Disease-causing mutations in KLHL3 impair its effect on WNK4 degradation

•KLHL3 decreases protein abundance of WNK4 but not that of NCC.•KLHL3 reduces the level of phosphorylated OSR1 but not that of total OSR1 protein.•Both cullin 3 and KLHL3 are involved in the degradation of WNK4.•KLHL3 increases the ubiquitination of WNK4 protein.•Mutations in KLHL3 reduce the inhibitory effect of KLHL3 on WNK4 protein abundance.

Mutations in with-no-lysine (K) kinase 4 (WNK4) and a ubiquitin E3 ligase complex component kelch-like 3 (KLHL3) both cause pseudohypoaldosteronism II (PHAII), a hereditary form of hypertension. We determined whether WNK4 or its effector is regulated by KLHL3 in Xenopus oocytes. KLHL3 inhibited the positive effect of WNK4 on Na+–Cl− cotransporter (NCC) by decreasing WNK4 protein abundance without decreasing that of NCC and the downstream kinase OSR1 directly. Ubiquitination and degradation of WNK4 were induced by KLHL3. The effect of KLHL3 on WNK4 degradation was blocked by a dominant negative form of cullin 3. All five PHAII mutations of KLHL3 tested disrupted the regulation on WNK4. We conclude that KLHL3 is a substrate adaptor for WNK4 in a ubiquitin E3 ligase complex.

Structured summary of protein interactions:CUL3physically interacts with KLHL3 by pull down (View interaction)WNK4physically interacts with KLHL3 by pull down (View interaction)