The R1185C mutation of WNK4 alters its interaction with calmodulin and phosphorylation by SGK1

Abstract

Different from mutations within the acidic motif of WNK4 that cause pseudohypoaldosteronism type II (PHA II), blood pressure is usually normal in patients with R1185C mutation. To understand the molecular pathogenesis of PHA II, we looked into changes brought to WNK4 by the R1185C mutation. We found that R1185 is within a potential calmodulin (CaM) binding site (amino acids 1175–1194) in the C-terminal region of WNK4. The GST-WNK4 construct (amino acids1163–1243) was capable of binding to S-35 labeled CaM in a calcium dependent manner. Compared to the wild-type, CaM binding was greatly decreased in the R1185C mutant. Interestingly, phosphorylation of WNK4 C-terminal by SGK1 was affected by CaM. Among the 3 SGK1 phosphorylation sites in the C-terminal of WNK4, including S1190, S1201 and S1217, CaM specifically inhibited the phosphorylation of S1201 by SGK1. Among the 3 SGK1 sites, phosphorylation state of a certain site affected SGK1-mediated phosphorylation of neighboring sites. In the presence of R1185C mutation, SGK1-mediated phosphorylation of S1190 was completely inhibited. As a result, the phosphorylation of S1201 was enhanced and that of S1217 was decreased in the R1185C mutant. Thus, the R1185C mutation resulted in weakened CaM binding and differential phosphorylation of SGK1 sites at WNK4 C-terminal. These alterations likely contribute to the pathogenesis of PHA II in patients carrying R1185C mutation.