Wilson, F. H. et al. Molecular pathogenesis of inherited hypertension with hyperkalemia: the Na-Cl cotransporter is inhibited by wild-type but not mutant WNK4. Proc. Natl Acad. Sci. USA 100, 680-684

Department of Genetics, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06510, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 01/2003; 100(2):680-4. DOI: 10.1073/pnas.242735399
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Mutations in the serine-threonine kinases WNK1 and WNK4 [with no lysine (K) at a key catalytic residue] cause pseudohypoaldosteronism type II (PHAII), a Mendelian disease featuring hypertension, hyperkalemia, hyperchloremia, and metabolic acidosis. Both kinases are expressed in the distal nephron, although the regulators and targets of WNK signaling cascades are unknown. The Cl(-) dependence of PHAII phenotypes, their sensitivity to thiazide diuretics, and the observation that they constitute a "mirror image" of the phenotypes resulting from loss of function mutations in the thiazide-sensitive Na-Cl cotransporter (NCCT) suggest that PHAII may result from increased NCCT activity due to altered WNK signaling. To address this possibility, we measured NCCT-mediated Na(+) influx and membrane expression in the presence of wild-type and mutant WNK4 by heterologous expression in Xenopus oocytes. Wild-type WNK4 inhibits NCCT-mediated Na-influx by reducing membrane expression of the cotransporter ((22)Na-influx reduced 50%, P < 1 x 10(-9), surface expression reduced 75%, P < 1 x 10(-14) in the presence of WNK4). This inhibition depends on WNK4 kinase activity, because missense mutations that abrogate kinase function prevent this effect. PHAII-causing missense mutations, which are remote from the kinase domain, also prevent inhibition of NCCT activity, providing insight into the pathophysiology of the disorder. The specificity of this effect is indicated by the finding that WNK4 and the carboxyl terminus of NCCT coimmunoprecipitate when expressed in HEK 293T cells. Together, these findings demonstrate that WNK4 negatively regulates surface expression of NCCT and implicate loss of this regulation in the molecular pathogenesis of an inherited form of hypertension.

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Available from: Maria D Lalioti, Sep 24, 2014
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    • "After the identification of WNK1 and WNK4 as the genes underlying PHAII, numerous investigations of the effects of coexpressing WNK1 and WNK4 with transporters, including NCC, were published (Kahle et al., 2003; Wilson et al., 2003; Yang et al., 2003; Kahle et al., 2004; Yamauchi et al., 2004, 2005; Cai et al., 2006; Gamba, 2006; Garzon-Muvdi et al., 2007; Ring et al., 2007; Yang et al., 2007a). "
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    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 · 3.51 Impact Factor
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    • "Recent exome sequencing approaches identified numerous recessive and dominant mutations in CUL3 and KLHL3 genes in patients suffering from type II pseudohypoaldosteronism (PHAII) or Gordon's syndrome, a rare disease featuring hypertension due to misbalance between renal salt reabsorption and electrolyte excretion (Boyden et al, 2012; Louis-Dit- Picard et al, 2012). Previously, mutations in WNK ('with no lysine') kinases have been correlated with this pathological condition (Wilson et al, 2003). Interestingly, 9 of 16 dominant mutations were found to cluster within the Kelch propeller of KLHL3 and in the vicinity of the other sites implicated in direct substrate binding, suggesting that KLHL3 mutations may abrogate binding and ubiquitylation of targets normally required for modulation of renal salt K þ and H þ handling in response to physiological challenge (Boyden et al, 2012). "
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    ABSTRACT: Protein ubiquitylation is a post-translational modification that controls all aspects of eukaryotic cell functionality, and its defective regulation is manifested in various human diseases. The ubiquitylation process requires a set of enzymes, of which the ubiquitin ligases (E3s) are the substrate recognition components. Modular CULLIN-RING ubiquitin ligases (CRLs) are the most prevalent class of E3s, comprising hundreds of distinct CRL complexes with the potential to recruit as many and even more protein substrates. Best understood at both structural and functional levels are CRL1 or SCF (SKP1/CUL1/F-box protein) complexes, representing the founding member of this class of multimeric E3s. Another CRL subfamily, called CRL3, is composed of the molecular scaffold CULLIN3 and the RING protein RBX1, in combination with one of numerous BTB domain proteins acting as substrate adaptors. Recent work has firmly established CRL3s as major regulators of different cellular and developmental processes as well as stress responses in both metazoans and higher plants. In humans, functional alterations of CRL3s have been associated with various pathologies, including metabolic disorders, muscle, and nerve degeneration, as well as cancer. In this review, we summarize recent discoveries on the function of CRL3s in both metazoans and plants, and discuss their mode of regulation and specificities.
    The EMBO Journal 08/2013; 32(17). DOI:10.1038/emboj.2013.173 · 10.43 Impact Factor
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    • "Unless stated otherwise, the oocytes will be assayed 2 days (36–48 h) after injection by radiotracer 22 Na + uptake or by extracting proteins for Western blot analysis. NCC activity was assessed by 22 Na + tracer uptake in isotonic uptake solution as reported [11] [12] [17], with or without overnight Cl À free treatment. After uptake, oocytes were washed six times and then lysed in 10% SDS solution. "
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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.
    FEBS letters 05/2013; 587(12). DOI:10.1016/j.febslet.2013.04.032 · 3.17 Impact Factor
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