Article

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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    • ") 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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    • "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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    • "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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