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Kinase docking to NKCC1 is essential for function
Author(s) -
Gag Kenneth B.,
England Roger,
Delpire Eric
Publication year - 2007
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.21.5.a532-b
Subject(s) - cotransporter , kinase , serine , phosphorylation , biochemistry , threonine , microbiology and biotechnology , docking (animal) , alanine , chemistry , protein kinase a , biology , amino acid , medicine , nursing , organic chemistry , sodium
The secretory Na‐K‐2Cl cotransporter, NKCC1, is highly expressed on the basolateral membrane of Cl − ‐secreting epithelia such as salivary, sweat and lacrimal glands as well as lung, stomach, colon, where it participates to salt/fluid secretion. In all cells where the cotransporter is expressed, NKCC1 is activated by cell shrinkage and participates in cell volume maintenance and regulation. The isosmotic activation NKCC1 by the serine/threonine kinases SPAK (Ste20p‐related proline alanine‐rich kinase) and WNK4 (with no lysine (K)) requires a functional interaction between the kinases and the cotransporter. The specificity of a kinase to a particular substrate can be significantly increased by a physical docking between the two proteins. We previously identified a single putative SPAK binding motif (R/KFxV/I) in the carboxy terminus of WNK4, and two SPAK binding motifs in the amino terminus of NKCC1. In this study, we demonstrate that a single RFxV motif in the N‐terminal tail of NKCC1 is necessary for isotonic cotransporter activation, and that an alanine substitution of the conserved phenylalanine (F) residue within the motif abrogates cotransporter activity in NKCC1‐injected Xenopus laevis oocytes. We also correlate the in vitro phosphorylation of NKCC1 by SPAK with the in vivo isosmotic activation of the cotransporter. Indeed, in the absence of kinase docking to the amino terminus of NKCC1, the rate of kinase phosphorylation of the cotransporter is markedly reduced. Therefore, our data demonstrate that SPAK binding (or docking) to the N‐terminus of NKCC1 is a pre‐requisite for cotransporter stimulation by the kinase. Interestingly, whereas physical binding is necessary for cotransporter activation, the distance between the docking site and the phosphorylation sites appears to be unimportant.