With No Lysine [K Research Articles

WNKs regulate mouse behavior and alter central nervous system glucose uptake and insulin signaling.

Certain areas of the brain involved in episodic memory and behavior, such as the hippocampus, express high levels of insulin receptors and glucose transporter-4 (GLUT4) and are responsive to insulin. Insulin and neuronal glucose metabolism improve cognitive functions and regulate mood in humans. Insulin-dependent GLUT4 trafficking has been extensively studied in muscle and adipose tissue, but little work has demonstrated either how it is controlled in insulin-responsive brain regions or its mechanistic connection to cognitive functions. In this study, we demonstrate that inhibition of WNK (With-No-lysine (K)) kinases improves learning and memory in mice. Neuronal inhibition of WNK enhances in vivo hippocampal glucose uptake. Inhibition of WNK enhances insulin signaling output and insulin-dependent GLUT4 trafficking to the plasma membrane in mice primary neuronal cultures and hippocampal slices. Therefore, we propose that the extent of neuronal WNK kinase activity has an important influence on learning, memory and anxiety-related behaviors, in part, by modulation of neuronal insulin signaling.

The Atypical Protein Kinase WNK1 Controls Leukemia Progression in TAL/LMO T-Cell Acute Lymphoblastic Leukemia

Genomic and proteomic approaches have been crucial in identifying genetic mutations and protein expression profiles underlying leukemia cell growth and survival, especially in heterogeneous diseases like T-cell acute lymphoblastic leukemia (T-ALL). T-ALL is mainly driven by “poorly druggable” transcription factors along with “fairly druggable” secondary events. However, only a few kinases, such as JAK2 and LCK, have shown dependency for successfully matched therapies, limiting treatment options for T-ALL patients. To identify druggable active pathways in T-ALL, we profiled 31 phosphorylated kinases in T-ALL cell lines, clinical T-ALL blasts, and T cells, and showed that WNK1 exhibited the most significant difference between T-ALL and T cells (p < 0.0001). WNK (with no lysine = K) proteins (WNK1-4) are atypical serine-threonine kinases in which the catalytic lysine, necessary for ATP binding, is replaced by cysteine and situated in subdomain I instead of II. WNK proteins regulate ion flux transport across cellular membranes and facilitate the release/recycling of carrier vesicles; however, their role in leukemia remains uncharacterized. From the analysis of different transcriptome datasets, we demonstrated that WNK1 is the most highly expressed WNK isoform in T-ALL samples, T-ALL cell lines, and patient-derived leukemia xenografts (PDLX) (p < 0.0001). WNK1 and p-WNK1 are predominately expressed in lymphoblasts compared to normal thymus or lymph nodes, indicating the involvement of disease-causing events that lead to the activation of this pathway. To correlate phosphorylation levels with protein and mRNA expression, we intersected the FORALL database, which encompasses ALL's proteomic, transcriptomic, and pharmacoproteomic profiling with the functional Cancer DepMap dataset. WNK1 emerged as a T-ALL lineage transcriptional dependency, with a high correlation between WNK1 protein and mRNA expression (Pearson r= 0.64, p=0.00123, n=22), providing additional support for targeting WNK1 in T-ALL. The next question is whether this level of WNK1 expression can identify a specific T-ALL subgroup. From two independent T-ALL datasets, we found that WNK1 is overexpressed in the TAL/ LMO-related (TAL-R) subgroups (p < 0.01), where PTEN/AKT mutations are notably abundant. An outlier analysis of WNK1 expression revealed a correlation with 6q14-q16 deletions (Fisher's exact test = 0.0268), a privileged abnormality in the TAL/LMO subgroup, suggesting a potential area for clinical exploitation in poor-prognosis T-ALL. To understand the contribution of WNK1 in leukemogenesis, we performed a network-based analysis (NetBID2) from RNA-seq data of T-ALL cells lacking WNK1 expression or treated with WNK463, an orally bioavailable pan-WNKs inhibitor. Results showed that modulation of WNK1 enriched signatures associated with the G2M checkpoint and mitotic spindle control (p < 0.0001). Consistently targeting WNK1 with shRNA or sgRNA-CRISPR Cas9 in TAL-R cell lines promoted polyploidy, resulting in cell cycle arrest and inhibition of cell proliferation in vitro. Similarly, treatment with WNK463 led to morphometric changes, such as incomplete cell division or chromosome segregation through mitotic spindles and abscission defects. Due to the potential translational significance of our findings, we established a PDLX TAL-R model by serially transplanting SIL-TAL1 + leukemia cells into NOD/SCID mice for in vivo drug treatment. Mice (n=10) were exposed to WNK463 at 1.5 mg/Kg/day for 26 days. At the control group endpoint, we sacrificed four mice per group for efficacy studies to reveal a decrease of leukemic cells infiltration in the bone marrow and spleen (p=0.0286), an effect pharmacodynamically associated with a reduction in the WNK1 phosphorylation levels. The remaining mice were observed for survival analysis. Treated mice showed a significant survival advantage (p=0.0009, log-rank Mantel-Cox test) compared to vehicle control. No relevant drug-related toxicity was recorded in the treatment group. In summary, our work demonstrates the significant contribution of WNK1 kinase in the leukemia progression in TAL/ LMO T-ALL cases. The lack of sequence similarity in the ATP binding domain compared to other kinases makes the identification of leukemia's dependence on atypical WNK1 kinase particularly promising for the development of selective inhibitors as a potential treatment for T-ALL.

