wnk1 destroys this rescue capability. The rescue experiments provide evidence that wnk1 is a downstream target for Vegfr2 (vascular endothelial growth factor receptor-2) and Akt/PI3K signaling and thereby affects angiogenesis in zebrafish embryos. Furthermore, we found that knockdown of vascular endothelial growth factor receptor-2 (flk1/vegfr2) or vascular endothelial growth factor receptor-3 (flt4/vegfr3) results in a decrease in wnk1a expression, as assessed by in situ hybridization and q-RT-PCR analysis. Thus, the Vegf/Vegfr signaling pathway controls angiogenesis in zebrafish via Akt kinase-mediated phosphorylation and activation of Wnk1 as well as transcriptional regulation of wnk1 expression.
    Full-text · Article · Aug 2014 · PLoS ONE
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    • "WNK4 is an integrative regulator of renal electrolyte transport with the thiazide-sensitive Na + –Cl À cotransporter (NCC) as the main target [8] [9] [10]. The effects of WNK4 on NCC are complex: on the one hand, WNK4 inhibits NCC and increases its lysosomal degradation [11] [12] [13] [14]; on the other, NCC is stimulated by WNK4 via proline–alanine-rich STE20-related kinase (SPAK) and oxidative stress-responsive 1 (OSR1) in a phosphorylation cascade [10] [15] [16]. Furthermore, in the presence of angiotensin II, the positive effect overtakes the inhibitory effect of WNK4 in a SPAKdependent manner [17]. "
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    ABSTRACT: Mutations in with-no-lysine (K) kinase 4 (WNK4) and a ubiquitin E3 ligase complex component kelch-like 3 (KLHL3) both cause pseudohypoaldosteronism II (PHAII), a hereditary form of hypertension. We determined whether WNK4 or its effector is regulated by KLHL3 in Xenopus oocytes. KLHL3 inhibited the positive effect of WNK4 on Na(+)-Cl(-) cotransporter (NCC) by decreasing WNK4 protein abundance without decreasing that of NCC and the downstream kinase OSR1 directly. Ubiquitination and degradation of WNK4 were induced by KLHL3. The effect of KLHL3 on WNK4 degradation was blocked by a dominant negative form of cullin 3. All five PHAII mutations of KLHL3 tested disrupted the regulation on WNK4. We conclude that KLHL3 is a substrate adaptor for WNK4 in a ubiquitin E3 ligase complex.
    Full-text · Article · May 2013 · FEBS letters
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