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Related Experiment Video

Updated: May 8, 2026

Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

Published on: August 7, 2018

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

Yutaro Mori¹, Mai Wakabayashi, Takayasu Mori

¹Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo, Tokyo 113-8519, Japan.

Biochemical and Biophysical Research Communications

|August 22, 2013

Summary

Mutations in KLHL3 (Kelch-like protein 3) impair its stability or binding to CUL3 (Cullin 3) or WNK4 (With No Lysine 4). This reduces WNK4 ubiquitination, increasing WNK4 levels and causing pseudohypoaldosteronism type II (PHAII).

Keywords:

Kelch-like protein Pseudohypoaldosteronism type II Ubiquitination WNK kinase

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Area of Science:

Biochemistry
Molecular Biology
Genetics

Background:

Pseudohypoaldosteronism type II (PHAII) is linked to mutations in WNK4, KLHL3, and CUL3.
KLHL3-CUL3 complexes ubiquitinate WNK4, and impaired WNK4 ubiquitination is a common PHAII mechanism.
Previous work showed KLHL3 R528H mutation decreases WNK4 binding, reducing ubiquitination.

Purpose of the Study:

To investigate the pathogenic mechanisms of PHAII caused by other KLHL3 mutations.
To examine the effects of three PHAII-causing KLHL3 mutations in different domains.

Main Methods:

HEK293T cell transfections to assess protein levels of KLHL3 mutants.
Cycloheximide chase assays to determine intracellular stability.
In vitro and in vivo assays to evaluate binding affinities and ubiquitination activity.

Main Results:

KLHL3 mutants S410L, E85A, and C164F exhibited altered protein levels and/or stability.
S410L mutation decreased KLHL3 intracellular stability.
E85A and C164F mutations reduced KLHL3 binding to CUL3; S410L and R528H reduced KLHL3 binding to WNK4.
All tested KLHL3 mutants decreased WNK4 ubiquitination and increased intracellular WNK4 levels.

Conclusions:

PHAII-causing KLHL3 mutants have reduced ability to ubiquitinate WNK4.
This impairment stems from decreased KLHL3 stability and/or reduced binding to CUL3 or WNK4.
These findings elucidate mechanisms of PHAII linked to KLHL3 dysfunction.