Characterization of SPAK and OSR1, regulatory kinases of the Na-K-2Cl cotransporter. Mol Cell Biol

Department of Anesthesiology, Vanderbilt University Medical Center, T-4202 Medical Center North, 1161 21st Avenue South, Nashville, Tennessee 37232, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 02/2006; 26(2):689-98. DOI: 10.1128/MCB.26.2.689-698.2006
Source: PubMed


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.


Available from: Kenneth Bradley Gagnon, Jun 27, 2014
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    • "WNK1 activates the downstream protein kinases STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) and oxidative stress responsive 1 (OSR1) through phosphorylation of the t-loop in the catalytic domain [12], [13], [14], [15]. Activated SPAK and OSR1 associate and then phosphorylate and activate other ion co-transporters, including Na+/K+/2Cl− co-transporter 1 (NKCC1) [16], [17]. NKCC1 is a ubiquitous ion transporter [18] that controls cell volume and maintains osmostasis through absorption of Na+, K+ and Cl− ions [19], [20], suggesting that under hyperosmotic conditions, WNK1 can regulate the activity of NKCC1 through SPAK and OSR1 [21]. "
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    ABSTRACT: The WNK1 (WNK lysine deficient protein kinase 1) protein is a serine/threonine protein kinase with emerging roles in cancer. WNK1 causes hypertension and hyperkalemia when overexpressed and cardiovascular defects when ablated in mice. In this study, the role of Wnk1 in angiogenesis was explored using the zebrafish model. There are two zebrafish wnk1 isoforms, wnk1a and wnk1b, and both contain all the functional domains found in the human WNK1 protein. Both isoforms are expressed in the embryo at the initiation of angiogenesis and in the posterior cardinal vein (PCV), similar to fms-related tyrosine kinase 4 (flt4). Using morpholino antisense oligonucleotides against wnk1a and wnk1b, we observed that wnk1 morphants have defects in angiogenesis in the head and trunk, similar to flk1/vegfr2 morphants. Furthermore, both wnk1a and wnk1b mRNA can partially rescue the defects in vascular formation caused by flk1/vegfr2 knockdown. Mutation of the kinase domain or the Akt/PI3K phosphorylation site within wnk1 destroys this rescue capability. The rescue experiments provide evidence that wnk1 is a downstream target for Vegfr2 (vascular endothelial growth factor receptor-2) and Akt/PI3K signaling and thereby affects angiogenesis in zebrafish embryos. Furthermore, we found that knockdown of vascular endothelial growth factor receptor-2 (flk1/vegfr2) or vascular endothelial growth factor receptor-3 (flt4/vegfr3) results in a decrease in wnk1a expression, as assessed by in situ hybridization and q-RT-PCR analysis. Thus, the Vegf/Vegfr signaling pathway controls angiogenesis in zebrafish via Akt kinase-mediated phosphorylation and activation of Wnk1 as well as transcriptional regulation of wnk1 expression.
    PLoS ONE 08/2014; 9(8):e106129. DOI:10.1371/journal.pone.0106129 · 3.23 Impact Factor
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    • ") and in Xenopus oocytes (Strange et al., 2000; Song et al., 2002; Gagnon et al., 2006a). In contrast to KCC2, several studies revealed no significant cotransporter activity of KCC4 under isotonic conditions, but found that KCC4 could be strongly (about 20-fold) activated by hypotonic swelling (Mercado et al., 2000; Song et al., 2002). "
