Comparison of WNK4 and WNK1 kinase and inhibiting activities

WNK kinases are novel serine/threonine protein kinases. Mutations in two members of the WNK family, WNK1 and WNK4, cause familial hyperkalemic hypertension. These kinases regulate ion transport across diverse epithelia; WNK4 reduces activity of the Na-Cl cotransporter activity and the potassium channel, ROMK, by reducing their appearance at the plasma membrane. We examined the kinase activity of WNK1 and WNK4 in vitro. A glutathione S-transferase (GST) fusion protein of the WNK1 kinse domain phosphorylated itself and a substrate protein, as reported previously. A longer construct, containing the autoinhibitory domain, did not. A GST WNK4 kinase domain construct demonstrated no kinase activity, in vitro or in HEK 293 cells. WNK4 constructs that included a region homologous to the autoinhibitory domain of WNK1 inhibited WNK1 kinase activity. Inhibition by a short WNK4 segment, WNK4 (444-518), was greater than inhibition by WNK4 (444-563). Together, these results suggest that WNK4 must be activated by currently unknown factors to exhibit kinase activity and that WNK4 contains an inhibitory domain that can inhibit the kinase activity of WNK1.     

WNK1 and WNK4 modulate CFTR activity

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-gated chloride channel. WNK kinases are widely expressed modulators of ion transport. WNK1 and WNK4, two WNK kinases that are mutated in familial hyperkalemic hypertension (FHHt), are co-expressed with CFTR in several organs, raising the possibility that WNK kinases might alter CFTR activity in vivo or that CFTR could be involved in the pathogenesis of FHHt. Here, we report that WNK1 co-localizes with CFTR protein in pulmonary epithelial cells. Co-expression of WNK1 or WNK4 with CFTR in Xenopus laevis oocytes suppresses chloride channel activity. The effect of WNK4 is dose dependent and occurs, at least in part, by reducing CFTR protein abundance at the plasma membrane. This effect is independent of WNK4 kinase activity. In contrast, the effect of WNK1 on CFTR activity requires intact WNK1 kinase activity. Moreover WNK1 and WNK4 exhibit additive CFTR inhibition. Previous reports suggest that patients with FHHt exhibit mild changes in nasal potential difference that resemble the more severe changes that occur in cystic fibrosis. We report that the FHHt-causing mutant WNK4 Q562E is a more potent inhibitor of CFTR activity than is the wild-type WNK4. Taken together, these results suggest that WNK1 and WNK4 may modulate CFTR activity; they further suggest that WNK kinases may be potential therapeutic targets for cystic fibrosis.     

Properties of WNK1 and implications for other family members

WNKs are large serine/threonine protein kinases structurally distinct from all other members of the protein kinase superfamily. Of the four human WNK family members, WNK1 and WNK4 have been linked to a hereditary form of hypertension, pseudohypoaldosteronism type II. We characterized the biochemical properties and regulation of WNK1 that may contribute to its physiological activities and abnormal function in disease. We showed that WNK1 is activated by hypertonic stress in kidney epithelial cells and in breast and colon cancer cell lines. In addition, hypotonic stress also led to a modest increase in WNK1 activity. Gel filtration suggested that WNK1 exists as a tetramer, and yeast two-hybrid data showed that the N terminus of WNK1 (residues 1-222) interacts with residues 481-660, which includes the WNK1 autoinhibitory domain and a C-terminal coiled-coil domain. Although cell biological studies have suggested a functional interaction between WNK1 and WNK4, we found no evidence of stable interactions between these kinases. However, WNK1 phosphorylated both WNK4 and WNK2. In addition, the WNK1 autoinhibitory domain inhibited the catalytic activity of these WNKs. These findings suggest potential mechanisms for interconnected regulation of WNK family members.     

