Kelch‐like 3 (KLHL3) controls WNK4 ubiquitination and degradation

Abstract

Mutations in with‐no‐lysine (K) kinase 4 (WNK4) and ubiquitin E3 ligase components KLHL3 and Cullin 3 (CUL3) cause pseudohypoaldosteronism II (PHAII), a hereditary form of hypertension. WNK4 regulates Na‐Cl cotransporter (NCC) and so does KLHL3. However, it is unclear whether NCC or a regulator of NCC is a substrate of the KLHL3‐CUL3 E3 ligase complex. The objective of this study is to identify which protein is the true substrate of KLHL3‐CUL3 E3 ligase. We expressed KLHL3 and/or CUL3 together with Na‐K‐2Cl cotransporter 2 (NKCC2) and WNK4 in Xenopus oocytes. KLHL3 significantly decreased NKCC2‐mediated Na uptake and the positive effect of WNK4 on NKCC2. However, the protein abundance of NKCC2 was not decreased, but the level of WNK4 was robustly decreased by KLHL3. KLHL3 also did not substantially reduce the protein abundance of NCC or oxidative stress‐responsive 1 (OSR1) in a similar setting. KLHL3 significantly increased the ubiquitination and degradation of WNK4 protein. Although CUL3 alone didn't have significant effect on WNK4, a dominant negative CUL3 construct alleviated the effect of KLHL3 on WNK4, suggesting that CUL3 works together with KLHL3 to regulate WNK4 stability. All five PHAII mutations in KLHL3 tested decreased the ability of KLHL3 to reduce WNK4 protein abundance. We conclude that WNK4 is a substrate of KLHL3‐CUL3 ubiquitin E3 ligase complex and PHAII mutations in KLHL3 affect electrolyte transporters indirectly via WNK4.