Disease-causing R1185C mutation of WNK4 disrupts a regulatory mechanism involving calmodulin binding and SGK1 phosphorylation sites.
The R1185C mutation in WNK4 is associated with pseudohypoaldosteronism type II (PHAII). Unlike other PHAII-causing mutations in the acidic motif, the R1185C mutation is located in the COOH-terminal region of WNK4. The goal of the study is to determine what properties of WNK4 are disrupted by the R1185C mutation. We found that the R1185C mutation is situated in the middle of a calmodulin (CaM) binding site and the mutation reduces the binding of WNK4 to Ca(2+)/CaM. The R1185C mutation is also close to serum- and glucocorticoid-induced protein kinase (SGK1) phosphorylation sites S1190 and S1217. In addition, we identified a novel SGK1 phosphorylation site (S1201) in WNK4, and phosphorylation at this site is reduced by Ca(2+)/CaM. In the wild-type WNK4, the level of phosphorylation at S1190 is the lowest and that at S1217 is the highest. In the R1185C mutant, phosphorylation at S1190 is eliminated and that at S1201 becomes the strongest. The R1185C mutation enhances the positive effect of WNK4 on the Na(+)-K(+)-2Cl(-) cotransporter 2 (NKCC2) as tested in Xenopus laevis oocytes. Deletion of the CaM binding site or phospho-mimicking at two or three of the SGK1 sites enhances the WNK4 effects on NKCC2. These results indicate that the R1185C mutation disrupts an inhibitory domain as part of the suppression mechanism of WNK4, leading to an elevated WNK4 activity at baseline. The presence of CaM binding and SGK1 phosphorylation sites in or close to the inhibitory domain suggests that WNK4 activity is subject to the regulation by intracellular Ca(2+) and phosphorylation.
Nephrology Research and Training Center, Division of Nephrology, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294-0006, USA.
Tao
T
Na
Guojin
G
Wu
Wei
W
Zhang
Wen-Ji
WJ
Dong
Ji-Bin
JB
Peng
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D011546:Pseudohypoaldosteronism / Q000235:genetics; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism; D064466:Solute Carrier Family 12, Member 1; D014982:Xenopus laevis; D000099184:Serum-Glucocorticoid Regulated Kinases
D002118:Calcium; D002147:Calmodulin; D017874:Immediate-Early Proteins; D017346:Protein Serine-Threonine Kinases; D028021:Sodium-Potassium-Chloride Symporters; D064466:Solute Carrier Family 12, Member 1; D000099184:Serum-Glucocorticoid Regulated Kinases; C578714:SLC12A1 protein, human; C433806:WNK4 protein, human