The thiazide-sensitive Na-Cl cotransporter is regulated by a WNK kinase signaling complex (original) (raw)
WNK3 stimulates NCC activity via its carboxyterminal domain. Kinase-active WNK3 has been reported to stimulate NCC activity, whereas kinase-dead WNK3 has been reported to inhibit it (20). We confirmed these results by expressing full-length and kinase-dead WNK3 with NCC in Xenopus oocytes and measuring sodium uptake. The results (Figure 1A) suggest that the kinase activity of WNK3 is essential for its effects on NCC and raise the possibility that WNK3 might phosphorylate NCC directly. We tested whether WNK3 exhibits kinase activity in vitro. WNK3 2–420 phosphorylated itself (autophosphorylation) and the substrate protein, histone (Figure 1B). We could not, however, detect any phosphorylation of either the amino-terminal or the carboxyterminal cytoplasmic domains of NCC by WNK3 2–420 (Figure 1B).
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.
WNK1 contains an autoinhibitory domain, just C-terminal to its kinase domain (24), which inhibits its activity. Thus, the construct WNK1 1–555 is relatively kinase inactive, in contrast to the shorter construct WNK1 1–491 (21, 24). A longer construct, WNK1 1–639, containing additional amino acid residues, including a coiled-coil domain, however, does exhibit kinase activity. This indicates that the WNK1 region between 555 and 639 represses the autoinhibitory domain (24). To test whether WNK3 contains a similar domain structure, we expressed the longer construct glutathione S transferase–WNK3 (GST-WNK3) 2–586, which contains both the putative autoinhibitory domain and the adjacent and coiled-coil domain (see schematic, Figure 1F). This construct did not demonstrate any detectable kinase activity (Figure 1B), indicating that the WNK3 coiled-coil domain does not repress the autoinhibitory domain. WNK3 2–420 also phosphorylated a kinase-dead WNK1 (WNK1 1–491 D368A) and the kinase-inactive WNK3 2–586, indicating that WNK3 is capable of phosphorylating other members of the WNK family and cross-phosphorylating WNK3 (Figure 1B).
Because the actions of WNK3 on NCC appeared to be dependent functionally on WNK3 kinase activity (Figure 1A and ref. 20), we used a deletion strategy to test whether the kinase domain of WNK3 can stimulate NCC. Surprisingly, the WNK3 kinase domain (WNK3 2–420), a construct that exhibits full kinase activity in vitro (Figure 1B), had no effect on NCC activity (uptake: 102% ± 15% of NCC alone; P = NS). In contrast, the carboxyl terminus, WNK3 421–1,743, a construct devoid of kinase activity, had the same stimulatory effect on NCC as full-length WNK3 (Figure 1C). The ability of WNK3 to stimulate NCC was fully inhibited by hydrochlorothiazide (HCTZ), indicating that it results from increased NCC activity. In addition, neither WNK3 nor the amino or carboxyl terminal WNK3 constructs stimulated Na uptake in the absence of NCC, further confirming that the observed stimulation is dependent entirely on NCC activation. All of the WNK3 constructs were expressed at the protein level, indicating that differential WNK3 protein expression does not explain these results (Figure 1D).
We confirmed that the effects on NCC activity were associated with parallel changes in NCC surface abundance (20), as documented by immunofluorescence of fixed oocytes (Figure 1E). There was no detectable effect of WNK3 on total abundance (Figure 1E).
WNK3 and WNK1 phosphorylate WNK4. In view of the evidence that the effects of WNK3 on NCC are separable from the activity of WNK3 as a kinase, we were interested in seeking the molecular basis of WNK kinase interactions. Previously, we showed that WNK1 and WNK4 interact to regulate NCC activity (15, 21, 22), and Cobb and colleagues showed that WNK1 can phosphorylate WNK4 within the kinase domain (25). Here, we tested whether WNK1 could phosphorylate WNK4 outside of the kinase domain. Figure 2, A and B, shows that WNK1 is capable of phosphorylating both the carboxy- (WNK4 1,172–1,222) and amino-terminal domains (WNK4 1–167). Interestingly, WNK1 was not capable of phosphorylating claudin 4 (Figure 2A). We then tested whether WNK3 exhibits similar activity. Figure 2C shows that WNK3 2–420 phosphorylated both the amino- and carboxyl termini of WNK4 in vitro. Thus, both WNK1 and WNK3 are capable of phosphorylating WNK4 in vitro.
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.
