The WNK1 and WNK4 protein kinases that are mutated in Gordon's hypertension syndrome phosphorylate and activate SPAK and OSR1 protein kinases.

MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, UK.
Biochemical Journal (Impact Factor: 4.78). 11/2005; 391(Pt 1):17-24. DOI: 10.1042/BJ20051180
Source: PubMed

ABSTRACT Mutations in the human genes encoding WNK1 [with no K (lysine) protein kinase-1] and the related protein kinase WNK4 are the cause of Gordon's hypertension syndrome. Little is known about the molecular mechanism by which WNK isoforms regulate cellular processes. We immunoprecipitated WNK1 from extracts of rat testis and found that it was specifically associated with a protein kinase of the STE20 family termed 'STE20/SPS1-related proline/alanine-rich kinase' (SPAK). We demonstrated that WNK1 and WNK4 both interacted with SPAK as well as a closely related kinase, termed 'oxidative stress response kinase-1' (OSR1). Wildtype (wt) but not catalytically inactive WNK1 and WNK4 phosphorylated SPAK and OSR1 to a much greater extent than with other substrates utilized previously, such as myelin basic protein and claudin-4. Phosphorylation by WNK1 or WNK4 markedly increased SPAK and OSR1 activity. Phosphopeptide mapping studies demonstrated that WNK1 phosphorylated kinase-inactive SPAK and OSR1 at an equivalent residue located within the T-loop of the catalytic domain (Thr233 in SPAK, Thr185 in OSR1) and a serine residue located within a C-terminal non-catalytic region (Ser373 in SPAK, Ser325 in OSR1). Mutation of Thr185 to alanine prevented the activation of OSR1 by WNK1, whereas mutation of Thr185 to glutamic acid (to mimic phosphorylation) increased the basal activity of OSR1 over 20-fold and prevented further activation by WNK1. Mutation of Ser325 in OSR1 to alanine or glutamic acid did not affect the basal activity of OSR1 or its ability to be activated by WNK1. These findings suggest that WNK isoforms operate as protein kinases that activate SPAK and OSR1 by phosphorylating the T-loops of these enzymes, resulting in their activation. Our analysis also describes the first facile assay that can be employed to quantitatively assess WNK1 and WNK4 activity.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Gordon Syndrome (GS) is a rare familial hypertension syndrome with a characteristic hyperkalaemia which distinguishes it from other syndromic forms of hypertension that typically cause hypokalaemia. Patients with GS respond to aggressive salt-restriction or relatively small doses of thiazide diuretics, which suggests that activation of the thiazide-sensitive Na/Cl cotransporter (NCC) in the distal nephron is to blame. However, the mechanism has proved to be complex. In 2001, mutations in genes encoding two serine/threonine kinases, WNK1 and WNK4, were identified as causing GS. However, it took several years to appreciate that these kinases operated in a cascade with downstream serine/threonine kinases (SPAK and OSR1) actually phosphorylating and activating NCC and the closely related cotransporters NKCC1 and NKCC2. The hyperkalaemia in GS arises from an independent action of WNK1/WNK4 to reduce cell-surface expression of ROMK, the secretory K-channel in the collecting ducts. However, mutations in WNK1/4 are present in a small minority of GS families, and further genes have emerged (CUL3 and KLHL3) that code for Cullin-3 (a scaffold protein in an ubiquitin-E3 ligase) and an adaptor protein, Kelch3, respectively. These new players regulate the ubiquitination and proteasomal degradation of WNK kinases, thereby adding to the complex picture we now have of NCC regulation in the distal nephron.
    Pediatric Nephrology 12/2014; DOI:10.1007/s00467-014-2956-7 · 2.88 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Dietary potassium deficiency, common in modern diets, raises blood pressure and enhances salt sensitivity. Potassium homeostasis requires a molecular switch in the distal convoluted tubule (DCT), which fails in familial hyperkalemic hypertension (pseudohypoaldosteronism type 2), activating the thiazide-sensitive NaCl cotransporter, NCC. Here, we show that dietary potassium deficiency activates NCC, even in the setting of high salt intake, thereby causing sodium retention and a rise in blood pressure. The effect is dependent on plasma potassium, which modulates DCT cell membrane voltage and, in turn, intracellular chloride. Low intracellular chloride stimulates WNK kinases to activate NCC, limiting potassium losses, even at the expense of increased blood pressure. These data show that DCT cells, like adrenal cells, sense potassium via membrane voltage. In the DCT, hyperpolarization activates NCC via WNK kinases, whereas in the adrenal gland, it inhibits aldosterone secretion. These effects work in concert to maintain potassium homeostasis. Copyright © 2015 Elsevier Inc. All rights reserved.
    Cell Metabolism 01/2015; 21(1):39-50. DOI:10.1016/j.cmet.2014.12.006 · 16.75 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Purpose:To identify whether kinases that regulate the activity of Cation Chloride Cotransporters (CCC) in other tissues are also expressed in rat and human lenses. Methods:The expression of With-No-Lysine Kinase (WNK 1, 3, 4), Oxidative Stress Response kinase 1 (OSR1) and Ste20-like Proline Alanine rich Kinase (SPAK) were determined at either the transcript or protein levels in rat and human lenses by reverse-transcriptase PCR and/or Western blotting, respectively. Selected kinases were regionally and subcellularly characterised in rat and human lenses. The transparency, wet weight and tissue morphology of lenses extracted from SPAK knock out animals was compared to wild type lenses. Results:WNK 1, 3, 4, SPAK, and OSR1 were identified at the transcript level in rat lenses and WNK1, 4, SPAK and OSR1 expression confirmed at the protein level in rat and human lenses.SPAK and OSR1 were found to associate with membranes as peripheral proteins and exhibited distinct subcellular and region-specific expression profiles throughout the lens. No significant difference in the wet weight of SPAK knock out lenses was detected relative to wild type lenses. However, SPAK knock out lenses showed an increased susceptibility to opacification. Conclusions:Our results show that the WNK-SPAK/OSR1 signalling system known to play a role in regulating the phosphorylation status and hence activity of CCCs in other tissues, is also present in the rat and human lenses. The increased susceptibility of SPAK lenses to opacification suggests that disruption of this signalling pathway may compromise the ability of the lens to control its volume and hence maintain transparency. Copyright © 2014 by Association for Research in Vision and Ophthalmology.
    Investigative Ophthalmology &amp Visual Science 12/2014; 56(1). DOI:10.1167/iovs.14-15911 · 3.66 Impact Factor

Full-text (2 Sources)

Available from
Jun 1, 2014