Decrease of WNK4 ubiquitination by disease-causing mutations of KLHL3 through different molecular mechanisms

Recently, we demonstrated that WNK4 is a substrate for KLHL3-Cullin3 (CUL3) E3 ubiquitin ligase complexes and that impaired WNK4 ubiquitination is a common mechanism for pseudohypoaldosteronism type II (PHAII) caused by WNK4, KLHL3, and CUL3 mutations. Among the various KLHL3 mutations that cause PHAII, we demonstrated that the R528H mutation in the Kelch domain decreased the binding to WNK4, thereby causing less ubiquitination and increased intracellular levels of WNK4. However, the pathogenic mechanisms of PHAII caused by other KLHL3 mutants remain to be determined. In this study, we examined the pathogenic effects of three PHAII-causing mutations in different KLHL3 domains; the protein levels of these mutants significantly differed when they were transiently expressed in HEK293T cells. In particular, S410L expression was low even with increased plasmid expression. The cycloheximide chase assay revealed that an S410L mutation in the Kelch domain significantly decreased the intracellular stability. Mutations in E85A in the BTB domain and C164F in the BACK domain decreased the binding to CUL3, and S410L as well as R528H demonstrated less binding to WNK4. In vitro and in vivo assays revealed that these mutants decreased the ubiquitination and increased the intracellular levels of WNK4 compared with wild-type KLHL3. Therefore, the KLHL3 mutants causing PHAII investigated in this study exhibited less ability to ubiquitinate WNK4 because of KLHL3's low stability and/or decreased binding to CUL3 or WNK4.