Serum and Glucocorticoid-induced Kinase (SGK) 1 and the Epithelial Sodium Channel Are Regulated by Multiple with No Lysine (WNK) Family Members

The four WNK (with no lysine (K)) protein kinases affect ion balance and contain an unusual protein kinase domain due to the unique placement of the active site lysine. Mutations in two WNKs cause a heritable form of ion imbalance culminating in hypertension. WNK1 activates the serum- and glucocorticoid-induced protein kinase SGK1; the mechanism is noncatalytic. SGK1 increases membrane expression of the epithelial sodium channel (ENaC) and sodium reabsorption via phosphorylation and sequestering of the E3 ubiquitin ligase neural precursor cell expressed, developmentally down-regulated 4-2 (Nedd4-2), which otherwise promotes ENaC endocytosis. Questions remain about the intrinsic abilities of WNK family members to regulate this pathway. We find that expression of the N termini of all four WNKs results in modest to strong activation of SGK1. In reconstitution experiments in the same cell line all four WNKs also increase sodium current blocked by the ENaC inhibitor amiloride. The N termini of the WNKs also have the capacity to interact with SGK1. More detailed analysis of activation by WNK4 suggests mechanisms in common with WNK1. Further evidence for the importance of WNK1 in this process comes from the ability of Nedd4-2 to bind to WNK1 and the finding that endogenous SGK1 has reduced activity if WNK1 is knocked down by small interfering RNA.     

WNK1 regulates phosphorylation of cation-chloride-coupled cotransporters via the STE20-related kinases, SPAK and OSR1

The WNK1 and WNK4 genes have been found to be mutated in some patients with hyperkalemia and hypertension caused by pseudohypoaldosteronism type II. The clue to the pathophysiology of pseudohypoaldosteronism type II was its striking therapeutic response to thiazide diuretics, which are known to block the sodium chloride cotransporter (NCC). Although this suggests a role for WNK1 in hypertension, the precise molecular mechanisms are largely unknown. Here we have shown that WNK1 phosphorylates and regulates the STE20-related kinases, Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress response 1 (OSR1). WNK1 was observed to phosphorylate the evolutionary conserved serine residue located outside the kinase domains of SPAK and OSR1, and mutation of the OSR1 serine residue caused enhanced OSR1 kinase activity. In addition, hypotonic stress was shown to activate SPAK and OSR1 and induce phosphorylation of the conserved OSR1 serine residue, suggesting that WNK1 may be an activator of the SPAK and OSR1 kinases. Moreover, SPAK and OSR1 were found to directly phosphorylate the N-terminal regulatory regions of cation-chloride-coupled cotransporters including NKCC1, NKCC2, and NCC. Phosphorylation of NCC was induced by hypotonic stress in cells. These results suggested that WNK1 and SPAK/OSR1 mediate the hypotonic stress signaling pathway to the transporters and may provide insights into the mechanisms by which WNK1 regulates ion balance.     

Interactions with WNK (With No Lysine) Family Members Regulate Oxidative Stress Response 1 and Ion Co-transporter Activity

Two of the four WNK (with no lysine (K)) protein kinases are associated with a heritable form of ion imbalance culminating in hypertension. WNK1 affects ion transport in part through activation of the closely related Ste20 family protein kinases oxidative stress-responsive 1 (OSR1) and STE20/SPS1-related proline-, alanine-rich kinase (SPAK). Once activated by WNK1, OSR1 and SPAK phosphorylate and stimulate the sodium, potassium, two chloride co-transporters, NKCC1 and NKCC2, and also affect other related ion co-transporters. We find that WNK1 and OSR1 co-localize on cytoplasmic puncta in HeLa and other cell types. We show that the C-terminal region of WNK1 including a coiled coil is sufficient to localize the fragment in a manner similar to the full-length protein, but some other fragments lacking this region are mislocalized. Photobleaching experiments indicate that both hypertonic and hypotonic conditions reduce the mobility of GFP-WNK1 in cells. The four WNK family members can phosphorylate the activation loop of OSR1 to increase its activity with similar kinetic constants. C-terminal fragments of WNK1 that contain three RFXV interaction motifs can bind OSR1, block activation of OSR1 by sorbitol, and prevent the OSR1-induced enhancement of ion co-transporter activity in cells, further supporting the conclusion that association with WNK1 is required for OSR1 activation and function at least in some contexts. C-terminal WNK1 fragments can be phosphorylated by OSR1, suggesting that OSR1 catalyzes feedback phosphorylation of WNK1.     

