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.

Download full-text


Available from: Nick Morrice, Jan 24, 2014
  • Source
    • "Review identified that oxidative stress-responsive gene 1 (OSR1) and Ste20-related proline-alanine-rich kinase (SPAK) were substrates of WNK1 and WNK4 (Moriguchi et al., 2005; Vitari et al., 2005). OSR1 and SPAK are related serine–threonine kinases that possess an N-terminal catalytic domain similar to those of other members of the Ste20 kinase subfamily , and two conserved regions known as the serine motif (S motif) and conserved C-terminal (CCT) domain . "
    [Show abstract] [Hide abstract]
    ABSTRACT: In 2001, with-no-lysine (WNK) kinases were identified as the genes responsible for the human hereditary hypertensive disease pseudohypoaldosteronism type II (PHAII). It took a further 6 years to clarify that WNK kinases participate in a signaling cascade with oxidative stress-responsive gene 1 (OSR1), Ste20-related proline-alanine-rich kinase (SPAK), and Thiazide-sensitive NaCl cotransporter (NCC) in the kidney and the constitutive activation of this signaling cascade is the molecular basis of PHAII. Since this discovery, the WNK-OSR1/SPAK-NCC signaling cascade has been shown to be involved not only in PHAII but also in the regulation of blood pressure under normal and pathogenic conditions, such as hyperinsulinemia. However, the molecular mechanisms of WNK kinase regulation by dietary and hormonal factors and by PHAII-causing mutations remain poorly understood. In 2012, two additional genes responsible for PHAII, Kelch-like 3 (KLHL3) and Cullin3, were identified. At the time of their discovery, the molecular mechanisms underlying the interaction between these genes and their involvement in PHAII were unknown. Here we review the pathophysiological roles of the WNK signaling cascade clarified to date and introduce a new mechanism of WNK kinase regulation by KLHL3 and Cullin3, which provides insight on previously unknown mechanisms of WNK kinase regulation. This article is protected by copyright. All rights reserved.
    Biology of the Cell 12/2013; 106(2). DOI:10.1111/boc.201300069 · 3.87 Impact Factor
  • Source
    • "This effect was abolished when oocytes were treated in a Cl À free medium overnight before uptake experiments (Fig. S1A). Consistent with previous report [15], the level of phosphorylated serine 325 (p-S325) in OSR1 was increased by WNK4 without Cl À depletion ; it was also elevated by Cl À depletion in the absence of exogenous WNK4 (Fig. S1B). Thus, the WNK4–OSR1/SPAK–NCC pathway might have been largely masked by Cl À depletion. "
    [Show abstract] [Hide abstract]
    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.
    FEBS letters 05/2013; 587(12). DOI:10.1016/j.febslet.2013.04.032 · 3.34 Impact Factor
  • Source
    • "no. AF099989) has been described previously [22]. The full-length SPAK cDNA has been cloned C-terminally from the Dac-tag using BamHI-EcoRV-NotI three-way ligation. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Penicillin-binding protein 5 (PBP5), a product of the Escherichia coli gene dacA, possesses some β-lactamase activity. On binding to penicillin or related antibiotics via an ester bond, it deacylates and destroys them functionally by opening the β-lactam ring. This process takes several minutes. We exploited this process and showed that a fragment of PBP5 can be used as a reversible and monomeric affinity tag. At ambient temperature (e.g., 22°C), a PBP5 fragment binds rapidly and specifically to ampicillin Sepharose. Release can be facilitated either by eluting with 10mM ampicillin or in a ligand-free manner by incubation in the cold (1-10°C) in the presence of 5% glycerol. The "Dac-tag", named with reference to the gene dacA, allows the isolation of remarkably pure fusion protein from a wide variety of expression systems, including (in particular) eukaryotic expression systems.
    Analytical Biochemistry 06/2012; 428(1):64-72. DOI:10.1016/j.ab.2012.06.007 · 2.22 Impact Factor
Show more