Characterization of SPAK and OSR1, regulatory kinases of the Na-K-2Cl cotransporter

Our recent studies demonstrate that SPAK (Ste20p-related Proline Alanine-rich Kinase), in combination with WNK4 [With No lysine (K) kinase], phosphorylates and stimulates the Na-K-2Cl cotransporter (NKCC1), whereas catalytically inactive SPAK (K104R) fails to activate the cotransporter. The catalytic domain of SPAK contains an activation loop between the well-conserved DFG and APE motifs. We speculated that four threonine residues (T231, T236, T243, and T247) in the activation loop might be sites of phosphorylation and kinase activation; therefore, we mutated each residue into an alanine. In this report, we demonstrate that coexpression of SPAK (T243A) or SPAK (T247A) with WNK4 not only prevented, but robustly inhibited, cotransporter activity in NKCC1-injected Xenopus laevis oocytes. These activation loop mutations produced an effect similar to that of the SPAK (K104R) mutant. In vitro phosphorylation experiments demonstrate that both intramolecular autophosphorylation of SPAK and phosphorylation of NKCC1 are significantly stronger in the presence of Mn2+ rather than Mg2+. We also show that SPAK activity is markedly inhibited by staurosporine and K252a, partially inhibited by N-ethylmaleimide and diamide, and unaffected by arsenite. OSR1, a kinase closely related to SPAK, exhibited similar kinase properties and similar functional activation of NKCC1 when coexpressed with WNK4.     

Apoptosis-associated tyrosine kinase scaffolding of protein phosphatase 1 and SPAK reveals a novel pathway for Na-K-2C1 cotransporter regulation

Previous work from our laboratory and others has established that Ste-20-related proline-alanine-rich kinase (SPAK/PASK) is central to the regulation of NKCC1 function. With no lysine (K) kinase (WNK4) has also been implicated in the regulation of NKCC1 activity through upstream activation of SPAK. Because previous studies from our laboratory also demonstrated a protein-protein interaction between SPAK and apoptosis-associated tyrosine kinase (AATYK), we explore here the possibility that AATYK is another component of the regulation of NKCC1. Heterologous expression of AATYK1 in NKCC1-injected Xenopus laevis oocytes markedly inhibited cotransporter activity under isosmotic conditions. Interestingly, mutation of key residues in the catalytic domain of AATYK1 revealed that the kinase activity does not play a role in the suppression of NKCC1 function. However, mutagenesis of the two SPAK-binding motifs in AATYK1 completely abrogated this effect. As protein phosphatase 1 (PP1) also plays a central role in the dephosphorylation and inactivation of NKCC1, we investigated the possibility that AATYK1 interacts with the phosphatase. We identified a PP1 docking motif in AATYK1 and demonstrated interaction using yeast-2-hybrid analysis. Mutation of a key valine residue (V1175) within this motif prevented protein-protein interaction. Furthermore, the physical interaction between PP1 and AATYK was required for inhibition of NKCC1 activity in Xenopus laevis oocytes. Taken together, our data are consistent with AATYK1 indirectly inhibiting the SPAK/WNK4 activation of the cotransporter by scaffolding an inhibitory phosphatase in proximity to a stimulatory kinase. ion fluxes; Xenopus laevis oocytes; yeast-2 hybrid; phosphorylation     

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

Activity of heterologously expressed NKCC1 was analyzed under basal and activated conditions in the presence and absence of binding of Ste20-related proline-alanine-rich kinase (SPAK). Mutant NKCC1 that lacks the ability to bind to this kinase showed K+ transport function identical to wild-type NKCC1. Thus, preventing the binding of the kinase to the cotransporter does not affect cotransporter function. In contrast, several experiments suggest a possible role for SPAK as a scaffolding protein. First, Western blot analysis revealed the presence, and in some tissues abundance, of truncated forms of SPAK and OSR1 in which the kinase domains are affected and thus lack kinase activity. Second, a yeast two-hybrid screen of proteins that interact with the regulatory (binding) domain of SPAK identified several proteins all involved in cellular stress pathways. Third, p38, one of the three major MAPKs, can be coimmunoprecipitated with SPAK and with NKCC1 in an activity-dependent manner. The amount of p38 coimmunoprecipitated with the kinase and the cotransporter significantly decreases upon cellular stress, whereas the interaction of the kinase with NKCC1 remains unchanged. These findings suggest that cation-chloride cotransporters might act as "sensors" for cellular stress, and SPAK, by interacting with the cotransporter, serves as an intermediate in the response to cellular stress.     

