Oxidative Stress-responsive Kinase Research Articles

Regulation of erythrocyte Na+/K+/2Cl− cotransport by an oxygen-switched kinase cascade

Many erythrocyte processes and pathways, including glycolysis, the pentose phosphate pathway (PPP), KCl cotransport, ATP release, Na+/K+-ATPase activity, ankyrin-band 3 interactions, and nitric oxide (NO) release, are regulated by changes in O2 pressure that occur as a red blood cell (RBC) transits between the lungs and tissues. The O2 dependence of glycolysis, PPP, and ankyrin-band 3 interactions (affecting RBC rheology) are controlled by O2-dependent competition between deoxyhemoglobin (deoxyHb), but not oxyhemoglobin (oxyHb), and other proteins for band 3. We undertook the present study to determine whether the O2 dependence of Na+/K+/2Cl- cotransport (catalyzed by Na+/K+/2Cl- cotransporter 1 [NKCC1]) might similarly originate from competition between deoxyHb and a protein involved in NKCC1 regulation for a common binding site on band 3. Using three transgenic mouse strains having mutated deoxyhemoglobin-binding sites on band 3, we found that docking of deoxyhemoglobin at the N terminus of band 3 displaces the protein with no lysine kinase 1 (WNK1) from its overlapping binding site on band 3. This displacement enabled WNK1 to phosphorylate oxidative stress-responsive kinase 1 (OSR1), which, in turn, phosphorylated and activated NKCC1. Under normal solution conditions, the NKCC1 activation increased RBC volume and thereby induced changes in RBC rheology. Because the deoxyhemoglobin-mediated WNK1 displacement from band 3 in this O2 regulation pathway may also occur in the regulation of other O2-regulated ion transporters, we hypothesize that the NKCC1-mediated regulatory mechanism may represent a general pattern of O2 modulation of ion transporters in erythrocytes.

The WNK-SPAK/OSR1 Kinases and the Cation-Chloride Cotransporters as Therapeutic Targets for Neurological Diseases

In recent years, cation-chloride cotransporters (CCCs) have drawn attention in the medical neuroscience research. CCCs include the family of Na+-coupled Cl- importers (NCC, NKCC1, and NKCC2), K+-coupled Cl- exporters (KCCs), and possibly polyamine transporters (CCC9) and CCC interacting protein (CIP1). For decades, CCCs have been the targets of several commonly used diuretic drugs, including hydrochlorothiazide, furosemide, and bumetanide. Genetic mutations of NCC and NKCC2 cause congenital renal tubular disorders and lead to renal salt-losing hypotension, secondary hyperreninemia, and hypokalemic metabolic alkalosis. New studies reveal that CCCs along with their regulatory WNK (Kinase with no lysine (K)), and SPAK (Ste20-related proline-alanine-rich kinase)/OSR1(oxidative stress-responsive kinase-1) are essential for regulating cell volume and maintaining ionic homeostasis in the nervous system, especially roles of the WNK-SPAK-NKCC1 signaling pathway in ischemic brain injury and hypersecretion of cerebrospinal fluid in post-hemorrhagic hydrocephalus. In addition, disruption of Cl- exporter KCC2 has an effect on synaptic inhibition, which may be involved in developing pain, epilepsy, and possibly some neuropsychiatric disorders. Interference with KCC3 leads to peripheral nervous system neuropathy as well as axon and nerve fiber swelling and psychosis. The WNK-SPAK/OSR1-CCCs complex emerges as therapeutic targets for multiple neurological diseases. This review will highlight these new findings.

The Photosensitising Clinical Agent Verteporfin Is an Inhibitor of SPAK and OSR1 Kinases.

STE20/SPS1-related proline/alanine-rich kinase (SPAK) and oxidative-stress-responsive kinase 1 (OSR1) are two serine/threonine protein kinases that play key roles in regulating ion homeostasis. Various SPAK and OSR1 mouse models exhibited reduced blood pressure. Herein, the discovery of verteporfin, a photosensitising agent used in photodynamic therapy, as a potent inhibitor of SPAK and OSR1 kinases is reported. It is shown that verteporfin binds the kinase domains of SPAK and OSR1 and inhibits their catalytic activity in an adenosine triphosphate (ATP)-independent manner. In cells, verteporfin was able to suppress the phosphorylation of the ion co-transporter NKCC1; a downstream physiological substrate of SPAK and OSR1 kinases. Kinase panel screening indicated that verteporfin inhibited a further eight protein kinases more potently than that of SPAK and OSR1. Although verteporfin has largely been studied as a modifier of the Hippo signalling pathway, this work indicates that the WNK-SPAK/OSR1 signalling cascade is also a target of this clinical agent. This finding could explain the fluctuation in blood pressure noted in patients and animals treated with this drug.