NF-κB Signaling-Mediated Activation of WNK-SPAK-NKCC1 Cascade in Worsened Stroke Outcomes of Ang II-Hypertensive Mice.

Worsened stroke outcomes with hypertension comorbidity are insensitive to blood pressure-lowering therapies. In an experimental stroke model with comorbid hypertension, we investigated causal roles of ang II (angiotensin II)-mediated stimulation of the brain WNK (with no lysine [K] kinases)-SPAK (STE20/SPS1-related proline/alanine-rich kinase)-NKCC1 (Na-K-Cl cotransporter) complex in worsened outcomes. Saline- or ang II-infused C57BL/6J male mice underwent stroke induced by permanent occlusion of the distal branches of the middle cerebral artery. Mice were randomly assigned to receive either vehicle dimethyl sulfoxide/PBS (2 mL/kg body weight/day, IP), a novel SPAK inhibitor, 5-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)-2-hydroxybenzamide (ZT-1a' 5 mg/kg per day, IP) or a NF-κB (nuclear factor-κB) inhibitor TAT-NBD (transactivator of transcription-NEMO-binding domain' 20 mg/kg per day, IP). Activation of brain NF-κB and WNK-SPAK-NKCC1 cascade as well as ischemic stroke outcomes were examined. Stroke triggered a 2- to 5-fold increase of WNK (isoforms 1, 2, 4), SPAK/OSR1 (oxidative stress-responsive kinase 1), and NKCC1 protein in the ang II-infused hypertensive mouse brains at 24 hours after stroke, which was associated with increased nuclear translocation of phospho-NF-κB protein in the cortical neurons (a Pearson correlation r of 0.77, P<0.005). The upregulation of WNK-SPAK-NKCC1 cascade proteins resulted from increased NF-κB recruitment on Wnk1, Wnk2, Wnk4, Spak, and Nkcc1 gene promoters and was attenuated by NF-κB inhibitor TAT-NBD. Poststroke administration of SPAK inhibitor ZT-1a significantly reduced WNK-SPAK-NKCC1 complex activation, brain lesion size, and neurological function deficits in the ang II-hypertensive mice without affecting blood pressure and cerebral blood flow. The ang II-induced stimulation of NF-κB transcriptional activity upregulates brain WNK-SPAK-NKCC1 cascade and contributes to worsened ischemic stroke outcomes, illustrating the brain WNK-SPAK-NKCC1 complex as a therapeutic target for stroke with comorbid hypertension.

