The thiazide-sensitive Na-Cl cotransporter is regulated by a WNK kinase signaling complex

Abstract

The pathogenesis of essential hypertension remains unknown, but thiazide diuretics are frequently recommended as first-line treatment. Recently, familial hyperkalemic hypertension (FHHt) was shown to result from activation of the thiazide-sensitive Na-Cl cotransporter (NCC) by mutations in WNK4, although the mechanism for this effect remains unknown. WNK kinases are unique members of the human kinome, intimately involved in maintaining electrolyte balance across cell membranes and epithelia. Previous work showed that WNK1, WNK4, and a kidney-specific isoform of WNK1 interact to regulate NCC activity, suggesting that WNK kinases form a signaling complex. Here, we report that WNK3, another member of the WNK kinase family expressed by distal tubule cells, interacts with WNK4 and WNK1 to regulate NCC in both human kidney cells and Xenopus oocytes, further supporting the WNK signaling complex hypothesis. We demonstrate that physiological regulation of NCC in oocytes results from antagonism between WNK3 and WNK4 and that FHHt-causing WNK4 mutations exert a dominant-negative effect on wild-type (WT) WNK4 to mimic a state of WNK3 excess. The results provide a mechanistic explanation for the divergent effects of WT and FHHt-mutant WNK4 on NCC activity, and for the dominant nature of FHHt in humans and genetically modified mice.

Figure 1. WNK3 stimulates but does not phosphorylate NCC.

(A) Full-length WNK3-stimulated Na uptake by Xenopus oocytes (expressed as percentage of Na uptake by oocytes injected with NCC alone), whereas kinase-dead WNK3 D294A downregulated Na uptake, compared with NCC alone. n = 4. (B) GST-WNK3 2–420 phosphorylated itself and the substrate histone, whereas GST-WNK3 2–586 was inactive. WNK3 2–420 also phosphorylated the kinase-inactive GST-WNK3 2–586 and GST-WNK1 1–491 D368A. WNK3 did not phosphorylate NCC, within either the N terminus (GST-NCC 1–136) or the C terminus (GST-NCC 600-1,001). Top: kinase assay; bottom: Coomassie-stained gel. Results are representative of experiments performed in triplicate. (C) The C terminus of WNK3 (WNK3 421–1,743) increased NCC activity in a manner similar to full-length WNK3. The NCC stimulation was inhibited by hydrochlorothiazide (HCTZ). Neither WNK3 alone nor WNK3 421–1,743 alone stimulated Na uptake in the absence of NCC. WNK3 2–420 had no effect on NCC activity (data not shown); n = 5. (D) WNK3 constructs were all expressed at the protein level (Western blot of Xenopus oocyte lysate). (E) WNK3 421–1,743, but not the kinase domain, WNK3 2–420, increased the abundance of NCC at the plasma membrane of oocytes, as detected by immunocytochemistry (results are representative of experiments performed in triplicate). Original magnification, ×400. Western blot of oocyte lysate showed no effect on total NCC. (F) Comparison of the domain structure of WNK1 and constructs employed in the present experiments. AID, autoinhibitory domain; CCD, coiled-coil domain. Sequences of autoinhibitory domains of WNK kinases are compared. Key phenylalanines shown to be essential for autoinhibition of WNK1 (24) are highlighted in blue.

Figure 2. WNK kinase and inhibitory activity.

(A) WNK1 1–491 phosphorylated the WNK4 carboxyl terminus, but not claudin 4. (B) WNK1 1–491 phosphorylated histone and the amino-terminal domain of WNK4 (WNK4 1–167). (C) WNK3 2–420, but not WNK3 2–420 D294A, phosphorylated both the amino and carboxyl termini of WNK4. Results are representative of 5 identical experiments. (D) KS-WNK1 2–84 inhibited WNK3 kinase activity in a dose-dependent manner, whereas GST alone had no effect. Results are representative of experiments performed in triplicate. (E) GST-WNK4 445–518 inhibited WNK3 phosphorylation of itself and of histone in a dose-dependent manner. WNK4 445-563, which extends beyond the autoinhibitory domain, had no effect. Results are representative of experiments performed in triplicate. (F) KS-WNK1 2–84 did not inhibit cAMP-activated PKA activity, as detected by phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) R domain.

