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
Source: PubMed

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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    ABSTRACT: The oxidative stress-responsive 1 (OSR1) kinase belongs to the mammalian STE20-like kinase family. OSR1 is activated by with no lysine [K] (WNKs) kinases, and then it phosphorylates cation-coupled Cl-cotransporters, regulating ion homeostasis and cell volume in mammalian cells. However, the specific mechanisms of OSR1 activation remains poorly defined, largely due to its extremely low basal activity. Here, we dissect in detail the regulatory mechanisms of OSR1 activation from the aspects of autoinhibition, upstream kinase WNK, and the newly identified master regulator mouse protein-25 (MO25). Based on our structural and biochemical studies, we propose a ″double lock″ model, accounting for the tight autoinhibition of OSR1, an effect that has to be removed by WNK before MO25 further activates OSR1. Particularly, the conserved carboxy-terminal (CCT) domain and αAL helix act together to strongly suppress OSR1 basal activity. WNKs bind to the CCT and trigger its conformational rearrangement to release the kinase domain of OSR1, allowing for MO25 binding and full activation. Finally, the regulatory mechanisms of OSR1 activation were further corroborated by cellular studies of OSR1-regulated cell volume control through WNK-OSR1 signaling pathway. Collectively, these results provide insights into the OSR1 kinase activation to facilitate further functional study.
    Journal of Biological Chemistry 11/2014; 289(52). DOI:10.1074/jbc.M114.592097 · 4.60 Impact Factor
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    ABSTRACT: Purpose:To identify whether kinases that regulate the activity of Cation Chloride Cotransporters (CCC) in other tissues are also expressed in rat and human lenses. Methods:The expression of With-No-Lysine Kinase (WNK 1, 3, 4), Oxidative Stress Response kinase 1 (OSR1) and Ste20-like Proline Alanine rich Kinase (SPAK) were determined at either the transcript or protein levels in rat and human lenses by reverse-transcriptase PCR and/or Western blotting, respectively. Selected kinases were regionally and subcellularly characterised in rat and human lenses. The transparency, wet weight and tissue morphology of lenses extracted from SPAK knock out animals was compared to wild type lenses. Results:WNK 1, 3, 4, SPAK, and OSR1 were identified at the transcript level in rat lenses and WNK1, 4, SPAK and OSR1 expression confirmed at the protein level in rat and human lenses.SPAK and OSR1 were found to associate with membranes as peripheral proteins and exhibited distinct subcellular and region-specific expression profiles throughout the lens. No significant difference in the wet weight of SPAK knock out lenses was detected relative to wild type lenses. However, SPAK knock out lenses showed an increased susceptibility to opacification. Conclusions:Our results show that the WNK-SPAK/OSR1 signalling system known to play a role in regulating the phosphorylation status and hence activity of CCCs in other tissues, is also present in the rat and human lenses. The increased susceptibility of SPAK lenses to opacification suggests that disruption of this signalling pathway may compromise the ability of the lens to control its volume and hence maintain transparency. Copyright © 2014 by Association for Research in Vision and Ophthalmology.
    Investigative Ophthalmology &amp Visual Science 12/2014; 56(1). DOI:10.1167/iovs.14-15911 · 3.66 Impact Factor
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    ABSTRACT: The NaCl cotransporter (NCC) of the renal distal convoluted tubule is stimulated by low K+ diet by an unknown mechanism. Since recent work has shown that the STE20/SPS-1-related proline-alanine-rich protein kinase (SPAK) can function to stimulate NCC by phosphorylation of specific N-terminal sites, we investigated if the NCC response to low K+ diet is mediated by SPAK. Using phospho-specific antibodies in Western blot and immunolocalization studies of wild-type (WT) and SPAK knock-out (SPAK-/-) mice fed a low K+ or control diet for 4 days, we found that low K+ diet strongly increased total NCC expression and phosphorylation of NCC. This was associated with an increase in total SPAK expression in cortical homogenates and an increase in phosphorylation of SPAK at the S383 activation site. The increased pNCC in response to low K+ diet was blunted but not completely inhibited in SPAK-/- mice. These findings reveal that SPAK is an important mediator of the increased NCC activation by phosphorylation that occurs in the DCT in response to a low K+ diet, but other low potassium-activated kinases are likely to be involved. Copyright © 2014, American Journal of Physiology - Renal Physiology.
    American journal of physiology. Renal physiology 01/2015; 308(8):ajprenal.00388.2014. DOI:10.1152/ajprenal.00388.2014 · 3.30 Impact Factor

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