WNK1 regulates skeletal muscle cell hypertrophy by modulating the nuclear localization and transcriptional activity of FOXO4

With-no-lysine (K) (WNK) kinases, which are mutated in the inherited form of hypertension pseudohypoaldosteronism type II, are essential regulators of membrane ion transporters. Here, we report that WNK1 positively regulates skeletal muscle cell hypertrophy via mediating the function of the pro-longevity transcription factor forkhead box protein O4 (FOXO4) independent of the conventional WNK signaling pathway linking SPS/STE20-related proline-alanine–rich kinase (SPAK)/oxidative stress response kinase 1 (OSR1) to downstream effector ion transporters. Small interfering RNA (siRNA)-mediated silencing of WNK1, but not SPAK/OSR1 kinases, induced myotube atrophy and remarkable increases in the mRNA expression of the muscle atrophy ubiquitin ligases MAFbx and MuRF1 in C2C12 mouse skeletal muscle cells. WNK1 silencing also increased FOXO4 nuclear localization, and co-transfection of Foxo4-targeted siRNA completely reversed the myotube atrophy and upregulation of atrogene transcription induced by WNK1 silencing. We further illustrated that WNK1 protein abundance in skeletal muscle was increased by chronic voluntary wheel running exercise (hypertrophic stimulus) and markedly decreased by adenine-induced chronic kidney disease (atrophic stimulus) in mice. These findings suggest that WNK1 is involved in the physiological regulation of mammalian skeletal muscle hypertrophy and atrophy via interactions with FOXO4. The WNK1-FOXO4 axis may be a potential therapeutic target in human diseases causing sarcopenia.

Role of nitric oxide and WNK-SPAK/OSR1-KCC2 signaling in daily changes in GABAergic inhibition in the rat dorsal raphe neurons

Serotonergic neurons in the dorsal raphe nucleus (DRN) act as wake-inducing neurons in the sleep-wake cycle and are controlled by gamma-aminobutyric acid (GABA) synaptic inputs. We investigated daily changes in GABAergic inhibition of the rat DRN neurons and the role of nitric oxide (NO) and cation-chloride co-transporters in the GABAergic action. Neuronal NO synthase (nNOS) was co-expressed in 74% of serotonergic DRN neurons and nNOS expression was higher during daytime (the sleep cycle) than that during nighttime (the wake cycle). GABAergic hyperpolarization of DRN neurons produced by GABAA receptor agonist muscimol was greater and the equilibrium potential of muscimol showed a hyperpolarizing shift during daytime compared to that during nighttime. Expression levels of potassium-chloride co-transporter 2 (KCC2) were higher during daytime than that during nighttime, whereas there were no changes in sodium-potassium-chloride co-transporter 1 (NKCC1) expression. With-no-lysine kinase (WNK) isoform 1 was more highly expressed during daytime than that during nighttime. Total Ste20-related proline alanine rich kinase (SPAK) and oxidative stress response kinase 1 (OSR1) were also higher during daytime than during nighttime, while there were no changes in phosphorylated SPAK and OSR1. Consistent with the findings during the sleep-wake cycle, ex vivo treatment of DRN slices with a NO donor sodium nitroprusside (SNP) increased the expression of KCC2, WNK1, WNK2, WNK3, SPAK, and OSR1, whilst decreasing phosphorylated SPAK. These results suggest that GABAergic synaptic inhibition of DRN serotonergic neurons shows daily changes during the sleep-wake cycle, which might be regulated by daily changes in nNOS-derived NO and WNK-SPAK/OSR1-KCC2 signaling.

Mechanisms and controversies in mutant Cul3-mediated familial hyperkalemic hypertension.

