WNK kinases comprise a recently identified group of serine and threonine kinases in which the location of the lysine required for ATP binding is unique (2
). Mutations in the genes encoding 2 WNKs, WNK1 and WNK4, cause FHHt in humans, indicating that these kinases play essential roles in regulating renal electrolyte homeostasis (1
). WNK kinases are expressed in multicellular organisms, including plants, Caenorhabditis elegans
, and Drosophila
, but not in the unicellular Saccharomyces cerevisiae
). Mammalian WNK kinases are expressed by epithelial tissues including the distal nephron of mammalian kidney, where they are believed to regulate solute movement (1
). Although the substrates and physiological functions of WNK kinases remain subjects of ongoing investigation, progress in understanding the role they play in regulating epithelial solute transport has been made. When expressed using heterologous expression systems, WNK4 inhibits NCC function by reducing NCC abundance at the plasma membrane (4
). WNK4 also reduces the membrane abundance of the inwardly rectifying K channel (ROMK) (16
) and other transport proteins (17
), which suggests a general role for WNK4 in regulating epithelial solute transport. The effect of WNK4 to reduce NCC activity has been reported to depend on its kinase activity (5
), whereas the effect of WNK4 to reduce the membrane abundance of ROMK (16
). Recently, WNK4 has been shown to regulate paracellular ion permeability, perhaps by interacting with claudins (18
WNK1 and WNK4 are both expressed by distal convoluted tubule and connecting tubule cells in the kidney (1
). The clinical phenotypes of FHHt resulting from mutations of WNK1 and WNK4 are similar (1
). Together, these findings suggest that WNK1 and WNK4 converge on a common pathway in the kidney that regulates blood pressure and electrolyte homeostasis. We showed previously that WNK1, though unable by itself to inhibit NCC activity, suppresses the effects of WNK4 on NCC (4
). This indicates that WNK kinases interact and suggests a potential mechanism underlying the final common pathway effects of these distinct kinases. The molecular mechanisms by which WNK kinases regulate ion transport proteins are unknown, although it has been reported that WNK4 regulates ROMK through an effect on clathrin-coated vesicle–mediated endocytosis (16
). The first goal of the present experiments, therefore, was to investigate mechanisms by which WNK4 regulates NCC activity. The current results show that the carboxyterminal 200 amino acids of NCC associate in a protein complex with WNK4; in contrast, the amino-terminal cytoplasmic domain of NCC does not interact with WNK4. These results extend those of Wilson and colleagues, who found that WNK4 associates with the carboxyterminal 416 amino acid residues of NCC (5
Our results also indicate that the carboxyterminal 222 amino acids of WNK4 are sufficient to inhibit NCC activity. They further indicate that the carboxyterminal 47 amino acid residues of WNK4 are required to inhibit NCC activity, even though they are not essential for NCC binding. This suggests that the 47 carboxyterminal amino acid residues of WNK4 comprise an essential region for negative regulatory signaling. They also suggest that the association of WNK4 and NCC in a protein complex plays a role in regulating NCC membrane abundance; WNK4 constructs that inhibit NCC activity (such as WNK4-[808–1222]) (Figure B) bind to the transport protein avidly. These experiments emphasize that physical association may be necessary for functional effects but is not sufficient. Although Wilson and colleagues (5
) reported that a putative kinase-dead WNK4 (WNK4-[D318A]) does not inhibit NCC activity, the present results indicate that WNK4 kinase activity is not required to suppress NCC. To confirm this, a highly active WNK4 construct was mutated at amino acid 318. WNK4-(D318A)-(168–1222) inhibited NCC activity as efficiently as its wild-type homolog. Because a WNK4 fragment that does not include the kinase domain can fully inhibit NCC activity, it appears that WNK4 regulates NCC primarily via protein-protein interactions rather than by phosphorylation. Interestingly, introducing a mutation that causes FHHt into a truncated WNK4, WNK4-(R1164C)-(445–1222), did not affect its ability to inhibit NCC. This raises the possibility that interactions between WNK4, NCC, and another unidentified protein affect ion transport and emphasizes the potentially complicated WNK interactions that may underlie the human disease.
