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The N‐terminal tail of the Na‐K‐2Cl cotransporter serves as a regulatory scaffold
Author(s) -
Gag Kenneth B.E.,
Delpire Eric
Publication year - 2009
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.23.1_supplement.797.8
Subject(s) - cotransporter , xenopus , phosphorylation , phosphatase , kinase , microbiology and biotechnology , protein phosphatase 1 , serine , scaffold protein , biology , protein kinase a , threonine , heterologous expression , biochemistry , chemistry , signal transduction , gene , recombinant dna , sodium , organic chemistry
The Na‐K‐2Cl cotransporter (NKCC1) participates in cell volume maintenance and regulation by mediating the movement of ions and water across the plasma membrane. Our laboratory identified two Ste20p‐like serine/threonine kinases (SPAK/OSR1) which mediate the phosphorylation and activation of NKCC1. In addition to two putative kinase binding motifs, the N‐terminal tail of NKCC1 contains a putative binding motif for protein phosphatase 1 (PP1). Here we show that heterologous expression of PP1 and NKCC1 in Xenopus laevis oocytes reduces hyperosmotically‐stimulated cotransporter activity to the level of water‐injected oocytes. Immunofluorescence microscopy of oocytes injected with PP1 and EGFP‐tagged NKCC1 demonstrate that the phosphatase does not alter membrane expression of the cotransporter. Mutation of key residues in the PP1 binding motif partially ameliorates the inhibition of hyperosmotically‐stimulated NKCC1 in Xenopus laevis oocytes. In vitro phosphorylation experiments demonstrate that both NKCC1 and SPAK are de‐phosphorylated by PP1, as de‐phosphorylation of SPAK is significantly greater when the N‐terminal tail of NKCC1 is present in the in vitro reaction to scaffold the phosphatase and kinase in proximity to one another. Taken together, our data are consistent with the N‐terminal tail of NKCC1 scaffolding the regulatory phosphatase, PP1, in proximity to the activating kinase, SPAK.