Autosomal dominant mutations in cullin-3 ( Cul3) cause the most severe form of familial hyperkalemic hypertension (FHHt). Cul3 mutations cause skipping of exon 9, which results in an internal deletion of 57 amino acids from the CUL3 protein (CUL3-∆9). The precise mechanism by which this altered form of CUL3 causes FHHt is controversial. CUL3 is a member of the cullin-RING ubiquitin ligase family that mediates ubiquitination and thus degradation of cellular proteins, including with-no-lysine [K] kinases (WNKs). In CUL3-∆9-mediated FHHt, proteasomal degradation of WNKs is abrogated, leading to overactivation of the WNK targets sterile 20/SPS-1 related proline/alanine-rich kinase and oxidative stress-response kinase-1, which directly phosphorylate and activate the thiazide-sensitive Na+-Cl- cotransporter. Several groups have suggested different mechanisms by which CUL3-∆9 causes FHHt. The majority of these are derived from in vitro data, but recently the Kurz group (Schumacher FR, Siew K, Zhang J, Johnson C, Wood N, Cleary SE, Al Maskari RS, Ferryman JT, Hardege I, Figg NL, Enchev R, Knebel A, O'Shaughnessy KM, Kurz T. EMBO Mol Med 7: 1285-1306, 2015) described the first mouse model of CUL3-∆9-mediated FHHt. Analysis of this model suggested that CUL3-∆9 is degraded in vivo, and thus Cul3 mutations cause FHHt by inducing haploinsufficiency. We recently directly tested this model but found that other dominant effects of CUL3-∆9 must contribute to the development of FHHt. In this review, we focus on our current knowledge of CUL3-∆9 action gained from in vitro and in vivo models that may help unravel this complex problem.

TRIM48 Promotes ASK1 Activation and Cell Death through Ubiquitination-Dependent Degradation of the ASK1-Negative Regulator PRMT1

Apoptosis signal-regulating kinase 1 (ASK1) is an oxidative stress-responsive kinase that is regulated by various interacting molecules and post-translational modifications. However, how these molecules and modifications cooperatively regulate ASK1 activity remains largely unknown. Here, we showed that tripartite motif 48 (TRIM48) orchestrates the regulation of oxidative stress-induced ASK1 activation. A pull-down screen identified a TRIM48-interacting partner, protein arginine methyltransferase 1 (PRMT1), which negatively regulates ASK1 activation by enhancing its interaction with thioredoxin (Trx), another ASK1-negative regulator. TRIM48 facilitates ASK1 activation by promoting K48-linked polyubiquitination and degradation of PRMT1. TRIM48 knockdown suppressed oxidative stress-induced ASK1 activation and cell death, whereas forced expression promoted cancer cell death in mouse xenograft model. These results indicate that TRIM48 facilitates oxidative stress-induced ASK1 activation and cell death through ubiquitination-dependent degradation of PRMT1. This study provides a cell death mechanism fine-tuned by the crosstalk between enzymes that engage various types of post-translational modifications.

Development of WNK signaling inhibitors as a new class of antihypertensive drugs

Pseudohypoaldosteronism type II (PHAII) is characterized by hyperkalemia and hypertension despite a normal glomerular filtration rate. Abnormal activation of the signal cascade of with-no-lysine kinase (WNK) with OSR1 (oxidative stress-responsive kinase 1)/SPAK (STE20/SPS1-related proline/alanine-rich kinase) and NCC (NaCl cotransporter) results in characteristic salt-sensitive hypertension. Thus, inhibitors of the WNK-OSR1/SPAK-NCC cascade are candidates for a new class of antihypertensive drugs. In this study, we developed novel inhibitors of this signal cascade from the 9-aminoacridine lead compound 1, one of the hit compounds obtained by screening our chemical library for WNK-SPAK binding inhibitors. Among the synthesized acridine derivatives, several acridine-3-amide and 3-urea derivatives, such as 10 (IC50: 6.9μM), 13 (IC50: 2.6μM), and 20 (IC50: 4.8μM), showed more potent inhibitory activity than the lead compound 1 (IC50: 15.4μM). Compounds 10 and 20 were confirmed to inhibit phosphorylation of NCC in vivo.