Volume sensitivity of cation-Cl- cotransporters is modulated by the interaction of two kinases: Ste20-related proline-alanine-rich kinase and WNK4

In the present study, we have demonstrated functional interaction between Ste20-related proline-alanine-rich kinase (SPAK), WNK4 [with no lysine (K)], and the widely expressed Na⁺-K⁺-2Cl^– cotransporter type 1 (NKCC1). NKCC1 function, which we measured in Xenopus laevis oocytes under both isosmotic (basal) and hyperosmotic (stimulated) conditions, was unaffected when SPAK and WNK4 were expressed alone. In contrast, expression of both kinases with NKCC1 resulted in a significant increase in cotransporter activity and an insensitivity to external osmolarity or cell volume. NKCC1 activation is dependent on the catalytic activity of SPAK and likely also of WNK4, because mutations in their catalytic domains result in an absence of cotransporter stimulation. The results of our yeast two-hybrid experiments suggest that WNK4 does not interact directly with NKCC1 but does interact with SPAK. Functional experiments demonstrated that the binding of SPAK to WNK4 was also required because a SPAK-interaction-deficient WNK4 mutant (Phe997Ala) did not increase NKCC1 activity. We also have shown that the transport function of K⁺-Cl^– cotransporter type 2 (KCC2), a neuron-specific KCl cotransporter, was diminished by the expression of both kinases under both isosmotic and hyposmotic conditions. Our data are consistent with WNK4 interacting with SPAK, which in turn phosphorylates and activates NKCC1 and phosphorylates and deactivates KCC2. bumetanide; Na⁺-K⁺-2Cl^– cotransporter; K⁺-Cl^– cotransporter; Xenopus oocytes     

A Novel Ste20-related Proline/Alanine-rich Kinase (SPAK)-independent Pathway Involving Calcium-binding Protein 39 (Cab39) and Serine Threonine Kinase with No Lysine Member 4 (WNK4) in the Activation of Na-K-Cl Cotransporters

Na⁺-dependent chloride cotransporters (NKCC1, NKCC2, and NCC) are activated by phosphorylation to play critical roles in diverse physiological responses, including renal salt balance, hearing, epithelial fluid secretion, and volume regulation. Serine threonine kinase WNK4 (With No K = lysine member 4) and members of the Ste20 kinase family, namely SPAK and OSR1 (Ste20-related proline/alanine-rich kinase, Oxidative stress-responsive kinase) govern phosphorylation. According to present understanding, WNK4 phosphorylates key residues within SPAK/OSR1 leading to kinase activation, allowing SPAK/OSR1 to bind to and phosphorylate NKCC1, NKCC2, and NCC. Recently, the calcium-binding protein 39 (Cab39) has emerged as a binding partner and enhancer of SPAK/OSR1 activity, facilitating kinase autoactivation and promoting phosphorylation of the cotransporters. In the present study, we provide evidence showing that Cab39 differentially interacts with WNK4 and SPAK/OSR1 to switch the classic two kinase cascade into a signal kinase transduction mechanism. We found that WNK4 in association with Cab39 activates NKCC1 in a SPAK/OSR1-independent manner. We discovered that WNK4 possesses a domain that bears close resemblance to the SPAK/OSR1 C-terminal CCT/PF2 domain, which is required for physical interaction between the Ste20 kinases and the Na⁺-driven chloride cotransporters. Modeling, yeast two-hybrid, and functional data reveal that this PF2-like domain located downstream of the catalytic domain in WNK4 promotes the direct interaction between the kinase and NKCC1. We conclude that in addition to SPAK and OSR1, WNK4 is able to anchor itself to the N-terminal domain of NKCC1 and to promote cotransporter activation.     

