Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
J Biol Chem
2011 Aug 26;28634:30171-80. doi: 10.1074/jbc.M111.222893.
Show Gene links
Show Anatomy links
WNK2 kinase is a novel regulator of essential neuronal cation-chloride cotransporters.
Rinehart J, Vázquez N, Kahle KT, Hodson CA, Ring AM, Gulcicek EE, Louvi A, Bobadilla NA, Gamba G, Lifton RP.
???displayArticle.abstract??? 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/OSR1Ste20-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 (86)Rb(+) 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. TiO(2) 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.
FIGURE 1. Analysis of WNK2 mRNA expression in mouse brain and kidney as compared with other WNK kinases. WNK2 localization (blue) by in situ hybridization shows a high level of expression in mouse brain both in embryonic development (E14.5) and in the adult (top two panels). A representative coronal section of the E14.5 brain (top left) is compared with sections of the adult brain (top right). Whole sections are shown in the inset on top of representative magnified region of the cortex. WNK2 is highly expressed in post-mitotic neurons in the cortical plate (CP) and intermediate zone (IZ) at E14.5. WNK2 is expressed in all cortical layers in the adult cortex (ctx) (top right). WNK2 expression in the adult cerebellum (bottom left). A whole section is shown in the inset on top of a magnified region of the cerebellum. Purkinje cells (pjk), the granular cell layer (gr), and the white matter (wm) are indicated. WNK2, WNK1, and WNK4 mRNA expression in the mouse kidney (bottom right). A whole sagittal section is shown in the insets below images of a magnified region of the cortex (ctx) for each WNK kinase indicated.
FIGURE 2. Characterization of WNK2-expressing cells in mouse brain. SMI-32 co-localization (brown) highlights WNK2 expression in pyramidal neurons (pyr) in the developing neocortex (top panel). GFAP-expressing astrocytes do not express Wnk2 (middle). The arrowhead marks an astrocyte (ast). WNK2 mRNA is expressed in the Purkinje cells (pc) in the cerebellum, labeled with calbindin (bottom). The molecular layer (mol) and granular layer (gr) border the Purkinje cell layer in the cerebellum.
FIGURE 3. Modulation of KCC2, KCC4, and NKCC1 activity by WNK2. Xenopus laevis oocytes were injected with (A) water or 10 ng/oocyte of KCC2 cRNA, (B) water or 10 ng/oocyte of KCC2 or KCC4 cRNA, and (C) water, or 10 ng/oocyte of NKCC1 cRNA. In all figures, cotransporters cRNA were injected alone or together with 10 ng/oocyte of WNK2 or WNK3 cRNA, as stated. Four days later, 86Rb+ influx experiments were performed in isotonic (A and C) or hypotonic conditions (B) in the presence (open bars) or absence (closed bars) of extracellular chloride (A and B) or in the absence (open bars) or presence (closed bars) of 100 μm bumetanide (C). *, significantly different from the uptake observed in corresponding control. Each figure depicts combined data of at least three different experiments.
FIGURE 4. Effect of WNK2 and WNK3 on the protein level and surface expression of NKCC1 and KCC4. Western blot analysis of the total and biotinylated fraction of proteins extracted from oocytes injected with NKCC1 (A) or KCC4 (B) cRNA in the absence or presence of WNK2 or WNK3 cRNA. C and D depict the results of the functional expression performed the same day using oocytes from the same batch for NKCC1 or KCC4, respectively, expressed as bumetanide-sensitive (C) or chloride-dependent (D) 86Rb+ uptake.