Chloride oscillation in pacemaker neurons regulates circadian rhythms through a chloride-sensing WNK kinase signaling cascade

Central pacemaker neurons regulate circadian rhythms and undergo diurnal variation in electrical activity in mammals and flies.1,2 Circadian variation in the intracellular chloride concentration of mammalian pacemaker neurons has been proposed to influence the response to GABAergic neurotransmission through GABAA receptor chloride channels.3 However, results have been contradictory,4-9 and a recent study demonstrated circadian variation in pacemaker neuron chloride without an effect on GABA response.10 Therefore, whether and how intracellular chloride regulates circadian rhythms remains controversial. Here, we demonstrate a signaling role for intracellular chloride in the Drosophila small ventral lateral (sLNv) pacemaker neurons. In control flies, intracellular chloride increases in sLNvs over the course of the morning. Chloride transport through sodium-potassium-2-chloride (NKCC) and potassium-chloride (KCC) cotransporters is a major determinant of intracellular chloride concentrations.11Drosophila melanogaster with loss-of-function mutations in the NKCC encoded by Ncc69 have abnormally low intracellular chloride 6h after lights on, loss of morning anticipation, and a prolonged circadian period. Loss of kcc, which is expected to increase intracellular chloride, suppresses the long-period phenotype of Ncc69 mutant flies. Activation of a chloride-inhibited kinase cascade, consisting of WNK (with no lysine [K]) kinase and its downstream substrate, Fray, is necessary and sufficient to prolong period length. Fray activation of an inwardly rectifying potassium channel, Irk1, is also required for the long-period phenotype. These results indicate that the NKCC-dependent rise in intracellular chloride in Drosophila sLNv pacemakers restrains WNK-Fray signaling and overactivation of an inwardly rectifying potassium channel to maintain normal circadian period length.

CCT and CCT-Like Modular Protein Interaction Domains in WNK Signaling.

The WNK [with no lysine (K)] kinases and their downstream effector kinases, oxidative stress responsive 1 (OSR1) and SPS/STE20-related proline-alanine-rich kinase (SPAK), have well established functions in the maintenance of cell volume and ion homeostasis. Mutations in these kinases have been linked to an inherited form of hypertension, neurologic defects, and other pathologies. A rapidly expanding body of evidence points to the involvement of WNKs in regulating multiple diverse cellular processes as well as the progression of some forms of cancer. How OSR1 and SPAK contribute to these processes is well understood in some cases but completely unknown in others. OSR1 and SPAK are targeted to both WNKs and substrates via their conserved C-terminal (CCT) protein interaction domains. Considerable effort has been put forth to understand the structure, function, and interaction specificity of the CCT domains in relation to WNK signaling, and multiple inhibitors of WNK signaling target these domains. The domains bind RFxV and RxFxV protein sequence motifs with the consensus sequence R-F-x-V/I or R-x-F-x-V/I, but residues outside the core motif also contribute to specificity. CCT interactions are required for OSR1 and SPAK activation and deactivation as well as cation-chloride cotransporter substrate phosphorylation. All four WNKs also contain CCT-like domains that have similar structures and conserved binding residues when compared with CCT domains, but their functions and interaction specificities are mostly unknown. A better understanding of the varied actions of these domains and their interactions will better define the known signaling mechanisms of the WNK pathway as well as uncover new ones. SIGNIFICANCE STATEMENT: WNK [with no lysine (K)] kinases and their downstream effector kinases, oxidative stress responsive 1 (OSR1) and SPS/STE20-related proline-alanine-rich kinase (SPAK), have been shown to be involved in an array of diverse cellular processes. Here we review the function of modular protein interaction domains found in OSR1 and SPAK as well as related domains found in WNKs.