Up-regulation of the clusterin gene after proteotoxic stress: implication of HSF1-HSF2 heterocomplexes

The SPAK (STE20/SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase-1) kinases interact and phosphorylate NKCC1 (Na+-K+-2Cl- co-transporter-1), leading to its activation. Recent studies indicated that SPAK and OSR1 are phosphorylated and activated by the WNK1 [with no K (lysine) protein kinase-1] and WNK4, genes mutated in humans affected by Gordon's hypertension syndrome. In the present study, we have identified three residues in NKCC1 (Thr175/Thr179/Thr184 in shark or Thr203/Thr207/Thr212 in human) that are phosphorylated by SPAK and OSR1, and have developed a peptide substrate, CATCHtide (cation chloride co-transporter peptide substrate), to assess SPAK and OSR1 activity. Exposure of HEK-293 (human embryonic kidney) cells to osmotic stress, which leads to phosphorylation and activation of NKCC1, increased phosphorylation of NKCC1 at the sites targeted by SPAK/OSR1. The residues on NKCC1, phosphorylated by SPAK/OSR1, are conserved in other cation co-transporters, such as the Na+-Cl- co-transporter, the target of thiazide drugs that lower blood pressure in humans with Gordon's syndrome. Furthermore, we characterize the properties of a 92-residue CCT (conserved C-terminal) domain on SPAK and OSR1 that interacts with an RFXV (Arg-Phe-Xaa-Val) motif present in the substrate NKCC1 and its activators WNK1/WNK4. A peptide containing the RFXV motif interacts with nanomolar affinity with the CCT domains of SPAK/OSR1 and can be utilized to affinity-purify SPAK and OSR1 from cell extracts. Mutation of the arginine, phenylalanine or valine residue within this peptide abolishes binding to SPAK/OSR1. We have identified specific residues within the CCT domain that are required for interaction with the RFXV motif and have demonstrated that mutation of these in OSR1 inhibited phosphorylation of NKCC1, but not of CATCHtide which does not possess an RFXV motif. We establish that an intact CCT domain is required for WNK1 to efficiently phosphorylate and activate OSR1. These data establish that the CCT domain functions as a multipurpose docking site, enabling SPAK/OSR1 to interact with substrates (NKCC1) and activators (WNK1/WNK4).     

WNK3-SPAK interaction is required for the modulation of NCC and other members of the SLC12 family

The serine/threonine with no lysine kinase 3 (WNK3) modulates the activity of the electroneutral cation-coupled chloride cotransporters (CCC) to promote Cl(-) influx and prevent Cl(-) efflux, thus fitting the profile for a putative "Cl(-)-sensing kinase". The Ste20-type kinases, SPAK/OSR1, become phosphorylated in response to reduction in intracellular chloride concentration and regulate the activity of NKCC1. Several studies have now shown that WNKs function upstream of SPAK/OSR1. This study was designed to analyze the role of WNK3-SPAK interaction in the regulation of CCCs with particular emphasis on NCC. In this study we used the functional expression system of Xenopus laevis oocytes to show that different SPAK binding sites in WNK3 ((241, 872, 1336)RFxV) are required for the kinase to have effects on CCCs. WNK3-F1337A no longer activated NKCC2, but the effects on NCC, NKCC1, and KCC4 were preserved. In contrast, the effects of WNK3 on these cotransporters were prevented in WNK3-F242A. The elimination of F873 had no consequence on WNK3 effects. WNK3 promoted NCC phosphorylation at threonine 58, even in the absence of the unique SPAK binding site of NCC, but this effect was abolished in the mutant WNK3-F242A. Thus, our data support the hypothesis that the effects of WNK3 upon NCC and other CCCs require the interaction and activation of the SPAK kinase. The effect is dependent on one of the three binding sites for SPAK that are present in WNK3, but not on the SPAK binding sites on the CCCs, which suggests that WNK3 is capable of binding both SPAK and CCCs to promote their phosphorylation.     