Distinct pathways for the involvement of WNK4 in the signaling of hypertonicity and EGF

WNK4 kinase mutations produce the autosomal dominant disorder familial hyperkalemia and hypertension (FHH), also known as pseudohypoaldosteronism type II, by a molecular mechanism that is not completely understood. In vitro experiments in frog oocytes showed that WNK4 affects ion transport systems such as the Na-Cl cotransporter and the renal outer medullary potassium channel. Some features of FHH suggest that long-term effects are involved in WNK4 signaling. In addition, WNK1 and WNK2, paralogs of WNK4, were shown to be involved in MAP kinase signaling. We therefore investigated possible WNK4 involvement in MAP kinase signaling. We stimulated HEK 293 cells overexpressing WNK4 by hypertonicity or using EGF, and measured phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and p38. WNK4 augmented the phosphorylation of ERK1/2 and p38 in response to both hypertonicity and EGF. The FHH-producing and kinase-deficient mutants behaved similarly to wild-type WNK4. Hypertonicity stimulation was accompanied by cellular relocalization of WNK4 as manifested by its reversible disappearance from the supernatant fraction following extraction with a detergent-containing buffer. Live-cell microscopy showed that the cytoplasmic-soluble WNK4 redistributes rapidly to membrane-bound organelles, which, in the case of WNK1 kinase, were recently shown to represent trans-Golgi network/recycling endosomes. In contrast, EGF stimulation was not accompanied by redistribution of WNK4 as determined by cell fractionation or cell microscopy. The observation that WNK4-induced MAP kinase stimulation caused by hypertonicity, but not that caused by EGF, is associated with WNK4 subcellular redistribution suggests that this redistribution has a role in WNK4 signaling.     

WNK1 and WNK4, new players in salt and water homeostasis

Arterial hypertension is a complex trait influenced by a variety of environmental and genetic factors. Several approaches can be used to identify its susceptibility genes : one is to study rare monogenic forms of hypertension, like familial hyperkalemic hypertension (FHH). Also known as pseudohypoaldosteronism type 2 or Gordon syndrome, FHH is characterized by hypertension, hyperkalemia despite normal renal glomerular filtration rate, abnormalities which are particularly sensitive to thiazide diuretics. Mild hyperchloremia, metabolic acidosis, and suppressed plasma renin activity are associated findings. Despite its phenotypic and genetic heterogeneity, mutations in two related genes, WNK1 and WNK4, were recently identified. These genes belong to a newly identified family of serine-threonine (with no lysine [K]) kinases. Both are highly expressed in the kidney and in a variety of epithelia involved in chloride transport. It has thus been postulated that these two kinases could be implicated in a new pathway of ionic transport regulation. Several studies have very recently confirmed this hypothesis in vitro, in Xenopus oocytes or kidney cell lines. They have shown that, in the renal distal tubule, WNK4 inhibits sodium reabsorption and potassium secretion, via inhibition of NCC (thiazide-sensitive Na+-Cl- cotransporter) and K+ channel ROMK activity, respectively. Interestingly, FHH mutations have opposite effects : while they lead to loss of NCC inhibition, they increase ROMK inhibition. Moreover, they also increase paracellular permeability to chloride of MDCK cells. WNK4 also inhibits apical and basal chloride transporters present in extra-renal epithelia, such as CFEX and Na+-K+-2 Cl-, respectively. It is also interesting to note that the WNK4-mediated negative regulation of NCC activity is in turn inhibited by WNK1. By its role on several transporters, WNK4 appears as a putative key regulator of ionic transport and blood pressure.     

