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     

A regulatory locus of phosphorylation in the N terminus of the Na-K-Cl cotransporter,NKCC1

The secretory Na-K-Cl cotransporter NKCC1 is activated by secretagogues through a phosphorylation-dependent mechanism. We found a phosphorylation stoichiometry of 3.0 +/- 0.4 phosphorylated residues/NKCC1 protein harvested from shark rectal gland tubules maximally stimulated with forskolin and calyculin A, showing that at least three sites on the cotransporter are phosphorylated upon stimulation. Three phosphoacceptor sites were identified in the N-terminal domain of the protein (at Thr(184), Thr(189), and Thr(202)) using high pressure liquid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry to analyze tryptic fragments of the radiolabeled cotransporter. None of these residues occurs in the context of strong consensus sites for known Ser/Thr kinases. The threonines and the surrounding amino acids are highly conserved between NKCC1 and NKCC2, and similarities are also present in the Na-Cl cotransporter NCC (or TSC). This strongly suggests that the phosphoregulatory mechanism is conserved among isoforms. Through expression of shark NKCC1 mutants in HEK-293 cells, Thr(189) was found to be necessary for activation of the protein, whereas phosphorylation at Thr(184) and Thr(202) was modulatory, but not required. In conjunction with the recent finding (Darmen, R. B., Flemmer, A., and Forbush, B. (2001) J. Biol. Chem. 276, 34359-34362) that protein phosphatase-1 binds to residues 107-112 in the shark NKCC1 sequence, these results demonstrate that the N terminus of NKCC1 constitutes a phosphoregulatory domain of the transporter.     

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.     

Na(+)/K(+)/Cl(-) cotransporter activates mitogen-activated protein kinase in fibroblasts and lymphocytes.

In a previous work, we have shown that overexpression of the Na(+)/K(+)/Cl(-) cotransporter (NKCC1) induces cell proliferation and transformation. We investigate in the present study the role of the NKCC1 in the mitogenic signal transduction. We show that overexpression of the cotransporter gene (NKCC1) in stablely transfected cells (Balb/c-NKCC1), resulted in enhanced phosphorylation of the extracellular regulated kinase (ERK) to produce double phosphorylated ERK (DP-ERK). Furthermore, the level of DP-ERK was reduced by 50-80% following the addition of bumetanide, a specific inhibitor of the Na(+)/K(+)/Cl(-) cotransporter, in quiescent as well as in proliferating cultures of the Balb/c-NKCC1 clone. In order to explore further the role of the Na(+)/K(+)/Cl(-) cotransporter in mitogenic signal transduction, we measured the effect of the two specific inhibitors of the cotransporter; bumetanide and furosemide, on DP-ERK level in immortalized non-transformed cells. In Balb/c 3T3 fibroblasts stimulated with FGF, bumetanide, and furosemide inhibited 50-60% of the ERK 1/2 phosphorylation. The inhibitor concentration needed for maximal inhibition of ERK 1/2 phosphorylation was similar to the concentration needed to block the K(+) influx mediated by the Na(+)/K(+)/Cl(-) cotransporter in these cells. To analyze whether the Na(+)/K(+)/Cl(-) cotransporter has a role in the mitogenic signal of normal cells, we measured the effect of bumetanide on ERK phosphorylation in human peripheral blood lymphocytes. The phosphorylation of ERK 1/2 in resting human lymphocytes, as well as in lymphocytes stimulated with phytohemagglutinin (PHA) was inhibited by bumetanide. The effect of bumetanide on ERK 2 phosphorylation was much lower than that of ERK 1 phosphorylation. The finding that the Na(+)/K(+)/Cl(-) cotransporter controls the ERK/MAPK (mitogen-activated protein kinase) signal transduction pathway, support our hypothesis that Na(+) and K(+) influxes mediated by this transporter plays a central role in the control of normal cell proliferation. Exploring the cellular ionic currents and levels, mediated by the Na(+)/K(+)/Cl(-) cotransporter, should lead to a better comprehension of cell proliferation and transformation machinery.     

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.     