The carboxyl terminus of WNK4 was shown to associate in a protein complex with ROMK and be required to inhibit its membrane abundance (16
). Because those experiments used a longer WNK4 construct that includes both coiled-coil domains, it is not clear whether the same WNK4 region interacts with both ROMK and NCC. Yet the observation that WNK4 mutations have disparate effects on NCC (4
) and ROMK (16
), together with the evidence that clathrin-coated pit mechanisms regulate ROMK (16
) but not NCC (5
), suggest that the effects of WNK4 on transport proteins may involve distinct, yet physically contiguous, domains.
The present results confirm our previous observation that WNK1 suppresses WNK4-mediated NCC inhibition and indicate that WNK1 and WNK4 can associate in a protein complex. They indicate that the interaction between WNK4 and WNK1 involves the amino-terminal (kinase) domains of each protein, regions that are highly homologous (1
). The current results also indicate that this interaction between WNK1 and WNK4 is essential for WNK1 suppression of WNK4-mediated NCC inhibition. Interestingly, the amino terminus of WNK4 is required to interact with WNK1, even though the carboxyl terminus of WNK4 can inhibit NCC fully when expressed alone. These conclusions are illustrated schematically in Figure C. The current results also indicate that WNK1-(D368A) is devoid of WNK4-inhibitory activity. This mutant was shown to lack kinase activity by Xu and colleagues (2
), suggesting that both physical association and catalytic activity are required for WNK1 to inhibit WNK4 (as shown in Figure C). Partial-length WNK1 constructs, either amino-terminal or carboxyterminal, do not suppress WNK4 effects on NCC.
Our data reveal different patterns of regulation between WNK1 and WNK4. With regard to WNK4, deleting the kinase domain preserves the full inhibitory effect of the protein. Thus, WNK4-mediated NCC inhibition appears to be strongly dependent on protein-protein interactions. In contrast, the WNK1 effect on WNK4 does not occur if the carboxyl terminus of WNK1 is expressed alone, or if a kinase-dead mutant is employed. Surprisingly, despite the requirement for kinase activity, WNK1 constructs that contain the kinase domain alone (WNK1-[1–491]) and WNK1 constructs that contain the first coiled-coil domain (WNK1-[1–1036]) do not suppress WNK4-mediated NCC inhibition. This is despite the fact that this fragment has been demonstrated to possess full kinase activity in vitro (7
). One hypothesis to explain these results is that the WNK1 action on WNK4 may require tetramer formation (8
). Deletion of the WNK1 carboxyl terminus would be expected to interfere with such multimer formation.
The observation that a kinase-dead WNK1 does not suppress WNK4-inhibiting activity may be relevant to the pathogenesis of FHHt. WNK1 is subject to complex regulatory alternative splicing, generating kinase-active and kinase-deficient WNK1 isoforms (9
). In the kidney, the predominant isoform appears to contain exon 4a and exon 12 and to lack exons 1–4 and exon 11; it is, therefore, kinase deficient (9
). This WNK1 isoform is generated by a separate promoter and by alternative splicing. There are also other renal WNK1 products. These include WNK1 proteins that lack exon 12, as well as kinase-active full-length WNK1 isoforms (9
). Although the effects of the kinase-deficient kidney-specific WNK1 isoform (KS-WNK1) have not been tested, it would be anticipated that this kinase-deficient kidney-specific WNK1 splice isoform would not inhibit WNK4, because WNK1 kinase activity is required (as shown in Figures and ). Under normal conditions, if the kinase-sufficient, full-length WNK1 is expressed at low levels along the distal nephron, then the ability of WNK4 to inhibit NCC activity will be manifest. If FHHt-causing WNK1 intronic mutations (1
) increase expression of a kinase-sufficient WNK1 isoform along the distal nephron, this would suppress the constitutive effects of WNK4 on NCC, thereby increasing renal Na and Cl reabsorption and inhibiting K secretion. Although WNK4 has been shown to regulate ROMK (16
), the effects of WNK1 isoforms on this transport protein have not been reported.
These data provide further support for the hypothesis that WNK1 and WNK4 converge, at least in part, on a common pathway in the distal nephron, where they regulate solute transport. As discussed above, WNK1 deficiency reduces blood pressure (3
). The current results indicate that the ability to suppress WNK4 activity is generated by the catalytically active kinase domain of WNK1, but that the intact WNK1 protein is required for such an effect to occur. These observations raise the possibility that novel antihypertensive drugs may be developed that enhance WNK4-mediated effects on renal NaCl transport and on blood pressure.