WNK4 kinase inhibits Maxi K channel activity by a kinase-dependent mechanism

WNK [with no lysine (k)] kinase is a serine/threonine kinase subfamily. Mutations in two of the WNK kinases result in pseudohypoaldosteronism type II (PHA II) characterized by hypertension, hyperkalemia, and metabolic acidosis. Recent studies showed that both WNK1 and WNK4 inhibit ROMK activity. However, little is known about the effect of WNK kinases on Maxi K, a large-conductance Ca(2+) and voltage-activated potassium (K) channel. Here, we report that WNK4 wild-type (WT) significantly inhibits Maxi K channel activity in HEK αBK stable cell lines compared with the control group. However, a WNK4 dead-kinase mutant, D321A, has no inhibitory effect on Maxi K activity. We further found that WNK4 inhibits total and cell surface protein expression of Maxi K equally compared with control groups. A dominant-negative dynamin mutant, K44A, did not alter the WNK4-mediated inhibitory effect on Maxi K surface expression. Treatment with bafilomycin A1 (a proton pump inhibitor) and leupeptin (a lysosomal inhibitor) reversed WNK4 WT-mediated inhibition of Maxi K total protein expression. These findings suggest that WNK4 WT inhibits Maxi K activity by reducing Maxi K protein at the membrane, but that the inhibition is not due to an increase in clathrin-mediated endocytosis of Maxi K, but likely due to enhancing its lysosomal degradation. Also, WNK4's inhibitory effect on Maxi K activity is dependent on its kinase activity.     

WNK1 phosphorylates synaptotagmin 2 and modulates its membrane binding

WNK (with no lysine [K]) protein kinases were named for their unique active site organization. Mutations in WNK1 and WNK4 cause a familial form of hypertension by undefined mechanisms. Here, we report that WNK1 selectively binds to and phosphorylates synaptotagmin 2 (Syt2) within its calcium binding C2 domains. Endogenous WNK1 and Syt2 coimmunoprecipitate and colocalize on a subset of secretory granules in INS-1 cells. Phosphorylation by WNK1 increases the amount of Ca2+ required for Syt2 binding to phospholipid vesicles; mutation of threonine 202, a WNK1 phosphorylation site, partially prevents this change. These findings suggest that phosphorylation of Syts by WNK1 can regulate Ca2+ sensing and the subsequent Ca2+-dependent interactions mediated by Syt C2 domains. These findings provide a biochemical mechanism that could lead to the retention or insertion of proteins in the plasma membrane. Interruption of this regulatory pathway may disturb membrane events that regulate ion balance.     

WNK4 phosphorylates ser(206) of claudin-7 and promotes paracellular Cl(-) permeability

Mutations in WNK4 have been linked to hypertension in PHAII. Paracellular ion transport has been reported to be involved in this disease process; however, the specific molecular target has not been identified. In this study, we found that TJ protein claudin-7 and WNK4 were partially co-localized in renal tubules of rat kidney and co-immunoprecipitated in kidney epithelial cells. The wild-type and PHAII-causing mutant, but not the kinase-dead mutant, phosphorylated claudin-7. We have identified ser(206) in the COOH-terminus of claudin-7 as a putative phosphorylation site for WNK4. More importantly, disease-causing mutant enhanced claudin-7 phosphorylation and significantly increased paracellular permeability to Cl(-).     

WNK kinases and the kidney

In the kidney, the renal tubule plays a major role in maintaining fluid and electrolyte balance. This balance is achieved by an interplay between various hormones and nerves that signal changes throughout the body and transfer these signals to transport proteins. Increased or reduced activity of these transporters helps to restore homeostasis, but can also contribute to disease (e.g. sodium retention in hypertension). In recent years, it has become clear that the signal transfer to transporters is largely mediated by kinases. Among these, WNK kinases (With No lysine=K) stand out, because they regulate the major sodium and potassium transporters in the distal nephron. Moreover, mutations in genes encoding WNK kinases result in an inherited form of salt-sensitive hypertension with hyperkalemia, illustrating their important role in sodium, potassium, and blood pressure regulation. More recently, WNK kinases were found to play a role in acquired forms of hypertension as well. Together, the evolving insight in the kinase regulation of ion transport is providing new insights in the longstanding question how salt and blood pressure are related. Here, we review the current models of how WNK kinases regulate the various transport proteins and which roles they play in health and disease.     