PKCdelta acts upstream of SPAK in the activation of NKCC1 by hyperosmotic stress in human airway epithelial cells

Airway epithelial Na-K-2Cl (NKCC1) cotransport is activated through hormonal stimulation and hyperosmotic stress via a protein kinase C (PKC) delta-mediated intracellular signaling pathway. Down-regulation of PKCdelta prevents activation of NKCC1 expressed in Calu-3 cells. Previous studies of this signaling pathway identified coimmunoprecipitation of PKCdelta with SPAK (Ste20-related proline alanine-rich kinase). We hypothesize that endogenous PKCdelta activates SPAK, which subsequently activates NKCC1 through phosphorylation. Double-stranded silencing RNA directed against SPAK reduced SPAK protein expression by 65.8% and prevented increased phosphorylation of NKCC1 and functional activation of NKCC1 during hyperosmotic stress, measured as bumetanide-sensitive basolateral to apical (86)Rb flux. Using recombinant proteins, we demonstrate direct binding of PKCdelta to SPAK, PKCdelta-mediated activation of SPAK, binding of SPAK to the amino terminus of NKCC1 (NT-NKCC1, amino acids 1-286), and competitive inhibition of SPAK-NKCC1 binding by a peptide encoding a SPAK binding site on NT-NKCC1. The carboxyl terminus of SPAK (amino acids 316-548) pulls down endogenous NKCC1 from Calu-3 total cell lysates and glutathione S-transferase-tagged NT-NKCC1 pulls down endogenous SPAK. In intact cells, hyperosmotic stress increased phosphorylated PKCdelta, indicating activation of PKCdelta, and activity of endogenous SPAK kinase. Inhibition of PKCdelta activity with rottlerin blocked the increase in SPAK kinase activity. The results indicate that PKCdelta acts upstream of SPAK to increase activity of NKCC1 during hyperosmotic stress.     

Cation chloride cotransporters interact with the stress-related kinases Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress response 1 (OSR1)

Cells respond to stress stimuli by mounting specific responses. During osmotic and oxidative stress, cation chloride cotransporters, e.g. Na-K-2Cl and K-Cl cotransporters, are activated to maintain fluid/ion homeostasis. Here we report the interaction of the stress-related serine-threonine kinases Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress response 1 (OSR1) with the cotransporters KCC3, NKCC1, and NKCC2 but not KCC1 and KCC4. The interaction was identified using yeast two-hybrid assays and confirmed via glutathione S-transferase pull-down experiments. Evidence for in vivo interaction was established by co-immunoprecipitation of SPAK from mouse brain with anti-NKCC1 antibody. The interacting region of both kinases comprises the last 100 amino acids of the protein. The SPAK/OSR1 binding motif on the cotransporters consists of nine residues, starting with an (R/K)FX(V/I) sequence followed by five additional residues that are essential for binding but for which no consensus was found. Immunohistochemical analysis of choroid plexus epithelium revealed co-expression of NKCC1 and SPAK on the apical membrane. In contrast, in choroid plexus epithelium from NKCC1 null mice, SPAK immunostaining was found in the cytoplasm. We conclude that several cation chloride co-transporters interact with SPAK and/or OSR1, and we hypothesize that this interaction might play a role during the initiation of the cellular stress response.     

SPAK Isoforms and OSR1 Regulate Sodium-Chloride Co-transporters in a Nephron-specific Manner

STE20/SPS-1-related proline-alanine-rich protein kinase (SPAK) and oxidative stress-related kinase (OSR1) activate the potassium-dependent sodium-chloride co-transporter, NKCC2, and thiazide-sensitive sodium-chloride cotransporter, NCC, in vitro, and both co-localize with a kinase regulatory molecule, Cab39/MO25α, at the apical membrane of the thick ascending limb (TAL) and distal convoluted tubule (DCT). Yet genetic ablation of SPAK in mice causes a selective loss of NCC function, whereas NKCC2 becomes hyperphosphorylated. Here, we explore the underlying mechanisms in wild-type and SPAK-null mice. Unlike in the DCT, OSR1 remains at the TAL apical membrane of KO mice where it is accompanied by an increase in the active, phosphorylated form of AMP-activated kinase. We found an alterative SPAK isoform (putative SPAK2 form), which modestly inhibits co-transporter activity in vitro, is more abundant in the medulla than the cortex. Thus, enhanced NKCC2 phosphorylation in the SPAK knock-out may be explained by removal of inhibitory SPAK2, sustained activity of OSR1, and activation of other kinases. By contrast, the OSR1/SPAK/M025α signaling apparatus is disrupted in the DCT. OSR1 becomes largely inactive and displaced from M025α and NCC at the apical membrane, and redistributes to dense punctate structures, containing WNK1, within the cytoplasm. These changes are paralleled by a decrease in NCC phosphorylation and a decrease in the mass of the distal convoluted tubule, exclusive to DCT1. As a result of the dependent nature of OSR1 on SPAK in the DCT, NCC is unable to be activated. Consequently, SPAK^−/− mice are highly sensitive to dietary salt restriction, displaying prolonged negative sodium balance and hypotension.     

