A single binding motif is required for SPAK activation of the Na-K-2Cl cotransporter.
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, (32)P-ATP in vitro phosphorylation assays, and (86)Rb(+) 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.


CONCLUSIONS
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.
Gagnon
Kenneth B E
KB
Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2520, USA.
England
Roger
R
Delpire
Eric
E
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D005260:Female; D066298:In Vitro Techniques; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D064506:Solute Carrier Family 12, Member 2; D020798:Two-Hybrid System Techniques; D014982:Xenopus laevis
D011994:Recombinant Proteins; C578878:Slc12a2 protein, mouse; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; C486407:Stk39 protein, mouse; D017346:Protein Serine-Threonine Kinases