KS-WNK1 and WNK4 inhibit WNK3 kinase activity. We reported previously that KS-WNK1, a kidney-specific kinase-deficient isoform of WNK1, exerts a dominant-negative effect on WNK1 (23). KS-WNK1 also inhibits WNK1 effects on the renal outer medullary K channel (ROMK; Kir 1.1) (26, 27), suggesting that this is a general action of KS-WNK1. We also reported that the autoinhibitory domain of WNK4 suppresses WNK1 kinase activity in vitro (21), providing evidence that WNK kinases form a regulatory complex. Here, we tested whether KS-WNK1 and WNK4 could inhibit WNK3 kinase activity, as they do for WNK1. The results (Figure 2D) showed that the unique KS-WNK1 amino-terminal domain (KS-WNK1 2–84) inhibited WNK3 kinase activity in a dose-dependent manner. We (21) and others (25) have shown previously that the WNK4 autoinhibitory domain suppresses the kinase activity of WNK1. Here, we show that the WNK3 kinase activity can also be suppressed by the WNK4 autoinhibitory domain (WNK4 445–518) (Figure 2E). A longer WNK4 that does not possess autoinhibitory activity did not inhibit WNK3 kinase activity (Figure 2E). These inhibitor effects are specific for WNK3, because KS-WNK1 2–84 does not inhibit cAMP-dependent PKA activity (Figure 2F). The results provide further evidence that WNK kinase functional domains regulate not only themselves but other members of the WNK kinase family as a signaling complex.
WNK3 associates with WNK4 in a protein complex. WNK1 has been reported to form homotetramers (25). We reported previously that WNK1 and WNK4 form protein complexes when expressed in oocytes (22), suggesting that heteromeric complexes may also occur. WNK1, WNK4, and WNK3 are all expressed by DCT cells (20, 28). Here, we tested whether WNK3 and WNK4 associate in a protein complex. Figure 3A shows that anti-myc precipitated WNK4 and that anti-WNK4 can precipitate myc-WNK3 when the proteins are expressed in Xenopus oocytes or in a mammalian cell line (HEK293t). A mutant WNK4 that causes FHHt (WNK4 Q562E) retains its ability to associate with WNK3, as does kinase-dead WNK4 D318A. Surprisingly, WNK3 and WNK4 interact via their carboxyl termini (Figure 3B), whereas WNK1 and WNK4 interact via their amino-terminal domains (22).
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).
Coiled-coil domains are protein-protein interaction motifs. We tested whether WNK4 coiled-coil domains are involved in the association with WNK3. Figure 3C shows that deleting amino acid residues between 1,136 and 1,174 (containing the second coiled-coil domain) abrogates the ability of WNK4 to interact with WNK3. These results indicate that the WNK4 amino acid residues that interact with WNK3 are within, or immediately adjacent to, the second coiled-coil domain (Figure 3C). The results of transfection experiments may not mimic protein interactions that occur in situ. We therefore tested whether GST-WNK4 could pull down endogenous WNK3 from HEK293 cells. Figure 3D shows the carboxyl terminus of WNK4 pulled down endogenous WNK3 from HEK cells; in contrast, the amino terminus did not.
WNK1 forms a protein complex with WNK4 (22). We tested whether WNK1 might compete with WNK3 for WNK4 interaction, but expressing WNK1 did not decrease the ability of WNK4 to immunoprecipitate WNK3 (Figure 3E). Figure 3F shows schematically that WNK4 interacts with both WNK1 and WNK3, but via different domains.
WNK3 and WNK4 compete to regulate NCC. In view of the fact that WNK3 and WNK4 form protein complexes, and because they exert opposite effects on NCC trafficking and activity, we tested whether WNK kinases interact functionally, with respect to NCC. We confirmed that the carboxyl terminus of WNK3 (WNK3 421–1,743) stimulates NCC activity, as shown above. We also confirmed our previous report (22) that the carboxyl terminus of WNK4 (WNK4 445–1,222) inhibits NCC activity when coexpressed in Xenopus oocytes. Interestingly, WNK4 445–1,222 inhibited WNK3’s stimulatory effect on NCC in a dose-dependent manner (Figure 4A). Similarly, WNK3 421–1,743 inhibited WNK4’s effect on NCC in a dose-dependent manner (Figure 4A). Note that the range of NCC activity is greater when WNK3 and WNK4 are expressed together than when either WNK3 or WNK4 is expressed alone.
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.
WNK4 mutations that cause FHHt exhibit some loss of their ability to inhibit NCC activity in oocytes (15–17, 29). Yet a simple loss of WNK4 action on NCC cannot explain the FHHt phenotype. Animals transgenic for FHHt-mutant WNK4 exhibit increased NCC activity, despite the presence of 2 wild-type WNK4 alleles (6); this result is not compatible with simple loss of function with respect to NCC. In view of the data indicating that WNK4 also regulates NCC activity through effects on WNK3, we tested the hypothesis that FHHt-causing WNK4 mutations affect the interaction between WNK4 and WNK3. Figure 4B shows that, unlike WNK4, KS-WNK1, WNK1, and kinase-dead WNK1 (WNK1 D368A) did not affect WNK3-mediated NCC stimulation. In contrast to wild-type WNK4, however, the FHHt-causing WNK4 Q562E completely lacked the ability to suppress WNK3 activation of NCC.
As noted, FHHt is inherited in an autosomal dominant manner; this is not compatible with a simple loss of WNK4 function. We therefore tested the hypothesis that FHHt-mutant WNK4 inhibits wild-type WNK4 actions on WNK3. Figure 4C shows that FHHt-mutant WNK4 Q562E inhibited WNK4-mediated suppression of WNK3 effects on NCC in a dose-dependent manner; these results indicate that FHHt-mutant WNK4 is a dominant-negative inhibitor of wild-type WNK4.