PASK (proline-alanine-rich STE20-related kinase), a regulatory kinase of the Na-K-Cl cotransporter (NKCC1)

Although the phosphorylation-dependent activation of the Na-K-Cl cotransporter (NKCC1) has been previously well documented, the identity of the kinase(s) responsible for this regulation has proven elusive. Recently, Piechotta et al. (Piechotta, K., Lu, J., and Delpire, E. (2002) J. Biol. Chem. 277, 50812-50819) reported the binding of PASK (also referred as SPAK (STE20/SPS1-related proline-alanine-rich kinase)) and OSR1 (oxidative stress response kinase) to cation-chloride cotransporters KCC3, NKCC1, and NKCC2. In this report, we show that overexpression of a kinase inactive, dominant negative (DN) PASK mutant drastically reduces both shark (60 +/- 5%) and human (80 +/- 3%) NKCC1 activation. Overexpression of wild type PASK causes a small (sNKCC1 22 +/- 8% p < 0.05, hNKCC1 12 +/- 3% p < 0.01) but significant increase in shark and human cotransporter activity in HEK cells. Importantly, DNPASK also inhibits the phosphorylation of two threonines, contained in the previously described N-terminal regulatory domain. We additionally show the near complete restoration of NKCC1 activity in the presence of the protein phosphatase type 1 inhibitor calyculin A, demonstrating that DNPASK inhibition results from an alteration in kinase/phosphatase dynamics rather than from a decrease in functional cotransporter expression. Coimmunoprecipitation assays confirm PASK binding to NKCC1 in transfected HEK cells and further suggest that this binding is not a regulated event; neither PASK nor NKCC1 activity affects the association. In cells preloaded with 32Pi, the phosphorylation of PASK, but not DNPASK, coincides with that of NKCC1 and increases 5.5 +/- 0.36-fold in low [Cl]e. These data conclusively link PASK with the phosphorylation and activation of NKCC1.     

WNK3 is a putative chloride-sensing kinase

The with-no-lysine kinase 3 (WNK3) is a serine/threonine kinase that modulates the activity of the electroneutral cation-coupled chloride cotransporters (CCC). Using the Xenopus laevis oocyte heterologous expression system, it has been shown that WNK3 activates the Na(+)-coupled chloride cotransporters NKCC1, NKCC2, and NCC and inhibits the K(+)-coupled chloride cotransporters KCC1 through KCC4. Interestingly, the effect of catalytically inactive WNK3 is opposite to that of wild type WNK3: inactive WNK3 inhibits NKCCs and activates KCCs. In doing so, wild type and catalytically inactive WNK3 bypass the tonicity requirement for activation/inhibition of the cotransporter. Thus, WNK3 modulation of the electroneutral cotransporters promotes Cl(-) influx and prevents Cl(-) efflux, thus fitting the profile for a putative "Cl(-)-sensing kinase". Other kinases that potentially have these properties are the Ste20-type kinases, SPAK/OSR1, which become phosphorylated in response to reductions in intracellular chloride concentration and regulate the activity of NKCC1. It has been demonstrated that WNKs lie upstream of SPAK/OSR1 and that the activity of these kinases is activated by phosphorylation of threonines in the T-loop by WNKs. It is possible that a protein phosphatase is also involved in the WNK3 effects on its associated cotransporters because activation of KCCs and inhibition of NKCCs by inactive WNK3 can be prevented by known inhibitors of protein phosphatases, such as calyculin A and cyclosporine, suggesting that a protein phosphatase is also involved in the protein complex.     

