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Autophosphorylation of SPAK and OSR1 are required for Na‐K‐2Cl cotransporter activation
Author(s) -
Gag Kenneth B.E.,
England Roger,
Delpire Eric
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a838-c
Subject(s) - cotransporter , autophosphorylation , phosphorylation , kinase , xenopus , chemistry , biochemistry , protein kinase a , serine , alanine , microbiology and biotechnology , biology , amino acid , sodium , organic chemistry , gene
Hormonal, oxidative, and osmotic stimuli activate the Na‐K‐2Cl cotransporter, NKCC1, through phosphorylation. Our recent in vivo studies demonstrate that co‐expression of SPAK ( S te20p related P roline A lanine rich K inase), and WNK4 ( W ith N o lysine ( K ) kinase)with the Na‐K‐2Cl cotransporter (NKCC1) in Xenopus laevis oocytes, phosphorylates and stimulates NKCC1, whereas catalytically‐inactive SPAK (K104R) fails to activate the cotransporter. In this study, we identified four threonine residues (T231, T236, T243, and T247) within the activation loop between the well‐conserved DFG and APE motifs, which we speculated might be sites of phosphorylation and kinase activation, therefore we mutated each residue into an alanine. Co‐expression 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 a similar effect to that of the catalytically‐inactive SPAK (K104R) mutant. In vitro phosphorylation experiments demonstrate that both intramolecular autophosphorylation of SPAK and phosphorylation of NKCC1 is significantly stronger in the presence of Mn 2+ , rather than Mg 2+ . Consistent with the in vivo heterologous expression studies, 32 P incorporation was significantly decreased in the T243A mutant and completely absent in the T247A and K104R mutants. We also show that SPAK activity is markedly inhibited by staurosporine and K252a, partially inhibited by N‐ethylmaleimide and diamide, and unaffected by arsenite or ML7. OSR1, a kinase closely related to SPAK, exhibited similar kinase properties and similar functional activation of NKCC1 when co‐expressed with WNK4. Together, these in vivo and in vitro data confirm that SPAK and OSR1 are kinases that phosphorylate and activate NKCC1.