SPAK/OSR1 regulate NKCC1 and WNK activity: analysis of WNK isoform interactions and activation by T-loop trans-autophosphorylation

Mutations in the WNK [with no lysine (K) kinase] family instigate hypertension and pain perception disorders. Of the four WNK isoforms, much of the focus has been on WNK1, which is activated in response to osmotic stress by phosphorylation of its T-loop residue (Ser382). WNK isoforms phosphorylate and activate the related SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1) protein kinases. In the present study, we first describe the generation of double-knockin ES (embryonic stem) cells, where SPAK and OSR1 cannot be activated by WNK1. We establish that NKCC1 (Na+/K+/2Cl- co-transporter 1), a proposed target of the WNK pathway, is not phosphorylated or activated in a knockin that is deficient in SPAK/OSR1 activity. We also observe that activity of WNK1 and WNK3 are markedly elevated in the knockin cells, demonstrating that SPAK/OSR1 significantly influences WNK activity. Phosphorylation of another regulatory serine residue, Ser1261, in WNK1 is unaffected in knockin cells, indicating that this is not phosphorylated by SPAK/OSR1. We show that WNK isoforms interact via a C-terminal CCD (coiled-coil domain) and identify point mutations of conserved residues within this domain that ablate the ability of WNK isoforms to interact. Employing these mutants, we demonstrate that interaction of WNK isoforms is not essential for their T-loop phosphorylation and activation, at least for overexpressed WNK isoforms. Moreover, we finally establish that full-length WNK1, WNK2 and WNK3, but not WNK4, are capable of directly phosphorylating Ser382 of WNK1 in vitro. This supports the notion that T-loop phosphorylation of WNK isoforms is controlled by trans-autophosphorylation. These results provide novel insights into the WNK signal transduction pathway and provide genetic evidence confirming the essential role that SPAK/OSR1 play in controlling NKCC1 function. They also reveal a role in which the downstream SPAK/OSR1 enzymes markedly influence the activity of the upstream WNK activators. The knockin ES cells lacking SPAK/OSR1 activity will be useful in validating new targets of the WNK signalling pathway.     

Regulation of WNK1 by an autoinhibitory domain and autophosphorylation

WNK family protein kinases are large enzymes that contain the catalytic lysine in a unique position compared with all other protein kinases. These enzymes have been linked to a genetically defined form of hypertension. In this study we introduced mutations to test hypotheses about the position of the catalytic lysine, and we examined mechanisms involved in the regulation of WNK1 activity. Through the analysis of enzyme fragments and sequence alignments, we have identified an autoinhibitory domain of WNK1. This isolated domain, conserved in all four WNKs, suppressed the activity of the WNK1 kinase domain. Mutation of two key residues in this autoinhibitory domain attenuated its ability to inhibit WNK kinase activity. Consistent with these results, the same mutations in a WNK1 fragment that contain the autoinhibitory domain increased its kinase activity. We also found that WNK1 expressed in bacteria is autophosphorylated; autophosphorylation on serine 382 in the activation loop is required for its activity.     