Structural insights into the recognition of substrates and activators by the OSR1 kinase

The oxidative-stress-responsive kinase 1 (OSR1) and the STE20/SPS1-related proline/alanine-rich kinase (SPAK) are key enzymes in a signalling cascade regulating the activity of Na(+)/K(+)/2Cl(-) co-transporters (NKCCs) in response to osmotic stress. Both kinases have a conserved carboxy-terminal (CCT) domain, which recognizes a unique peptide (Arg-Phe-Xaa-Val) motif present in OSR1- and SPAK-activating kinases (with-no-lysine kinase 1 (WNK1) and WNK4) as well as its substrates (NKCC1 and NKCC2). Here, we describe the structural basis of this recognition event as shown by the crystal structure of the CCT domain of OSR1 in complex with a peptide containing this motif, derived from WNK4. The CCT domain forms a novel protein fold that interacts with the Arg-Phe-Xaa-Val motif through a surface-exposed groove. An intricate web of interactions is observed between the CCT domain and an Arg-Phe-Xaa-Val motif-containing peptide derived from WNK4. Mutational analysis shows that these interactions are required for the CCT domain to bind to WNK1 and NKCC1. The CCT domain structure also shows how phosphorylation of a Ser/Thr residue preceding the Arg-Phe-Xaa-Val motif results in a steric clash, promoting its dissociation from the CCT domain. These results provide the first molecular insight into the mechanism by which the SPAK and OSR1 kinases specifically recognize their upstream activators and downstream substrates.     

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 (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.     

Shrinkage insensitivity of NKCC1 in myosin II-depleted cytoplasts from Ehrlich ascites tumor cells

Protein phosphorylation/dephosphorylation and cytoskeletal reorganization regulate the Na⁺-K⁺-2Cl^– cotransporter (NKCC1) during osmotic shrinkage; however, the mechanisms involved are unclear. We show that in cytoplasts, plasma membrane vesicles detached from Ehrlich ascites tumor cells (EATC) by cytochalasin treatment, NKCC1 activity evaluated as bumetanide-sensitive ⁸⁶Rb influx was increased compared with the basal level in intact cells yet could not be further increased by osmotic shrinkage. Accordingly, cytoplasts exhibited no regulatory volume increase after shrinkage. In cytoplasts, cortical F-actin organization was disrupted, and myosin II, which in shrunken EATC translocates to the cortical region, was absent. Moreover, NKCC1 activity was essentially insensitive to the myosin light chain kinase (MLCK) inhibitor ML-7, a potent blocker of shrinkage-induced NKCC1 activity in intact EATC. Cytoplast NKCC1 activity was potentiated by the Ser/Thr protein phosphatase inhibitor calyculin A, partially inhibited by the protein kinase A inhibitor H89, and blocked by the broad protein kinase inhibitor staurosporine. Cytoplasts exhibited increased protein levels of NKCC1, Ste20-related proline- and alanine-rich kinase (SPAK), and oxidative stress response kinase 1, yet they lacked the shrinkage-induced plasma membrane translocation of SPAK observed in intact cells. The basal phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was increased in cytoplasts compared with intact cells, yet in contrast to the substantial activation in shrunken intact cells, p38 MAPK could not be further activated by shrinkage of the cytoplasts. Together these findings indicate that shrinkage activation of NKCC1 in EATC is dependent on the cortical F-actin network, myosin II, and MLCK. F-actin; Na⁺-K⁺-2Cl^– cotransporter; myosin light chain kinase; protein kinase A     

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.     