WNK regulates Wnt signalling and \u03b2-Catenin levels by interfering with the interaction between \u03b2-Catenin and GID

β-Catenin is an important component of the Wnt signalling pathway. As dysregulation or mutation of this pathway causes many diseases, including cancer, the β-Catenin level is carefully regulated by the destruction complex in the Wnt signalling pathway. However, the mechanisms underlying the regulation of β-Catenin ubiquitination and degradation remain unclear. Here, we find that WNK (With No Lysine [K]) kinase is a potential regulator of the Wnt signalling pathway. We show that WNK protects the interaction between β-Catenin and the Glucose-Induced degradation Deficient (GID) complex, which includes an E3 ubiquitin ligase targeting β-Catenin, and that WNK regulates the β-Catenin level. Furthermore, we show that WNK inhibitors induced β-Catenin degradation and that one of these inhibitors suppressed xenograft tumour development in mice. These results suggest that WNK is a previously unrecognized regulator of β-Catenin and a therapeutic target of cancer.

WNK2 Inhibits Autophagic Flux in Human Glioblastoma Cell Line.

Autophagy is a cell-survival pathway with dual role in tumorigenesis, promoting either tumor survival or tumor death. WNK2 gene, a member of the WNK (with no lysine (K)) subfamily, acts as a tumor suppressor gene in gliomas, regulating cell migration and invasion; however, its role in autophagy process is poorly explored. The WNK2-methylated human glioblastoma cell line A172 WT (wild type) was compared to transfected clones A172 EV (empty vector), and A172 WNK2 (WNK2 overexpression) for the evaluation of autophagy using an inhibitor (bafilomycin A1—baf A1) and an inducer (everolimus) of autophagic flux. Western blot and immunofluorescence approaches were used to monitor autophagic markers, LC3A/B and SQSTM1/p62. A172 WNK2 cells presented a significant decrease in LC3B and p62 protein levels, and in LC3A/B ratio when compared with control cells, after treatment with baf A1 + everolimus, suggesting that WNK2 overexpression inhibits the autophagic flux in gliomas. The mTOR pathway was also evaluated under the same conditions, and the observed results suggest that the inhibition of autophagy mediated by WNK2 occurs through a mTOR-independent pathway. In conclusion, the evaluation of the autophagic process demonstrated that WNK2 inhibits the autophagic flux in glioblastoma cell line.

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.

WNK Kinases in Development and Disease.

WNK (With-No-Lysine (K)) kinases are serine-threonine kinases characterized by an atypical placement of a catalytic lysine within the kinase domain. Mutations in human WNK1 or WNK4 cause an autosomal dominant syndrome of hypertension and hyperkalemia, reflecting the fact that WNK kinases are critical regulators of renal ion transport processes. Here, the role of WNKs in the regulation of ion transport processes in vertebrate and invertebrate renal function, cellular and organismal osmoregulation, and cell migration and cerebral edema will be reviewed, along with emerging literature demonstrating roles for WNKs in cardiovascular and neural development, Wnt signaling, and cancer. Conserved roles for these kinases across phyla are emphasized.

New perspective on the role of WNK1 and WNK4 in the regulation ofNaCl reabsorption and K(+) secretion by the distal nephron

The study of Familial Hyperkalemic Hypertension (FHHt), a rare monogenic disease, allowed remarkable advances in the understanding of the mechanisms of regulation of NaCl reabsorption by the distal nephron. FHHt results from mutations in the genes encoding WNK1 and WNK4, two serine-threonine kinases of the WNK (With No lysine [K]) family. The clinical manifestations of FHHt are due, among others, to an increased activity of the Na(+)-Cl(-) cotransporter NCC. Several groups therefore tried to understand how WNK1 and WNK4 could regulate NCC. However, the data were often contradictory. Two of our recent studies allowed to partially explain these controversies and to propose a new model for the regulation of NCC by the WNKs.