WNK4 inhibits NCC protein expression through MAPK ERK1/2 signaling pathway

WNK [with no lysine (K)] kinase is a subfamily of serine/threonine kinases. Mutations in two members of this family (WNK1 and WNK4) cause pseudohypoaldosteronism type II featuring hypertension, hyperkalemia, and metabolic acidosis. WNK1 and WNK4 were shown to regulate sodium chloride cotransporter (NCC) activity through phosphorylating SPAK and OSR1. Previous studies including ours have also shown that WNK4 inhibits NCC function and its protein expression. A recent study reported that a phorbol ester inhibits NCC function via activation of extracellular signal-regulated kinase (ERK) 1/2 kinase. In the current study, we investigated whether WNK4 affects NCC via the MAPK ERK1/2 signaling pathway. We found that WNK4 increased ERK1/2 phosphorylation in a dose-dependent manner in mouse distal convoluted tubule (mDCT) cells, whereas WNK4 mutants with the PHA II mutations (E562K and R1185C) lost the ability to increase the ERK1/2 phosphorylation. Hypertonicity significantly increased ERK1/2 phosphorylation in mDCT cells. Knock-down of WNK4 expression by siRNA resulted in a decrease of ERK1/2 phosphorylation. We further showed that WNK4 knock-down significantly increases the cell surface and total NCC protein expressions and ERK1/2 knock-down also significantly increases cell surface and total NCC expression. These data suggest that WNK4 inhibits NCC through activating the MAPK ERK1/2 signaling pathway.     

WNK1： analysis of protein kinase structure, downstream targets, and potential roles in hypertension

The WNK kinases are a recently discovered family of serine-threonine kinases that have been shown to play an essential role in the regulation of electrolyte homeostasis, lntronic deletions in the WNK1 gene resuk in its overexpression and lead to pseudohypoaldosteronism type Ⅱ, a disease with salt-sensitive hypertension and hyperkalemia. This review focuses on the recent evidence elucidating the structure of the kinase domain of WNK1 and functions of these kinases in normal and disease physiology. Their functions have implications for understanding the biochemical mechanism that could lead to the retention or insertion of proteins in the plasma membrane. The WNK kinases may be able to influence ion homeostasis through its effects on synaptotagmin function.     

Disease-causing mutations in the acidic motif of WNK4 impair the sensitivity of WNK4 kinase to calcium ions

WNK4 is a serine/threonine protein kinase that is involved in pseudohypoaldosteronism type II (PHAII), a Mendelian form disorder featuring hypertension and hyperkalemia. Most of the PHAII-causing mutations are clustered in an acidic motif rich in negatively charged residues. It is unclear, however, whether these mutations affect the kinase activity in any way. In this study, we isolated kinase domain of WNK4 produced by Escherichia coli, and demonstrated its ability to phosphorylate the oxidative stress-responsive kinase-1 (OSR1) and the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) in vitro. Threonine 48 was identified as the WNK4 phosphorylation site at mouse NCC. The phospho-mimicking T48D mutant of mouse NCC increased its protein abundance and Na(+) uptake, and also enhanced the phosphorylation at the N-terminal region of NCC by OSR1. When the acidic motif was included in the WNK4 kinase construct, the kinase activity of WNK4 exhibited sensitivity to Ca(2+) ions with the highest activity at Ca(2+) concentration around 1 μM using kinase-inactive OSR1 as a substrate. All tested PHAII-causing mutations at the acidic motif exhibited impaired Ca(2+) sensitivity. Our results suggest that these PHAII-causing mutations disrupt a Ca(2+)-sensing mechanism around the acidic motif necessary for the regulation of WNK4 kinase activity by Ca(2+) ions.     

WNK1 activates ERK5 by an MEKK2/3-dependent mechanism

WNK1 belongs to a unique protein kinase family that lacks the catalytic lysine in its normal position. Mutations in human WNK1 and WNK4 have been implicated in causing a familial form of hypertension. Here we report that overexpression of WNK1 led to increased activity of cotransfected ERK5 in HEK293 cells. ERK5 activation was blocked by the MEK5 inhibitor U0126 and expression of a dominant negative MEK5 mutant. Expression of dominant negative mutants of MEKK2 and MEKK3 also blocked activation of ERK5 by WNK1. Moreover, both MEKK2 and MEKK3 coimmunoprecipitated with endogenous WNK1 from cell lysates. WNK1 phosphorylated both MEKK2 and -3 in vitro, and MEKK3 was activated by WNK1 in 293 cells. Finally, ERK5 activation by epidermal growth factor was attenuated by suppression of WNK1 expression using small interfering RNA. Taken together, these results place WNK1 in the ERK5 MAP kinase pathway upstream of MEKK2/3.