The role of volume-sensitive ion transport systems in regulation of epithelial transport

This review focuses on using the knowledge on volume-sensitive transport systems in Ehrlich ascites tumour cells and NIH-3T3 cells to elucidate osmotic regulation of salt transport in epithelia. Using the intestine of the European eel (Anguilla anguilla) (an absorptive epithelium of the type described in the renal cortex thick ascending limb (cTAL)) we have focused on the role of swelling-activated K+- and anion-conductive pathways in response to hypotonicity, and on the role of the apical (luminal) Na+-K+-2Cl- cotransporter (NKCC2) in the response to hypertonicity. The shrinkage-induced activation of NKCC2 involves an interaction between the cytoskeleton and protein phosphorylation events via PKC and myosin light chain kinase (MLCK). Killifish (Fundulus heteroclitus) opercular epithelium is a Cl(-)-secreting epithelium of the type described in exocrine glands, having a CFTR channel on the apical side and the Na+/K+ ATPase, NKCC1 and a K+ channel on the basolateral side. Osmotic control of Cl- secretion across the operculum epithelium includes: (i) hyperosmotic shrinkage activation of NKCC1 via PKC, MLCK, p38, OSR1 and SPAK; (ii) deactivation of NKCC by hypotonic cell swelling and a protein phosphatase, and (iii) a protein tyrosine kinase acting on the focal adhesion kinase (FAK) to set levels of NKCC activity.     

Regulation of erythrocyte Na-K-2Cl cotransport by threonine phosphorylation

A method is described to measure threonine phosphorylation of the Na-K-2Cl cotransporter in ferret erythrocytes using readily available antibodies. We show that most, if not all, cotransporter in these cells is NKCC1, and this was immunoprecipitated with T4. Cotransport rate, measured as 86Rb influx, correlates well with threonine phosphorylation of T4-immunoprecipitated protein. The cotransporter effects large fluxes and is significantly phosphorylated in cells under control conditions. Transport and phosphorylation increase 2.5- to 3-fold when cells are treated with calyculin A or Na+ arsenite. Both fall to 60% control when cell [Mg2+] is reduced below micromolar or when cells are treated with the kinase inhibitors, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine or staurosporine. Importantly, these latter interventions do not abolish either phosphorylation or transport suggesting that a phosphorylated form of the cotransporter is responsible for residual fluxes. Our experiments suggest protein phosphatase 1 (PrP-1) is extremely active in these cells and dephosphorylates key regulatory threonine residues on the cotransporter. Examination of the effects of kinase inhibition after cells have been treated with high concentrations of calyculin indicates that residual PrP-1 activity is capable of rapidly dephosphorylating the cotransporter. Experiments on cotransporter precipitation with microcystin sepharose suggest that PrP-1 binds to a phosphorylated form of the cotransporter.     

The Ste20 Kinases Ste20-related Proline-Alanine-rich Kinase and Oxidative-stress Response 1 Regulate NKCC1 Function in Sensory Neurons

