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Molecular and Cellular Biology, January 2006, p. 689-698, Vol. 26, No. 2
0270-7306/06/$08.00+0     doi:10.1128/MCB.26.2.689-698.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Characterization of SPAK and OSR1, Regulatory Kinases of the Na-K-2Cl Cotransporter

Kenneth B. E. Gagnon, Roger England, and Eric Delpire*

Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, Tennessee 37232

Received 3 September 2005/ Returned for modification 21 October 2005/ Accepted 29 October 2005

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.

* Corresponding author. Mailing address: Department of Anesthesiology, Vanderbilt University Medical Center, T-4202 Medical Center North, 1161 21st Avenue South, Nashville, TN 37232. Phone: (615) 343-7409. Fax: (615) 343-3916. E-mail: eric.delpire{at}vanderbilt.edu.

Molecular and Cellular Biology, January 2006, p. 689-698, Vol. 26, No. 2
0022-538X/06/$08.00+0     doi:10.1128/MCB.26.2.689-698.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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