Mechanisms of WNK1 and WNK4 interaction in the regulation of thiazide-sensitive NaCl cotransport (original) (raw)
NCC belongs to the cation chloride cotransporter gene family (SLC12). The mouse NCC comprises 1,001 amino acid residues, including 12 putative membrane-spanning domains, a 132-residue cytoplasmic amino terminus, and a 400–amino acid cytoplasmic carboxyl terminus. WNK kinases comprise novel serine/threonine kinases that share approximately 70% amino acid identity within their kinase domains. They all appear to possess a short amino-terminal domain and a longer carboxyl terminus, which includes 2 coiled-coil domains. We previously reported that WNK4 downregulates NCC activity and that WNK1 suppresses this effect (4). These effects reflect changes in NCC trafficking to the plasma membrane. Here we examined whether the functional effects reflect protein-protein interactions and, if so, what protein domains interact to determine physiological effects of WNK kinases.
WNK4 associates with NCC and WNK1. We demonstrate that full-length WNK4 interacted with full-length NCC when expressed in HEK293T cells and confirm results of others (5) showing further that WNK4 could associate with the carboxyterminal 200 amino acid residues of NCC (Figure 1, A and B). Although both amino-terminal (NCC-[1–132]) and carboxyterminal (NCC-[801–1001]) constructs were expressed at the protein level in HEK293T cells, only the carboxyterminal NCC fragment, and not the amino-terminal fragment, was precipitated with anti-WNK4 (Figure 1B).
WNK4 associates with NCC and with WNK1. (A) HEK293 cells were transfected with full-length WNK4 with or without full-length NCC. Lysates were immunoprecipitated with anti-WNK4 or a control IgG. An immunoprecipitated product was present only when both NCC and WNK4 were included. Shown is 1 of 3 similar experiments. (B) HEK293 cells were transfected with Flag-tagged NCC-(1–132) or Flag-tagged NCC-(801–1001) and WNK4. Lysates were immunoprecipitated using anti-WNK4. Immunoblots of cell lysates, detected with anti-Flag and anti-WNK4 (bottom), indicate expression of the expected proteins. Nonspecific bands appear in all lanes (middle). WNK4 immunoprecipitated NCC-(801–1001) but not NCC-(1–132) (top). Shown is 1 of 3 similar experiments. (C) Oocytes injected with cRNA encoding full-length WNK1, HA-WNK4, or water were subjected to immunoprecipitation with anti-WNK1, anti-WNK4, or anti-HA, and then blotted with anti-HA or anti-WNK1 as indicated. WNK4 was immunoprecipitated by WNK1. WNK1 was immunoprecipitated by both anti-WNK4 and anti-HA. Shown is 1 of 3 similar experiments. (D) Immunoprecipitation of WNK4 fragments by full-length WNK1. Oocytes were injected with WNK1 and the indicated HA-tagged WNK4. The blot shows cell lysates or material immunoprecipitated with either IgG (control) or anti-WNK1. The results show that both WNK4 fragments are precipitated by WNK1. Shown is 1 of 3 similar experiments.
We showed previously (4) that WNK1 could largely suppress WNK4-mediated NCC inhibition when expressed in Xenopus oocytes. In the current experiments, we used the same experimental system to determine whether WNK1 and WNK4 could associate in a protein complex. WNK1 precipitated WNK4, and vice versa, utilizing both anti-WNK4 and anti-HA antibodies (Figure 1C).
To identify the WNK4 domains responsible for interacting with WNK1, immunoprecipitation experiments were performed using full-length WNK1 and truncated WNK4 constructs. Both WNK4-(1–444) and WNK4-(1–608) interacted with WNK1 (Figure 1D), indicating that the amino-terminal WNK4 kinase domain interacts with WNK1.