NKCC1 is highly expressed in dorsal root ganglion neurons, where it is involved in gating sensory information. In a recent study, it was shown that peripheral nerve injury results in increased NKCC1 activity, not due to an increase in cotransporter expression, but to increased phosphorylation of the cotransporter (Pieraut, S., Matha, V., Sar, C., Hubert, T., Méchaly, I., Hilaire, C., Mersel, M., Delpire, E., Valmier, J., and Scamps, F. (2007) J. Neurosci. 27, 6751–6759). Our laboratory has also identified two Ste20-like kinases that bind and phosphorylate NKCC1: Ste20-related proline-alanine-rich kinase (SPAK) and oxidative-stress response 1 (OSR1). In this study, we show that both kinases are expressed at similar expression levels in spinal cord and dorsal root ganglion neurons, and that both kinases participate equally in the regulation of NKCC1. Using a novel fluorescence method to assay NKCC1 activity in single cells, we show a 50% reduction in NKCC1 activity in DRG neurons isolated from SPAK knockout mice, indicating that another kinase, e.g. OSR1, is present to phosphorylate and activate the cotransporter. Using a nociceptive dorsal root ganglion sensory neuronal cell line, which expresses the same cation-chloride cotransporters and kinases as native DRG neurons, and gene silencing via short hairpin RNA, we demonstrate a direct relationship between kinase expression and cotransporter activity. We show that inactivation of either kinase significantly affects NKCC1 activity, whereas inactivation of both kinases results in an additive effect. In summary, our study demonstrates redundancy of kinases in the regulation of NKCC1 in dorsal root ganglion neurons.The regulation of intracellular Cl^– in neurons is a critical determinant of inhibitory synaptic neurotransmission. Sensory or peripheral neurons express, in abundance, an inwardly poised Na⁺- and K⁺-dependent Cl^– transport mechanism, NKCC1,² whose activity drives the uphill accumulation of Cl^– ions (2, 3). High intracellular Cl^– concentration in dorsal root ganglion (DRG) neurons permits depolarizing γ-aminobutyric acid responses, which mediate presynaptic inhibition and filtration of sensory noise (4). Consequently, the knockout of NKCC1 exhibits a redistribution of internal Cl^– in DRG neurons and a pain perception phenotype (3, 5). In addition, peripheral inflammation or nerve injury results in increased NKCC1 function in primary afferents (6, 7). Using a phosphopeptide-specific NKCC1 antibody, it was recently shown that NKCC1 phosphorylation instead of expression level increased in DRG neurons upon nerve injury (1). This observation points to the importance of NKCC1 regulation in the neuropathic pain pathway.In recent work, our laboratory identified two Ste20-like kinases that directly bind to the cytosolic N-terminal tail of NKCC1 (8). The binding is a pre-requisite for NKCC1 phosphorylation and activation (9–13). The kinases, named SPAK (Ste20-related proline-alanine-rich kinase) and OSR1 (oxidative-stress response 1), share high homology in both their catalytic and regulatory domains. Expression of the two proteins has been examined in tissues using both Northern and Western blot analysis. These studies established that both kinases are widely expressed and that their expression pattern often overlaps (14–18). Co-expression of the kinases has been confirmed using Western blot analysis of well established cultured cell lines (17).Heterologous expression of SPAK and OSR1 in Xenopus laevis oocytes demonstrates that both kinases are able to bind and activate NKCC1 (10). Whether or not they equally participate to the regulation of NKCC1 in vivo under normal physiological conditions has not yet been determined. We chose to address the role of SPAK and OSR1 by using an established nociceptive dorsal root ganglion sensory neuronal cell line (19), as well as isolated mouse DRG neurons. We demonstrate that undifferentiated 50B11 cells express the same cotransporters and regulatory kinases as native DRG cells. To manipulate the kinases, we decreased their expression via shRNA knockdown and used a SPAK knockout mouse for the isolated DRG neurons. To measure NKCC1 activity, we used unidirectional ⁸⁶Rb-uptake with the established cell line and developed a novel fluorescence assay to assess cotransporter activity in single isolated DRG neurons. Our data demonstrate that both kinases are expressed to similar levels in both the cell line and isolated mouse DRG neurons, and that the kinases participate in concert to regulate NKCC1 function.     

The Na+:Cl- cotransporter is activated and phosphorylated at the amino-terminal domain upon intracellular chloride depletion

The renal Na(+):Cl(-) cotransporter rNCC is mutated in human disease, is the therapeutic target of thiazide-type diuretics, and is clearly involved in arterial blood pressure regulation. rNCC belongs to an electroneutral cation-coupled chloride cotransporter family (SLC12A) that has two major branches with inverse physiological functions and regulation: sodium-driven cotransporters (NCC and NKCC1/2) that mediate cellular Cl(-) influx are activated by phosphorylation, whereas potassium-driven cotransporters (KCCs) that mediate cellular Cl(-) efflux are activated by dephosphorylation. A cluster of three threonine residues at the amino-terminal domain has been implicated in the regulation of NKCC1/2 by intracellular chloride, cell volume, vasopressin, and WNK/STE-20 kinases. Nothing is known, however, about rNCC regulatory mechanisms. By using rNCC heterologous expression in Xenopus laevis oocytes, here we show that two independent intracellular chloride-depleting strategies increased rNCC activity by 3-fold. The effect of both strategies was synergistic and dose-dependent. Confocal microscopy of enhanced green fluorescent protein-tagged rNCC showed no changes in rNCC cell surface expression, whereas immunoblot analysis, using the R5-anti-NKCC1-phosphoantibody, revealed increased phosphorylation of rNCC amino-terminal domain threonine residues Thr(53) and Thr(58). Elimination of these threonines together with serine residue Ser(71) completely prevented rNCC response to intracellular chloride depletion. We conclude that rNCC is activated by a mechanism that involves amino-terminal domain phosphorylation.