FIGURE 5. Effect of kinase-dead (DA) WNK2 upon NKCC1 and KCCs cotransporters activity. A, 86Rb+ uptake in oocytes injected with water, NKCC1 cRNA alone or together with WNK2-DA or WNK3-DA cRNA. Uptake was performed in isotonic conditions in the absence (open bars) or presence (closed bars) of 100 μm bumetanide. *, significantly different from the uptake observed in NKCC1 cRNA group. B, oocytes were injected with water, KCC2 cRNA alone or together with kinase-dead WNK2 or WNK3, as stated. C, oocytes were injected with water, KCC2, or KCC4 cRNA alone or together with kinase-dead WNK2 or WNK3, as stated. 86Rb+ Uptake was performed in in the presence (open bars) or absence (closed bars) of extracellular Cl−, in hypotonic (B) or isotonic (C) conditions. *, significantly different from the uptake observed in corresponding control. D, representative Western blot analysis of protein homogenates extracted from oocytes injected with wild type or kinase-dead HA-WNK2 cRNA, together with NKCC1 or KCC2 cRNA, as stated. Western blots were then performed using anti-HA monoclonal antibodies. Similar results were observed in the absence of NKCC1 cRNA injections.
FIGURE 6. MS/MS analysis of WNK2 phosphorylation. A, phosphorylation sites are shown in the context of full-length human WNK2. The conserved kinase domain and two coiled-coiled regions are labeled. Phosphorylation sites identified in human WNK2 expressed in HEK cells are numbered on the bottom of the diagram. Sites unique to this study are in black, and sites in common with other studies are in red. Phosphorylation sites identified in native mouse WNK2 are numbered according to their position in the mouse sequence and mapped onto the corresponding position in human WNK2. Phosphorylation at Ser-1770 (mouse) and Ser-1862 (human) are homologous. B, SDS-PAGE analyisis of native WNK2 immunoprecipitates prepared from mouse brain for MS/MS. An example gel is shown with the results of the MS/MS analysis for each protein band shown to the left. Phosphorylation sites from full-length WNK2 and SPAK identified by MS/MS are listed (for additional peptide information see supplemental Tables S1 and S2. A brief Coomassie-staining method was used to reveal the proteins bands to the naked eye. Bands that were unique to brain IP and not visible in a control IP from mouse kidney (not shown) were cut and subjected to MS analysis. C, table of WNK2 phosphopeptides identified in MS/MS analysis of purified human and mouse WNK2. Mascot searches followed by manual MS/MS spectra interpretation places the possible phosphorylation site within each peptide. Unambiguous assignment of phosphorylation sites are indicated in underlined, red capital S, while ambiguous assignments have the second most likely site as a lowercase s. Kinase motifs corresponding to each phosphorylation site were identified with NetworKIN and NetPhorest analysis. Abbreviations are for Rho-associated protein kinase 1 (DMPK), cAMP-dependent protein kinase (PKA), casein kinase II (CKII), PKC protein kinase CΔ (PKC), p21-activated kinase (PAK), p38 mitogen-activated protein kinase (p38MAPK), and cyclin-dependent kinases (CDK2, 3, and 5).
FIGURE 7. Relationship between GABA excitatory and inhibitory effect and the expression of WNK2, WNK3, NKCC1, and KCC2 in the CNS from embryonic to postnatal life.
Arroyo,
Nedd4-2 modulates renal Na+-Cl- cotransporter via the aldosterone-SGK1-Nedd4-2 pathway.
2011, Pubmed,
Xenbase
Arroyo,
Nedd4-2 modulates renal Na+-Cl- cotransporter via the aldosterone-SGK1-Nedd4-2 pathway.
2011,
Pubmed
,
Xenbase Ben-Ari,
GABA: a pioneer transmitter that excites immature neurons and generates primitive oscillations.
2007,
Pubmed Blaesse,
Cation-chloride cotransporters and neuronal function.
2009,
Pubmed Bodenmiller,
Reproducible isolation of distinct, overlapping segments of the phosphoproteome.
2007,
Pubmed Breitwieser,
Osmotic stimulation of Na(+)-K(+)-Cl- cotransport in squid giant axon is [Cl-]i dependent.
1990,
Pubmed Byun,
Axonal and periaxonal swelling precede peripheral neurodegeneration in KCC3 knockout mice.
2007,
Pubmed Cai,
WNK4 kinase regulates surface expression of the human sodium chloride cotransporter in mammalian cells.
2006,
Pubmed
,
Xenbase Campbell,
Monoclonal antibody to neurofilament protein (SMI-32) labels a subpopulation of pyramidal neurons in the human and monkey neocortex.