Alpha1-AR-mediated activation of NKCC in rat cardiomyocytes involves ERK-dependent phosphorylation of the cotransporter

We studied molecular and functional characteristics as well as hormonal regulation of the Na-K-2Cl cotransporter (NKCC) in the isolated rat heart and cardiomyocytes. NKCC activity was measured as bumetanide-sensitive (86)Rb(+) influx in isolated perfused rat hearts and isolated cardiomyocytes. Stimulation of alpha(1)-adrenoceptors (AR) by phenylephrine (30 microM) increased (86)Rb(+) influx. The NKCC inhibitor bumetanide (50 microM) reduced the response to phenylephrine by 45 +/- 13% (n = 12, P < 0.01). PD-98059 (10 microM), an inhibitor of the activation of the mitogen-activated protein kinases extracellular signal-regulated protein kinase 1 and 2 (ERK1/2), reduced the total response to phenylephrine by 51 +/- 13% (n = 10, P < 0.01) and eliminated the bumetanide-sensitive component, indicating that alpha(1)-AR mediated stimulation of NKCC is dependent on activation of ERK1/2. Inhibitors of protein kinase C or phosphatidylinositol 3-kinase had no effect. The presence of NKCC mRNA and protein was demonstrated in isolated rat cardiomyocytes. Phosphorylation of NKCC after alpha(1)-AR stimulation was shown by immunoprecipitation of the phosphoprotein from (32)P(i) prelabeled cardiomyocytes. Increased phosphorylation of the NKCC protein was also abolished by PD-98059. We conclude that the NKCC is present in rat cardiomyocytes and that ion transport by the cotransporter is regulated by alpha(1)-AR stimulation through phosphorylation of this protein involving the ERK pathway.     

A single binding motif is required for SPAK activation of the Na-K-2Cl cotransporter.

BACKGROUND: SPAK (Ste20p-related proline alanine-rich kinase) phosphorylates and activates NKCC1 (Na-K-2Cl cotransporter) in the presence of another serine/threonine kinase WNK4 (With No lysine (K)). However, whether or not the docking of SPAK to NKCC1 is a requirement for cotransporter activation has not been fully resolved. METHODS: We mutated both SPAK binding motifs in the amino-terminal tail of NKCC1 and tested the interaction between SPAK and NKCC1 using a semi in vivo yeast two-hybrid assay, (32)P-ATP in vitro phosphorylation assays, and (86)Rb(+) uptake (a K(+) congener) assays in heterologously expressed Xenopus laevis oocytes. We also used site-directed mutagenesis to identify the principle phospho-regulatory threonine residues in the amino-terminal tail of NKCC1. RESULTS: A single SPAK binding motif is necessary for isotonic NKCC1 activation. Mutation of the phenylalanine (F) residue within the motif abrogates binding and function. Phosphorylation of the cotransporter is markedly reduced in the absence of SPAK docking to NKCC1. Truncations of internal regions of the amino-terminus of NKCC1 do not disrupt protein structure enough to affect cotransporter function. Threonine residues (T(206) and T(211)) are both identified as phospho-regulatory sites of NKCC1 function. CONCLUSION: We demonstrate that physical docking of SPAK to NKCC1 is necessary for cotransporter activity under both baseline and hyperosmotic conditions. We identify T(206) and T(211) as major phospho-acceptor sites involved in cotransporter function, with T(206) common to two separate regulatory pathways: one involving SPAK, the other involving a still unknown kinase that is responsive to forskolin/PKA stimulation.     

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     

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.     

Regulatory activation is accompanied by movement in the C terminus of the Na-K-Cl cotransporter (NKCC1)

The Na-K-Cl cotransporter (NKCC1) is expressed in most vertebrate cells and is crucial in the regulation of cell volume and intracellular chloride concentration. To study the structure and function of NKCC1, we tagged the transporter with cyan (CFP) and yellow (YFP) fluorescent proteins at two sites within the C terminus and measured fluorescence resonance energy transfer (FRET) in stably expressing human embryonic kidney cell lines. Both singly and doubly tagged NKCC1s were appropriately produced, trafficked to the plasma membrane, and exhibited (86)Rb transport activity. When both fluorescent probes were placed within the same C terminus of an NKCC1 transporter, we recorded an 11% FRET decrease upon activation of the transporter. This result clearly demonstrates movement of the C terminus during the regulatory response to phosphorylation of the N terminus. When we introduced CFP and YFP separately in different NKCC1 constructs and cotransfected these in HEK cells, we observed FRET between dimer pairs, and the fractional FRET decrease upon transporter activation was 46%. Quantitatively, this indicates that the largest FRET-signaled movement is between dimer pairs, an observation supported by further experiments in which the doubly tagged construct was cotransfectionally diluted with untagged NKCC1. Our results demonstrate that regulation of NKCC1 is accompanied by a large movement between two positions in the C termini of a dimeric cotransporter. We suggest that the NKCC1 C terminus is involved in transport regulation and that dimerization may play a key structural role in the regulatory process. It is anticipated that when combined with structural information, our findings will provide a model for understanding the conformational changes that bring about NKCC1 regulation.     