Phosphorylation by PKC and PKA regulate the kinase activity and downstream signaling of WNK4

With-no-lysine kinase 4 (WNK4) regulates electrolyte homeostasis and blood pressure. WNK4 phosphorylates the kinases SPAK (Ste20-related proline alanine-rich kinase) and OSR1 (oxidative stress responsive kinase), which then phosphorylate and activate the renal Na-Cl cotransporter (NCC). WNK4 levels are regulated by binding to Kelch-like 3, targeting WNK4 for ubiquitylation and degradation. Phosphorylation of Kelch-like 3 by PKC or PKA downstream of AngII or vasopressin signaling, respectively, abrogates binding. We tested whether these pathways also affect WNK4 phosphorylation and activity. By tandem mass spectrometry and use of phosphosite-specific antibodies, we identified five WNK4 sites (S47, S64, S1169, S1180, S1196) that are phosphorylated downstream of AngII signaling in cultured cells and in vitro by PKC and PKA. Phosphorylation at S64 and S1196 promoted phosphorylation of the WNK4 kinase T-loop at S332, which is required for kinase activation, and increased phosphorylation of SPAK. Volume depletion induced phosphorylation of these sites in vivo, predominantly in the distal convoluted tubule. Thus, AngII, in addition to increasing WNK4 levels, also modulates WNK4 kinase activity via phosphorylation of sites outside the kinase domain.

Future considerations based on the information from Barrter's and Gitelman's syndromes.

Bartter and Gitelman syndromes are typical normotensive salt losing hypokalaemic tubulopathies. Their pathogenesis was gradually deciphered in the past 5 decades, first by typical salt balance studies and histopathology, followed by genetic characterization and discovery of the affected different ion channels. Although the different genotypic subtypes were originally thought to show a similar phenotype, important clinical and biochemical differences can now be found. New findings on the regulation of these channels, as well as the recent discovery of newly affected genes, merit an update on this topic. Na-K-2CL cotransporter and NaCl cotransporter, the two main luminal channels in the thick ascending limb and distal convoluted tubule were found to be regulated by Ste 20-related proline alanine-rich kinase and oxidative stress response kinase. Knockout mice to these channels express a Bartter-like phenotype. MAGE-D2 is new gene found to cause severe polyhydramnios and transient postnatal Bartter-like syndrome. Variants in the different channels causing Bartter syndromes/Gitelman syndromes may also confer susceptibility for hypertension or protect against it. It remains to be determined if polymorphism or epigenetic changes in these genes and proteins may affect salt handling, explaining, apart from Bartter syndromes and Gitelman syndromes, also hypertension or stroke tendency, or both.

OSR1 and SPAK cooperatively modulate Sertoli cell support of mouse spermatogenesis.

We investigated the role of oxidative stress-responsive kinase-1 (OSR1) and STE20 (sterile 20)/SPS1-related proline/alanine-rich kinase (SPAK), upstream regulators of the Na+-K+-2Cl− cotransporter (NKCC1)—essential for spermatogenesis—in mouse models of male fertility. Global OSR1+/− gene mutations, but not global SPAK−/− or Sertoli cell (SC)-specific OSR1 gene knockout (SC-OSR1−/−), cause subfertility with impaired sperm function and are associated with reduced abundance of phosphorylated (p)-NKCC1 but increased p-SPAK expression in testicular tissue and spermatozoa. To dissect further in a SC-specific manner the compensatory effect of OSR1 and SPAK in male fertility, we generated SC-OSR1−/− and SPAK−/− double knockout (DKO) male mice. These are infertile with defective spermatogenesis, presenting a SC-only-like syndrome. Disrupted meiotic progression and increased germ cell apoptosis occurred in the first wave of spermatogenesis. The abundance of total and p-NKCC1 was significantly decreased in the testicular tissues of DKO mice. These results indicate that OSR1 and SPAK cooperatively regulate NKCC1-dependent spermatogenesis in a SC-restricted manner.

The regulation of Na+Cl- cotransporter by with-no-lysine kinase 4.

