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Mechanisms of WNK1 and WNK4 Interaction in the Regulation of Thiazide-Sensitive Nacl Cotransport

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Mechanisms of WNK1 and WNK4 Interaction in the Regulation of Thiazide-Sensitive Nacl Cotransport Mechanisms of WNK1 and WNK4 interaction in the regulation of thiazide-sensitive NaCl cotransport Chao-Ling Yang, … , Arohan R. Subramanya, David H. Ellison J Clin Invest. 2005;115(5):1379-1387. https://doi.org/10.1172/JCI22452. Article Cardiology With-no-lysine (WNK) kinases are highly expressed along the mammalian distal nephron. Mutations in either WNK1 or WNK4 cause familial hyperkalemic hypertension (FHHt), suggesting that the protein products converge on a final common pathway. We showed previously that WNK4 downregulates thiazide-sensitive NaCl cotransporter (NCC) activity, an effect suppressed by WNK1. Here we investigated the mechanisms by which WNK1 and WNK4 interact to regulate ion transport. We report that WNK1 suppresses the WNK4 effect on NCC activity and associates with WNK4 in a protein complex involving the kinase domains. Although a kinase-dead WNK1 also associates with WNK4, it fails to suppress WNK4-mediated NCC inhibition; the WNK1 kinase domain alone, however, is not sufficient to block the WNK4 effect. The carboxyterminal 222 amino acids of WNK4 are sufficient to inhibit NCC, but this fragment is not blocked by WNK1. Instead, WNK1 inhibition requires an intact WNK4 kinase domain, the region that binds to WNK1. In summary, these data show that: (a) the WNK4 carboxyl terminus mediates NCC suppression, (b) the WNK1 kinase domain interacts with the WNK4 kinase domain, and (c) WNK1 inhibition of WNK4 is dependent on WNK1 catalytic activity and an intact WNK1 protein. These findings provide insight into the complex interrelationships between WNK1 and WNK4 and provide a molecular basis for FHHt. Find the latest version: https://jci.me/22452/pdf Research article Mechanisms of WNK1 and WNK4 interaction in the regulation of thiazide-sensitive NaCl cotransport Chao-Ling Yang,1,2 Xiaoman Zhu,1,2 Zhaohong Wang,1 Arohan R. Subramanya,1 and David H. Ellison1,2,3 1Division of Nephrology and Hypertension, Oregon Health & Science University, Portland, Oregon, USA. 2Portland Veterans Affairs Medical Center, Portland, Oregon, USA. 3Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, Oregon, USA. With-no-lysine (WNK) kinases are highly expressed along the mammalian distal nephron. Mutations in either WNK1 or WNK4 cause familial hyperkalemic hypertension (FHHt), suggesting that the protein prod- ucts converge on a final common pathway. We showed previously that WNK4 downregulates thiazide-sen- sitive NaCl cotransporter (NCC) activity, an effect suppressed by WNK1. Here we investigated the mecha- nisms by which WNK1 and WNK4 interact to regulate ion transport. We report that WNK1 suppresses the WNK4 effect on NCC activity and associates with WNK4 in a protein complex involving the kinase domains. Although a kinase-dead WNK1 also associates with WNK4, it fails to suppress WNK4-mediated NCC inhibi- tion; the WNK1 kinase domain alone, however, is not sufficient to block the WNK4 effect. The carboxyter- minal 222 amino acids of WNK4 are sufficient to inhibit NCC, but this fragment is not blocked by WNK1. Instead, WNK1 inhibition requires an intact WNK4 kinase domain, the region that binds to WNK1. In sum- mary, these data show that: (a) the WNK4 carboxyl terminus mediates NCC suppression, (b) the WNK1 kinase domain interacts with the WNK4 kinase domain, and (c) WNK1 inhibition of WNK4 is dependent on WNK1 catalytic activity and an intact WNK1 protein. These findings provide insight into the complex inter- relationships between WNK1 and WNK4 and provide a molecular basis for FHHt. Introduction In contrast, WNK1 did not affect NCC activity directly, but instead Familial hyperkalemic hypertension (FHHt; also known as pseu- strongly suppressed the WNK4 effect on NCC (4); in other words, dohypoaldosteronism type II; Online Mendelian Inheritance in WNK1 coexpression with WNK4 returned NCC activity close to base- Man reference number #145260) is an autosomal dominant disease line levels. The mechanisms by which WNK kinases interact were not characterized by hypertension, hyperkalemia, and sensitivity to thia- elucidated by these experiments. Both kinase-dependent and kinase- zide diuretics. Wilson and colleagues (1) reported that mutations independent effects may be involved, as some (2, 6–8), but not all in 2 genes encoding homologous proteins, WNK1 (PRKWNK1) and (7, 9–11), WNK kinases possess kinase activity. Furthermore, all WNK WNK4 (PRKWNK4), can cause FHHt. The with-no-lysine (WNK) kinases contain coiled-coil domains that may participate in protein- kinases are novel serine/threonine kinases that were named because protein interactions. The current experiments investigated mecha- they lack lysine at a location previously thought to be essential for nisms by which WNK1 and WNK4 interact to regulate NCC activity. kinase activity (2). Both WNK1 and WNK4 are highly expressed in The results demonstrate that the carboxyl terminus of WNK4 is suf- the kidney. The FHHt-causing WNK1 mutations are deletions with- ficient to inhibit NCC but is not blocked by WNK1. WNK1-mediated in the first intron. These mutations increase WNK1 expression in WNK4 suppression is dependent on both binding and catalytic activ- leukocytes and were postulated to be gain-of-function mutations (1). ity. These results provide insight into mechanisms by which WNK Recently, heterozygous WNK1-deficient mice were shown to exhibit kinases converge on a common ion transport pathway in the distal lower blood pressure than wild-type controls (3), supporting the nephron and indicate that there are novel protein-protein interac- hypothesis that WNK1 exerts a positive effect on blood pressure. tions between different members of this kinase family. They also sug- The WNK4 mutations that cause FHHt are discrete missense muta- gest the potential development of novel antihypertensive agents. tions in 2 areas of the coding region (1). These mutations cause phenotypic features similar to those that result from WNK1 intron Results mutations (1), but the mechanisms involved may be different. NCC belongs to the cation chloride cotransporter gene family We showed previously that WNK4 inhibits the thiazide-sensitive (SLC12). The mouse NCC comprises 1,001 amino acid residues, NaCl cotransporter (NCC, gene symbol SLC12A3) when expressed including 12 putative membrane-spanning domains, a 132-residue in Xenopus oocytes (4); similar results were obtained by others (5). cytoplasmic amino terminus, and a 400–amino acid cytoplasmic carboxyl terminus. WNK kinases comprise novel serine/threo- nine kinases that share approximately 70% amino acid identity Nonstandard abbreviations used: FHHt, familial hyperkalemic hypertension; NCC, within their kinase domains. They all appear to possess a short thiazide-sensitive NaCl cotransporter; ROMK, inwardly rectifying K channel; WNK, with no lysine. amino-terminal domain and a longer carboxyl terminus, which Conflict of interest: The authors have declared that no conflict of interest exists. includes 2 coiled-coil domains. We previously reported that WNK4 Citation for this article: J. Clin. Invest. 115:1379–1387 (2005). downregulates NCC activity and that WNK1 suppresses this effect doi:10.1172/JCI200522452. (4). These effects reflect changes in NCC trafficking to the plasma The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 5 May 2005 1379 research article Figure 1 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. membrane. Here we examined whether the functional effects reflect A kinase-dead WNK1 associates with, but does not inhibit, WNK4. WNK1 protein-protein interactions and, if so, what protein domains inter- is a catalytically active kinase (2, 7, 8). Cobb and colleagues demon- act to determine physiological effects of WNK kinases. strated that the WNK1-(D368A) mutant is catalytically inactive (2). WNK4 associates with NCC and WNK1. We demonstrate that full- We examined whether WNK1 catalytic activity is necessary for its length WNK4 interacted with full-length NCC when expressed in effects on WNK4. Figure 2A confirms our previous observations HEK293T cells and confirm results of others (5) showing further that WNK4 inhibits NCC activity and that coexpression with WNK1 that WNK4 could associate with the carboxyterminal 200 amino suppresses the WNK4 effect (i.e., WNK1 inhibits WNK4). In con- acid residues of NCC (Figure 1, A and B). Although both amino- trast, coexpression of WNK1-(D368A) with WNK4 did not restore terminal (NCC-[1–132]) and carboxyterminal (NCC-[801–1001]) NCC activity to baseline levels. Therefore, kinase-dead WNK1 does constructs were expressed at the protein level in HEK293T cells, not suppress WNK4 inhibition of NCC. We then tested whether only the carboxyterminal NCC fragment, and not the amino-ter- WNK1-(D368A) associates with WNK4 in a protein complex, as minal fragment, was precipitated with anti-WNK4 (Figure 1B).
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