Disease‐causing mutation R1185C alters phosphorylation of WNK4 by SGK1 via a calmodulin binding site

Abstract

WNK4 is a protein serine/threonine kinase, which is linked to familial hyperkalemic hypertension (FHH). In this study, we evaluated 2 putative calmodulin (CaM) binding sites in WNK4 (amino‐acids 502–525 and 1175–1194) as predicted by a network based CaM target database. The first CaM binding site is close to a cluster of FHH‐causing mutations (E562K, D564A and Q565E) whereas the second contains a FHH‐causing mutation (R1185C) and S1190, a SGK1 phosphorylation site. The two GST‐fusion proteins containing CaM binding sites of WNK4, GST‐WNK4a (492–552) and GST‐WNK4b (1163–1212), exhibited CaM binding ability using pull down assay with CaM‐agarose. Reducing a positive charge in R1185C mutation and introducing a negative charge in phospho‐mimicking S1190D mutation resulted in a reduction of CaM binding ability in GST‐WNK4b. On the other hand, in vitro phosphorylation of GST‐WNK4b by SGK1 was inhibited by CaM in a Ca2+ dependent manner and it was less Ca2+dependent for the R1185C mutant. These results indicate Ca2+‐CaM is involved in the regulation of WNK4 and this regulation may be altered under FHH condition.