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
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.

0 Followers
 · 
109 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Upon activation by with-no-lysine kinases, STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) phosphorylates and activates SLC12A transporters such as the Na(+)-Cl(-) cotransporter (NCC) and Na(+)-K(+)-2Cl(-) cotransporter type 1 (NKCC1) and type 2 (NKCC2); these transporters have important roles in regulating BP through NaCl reabsorption and vasoconstriction. SPAK knockout mice are viable and display hypotension with decreased activity (phosphorylation) of NCC and NKCC1 in the kidneys and aorta, respectively. Therefore, agents that inhibit SPAK activity could be a new class of antihypertensive drugs with dual actions (i.e., NaCl diuresis and vasodilation). In this study, we developed a new ELISA-based screening system to find novel SPAK inhibitors and screened >20,000 small-molecule compounds. Furthermore, we used a drug repositioning strategy to identify existing drugs that inhibit SPAK activity. As a result, we discovered one small-molecule compound (Stock 1S-14279) and an antiparasitic agent (Closantel) that inhibited SPAK-regulated phosphorylation and activation of NCC and NKCC1 in vitro and in mice. Notably, these compounds had structural similarity and inhibited SPAK in an ATP-insensitive manner. We propose that the two compounds found in this study may have great potential as novel antihypertensive drugs.
    Journal of the American Society of Nephrology 11/2014; DOI:10.1681/ASN.2014060560 · 9.47 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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
  • Source
    [Show abstract] [Hide abstract]
    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

Full-text (2 Sources)

Download
25 Downloads
Available from
Jun 27, 2014