Abundant evidence supports that the NaCl cotransporter (NCC) activity is tightly regulated by the with-no-lysine (WNK) kinases. Here, we summarize the data regarding NCC regulation by WNKs, with a particular emphasis on WNK4. Several studies involving in-vivo and in-vitro models have provided paradoxical data regarding WNK4 regulation of the NCC. Although some studies show that WNK4 can activate the NCC, other equally compelling studies show that WNK4 inhibits the NCC. Recent studies have shown that WNK4 is regulated by the intracellular chloride concentration ([Cl]i), which could account for these paradoxical results. In conditions of high [Cl]i, WNK4 could act as an inhibitor via heterodimer formation with other WNKs. In contrast, when [Cl]i is low, WNK4 can activate Ste20-related, proline-alanine-rich kinase (SPAK)/oxidative stress responsive kinase 1 (OSR1) and thus the NCC. Modulation of WNK4 by [Cl]i has been shown to account for the potassium-sensing properties of the distal convoluted tubule. Other regulators of WNK4 include hormones and ubiquitination. Modulation of WNK4 activity by [Cl]i can account for its dual role on the NCC, and this has important physiological implications regarding the regulation of extracellular potassium concentration. Defective regulation of WNKs by ubiquitination explains most cases of familial hyperkalemic hypertension.

SPAK and OSR1 play essential roles in potassium homeostasis through actions on the distal convoluted tubule.

STE20 (Sterile 20)/SPS-1 related proline/alanine-rich kinase (SPAK) and oxidative stress-response kinase-1 (OSR1) phosphorylate and activate the renal Na(+) -K(+) -2Cl(-) cotransporter 2 (NKCC2) and Na(+) Cl(-) cotransporter (NCC). Mouse models suggest that OSR1 mainly activates NKCC2-mediated sodium transport along the thick ascending limb, while SPAK mainly activates NCC along the distal convoluted tubule, but the kinases may compensate for each other. We hypothesized that disruption of both kinases would lead to polyuria and severe salt-wasting, and generated SPAK/OSR1 double knockout mice to test this. Despite a lack of SPAK and OSR1, phosphorylated NKCC2 abundance was still high, suggesting the existence of an alternative activating kinase. Compensatory changes in SPAK/OSR1-independent phosphorylation sites on both NKCC2 and NCC and changes in sodium transport along the collecting duct were also observed. Potassium restriction revealed that SPAK and OSR1 play essential roles in the emerging model that NCC activation is central to sensing changes in plasma [K(+) ]. STE20 (Sterile 20)/SPS-1 related proline/alanine-rich kinase (SPAK) and oxidative stress-response kinase-1 (OSR1) activate the renal cation cotransporters Na(+) -K(+) -2Cl(-) cotransporter (NKCC2) and Na(+) -Cl(-) cotransporter (NCC) via phosphorylation. Knockout mouse models suggest that OSR1 mainly activates NKCC2, while SPAK mainly activates NCC, with possible cross-compensation. We tested the hypothesis that disrupting both kinases causes severe polyuria and salt-wasting by generating SPAK/OSR1 double knockout (DKO) mice. DKO mice displayed lower systolic blood pressure compared with SPAK knockout (SPAK-KO) mice, but displayed no severe phenotype even after dietary salt restriction. Phosphorylation of NKCC2 at SPAK/OSR1-dependent sites was lower than in SPAK-KO mice, but still significantly greater than in wild type mice. In the renal medulla, there was significant phosphorylation of NKCC2 at SPAK/OSR1-dependent sites despite a complete absence of SPAK and OSR1, suggesting the existence of an alternative activating kinase. The distal convoluted tubule has been proposed to sense plasma [K(+) ], with NCC activation serving as the primary effector pathway that modulates K(+) secretion, by metering sodium delivery to the collecting duct. Abundance of phosphorylated NCC (pNCC) is dramatically lower in SPAK-KO mice than in wild type mice, and the additional disruption of OSR1 further reduced pNCC. SPAK-KO and kidney-specific OSR1 single knockout mice maintained plasma [K(+) ] following dietary potassium restriction, but DKO mice developed severe hypokalaemia. Unlike mice lacking SPAK or OSR1 alone, DKO mice displayed an inability to phosphorylate NCC under these conditions. These data suggest that SPAK and OSR1 are essential components of the effector pathway that maintains plasma [K(+) ].

With no lysine L-WNK1 isoforms are negative regulators of the K+-Cl- cotransporters.