1989,
Pubmed Choate,
WNK1, a kinase mutated in inherited hypertension with hyperkalemia, localizes to diverse Cl- -transporting epithelia.
2003,
Pubmed Davies,
Somatic mutations of the protein kinase gene family in human lung cancer.
2005,
Pubmed de Los Heros,
WNK3 bypasses the tonicity requirement for K-Cl cotransporter activation via a phosphatase-dependent pathway.
2006,
Pubmed
,
Xenbase Delpire,
SPAK and OSR1: STE20 kinases involved in the regulation of ion homoeostasis and volume control in mammalian cells.
2008,
Pubmed Duebel,
Two-photon imaging reveals somatodendritic chloride gradient in retinal ON-type bipolar cells expressing the biosensor Clomeleon.
2006,
Pubmed Gagnon,
On the substrate recognition and negative regulation of SPAK, a kinase modulating Na+-K+-2Cl- cotransport activity.
2010,
Pubmed
,
Xenbase Gagnon,
Volume sensitivity of cation-Cl- cotransporters is modulated by the interaction of two kinases: Ste20-related proline-alanine-rich kinase and WNK4.
2006,
Pubmed
,
Xenbase Gamba,
Role of WNK kinases in regulating tubular salt and potassium transport and in the development of hypertension.
2005,
Pubmed Gamba,
Molecular physiology and pathophysiology of electroneutral cation-chloride cotransporters.
2005,
Pubmed Garzón-Muvdi,
WNK4 kinase is a negative regulator of K+-Cl- cotransporters.
2007,
Pubmed
,
Xenbase Geng,
The Ste20 kinases Ste20-related proline-alanine-rich kinase and oxidative-stress response 1 regulate NKCC1 function in sensory neurons.
2009,
Pubmed Golbang,
Regulation of the expression of the Na/Cl cotransporter by WNK4 and WNK1: evidence that accelerated dynamin-dependent endocytosis is not involved.
2006,
Pubmed
,
Xenbase Greenman,
Patterns of somatic mutation in human cancer genomes.
2007,
Pubmed Haas,
Inhibition of the Sodium-Potassium-Chloride Cotransporter Isoform-1 reduces glioma invasion.
2010,
Pubmed Hoffmann,
Physiology of cell volume regulation in vertebrates.
2009,
Pubmed Hong,
Epigenome scans and cancer genome sequencing converge on WNK2, a kinase-independent suppressor of cell growth.
2007,
Pubmed Jun,
Epigenetic silencing of the kinase tumor suppressor WNK2 is tumor-type and tumor-grade specific.
2009,
Pubmed Kahle,
WNK3 modulates transport of Cl- in and out of cells: implications for control of cell volume and neuronal excitability.
2005,
Pubmed
,
Xenbase Kahle,
WNK4 regulates apical and basolateral Cl- flux in extrarenal epithelia.
2004,
Pubmed
,
Xenbase Kahle,
Roles of the cation-chloride cotransporters in neurological disease.
2008,
Pubmed Kahle,
Molecular physiology of the WNK kinases.
2008,
Pubmed Kahle,
WNK protein kinases modulate cellular Cl- flux by altering the phosphorylation state of the Na-K-Cl and K-Cl cotransporters.
2006,
Pubmed Lenertz,
Properties of WNK1 and implications for other family members.
2005,
Pubmed Linding,
Systematic discovery of in vivo phosphorylation networks.
2007,
Pubmed Louvi,
Ectopic engrailed 1 expression in the dorsal midline causes cell death, abnormal differentiation of circumventricular organs and errors in axonal pathfinding.
2000,
Pubmed Lytle,
The Na-K-Cl cotransport protein of shark rectal gland. II. Regulation by direct phosphorylation.
1992,
Pubmed Lytle,
Coordinate modulation of Na-K-2Cl cotransport and K-Cl cotransport by cell volume and chloride.
2002,
Pubmed Marty,
Excitatory effects of GABA in established brain networks.
2005,
Pubmed McManus,
Regulation of cell volume in health and disease.
1995,
Pubmed Moniz,
Emerging roles for WNK kinases in cancer.