A kinase-dead WNK1 associates with, but does not inhibit, WNK4. WNK1 is a catalytically active kinase (2, 7, 8). Cobb and colleagues demonstrated that the WNK1-(D368A) mutant is catalytically inactive (2). We examined whether WNK1 catalytic activity is necessary for its effects on WNK4. Figure 2A confirms our previous observations that WNK4 inhibits NCC activity and that coexpression with WNK1 suppresses the WNK4 effect (i.e., WNK1 inhibits WNK4). In contrast, coexpression of WNK1-(D368A) with WNK4 did not restore NCC activity to baseline levels. Therefore, kinase-dead WNK1 does not suppress WNK4 inhibition of NCC. We then tested whether WNK1-(D368A) associates with WNK4 in a protein complex, as wild-type WNK1 does. Figure 2B shows that both wild-type WNK1 and WNK1-(D368A) associated with WNK4 in a protein complex when expressed in Xenopus oocytes. This suggests that kinase-dead WNK1 does not lose its inhibiting activity simply because it does not bind to WNK4. Additional experiments will be needed to determine whether WNK1 phosphorylates WNK4 directly.
WNK1 catalytic activity is required to inhibit, but not to bind, WNK4. (A) 22Na uptake by oocytes injected with NCC, WNK4, wild-type WNK1, or kinase-deficient WNK1 (WNK1-[D368A]). WNK4 inhibited NCC-mediated Na uptake; WNK1 suppressed the inhibition; WNK1-(D368A) did not suppress WNK4-mediated NCC inhibition. *P < 0.05 versus NCC alone. (B) Oocytes injected with wild-type or kinase-deficient WNK1 constructs and HA-WNK4 were lysed and precipitated with anti-WNK1. Blots of lysates show protein expression in expected lanes. Both wild-type WNK1 and kinase-deficient WNK1 (WNK1-[D368A]) immunoprecipitated WNK4.
The WNK4 carboxyl terminus inhibits NCC but does not interact with WNK1. To determine whether the WNK4 kinase domain is required to regulate NCC activity, WNK4 constructs that include the kinase domain (WNK4-[1–444] and WNK4-[168–1222]) and WNK4 constructs that do not include the kinase domain (WNK4-[445–1222]) were compared in the Na uptake assay. WNK4 constructs that include the carboxyl terminus inhibited NCC activity (Figure 3A). In contrast, the WNK4 construct that comprises only the kinase domain plus the short amino terminus, WNK4-(1–444), did not affect Na uptake. Although this indicates that the WNK4 carboxyl terminus, and not the kinase domain, is essential for NCC inhibition, the results with WNK1 coexpression were strikingly different. Whereas WNK1 suppressed the effect of WNK4-(168–1222) on NCC activity, it had no effect on WNK4-(445–1222)–mediated NCC suppression (Figure 3A). Therefore, the carboxyl terminus of WNK4 is essential for NCC inhibition, but the amino terminus of WNK4, which contains the kinase domain, is essential for WNK1 to suppress the WNK4 effect. Figure 3B shows that WNK1-(1–555) strongly immunoprecipitated WNK4-(1–444) but minimally immunoprecipitated WNK4-(445–1222). Therefore, the amino terminus of WNK1 associates in a protein complex with the amino terminus of WNK4 when expressed in oocytes.
The amino-terminal kinase domain of WNK4 is not required for NCC inhibition but is required for WNK1 interaction. (A) 22Na uptake by oocytes injected with cRNA encoding NCC and several WNK4 constructs, with or without WNK1. The WNK4 constructs are diagrammed below. Note that carboxyterminal WNK4 constructs (WNK4-[168–1222] and WNK4-[445–1222]) inhibited Na uptake compared with NCC alone, but a kinase-domain WNK4 construct (WNK4-[1–444]) did not. WNK1 blocked the effect of a WNK4 construct that contains both the kinase domain and the carboxyl terminus (WNK4-[168–1222]) but did not affect the inhibition by carboxyterminal constructs (WNK4-[445–1222]). *P < 0.05 versus NCC alone. cc, coiled coil domains. (B) Immunoprecipitation of the amino-terminal WNK4 domain by WNK1. Oocytes were injected with HA-WNK4 amino- and carboxyterminal constructs and with His-WNK1-(1–555). Only the amino-terminal WNK1 precipitated the amino-terminal WNK4 construct. Bottom blots confirm protein expression of all constructs. A faint band indicates that a much weaker association between the carboxyl terminus of WNK4 and the amino terminus of WNK1 could be detected. (C) Immunoprecipitation of WNK4 fragments by WNK1 kinase domain alone. The left 2 panels show expression of both protein constructs. The right panel shows that myc-WNK1-(218–491) precipitated only WNK4 constructs that contain the kinase domain. Shown is 1 of 3 similar experiments.
