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

Our recent studies demonstrate that (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 duanyu1842K (K104R) fails to activate the cotransporter. The catalytic domain of duanyu1842K 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 duanyu1842K (T243A) or duanyu1842K (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 duanyu1842K (K104R) mutant. In vitro phosphorylation experiments demonstrate that both intramolecular autophosphorylation of duanyu1842K and phosphorylation of NKCC1 are significantly stronger in the presence of Mn2+ rather than Mg2+. We also show that duanyu1842K 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 exhibited similar kinase properties and similar functional activation of NKCC1 when coexpressed with WNK4.

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