2010,
Pubmed Moriguchi,
WNK1 regulates phosphorylation of cation-chloride-coupled cotransporters via the STE20-related kinases, SPAK and OSR1.
2005,
Pubmed O'Reilly,
Dietary electrolyte-driven responses in the renal WNK kinase pathway in vivo.
2006,
Pubmed
,
Xenbase Piechotta,
Characterization of the interaction of the stress kinase SPAK with the Na+-K+-2Cl- cotransporter in the nervous system: evidence for a scaffolding role of the kinase.
2003,
Pubmed
,
Xenbase Pinkse,
Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns.
2004,
Pubmed Plotkin,
Expression of the Na(+)-K(+)-2Cl- cotransporter BSC2 in the nervous system.
1997,
Pubmed Plotkin,
Expression of the Na-K-2Cl cotransporter is developmentally regulated in postnatal rat brains: a possible mechanism underlying GABA's excitatory role in immature brain.
1997,
Pubmed Ponce-Coria,
Regulation of NKCC2 by a chloride-sensing mechanism involving the WNK3 and SPAK kinases.
2008,
Pubmed
,
Xenbase Rafiqi,
Role of the WNK-activated SPAK kinase in regulating blood pressure.
2010,
Pubmed Richardson,
The regulation of salt transport and blood pressure by the WNK-SPAK/OSR1 signalling pathway.
2008,
Pubmed Richardson,
Activation of the thiazide-sensitive Na+-Cl- cotransporter by the WNK-regulated kinases SPAK and OSR1.
2008,
Pubmed Rinehart,
WNK3 kinase is a positive regulator of NKCC2 and NCC, renal cation-Cl- cotransporters required for normal blood pressure homeostasis.
2005,
Pubmed
,
Xenbase Rinehart,
Sites of regulated phosphorylation that control K-Cl cotransporter activity.
2009,
Pubmed Rivera,
The K+/Cl- co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation.
1999,
Pubmed Rudnik-Schöneborn,
Andermann syndrome can be a phenocopy of hereditary motor and sensory neuropathy--report of a discordant sibship with a compound heterozygous mutation of the KCC3 gene.
2009,
Pubmed Schomberg,
Cross talk between the GABA(A) receptor and the Na-K-Cl cotransporter is mediated by intracellular Cl-.
2003,
Pubmed Shekarabi,
Mutations in the nervous system--specific HSN2 exon of WNK1 cause hereditary sensory neuropathy type II.
2008,
Pubmed Sjöblom,
The consensus coding sequences of human breast and colorectal cancers.
2006,
Pubmed Song,
Molecular, functional, and genomic characterization of human KCC2, the neuronal K-Cl cotransporter.
2002,
Pubmed
,
Xenbase Subramanya,
WNK4 diverts the thiazide-sensitive NaCl cotransporter to the lysosome and stimulates AP-3 interaction.
2009,
Pubmed
,
Xenbase Tole,
Regionalization of the developing forebrain: a comparison of FORSE-1, Dlx-2, and BF-1.
1995,
Pubmed Veríssimo,
WNK kinases, a novel protein kinase subfamily in multi-cellular organisms.
2001,
Pubmed Vitari,
The WNK1 and WNK4 protein kinases that are mutated in Gordon's hypertension syndrome phosphorylate and activate SPAK and OSR1 protein kinases.
2005,
Pubmed Williams,
The neuron-specific K-Cl cotransporter, KCC2. Antibody development and initial characterization of the protein.
1999,
Pubmed Wilson,
Human hypertension caused by mutations in WNK kinases.
2001,
Pubmed Wilson,
Molecular pathogenesis of inherited hypertension with hyperkalemia: the Na-Cl cotransporter is inhibited by wild-type but not mutant WNK4.
2003,
Pubmed
,
Xenbase Xu,
WNK1, a novel mammalian serine/threonine protein kinase lacking the catalytic lysine in subdomain II.
2000,
Pubmed Zagórska,
Regulation of activity and localization of the WNK1 protein kinase by hyperosmotic stress.
2007,
Pubmed
