Disease-causing mutations in the acidic motif of WNK4 impair the sensitivity of WNK4 kinase to calcium ions

► WNK4 kinase domain isolated from Escherichia coli exhibits kinase activity. ► WNK4 phosphorylates mouse Na-Cl cotransporter NCC at Thr48 in vitro. ► Phospho-mimicking at Thr48 increases NCC abundance and Na+ uptake activity. ► In the presence of the acidic motif, Ca2+ regulates the kinase activity of WNK4. ► PHAII mutations in the acidic motif disrupt a Ca2+-sensing mechanism of WNK4.