WNK3‐SPAK/OSR1‐NKCC1 Signaling Pathway in Ischemic Brain Injury

Ischemia-mediated activation of Na-K-Cl cotransporter (NKCC1) leads to Na+ overload, cytotoxic edema, and cell death. However, the molecular mechanisms underlying these processes are not understood. Here we investigated the role of WNK (with no lysine = K)-SPAK/OSR1 kinase signaling pathway in regulating the function of NKCC1 in cerebral ischemia. We show that genetic deletion of WNK3, a WNK family member highly expressed in brain, or siRNA-mediated knockdown of SPAK or OSR1, protects against ischemic neuroglial death triggered by oxygen-glucose deprivation/reoxygenation. Using phospho specific antibodies, we detected robust up regulation of p-NKCC1 as well as p-SPAK/p-OSR1expression in peri-infarct cortex, striatum and corpus callosum in wild type mice subjected to transient middle cerebral artery occlusion. In WNK3 knockout mice, ischemia-induced hyper-phosphorylations of both the SPAK/OSR1 catalytic T-loop and NKCC1 are abolished, infarct volume and axonal demyelination are reduced, and neurobehavioral recovery is accelerated. These data provide fresh insight into the roles of ion transporters and their regulatory kinases in ischemic neuroglial injury, and identify the WNK3-SPAK/OSR1 kinase complex as a compelling target for novel neuroprotective strategies following stroke. Support: NIH R01NS38118

AtWNK9 is regulated by ABA and dehydration and is involved in drought tolerance in Arabidopsis

WNK (with no lysine [K]) kinases play important regulatory roles in flowering, as well as salt and osmotic stress tolerance in plants. Here, we report that AtWNK9, a member of the Arabidopsis WNK gene family, was induced by exogenous abscisic acid (ABA) treatment and dehydration stress. Overexpression of AtWNK9 from the cauliflower mosaic virus 35S promoter in Arabidopsis resulted in increased sensitivity to ABA, strong inhibition of primary root elongation, increased proline accumulation, reduced stomatal aperture, and a reduced rate of water loss. In addition, plant survival under drought stress was improved compared to wild type. In contrast, a mutant with a T-DNA insertion in AtWNK9 showed reduced ABA sensitivity and an increased rate of water loss; further, it showed increased susceptibility to drought stress. The transcription of a number of ABA signaling components, including ABI1, ERA1, ABI3, and ABF3, was up-regulated in AtWNK9 transgenic plants and down-regulated in the wnk9 mutant in response to ABA. Some ABA-responsive and biosynthetic genes, as well as other drought-related genes, were altered at various levels in AtWNK9 transgenic plants and wnk9 mutants under dehydration stress. Overall, these findings suggest that AtWNK9 plays a positive role in ABA signaling and improves drought tolerance in transgenic Arabidopsis.

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.

A New Methodology for Quantification of Alternatively Spliced Exons Reveals a Highly Tissue-Specific Expression Pattern of WNK1 Isoforms

Mutations in the WNK1 gene, encoding a serine-threonine kinase of the WNK (With No lysine (K)) family, have been implicated in two rare human diseases, Familial Hyperkalemic Hypertension (FHHt) and Hereditary Sensory and Autonomic Neuropathy type 2 (HSAN2). Alternative promoters give rise to a ubiquitous isoform, L-WNK1, and a kidney-specific isoform, KS-WNK1. Several other isoforms are generated through alternative splicing of exons 9, 11 and 12 but their precise tissue distribution is not known. Two additional exons, 8b and HSN2, involved in HSAN2, are thought to be specifically expressed in the nervous system. The purpose of this study was to establish an exhaustive description of all WNK1 isoforms and to quantify their relative level of expression in a panel of human and mouse tissues and in mouse nephron segments. For the latter purpose, we developed a new methodology allowing the determination of the proportions of the different isoforms generated by alternative splicing. Our results evidenced a striking tissue-specific distribution of the different isoforms and the unexpected presence of exon HSN2 in many tissues other than the nervous system. We also found exon 26 to be alternatively spliced in human and identified two new exons, 26a and 26b, within intron 26, specifically expressed in nervous tissues both in humans and mice. WNK1 should therefore no longer be designated as a 28- but as a 32-exon gene, with 8 of them - 8b, HSN2, 9, 11, 12, 26, 26a and 26b - alternatively spliced in a tissue-specific manner. These tissue-specific isoforms must be considered when studying the different roles of this ubiquitous kinase.