JNK is a volume-sensitive kinase that phosphorylates the Na-K-2Cl cotransporter in vitro

Cell shrinkagephosphorylates and activates the Na-K-2Cl cotransporter (NKCC1),indicating the presence of a volume-sensitive protein kinase. Toidentify this kinase, extracts of normal and shrunken aorticendothelial cells were screened for phosphorylation of NKCC1 fusionproteins in an in-the-gel kinase assay. Hypertonic shrinkage activateda 46-kDa kinase that phosphorylated anNH₂-terminal fusion protein, withweaker phosphorylation of a COOH-terminal fusion protein. Thiscytosolic kinase was activated by both hypertonic and isosmoticshrinkage, indicating regulation by cell volume rather than osmolarity.Subsequent studies identified this kinase as c-JunNH₂-terminal kinase (JNK).Immunoblotting revealed increased JNK activity in shrunken cells; therewas volume-sensitive phosphorylation ofNH₂-terminal c-Jun fusion protein;immunoprecipitation of JNK from shrunken cells but not normal cellsphosphorylated NKCC1 in gel kinase assays; and treatment of cells withtumor necrosis factor, a known activator of JNK, mimicked the effect ofhypertonicity. We conclude that JNK is a volume-sensitive kinase inendothelial cells that phosphorylates NKCC1 in vitro. This is the firstdemonstration of a volume-sensitive protein kinase capable ofphosphorylating a volume-regulatory transporter.

     

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.     

Role for protein phosphatase 2A in the regulation of Calu-3 epithelial Na+-K+-2Cl-, type 1 co-transport function

Activity of Na+-K+-2Cl- co-transport (NKCC1) in epithelia is thought to be highly regulated through phosphorylation and dephosphorylation of the transporter. Previous functional studies from this laboratory suggested a role for protein phosphatase 2A (PP2A) as a serine/threonine protein phosphatase involved in the regulation of mammalian tracheal epithelial NKCC1. We expand on these studies to characterize serine/threonine protein phosphatase(s) necessary for regulation of NKCC1 function and the interaction of the phosphatase(s) with proteins associated with NKCC1. NKCC1 activity was measured as bumetanide-sensitive 86Rb uptake or basolateral to apical 86Rb flux in primary cultures of human tracheal epithelial cells or in Calu-3 airway epithelial cells grown on Transwell filter inserts. Preincubation with 0.1 nm okadaic acid, a PP2A > phosphatase 1 (PP1) inhibitor, increased NKCC1 activity 3.5-fold in human tracheal epithelial cells and 4.1-fold in Calu-3 cells. Calyculin, a PP1 > PP2A inhibitor, did not alter NKCC1 activity or percent bumetanide-sensitive flux. The effect of OA was dose-dependent with an IC50 of 0.4 nm. The alpha1-adrenergic agonist methoxamine increased NKCC1 activity and transiently increased PP2A activity 3.8-fold but did not alter PP1 activity. OA augmented methoxamine-dependent stimulation of NKCC1 activity. PP1, PP2A, and PP2C but not PP2B were detected in lysates from Calu-3 cells by immunoblot analysis. PP1 was not detected in immunoprecipitates of NKCC1 and vice versa. PP2A co-immunoprecipitated with NKCC1 and protein kinase C-delta (PKC-delta) and was pulled down by a recombinant N terminus of NKCC1 consisting of amino acids 1-286. One novel finding is co-precipitation of STE20-related proline-alanine-rich kinase, a regulatory kinase for NKCC1, with PP2A and PKC-delta. The results suggest a model of actin serving as a scaffold for binding and association of PKC-delta, PP2A, and STE20-related proline-alanine-rich kinase. The role of the complex of serine/threonine protein kinases and a protein phosphatase is probably the maintenance of optimal phosphorylation of NKCC1 coincident with its physiological function in epithelial absorption and secretion.     

Is myosin light-chain phosphorylation a regulatory signal for the osmotic activation of the Na+-K+-2Cl- cotransporter?