The K(+)-Cl(-) cotransporters (KCC1-KCC4) encompass a branch of the SLC12 family of electroneutral cation-coupled chloride cotransporters that translocate ions out of the cell to regulate various factors, including cell volume and intracellular chloride concentration, among others. L-WNK1 is an ubiquitously expressed kinase that is activated in response to osmotic stress and intracellular chloride depletion, and it is implicated in two distinct hereditary syndromes: the renal disease pseudohypoaldosteronism type II (PHAII) and the neurological disease hereditary sensory neuropathy 2 (HSN2). The effect of L-WNK1 on KCC activity is unknown. Using Xenopus laevis oocytes and HEK-293 cells, we show that the activation of KCCs by cell swelling was prevented by L-WNK1 coexpression. In contrast, the activity of the Na(+)-K(+)-2Cl(-) cotransporter NKCC1 was remarkably increased with L-WNK1 coexpression. The negative effect of L-WNK1 on the KCCs is kinase dependent. Elimination of the STE20 proline-alanine rich kinase (SPAK)/oxidative stress-responsive kinase (OSR1) binding site or the HQ motif required for the WNK-WNK interaction prevented the effect of L-WNK1 on KCCs, suggesting a required interaction between L-WNK1 molecules and SPAK. Together, our data support that NKCC1 and KCCs are coordinately regulated by L-WNK1 isoforms.

Abstract WP269: New Insights Into Worsened Cerebral Ischemia in Hypertensive Rats: Roles of WNK-SPAK/OSR1-NKCC1 Signaling

Background and Purpose: WNK kinases [with-no-lysine (K)], Ste20-related proline alanine-rich kinase (SPAK) and oxidative stress-responsive kinase-1 (OSR1) are a family of evolutionarily conserved regulators of ion transporter proteins. The kinases alter the phosphorylation state of ion transporter proteins and so play an important role in renal salt handling and in the pathogenesis of hypertension. We previously reported that pharmacological inhibition or genetic knockout of Na+-K+-2Cl- cotransporter 1 (NKCC1) is neuroprotective against ischemic injury. Our current study investigated whether WNK-SPAK/OSR1-NKCC1 signaling is augmented in cerebral ischemia in hypertension. Methods: Cerebral ischemia was induced in adult male Wistar Kyoto (WKY) or spontaneously hypertensive rats (SHR) by 2-hour occlusion of the left middle cerebral artery (MCAO) followed by reperfusion. Brain tissues were harvested for immunoblot assessment of expression levels of NKCC1, SPAK/OSR1 or WNK1-4. Another two cohorts of WKY and SHR were treated with saline or NKCC1 inhibitor bumetanide (BMT, 10 mg/kg/day, i.p.) after MCAO. In these animals, ischemic infarct volume was determined by TTC staining and behavioral deficits were analyzed by neurological scoring and turning-alley test. Results: Basal levels of total NKCC1 protein were significantly higher in the contralateral (CL) cortical tissues in SHR brains than WKY brains. Ischemia was associated with increased phosphorylation of NKCC1 in the ipsilateral (IL) cortex of SHR, but not WKY at 6 hours post-ischemia. WNK1 increased 6.1±1.5 fold and WNK4 increased 8.7±1.7 fold in IL cortex of SHR, but not in that of WKY, and phosphorylated SPAK/OSR1 was unchanged. Lastly, sensitivity to pharmacological NKCC1 inhibition was higher in SHR than WKY rats. BMT blunted the MCAO-induced infarction and improved neurobehavioral score and sensory motor function in SHR rats. Conclusion: These findings suggest that augmented WNK1/4-NKCC1 signaling is responsible, at least in part, for the increased susceptibility to ischemic brain damage associated with hypertension. They highlight the potential for inhibition of WNK1/4-NKCC1 signaling as part of anti-ischemic stroke therapy. Supported by NIH Grant R01 NS38118 (D Sun).

Osmotic stress induces the phosphorylation of WNK4 Ser575 via the p38MAPK-MK pathway.