Figure 3. WNK3 associates with WNK4 and WNK1.

(A) WNK3 associated with WNK4, FHHt mutant WNK4 (WNK4 Q562E), and kinase-dead WNK4 (D318A) in Xenopus oocytes and HEK293t cells. (B) WNK3 and WNK4 associate within their C termini. Left: myc-WNK3 expressed with HA-tagged fragments of WNK4. Anti-myc antibody precipitated only WNK4 fragments that included the carboxyl terminal domain (WNK4 445–1,222 and 808–1,222). Right: WNK4 precipitated only WNK3 fragments that contain the carboxyl terminal domain (WNK3 421–1,743 and 1,243–1,743). (C) Identification of the WNK4 region involved in interaction with WNK3. Progressive truncation of the WNK4 carboxyl terminus identified a region between residues 1,135 and 1,175 as essential for interaction. This region encompasses the second WNK4 coiled-coil domain, as shown schematically. The coiled-coil domains of WNK1, -3, and -4 are compared. (D) WNK1, WNK3, and WNK4 formed protein complexes in Xenopus oocytes. Myc-WNK3 and WNK4 were expressed with increasing amounts of WNK1 cRNA. The WNK3/WNK4 expression ratio was 1:1. The WNK1/WNK3 and WNK1/WNK4 ratios were 0.25:1 to 2:1. Lysates were precipitated using an anti-WNK4 antibody and detected using anti-myc and anti-WNK1. Increasing expression of WNK1 did not dissociate the complex. (E) GST-WNK4 1,122–1,222, but not GST-WNK4 1–167, pulls down endogenous WNK3 from HEK293 cells. Endogenous WNK3 is present in cell lysate. Results are representative of experiments performed in triplicate. (F) Schematic comparing sites of association between WNK4 with WNK3 (these studies) and WNK4 with WNK1 (22).

Figure 4. WNK3 and WNK4 compete to regulate NCC.

(A) Increasing WNK4 445–1,222 cRNA reverses WNK3 421–1,743–mediated NCC stimulation, whereas WNK3 overcame WNK4-mediated NCC inhibition. Note that the amplitude of NCC activity, when regulated by both WNK3 and WNK4, was considerably greater than when regulated by either WNK3 or WNK4 alone. Injected amounts were: 3 ng NCC, 0–9 ng WNK3, 0–9 ng WNK4; n = 6. (B) WNK4, but not FHHt mutant WNK4 (WNK4 Q562E), attenuated stimulation of NCC activity by WNK3. KS-WNK1, full-length WNK1, and kinase-dead WNK1 had no effect on WNK3-mediated NCC stimulation. n = 4. (C) FHHt-mutant WNK4 Q562E, but not kinase-dead WNK4 D318A, inhibited wild-type WNK4 inhibition of WNK3 effects on NCC. Amounts of injected WNK4 are shown. n = 3. (D) The current results show that NCC abundance at the plasma membrane (step 1) is determined by WNK3 and WNK4 directly and by interaction between WNK3 and WNK4. FHHt-mutant WNK4 Q562E acts as a dominant-negative regulator of WNK4 actions on WNK3. WNK1 also interacts with WNK4 to regulate NCC and in turn is regulated by KS-WNK1. KS-WNK1 also inhibits WNK3 kinase activity, although it is not clear whether this affects WNK3 actions on NCC. As noted in the text, NCC is also activated by phosphorylation by unknown kinases (step 2). WNK kinases may be involved in this process. +, positive regulation; –, negative regulation.