4.2 Article

A single binding motif is required for SPAK activation of the Na-K-2Cl cotransporter

Journal

CELLULAR PHYSIOLOGY AND BIOCHEMISTRY
Volume 20, Issue 1-4, Pages 131-142

Publisher

KARGER
DOI: 10.1159/000104161

Keywords

membrane transport; phosphorylation; stress kinases; yeast 2-hybrid; Xenopus oocytes

Funding

  1. NIGMS NIH HHS [GM04771] Funding Source: Medline
  2. NINDS NIH HHS [NS36758] Funding Source: Medline
  3. NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE [R01NS036758] Funding Source: NIH RePORTER

Ask authors/readers for more resources

Background: SPAK (Ste20p-related proline alanine-rich kinase) phosphorylates and activates NKCC1 (Na-K-2Cl cotransporter) in the presence of another serine/threonine kinase WNK4 (With No lysine (K). However, whether or not the docking of SPAK to NKCC1 is a requirement for cotransporter activation has not been fully resolved. Methods: We mutated both SPAK binding motifs in the amino-terminal tail of NKCC1 and tested the interaction between SPAK and NKCC1 using a semi in vivo yeast two-hybrid assay, P-32-ATP in vitro phosphorylation assays, and Rb-86(+) uptake (a K+ congener) assays in heterologously expressed Xenopus laevis oocytes. We also used site-directed mutagenesis to identify the principle phospho-regulatory threonine residues in the amino-terminal tail of NKCC1. Results: A single SPAK binding motif is necessary for isotonic NKCC1 activation. Mutation of the phenylalanine (F) residue within the motif abrogates binding and function. Phosphorylation of the cotransporter is markedly reduced in the absence of SPAK docking to NKCC1. Truncations of internal regions of the amino-terminus of NKCC1 do not disrupt protein structure enough to affect cotransporter function. Threonine residues (T-206 and T-211) are both identified as phospho-regulatory sites of NKCC1 function. Conclusion: We demonstrate that physical docking of SPAK to NKCC1 is necessary for cotransporter activity under both baseline and hyperosmotic conditions. We identify T-206 and T-211 as major phospho-acceptor sites involved in cotransporter function, with T-206 common to two separate regulatory pathways: one involving SPAK, the other involving a still unknown kinase that is responsive to forskolin/PKA stimulation. Copyright (c) 2007 S. Karger AG, Basel.

Authors

I am an author on this paper
Click your name to claim this paper and add it to your profile.

Reviews

Primary Rating

4.2
Not enough ratings

Secondary Ratings

Novelty
-
Significance
-
Scientific rigor
-
Rate this paper

Recommended

No Data Available
No Data Available