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    ABSTRACT: In the mammalian central nervous system (CNS), the inhibitory strength of chloride (Cl(-))-permeable GABAA and glycine receptors (GABAAR and GlyR) depends on the intracellular Cl(-) concentration ([Cl(-)]i). Lowering [Cl(-)]i enhances inhibition, whereas raising [Cl(-)]i facilitates neuronal activity. A neuron's basal level of [Cl(-)]i, as well as its Cl(-) extrusion capacity, is critically dependent on the activity of the electroneutral K(+)-Cl(-) cotransporter KCC2, a member of the SLC12 cation-Cl(-) cotransporter (CCC) family. KCC2 deficiency compromises neuronal migration, formation and the maturation of GABAergic and glutamatergic synaptic connections, and results in network hyperexcitability and seizure activity. Several neurological disorders including multiple epilepsy subtypes, neuropathic pain, and schizophrenia, as well as various insults such as trauma and ischemia, are associated with significant decreases in the Cl(-) extrusion capacity of KCC2 that result in increases of [Cl(-)]i and the subsequent hyperexcitability of neuronal networks. Accordingly, identifying the key upstream molecular mediators governing the functional regulation of KCC2, and modifying these signaling pathways with small molecules, might constitute a novel neurotherapeutic strategy for multiple diseases. Here, we discuss recent advances in the understanding of the mechanisms regulating KCC2 activity, and of the role these mechanisms play in neuronal Cl(-) homeostasis and GABAergic neurotransmission. As KCC2 mediates electroneutral transport, the experimental recording of its activity constitutes an important research challenge; we therefore also, provide an overview of the different methodological approaches utilized to monitor function of KCC2 in both physiological and pathological conditions.
    Frontiers in Cellular Neuroscience 02/2014; 8:27. DOI:10.3389/fncel.2014.00027 · 4.29 Impact Factor
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    • "Previous in vitro studies have demonstrated SPAK [STE20 (sterile 20)/SPS1-related proline/alanine-rich kinase] and OSR1 (Oxidative stress-responsive kinase-1) as downstream substrates of WNK kinases.[19]–[22] They belong to the same germinal center kinase VI family and share high sequence homology in their N-terminal catalytic and C-terminal regulatory domains. "
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    ABSTRACT: Stimulation of the OSR1 (Oxidative stress-responsive kinase-1)/SPAK [STE20 (sterile 20)/SPS1-related proline/alanine-rich kinase]-NCC (Na(+)-Cl(-) cotransporter) signaling cascade plays an important role in the WNK [With-No-Lysine (K)] kinase 4 D561A knock-in mouse model of pseudohypoaldosteronism type II (PHA II) characterized by salt-sensitive hypertension and hyperkalemia. The aim of this study was to investigate the respective roles of Osr1 and Spak in the pathogenesis of PHA II in vivo. Wnk4 (D561A/+) mice were crossed with kidney tubule-specific (KSP) Osr1 knockout (KSP-Osr1 (-/-)) and Spak knockout (Spak (-/-)) mice. Blood pressure, plasma and urine biochemistries, and the relevant protein expression in the kidneys were examined. Wnk4 (D561A/+), KSP-Osr1 (-/-), and Spak (-/-) mice recapitulated the phenotypes of PHA II, Bartter-like syndrome, and Gitelman syndrome, respectively. Wnk4 (D561A/+).KSP-Osr1 (-/-) remained phenotypically PHA II while Wnk4 (D561A/+).Spak (-/-) mice became normotensive and lacked the PHA II phenotype. Phosphorylated Spak and Ncc were similarly increased in both Wnk4 (D561A/+) and Wnk4 (D561A/+).KSP-Osr1 (-/-) mice while phosphorylated Ncc normalized in Wnk4 (D561A/+).Spak (-/-) mice. Furthermore, Wnk4 (D561A/+).KSP-Osr1 (-/-) mice exhibited exaggerated salt excretion in response to thiazide diuretics while Wnk4 (D561A/+).Spak (-/-) mice exhibited normal responses. Wnk4(D561A/+).Spak (-/-).KSP-Osr1 (-/-) triple mutant mice had low blood pressure and diminished phosphorylated Ncc. Both SPAK and OSR1 are important in the maintenance of blood pressure but activation of SPAK-NCC plays the dominant role in PHA II. SPAK may be a therapeutic target for disorders with salt-sensitive hypertension related to WNK4 activation.
    PLoS ONE 09/2013; 8(9):e72969. DOI:10.1371/journal.pone.0072969 · 3.23 Impact Factor
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