Overexpression of the soybean GmWNK1 altered the sensitivity to salt and osmotic stress in Arabidopsis

The WNK (With No Lysine K) serine-threonine kinases have been shown to be osmosensitive regulators and are critical for cell volume homeostasis in humans. We previously identified a soybean root-specific WNK homolog, GmWNK1, which is important for normal late root development by fine-tuning regulation of ABA levels. However, the functions of WNKs in plant osmotic stress response remains uncertain. In this study, we generated transgenic Arabidopsis plants with constitutive expression of GmWNK1. We found that these transgenic plants had increased endogenous ABA levels and altered expression of ABA-responsive genes, and exhibited a significantly enhanced tolerance to NaCl and osmotic stresses during seed germination and seedling development. These findings suggest that, in addition to regulating root development, GmWNK1 also regulates ABA-responsive gene expression and/or interacts with other stress related signals, thereby modulating osmotic stress responses. Thus, these results suggest that WNKs are members of an evolutionarily conserved kinase family that modulates cellular response to osmotic stresses in both animal and plants.

WNK2 Kinase Is a Novel Regulator of Essential Neuronal Cation-Chloride Cotransporters

NKCC1 and KCC2, related cation-chloride cotransporters (CCC), regulate cell volume and γ-aminobutyric acid (GABA)-ergic neurotranmission by modulating the intracellular concentration of chloride [Cl−]. These CCCs are oppositely regulated by serine-threonine phosphorylation, which activates NKCC1 but inhibits KCC2. The kinase(s) that performs this function in the nervous system are not known with certainty. WNK1 and WNK4, members of the WNK (with no lysine [K]) kinase family, either directly or via the downstream SPAK/OSR1 Ste20-type kinases, regulate the furosemide-sensitive NKCC2 and the thiazide-sensitive NCC, kidney-specific CCCs. What role the novel WNK2 kinase plays in this regulatory cascade, if any, is unknown. Here, we show that WNK2, unlike other WNKs, is not expressed in kidney; rather, it is a neuron-enriched kinase primarily expressed in neocortical pyramidal cells, thalamic relay cells, and cerebellar granule and Purkinje cells in both the developing and adult brain. Bumetanide-sensitive and Cl−-dependent 86Rb+ uptake assays in Xenopus laevis oocytes revealed that WNK2 promotes Cl− accumulation by reciprocally activating NKCC1 and inhibiting KCC2 in a kinase-dependent manner, effectively bypassing normal tonicity requirements for cotransporter regulation. TiO2 enrichment and tandem mass spectrometry studies demonstrate WNK2 forms a protein complex in the mammalian brain with SPAK, a known phosphoregulator of NKCC1. In this complex, SPAK is phosphorylated at Ser-383, a consensus WNK recognition site. These findings suggest a role for WNK2 in the regulation of CCCs in the mammalian brain, with implications for both cell volume regulation and/or GABAergic signaling.

Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2

Nedd4-2 is an archetypal HECT ubiquitin E3 ligase that disposes target proteins for degradation. Because of the proven roles of Nedd4-2 in degradation of membrane proteins, such as epithelial Na(+) channel, we examined the effect of Nedd4-2 on the apical Ca(2+) channel TRPV6, which is involved in transcellular Ca(2+) transport in the intestine using the Xenopus laevis oocyte system. We demonstrated that a significant amount of Nedd4-2 protein was distributed to the absorptive epithelial cells in ileum, cecum, and colon along with TRPV6. When co-expressed in oocytes, Nedd4-2 and, to a lesser extent, Nedd4 down-regulated the protein abundance and Ca(2+) influx of TRPV6 and TRPV5, respectively. TRPV6 ubiquitination was increased, and its stability was decreased by Nedd4-2. The Nedd4-2 inhibitory effects on TRPV6 were partially blocked by proteasome inhibitor MG132 but not by the lysosome inhibitor chloroquine. The rate of TRPV6 internalization was not significantly altered by Nedd4-2. The HECT domain was essential to the inhibitory effect of Nedd4-2 on TRPV6 and to their association. The WW1 and WW2 domains interacted with TRPV6 terminal regions, and a disruption of the interactions by D204H and D376H mutations in the WW1 and WW2 domains increased TRPV6 ubiquitination and degradation. Thus, WW1 and WW2 may serve as a molecular switch to limit the ubiquitination of TRPV6 by the HECT domain. In conclusion, Nedd4-2 may regulate TRPV6 protein abundance in intestinal epithelia by controlling TRPV6 ubiquitination.

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.

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 kinase family (WNK1 and WNK4) cause pseudohypoaldosteronism type II featuring hypertension, hyperkalemia and metabolic acidosis. WNK1 and WNK4 were shown to modulate NCC activity through SPAK and OSR1. Activation of PKC by a phorbol ester inhibits NCC function via activation of ERK1/2 kinase. To investigate whether WNK4 affects NCC via MAPK signaling pathway besides SPAK and OSR1, we first determine whether WNK4 phosphorylates ERK1/2 kinase. We found that WNK4 increases the phosphorylation of ERK1/2 in a dose‐dependent manner in mouse distal convoluted tubule (mDCT) cells, whereas WNK4 mutants with the PHA II mutations (E562K and R1185C) do not increase the ERK1/2 phosphorylation. We next showed that hypertonic stimulation significantly increased ERK1/2 phosphorylation in mDCT cells. The ERK1/2 activation by hypertonic stimulation was significantly attenuated by WNK4 siRNA knock‐down expression (23.19 ± 8.21 % in siWNK4 group vs 100 % in control group, p &lt;0.01, n=3). We further showed that WNK4 knock‐down significantly increases total NCC expression (100 ± 0 % vs 120.7 ± 6.2 %, p &lt;0.05, n=4) as well as NCC surface expression (100 ± 0 % vs 256.8 ± 50.3 %, p &lt;0.05, n=4). We conclude that the inhibitory effect of WNK4 on NCC is mediated by MAPK ERK 1/2 signaling pathway.Note: BZ and YZ contributed equally to this work.

LINGO-1 Interacts with WNK1 to Regulate Nogo-induced Inhibition of Neurite Extension

LINGO-1 is a component of the tripartite receptor complexes, which act as a convergent mediator of the intracellular signaling in response to myelin-associated inhibitors and lead to collapse of growth cone and inhibition of neurite extension. Although the function of LINGO-1 has been intensively studied, its downstream signaling remains elusive. In the present study, a novel interaction between LINGO-1 and a serine-threonine kinase WNK1 was identified by yeast two-hybrid screen. The interaction was further validated by fluorescence resonance energy transfer and co-immunoprecipitation, and this interaction was intensified by Nogo66 treatment. Morphological evidences showed that WNK1 and LINGO-1 were co-localized in cortical neurons. Furthermore, either suppressing WNK1 expression by RNA interference or overexpression of WNK1-(123-510) attenuated Nogo66-induced inhibition of neurite extension and inhibited the activation of RhoA. Moreover, WNK1 was identified to interact with Rho-GDI1, and this interaction was attenuated by Nogo66 treatment, further indicating its regulatory effect on RhoA activation. Taken together, our results suggest that WNK1 is a novel signaling molecule involved in regulation of LINGO-1 mediated inhibition of neurite extension.