To narrow the regions of WNK kinase interaction further, additional immunoprecipitation experiments were performed. The results, shown in Figure 3C, indicate that the kinase domain of WNK1 (myc-WNK1-[218–491]) specifically immunoprecipitated the WNK4 kinase domain (for example, HA-WNK4-[1–444]) but not protein fragments that contain only the amino-terminal pre-kinase domain or the carboxyl terminus of WNK4. These results indicate that the kinase domains of WNK1 and WNK4 associate with each other.
The results shown in Figure 3A indicate that a WNK4 region distal to the kinase domain is sufficient to inhibit NCC activity. To determine whether other WNK4 domains can inhibit NCC activity and to narrow the NCC inhibitory domain further, additional WNK4 constructs were generated and expressed. WNK4-(1–608) contains the amino terminus, the kinase domain, the autoinhibitory domain, and the first coiled-coil domain. It also contains sites of amino acid mutations in patients with FHHt (1). This construct did not affect NCC-mediated Na uptake when coexpressed in oocytes with NCC (see Figure 4A). In contrast, WNK4 constructs containing only the far carboxyterminal domain, WNK4-(808–1222) and WNK4-(1000–1222), fully inhibited NCC activity. As was the case for the longer carboxyterminal WNK4 construct, however, NCC inhibition by shorter carboxyterminal WNK4 constructs was insensitive to suppression by WNK1 (Figure 4A). These results identify a WNK4 domain that comprises the carboxyterminal 222 amino acids and includes the second coiled-coil domain as sufficient for NCC inhibition. Although this short segment could inhibit NCC, it did not interact with WNK1 functionally (i.e., WNK1 did not suppress its inhibitory effect on WNK4) (Figure 4A). To determine whether WNK4 domains that inhibit NCC associate with the transport protein in a complex, additional immunoprecipitation experiments were performed. The carboxyterminal WNK4 fragments that inhibited NCC strongly associated with the transporter (Figure 4B). In contrast, an ineffective amino-terminal WNK4 fragment associated much less avidly. Figure 4C indicates that the inability of WNK1 to block NCC inhibition mediated by WNK4-(1000–1222) can not be explained on the basis of deficient WNK1 protein expression; both WNK4 constructs and WNK1 were expressed by the oocytes.
A short carboxyterminal WNK4 domain binds to and mediates NCC suppression. (A) 22Na uptake by oocytes injected with cRNA encoding NCC and several WNK4 constructs, with or without WNK1. The WNK4 constructs are shown diagrammatically. WNK4 constructs that include a short carboxyterminal domain inhibited NCC activity. Note that inhibition by the carboxyl terminus of WNK4 (WNK4-[1000–1222]) was as potent as inhibition by full-length WNK4 (compare WNK4 in Figure 2A with WNK4-[808–1222]). This inhibition was also not suppressed by WNK1. The WNK4 construct containing the kinase domain and the first coiled-coil domain (WNK4-[1–608]) did not inhibit NCC. *P < 0.05 versus NCC alone. (B) Immunoprecipitation of the NCC carboxyl terminus by WNK4. Oocytes were injected with RNA encoding HA-tagged WNK4 constructs and a Flag-tagged NCC carboxyterminal domain. Although modest interaction between the amino-terminal WNK4 domain and NCC was detected, much stronger interaction between NCC carboxyterminal domain and WNK4 carboxyterminal domain was observed. Shown is 1 of 3 similar experiments. (C) Western blots showing that WNK1 and each of the WNK4 constructs were expressed, both alone and together. The WNK1 antibody used for this blot was Y-1606.
Localization of a negative regulatory signal region of WNK4. To narrow sites of NCC inhibition further, the ability of a carboxyterminal WNK4 construct to inhibit NCC activity was compared with that of a construct missing the carboxyterminal 47 amino acids. Figure 5A shows that the truncated construct (WNK4-[445–1175]) did not inhibit NCC activity, in contrast to the construct containing the carboxyterminal 47 amino acids (WNK4-[445–1222]). This identifies these amino acids as essential for inhibition. Interestingly, this region does not include a nearby amino acid shown to be mutated in some patients with FHHt (1). To determine whether the disease-causing WNK4 mutation at this site abrogates NCC-inhibiting activity, this mutant was tested. WNK4-(445–1222)-R1164C was as effective at inhibiting NCC activity as WNK4-(445–1222). Figure 5A also compares this essential WNK4 regulatory domain from three species and with the carboxyterminal 50 amino acids of WNK1.