Myosin light-chain (MLC) kinase (MLCK)-dependent increase in MLC phosphorylation has been proposed to be a key mediator of the hyperosmotic activation of the Na⁺-K⁺-2Cl^– cotransporter (NKCC). To address this hypothesis and to assess whether MLC phosphorylation plays a signaling or permissive role in NKCC regulation, we used pharmacological and genetic means to manipulate MLCK, MLC phosphorylation, or myosin ATPase activity and followed the impact of these alterations on the hypertonic stimulation of NKCC in porcine kidney tubular LLC-PK1 epithelial cells. We found that the MLCK inhibitor ML-7 suppressed NKCC activity independently of MLC phosphorylation. Notably, ML-7 reduced both basal and hypertonically stimulated NKCC activity without influencing MLC phosphorylation under these conditions, and it inhibited NKCC activation by Cl^– depletion, a treatment that did not increase MLC phosphorylation. Furthermore, prevention of the osmotically induced increase in MLC phosphorylation by viral induction of cells with a nonphosphorylatable, dominant negative MLC mutant (AA-MLC) did not affect the hypertonic activation of NKCC. Conversely, a constitutively active MLC mutant (DD-MLC) that mimics the diphosphorylated form neither stimulated isotonic nor potentiated hypertonic NKCC activity. Furthermore, a depolarization-induced increase in endogenous MLC phosphorylation failed to activate NKCC. However, complete abolition of basal MLC phosphorylation by K252a or the inhibition of myosin ATPase by blebbistatin significantly reduced the osmotic stimulation of NKCC without suppressing its basal or Cl^– depletion-triggered activity. These results indicate that an increase in MLC phosphorylation is neither a sufficient nor a necessary signal to stimulate NKCC in tubular cells. However, basal myosin activity plays a permissive role in the optimal osmotic responsiveness of NKCC. proline-alanine-rich STE20-related kinase     

Phosphorylation of the salivary Na(+)-K(+)-2Cl(-) cotransporter

Westudied the phosphorylation of the secretoryNa⁺-K⁺-2Cl cotransporter (NKCC1)in rat parotid acinar cells. We have previously shown that NKCC1activity in these cells is dramatically upregulated in response to-adrenergic stimulation and that this upregulation correlates withNKCC1 phosphorylation, possibly due to protein kinase A (PKA). We showhere that when ATP is added to purified acinar basolateral membranes(BLM), NKCC1 is phosphorylated as a result of membrane-associatedprotein kinase activity. Additional NKCC1 phosphorylation is seen whenPKA is added to BLMs, but our data indicate that this is due to aneffect of PKA on endogenous membrane kinase or phosphatase activities,rather than its direct phosphorylation of NKCC1. Also, phosphopeptidemapping demonstrates that these phosphorylations do not take place atthe site associated with the upregulation of NKCC1 by -adrenergicstimulation. However, this upregulatory phosphorylation can be mimickedby the addition of cAMP to permeabilized acini, and this effect can beblocked by a specific PKA inhibitor. These latter results provide good evidence that PKA is indeed involved in the upregulatoryphosphorylation of NKCC1 and suggest that an additional factor presentin the acinar cell but absent from isolated membranes is required to bring about the phosphorylation.

     

Structural bases of PAS domain-regulated kinase (PASK) activation in the absence of activation loop phosphorylation

Per-Arnt-Sim (PAS) domain-containing protein kinase (PASK) is an evolutionary conserved protein kinase that coordinates cellular metabolism with metabolic demand in yeast and mammals. The molecular mechanisms underlying PASK regulation, however, remain unknown. Herein, we describe a crystal structure of the kinase domain of human PASK, which provides insights into the regulatory mechanisms governing catalysis. We show that the kinase domain adopts an active conformation and has catalytic activity in vivo and in vitro in the absence of activation loop phosphorylation. Using site-directed mutagenesis and structural comparison with active and inactive kinases, we identified several key structural features in PASK that enable activation loop phosphorylation-independent activity. Finally, we used combinatorial peptide library screening to determine that PASK prefers basic residues at the P-3 and P-5 positions in substrate peptides. Our results describe the key features of the PASK structure and how those features are important for PASK activity and substrate selection.     

Exercise effects on muscle beta-adrenergic signaling for MAPK-dependent NKCC activity are rapid and persistent.

This study investigated exercise adaptation of signaling mechanisms that control Na(+)-K(+)-2Cl(-) cotransporter (NKCC) activity in rat skeletal muscle. An acute bout of exercise increased total and NKCC-mediated (86)Rb influx. Inhibition of extracellular signal-regulated kinase (ERK) activation abolished the exercise-induced NKCC upregulation. Treadmill training (20 m/min, 20% grade, 30 min/day, 5 days/wk) stimulated total (86)Rb influx and increased NKCC activity in the soleus muscle after 2 wk and in the plantaris muscle after 4 wk. Exercise-induced NKCC activity was associated with a 1.4- to 2-fold increase in ERK phosphorylation. Isoproterenol, which activates ERK and NKCC in sedentary muscle, caused a remarkable inhibition of the exercise-induced NKCC activity. Furthermore, isoproterenol inhibition of exercise-induced NKCC activity was accompanied with decreased ERK phosphorylation in the plantaris muscle. Akt (protein kinase B) phosphorylation on both Thr(308) and Ser(473), which activates Akt and inhibits NKCC activity in sedentary muscle, was stimulated by acute and chronic exercise. This Akt activation was unaffected by isoproterenol. These results indicate an immediate and persistent exercise adaptation of the signal pathways that participate in the control of potassium transport.     

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.     

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.