The With No lysine [K] (WNK)-Ste20-related proline/alanine-rich kinase (SPAK)/oxidative stress-responsive kinase 1 (OSR1) pathway has been reported to be a crucial signaling pathway for triggering pseudohypoaldosteronism type II (PHAII), an autosomal dominant hereditary disease that is characterized by hypertension. However, the molecular mechanism(s) by which the WNK-SPAK/OSR1 pathway is regulated remain unclear. In this report, we identified WNK4 as an interacting partner of a recently identified MAP3K, apoptosis signal-regulating kinase 3 (ASK3). We found that WNK4 is phosphorylated in an ASK3 kinase activity-dependent manner. By exploring the ASK3-dependent phosphorylation sites, we identified Ser575 as a novel phosphorylation site in WNK4 by LC-MS/MS analysis. ASK3-dependent WNK4 Ser575 phosphorylation was mediated by the p38MAPK-MAPK-activated protein kinase (MK) pathway. Osmotic stress, as well as hypotonic low-chloride stimulation, increased WNK4 Ser575 phosphorylation via the p38MAPK-MK pathway. ASK3 was required for the p38MAPK activation induced by hypotonic stimulation but was not required for that induced by hypertonic stimulation or hypotonic low-chloride stimulation. Our results suggest that the p38MAPK-MK pathway might regulate WNK4 in an osmotic stress-dependent manner but its upstream regulators might be divergent depending on the types of osmotic stimuli.

OSR1 and SPAK Sensitivity of Large-Conductance Ca2+ Activated K+ Channel

Background/Aims: The oxidative stress-responsive kinase 1 (OSR1) and the serine/threonine kinases SPAK (SPS1-related proline/alanine-rich kinase) are under the control of WNK (with-no-K [Lys]) kinases. OSR1 and SPAK participate in diverse functions including cell volume regulation and neuronal excitability. Cell volume and neuronal excitation are further modified by the large conductance Ca²⁺-activated K⁺ channels (maxi K⁺ channel or BK channels). An influence of OSR1 and/or SPAK on BK channel activity has, however, never been shown. The present study thus explored whether OSR1 and/or SPAK modify the activity of BK channels. Methods: cRNA encoding the Ca²⁺ insensitive BK channel mutant BK^{M513I+Δ899-903} was injected into Xenopus laevis oocytes without or with additional injection of cRNA encoding wild-type OSR1 or wild-type SPAK, constitutively active ^T185EOSR1, catalytically inactive ^D164AOSR1, constitutively active ^T233ESPAK or catalytically inactive ^D212ASPAK. K⁺ channel activity was measured utilizing dual electrode voltage clamp. Results: BK channel activity in BK^{M513I+Δ899-903} expressing oocytes was significantly decreased by co-expression of OSR1 or SPAK. The effect of wild-type OSR1/SPAK was mimicked by ^T185EOSR1 and ^T233ESPAK, but not by ^D164AOSR1 or ^D212ASPAK. Conclusions: OSR1 and SPAK suppress BK channels, an effect possibly contributing to cell volume regulation and neuroexcitability.

WNK-Cab39-NKCC1 signaling increases the susceptibility to ischemic brain damage in hypertensive rats

With-no-lysine kinase (WNK) and Na+-K+-2Cl− cotransporter 1 (NKCC1) are involved in the pathogenesis of hypertension. In this study, we investigated the WNK-NKCC1 signaling pathway in spontaneously hypertensive rats (SHR) and their associated susceptibility to stroke injury. Basal NKCC1 protein levels were higher in SHR than in normotensive Wistar Kyoto (WKY) rat brains. After inducing ischemic stroke, adult male WKY and SHR received either saline or NKCC1 inhibitor bumetanide (10 mg/kg/day, i.p.) starting at 3-h post-reperfusion. NKCC1 inhibition blunted the extent of ischemic infarction in SHR and improved their neurobehavioral functions. Interestingly, ischemia led to increased NKCC1 phosphorylation in SHR but not in WKY rats. Pronounced elevation of WNK1, WNK2 and WNK4 protein and downregulation of WNK3 were detected in ischemic SHR, but not in ischemic WKY rats. Upregulation of WNK-NKCC1 complex in ischemic SHR brain was associated with increased Ca2+-binding protein 39 (Cab39), without increases in Ste20-related proline alanine-rich kinase or oxidative stress-responsive kinase-1. Moreover, subacute middle cerebral artery stroke human brain autopsy exhibited increased expression of NKCC1 protein. We conclude that augmented WNK-Cab39-NKCC1 signaling in SHR is associated with an increased susceptibility to ischemic brain damage and may serve as a novel target for anti-hypertensive and anti-ischemic stroke therapy.

