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

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: 5.04). 02/2006; 26(2):689-98. DOI: 10.1128/MCB.26.2.689-698.2006
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ABSTRACT 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.

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Available from: Kenneth Bradley Gagnon, Jun 27, 2014
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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 · 4.18 Impact Factor
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    • "Existing data do not favour one model of activation over another. Two kinases, Ste20 proline–alanine-rich kinase (SPAK) and oxidative stress-response kinase (OSR1), have been shown to directly phosphorylate NKCC1 (Gagnon et al. 2006a; Moriguchi et al. 2006; Vitari et al. 2006) and NKCC2 (Gimenez & Forbush, 2005). These kinases belong to the family of mammalian Ste20-like protein kinases (Dan et al. 2001; Delpire, 2009). "
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    ABSTRACT: Na(+)-K(+)-2Cl(-) cotransport (NKCC) mediates the movement of two Cl(-) ions for one Na(+) and one K(+) ion. Under isosmotic conditions or with activation of the kinases SPAK/WNK4, the NKCC1-mediated Cl(-) uptake in Xenopus laevis oocytes, as measured using (36)Cl, is twice the value of K(+) uptake, as determined using (86)Rb. Under hyperosmotic conditions, there is a significant activation of the bumetanide-sensitive K(+) uptake with only a minimal increase in bumetanide-sensitive Cl(-) uptake. This suggests that when stimulated by hypertonicity, the cotransporter mediates K(+)/K(+) and Cl(-)/Cl(-) exchange. Although significant stimulation of K(+)/K(+) exchange was observed with NKCC1, a significantly smaller hyperosmotic stimulatory effect was observed with NKCC2. In order to identify the molecular determinant(s) of this NKCC1-specific activation, we created chimeras of the mouse NKCC1 and the rat NKCC2. Swapping the regulatory amino termini of the cotransporters neither conferred activation to NKCC2 nor prevented activation of NKCC1. Using unique restrictions sites, we created additional chimeric molecules and determined that the first intracellular loop between membrane-spanning domains one and two and the second extracellular loop between membrane-spanning domains three and four of NKCC1 are necessary components of the hyperosmotic stimulation of K(+)/K(+) exchange.
    The Journal of Physiology 09/2010; 588(Pt 18):3385-96. DOI:10.1113/jphysiol.2010.191932 · 4.54 Impact Factor
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    • "All known substrates and regulators of GCK-VI kinases require the C-terminal PF2 region (Anselmo et al., 2006; Choe and Strange, 2007; Gagnon et al., 2006b; Moriguchi et al., 2005; Piechotta et al., 2003; Vitari et al., 2006). The PF2 domain is sufficient for all known protein–protein interactions (Choe and Strange, 2007), but this domain is dispensable for in vitro GCK-VI kinase activity (Anselmo et al., 2006; Chen et al., 2004; Gagnon et al., 2006a; Moriguchi et al., 2005). In accordance with this, we find that all somatic functions of GCK-3 require kinase activity, and the phenotypes of animals expressing a C-terminal truncated form of GCK-3 are less severe than the phenotypes of animals with no GCK-3 protein. "
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    ABSTRACT: Ste20 kinases constitute a large family of serine/threonine kinases with a plethora of biological functions. Members of the GCK-VI subfamily have been identified as important regulators of osmohomeostasis across species functioning upstream of ion channels. Although the expression of the two highly similar mammalian GCK-VI kinases is eminent in a wide variety of tissues, which includes also the testis, their potential roles in development remain elusive. Caenorhabditis elegans contains a single ancestral ortholog termed GCK-3. Here, we report a comprehensive analysis of gck-3 function and demonstrate its requirement for several developmental processes independent of ion homeostasis, i.e., larval progression, vulva, and germ line formation. Consistent with a wide range of gck-3 function we find that endogenous GCK-3 is expressed ubiquitously. The serine/threonine kinase activity of GCK-3, but not its presumed C-terminal substrate interaction domain, is essential for gck-3 gene function. Although expressed in female germ cells, we find GCK-3 progressively accumulating during spermatogenesis where it promotes the first meiotic cell division and facilitates faithful chromosome segregation. In particular, we find that different levels of gck-3 activity appear to be important for various aspects of germ line development. Taken together, our findings suggest that members of the GCK-VI kinase subfamily may act as key regulators of many developmental processes and that this newly described role in meiotic progression might be conserved and an important part of sexual reproduction.
    Developmental Biology 08/2010; 344(2):758-71. DOI:10.1016/j.ydbio.2010.05.505 · 3.64 Impact Factor
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