Localization of a negative regulatory signal region of WNK4. (A) Oocytes were injected with RNA encoding WNK4 constructs and NCC. The WNK4 constructs are diagrammed below. WNK4 missing the carboxyterminal 47 amino acids did not inhibit uptake, in contrast to constructs that contained these amino acid residues (the homologous regions of WNK4 from mouse [m], human [h], and rat [r] are shown below, with the terminal domain of rat WNK1 for comparison). A WNK4 fragment containing a human disease-causing mutation (WNK4-[R1164C]-[445–1222]) inhibited uptake as effectively as the wild type. *P < 0.05 versus NCC alone. (B) Immunoprecipitation of carboxyterminal WNK4 fragments by NCC. Oocytes were injected with cRNA encoding a Flag-tagged carboxyterminal fragment of NCC and HA-tagged carboxyterminal WNK constructs. Both carboxyterminal WNK4 constructs were immunoprecipitated by anti-Flag (NCC) antibodies.
In view of the identification of the carboxyterminal domain as essential for WNK4’s inhibiting activity, we then tested whether these WNK4 constructs interacted with NCC in a protein complex. Immunoprecipitation experiments (Figure 5B) showed that both WNK4-(445–1222) and WNK4-(445–1175) interacted with NCC in a protein complex. Control experiments in which a nonspecific IgG was employed to immunoprecipitate showed that the results were specific_._ In combination, the results of the functional studies and the immunoprecipitation studies suggest the presence of discrete NCC-binding and NCC-regulatory WNK4 domains.
Catalytically active full-length WNK1 inhibits WNK4-mediated NCC inhibition. In view of the fact that WNK1 kinase activity is essential for WNK1 to inhibit WNK4, we tested whether WNK1 fragments containing the kinase domain alone could block the WNK4 effect. Surprisingly, Figure 6A shows that only a full-length WNK1 construct inhibited WNK4 mediated suppression of NCC activity. Neither fragments comprising the kinase domain alone (WNK1-[1–491]) (7), fragments containing the first coiled-coil domain (WNK1-[1–1036]), nor fragments lacking the kinase domain (WNK1-[640–2126]) were able to block WNK4-mediated NCC suppression. Figure 6B confirms that all WNK1 constructs that did not block the WNK4 effect were expressed at the protein level.
Only full-length WNK1 can inhibit WNK4 effects. (A) 22Na uptake by oocytes injected with cRNA encoding NCC, WNK4-(168–1222), WNK4-(D318A)-(168–1222), and several WNK1 constructs, as indicated. The WNK1 constructs are diagrammed below. Note that a putative kinase-deficient WNK4 construct inhibited Na uptake as effectively as a wild-type product. Only the full-length WNK1 construct suppressed the effect of WNK4 on NCC. *P < 0.05 versus NCC alone. (B) Blot showing that all WNK1 constructs were expressed by the oocytes at the protein level. (C) Schematic representation of major results. The carboxyl terminus of WNK4 associates with the carboxyl terminus of NCC. This interaction inhibits NCC activity (–). The 47 terminal WNK4 amino acids (shaded dark gray) function as a negative regulatory signal region but are not essential for binding. The amino-terminal domain of WNK1 associates with the amino-terminal domain of WNK4. This interaction inhibits WNK4 activity in a kinase-dependent manner. The functional effect requires full-length WNK1 (dotted line). Although catalytic activity is required, it is not clear whether WNK1 phosphorylates WNK4 directly.
A putative kinase-dead WNK4 was reported to lack NCC-inhibiting activity (5). In light of the current results showing NCC inhibition by the carboxyl terminus of WNK4, we tested whether WNK4-(D318A)-(168–1222) could inhibit NCC. WNK4-(168–1222) was shown to actively suppress NCC in Figure 3A. The results (Figure 6A) show that WNK4-(D318A)-(168–1222) inhibited NCC as effectively as did wild-type WNK4.