Development and regulation of chloride homeostasis in the central nervous system.

γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter of the mature central nervous system (CNS). The developmental switch of GABAergic transmission from excitation to inhibition is induced by changes in Cl− gradients, which are generated by cation-Cl− co-transporters. An accumulation of Cl− by the Na+-K+-2Cl− co-transporter (NKCC1) increases the intracellular Cl− concentration ([Cl−]i) such that GABA depolarizes neuronal precursors and immature neurons. The subsequent ontogenetic switch, i.e., upregulation of the Cl−-extruder KCC2, which is a neuron-specific K+-Cl− co-transporter, with or without downregulation of NKCC1, results in low [Cl−]i levels and the hyperpolarizing action of GABA in mature neurons. Development of Cl− homeostasis depends on developmental changes in NKCC1 and KCC2 expression. Generally, developmental shifts (decreases) in [Cl−]i parallel the maturation of the nervous system, e.g., early in the spinal cord, hypothalamus and thalamus, followed by the limbic system, and last in the neocortex. There are several regulators of KCC2 and/or NKCC1 expression, including brain-derived neurotrophic factor (BDNF), insulin-like growth factor (IGF), and cystic fibrosis transmembrane conductance regulator (CFTR). Therefore, regionally different expression of these regulators may also contribute to the regional developmental shifts of Cl− homeostasis. KCC2 and NKCC1 functions are also regulated by phosphorylation by enzymes such as PKC, Src-family tyrosine kinases, and WNK1–4 and their downstream effectors STE20/SPS1-related proline/alanine-rich kinase (SPAK)-oxidative stress responsive kinase-1 (OSR1). In addition, activation of these kinases is modulated by humoral factors such as estrogen and taurine. Because these transporters use the electrochemical driving force of Na+ and K+ ions, topographical interaction with the Na+-K+ ATPase and its modulators such as creatine kinase (CK) should modulate functions of Cl− transporters. Therefore, regional developmental regulation of these regulators and modulators of Cl− transporters may also play a pivotal role in the development of Cl− homeostasis.

Down-Regulation of Excitatory Amino Acid Transporters EAAT1 and EAAT2 by the Kinases SPAK and OSR1.

SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1) are cell volume-sensitive kinases regulated by WNK (with-no-K[Lys]) kinases. SPAK/OSR1 regulate several channels and carriers. SPAK/OSR1 sensitive functions include neuronal excitability. Orchestration of neuronal excitation involves the excitatory glutamate transporters EAAT1 and EAAT2. Sensitivity of those carriers to SPAK/OSR1 has never been shown. The present study thus explored whether SPAK and/or OSR1 contribute to the regulation of EAAT1 and/or EAAT2. To this end, cRNA encoding EAAT1 or EAAT2 was injected into Xenopus oocytes without or with additional injection of cRNA encoding wild-type SPAK or wild-type OSR1, constitutively active (T233E)SPAK, WNK insensitive (T233A)SPAK, catalytically inactive (D212A)SPAK, constitutively active (T185E)OSR1, WNK insensitive (T185A)OSR1 or catalytically inactive (D164A)OSR1. The glutamate (2 mM)-induced inward current (I Glu) was taken as a measure of glutamate transport. As a result, I Glu was observed in EAAT1- and in EAAT2-expressing oocytes but not in water-injected oocytes, and was significantly decreased by coexpression of SPAK and OSR1. As shown for EAAT2, SPAK, and OSR1 decreased significantly the maximal transport rate but significantly enhanced the affinity of the carrier. The effect of wild-type SPAK/OSR1 on EAAT1 and EAAT2 was mimicked by (T233E)SPAK and (T185E)OSR1, but not by (T233A)SPAK, (D212A)SPAK, (T185A)OSR1, or (D164A)OSR1. Coexpression of either SPAK or OSR1 decreased the EAAT2 protein abundance in the cell membrane of EAAT2-expressing oocytes. In conclusion, SPAK and OSR1 are powerful negative regulators of the excitatory glutamate transporters EAAT1 and EAAT2.