Impaired degradation of WNK1 and WNK4 kinases causes PHAII in mutant KLHL3 knock-in mice

Hum Mol Genet. 2014 Oct 1;23(19):5052-60. doi: 10.1093/hmg/ddu217. Epub 2014 May 12.


Pseudohypoaldosteronism type II (PHAII) is a hereditary disease characterized by salt-sensitive hypertension, hyperkalemia and metabolic acidosis, and genes encoding with-no-lysine kinase 1 (WNK1) and WNK4 kinases are known to be responsible. Recently, Kelch-like 3 (KLHL3) and Cullin3, components of KLHL3-Cullin3 E3 ligase, were newly identified as responsible for PHAII. We have reported that WNK4 is the substrate of KLHL3-Cullin3 E3 ligase-mediated ubiquitination. However, WNK1 and Na-Cl cotransporter (NCC) were also reported to be a substrate of KLHL3-Cullin3 E3 ligase by other groups. Therefore, it remains unclear which molecule is the target(s) of KLHL3. To investigate the pathogenesis of PHAII caused by KLHL3 mutation, we generated and analyzed KLHL3(R528H/+) knock-in mice. KLHL3(R528H/+) knock-in mice exhibited salt-sensitive hypertension, hyperkalemia and metabolic acidosis. Moreover, the phosphorylation of NCC was increased in the KLHL3(R528H/+) mouse kidney, indicating that the KLHL3(R528H/+) knock-in mouse is an ideal mouse model of PHAII. Interestingly, the protein expression of both WNK1 and WNK4 was significantly increased in the KLHL3(R528H/+) mouse kidney, confirming that increases in these WNK kinases activated the WNK-OSR1/SPAK-NCC phosphorylation cascade in KLHL3(R528H/+) knock-in mice. To examine whether mutant KLHL3 R528H can interact with WNK kinases, we measured the binding of TAMRA-labeled WNK1 and WNK4 peptides to full-length KLHL3 using fluorescence correlation spectroscopy, and found that neither WNK1 nor WNK4 bound to mutant KLHL3 R528H. Thus, we found that increased protein expression levels of WNK1 and WNK4 kinases cause PHAII by KLHL3 R528H mutation due to impaired KLHL3-Cullin3-mediated ubiquitination.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Animals
  • Disease Models, Animal
  • Epithelial Cells / metabolism
  • Gene Expression
  • Gene Order
  • Gene Targeting
  • Genetic Vectors / genetics
  • Genotype
  • Kidney / metabolism
  • Mice
  • Mice, Transgenic
  • Microfilament Proteins / genetics*
  • Minor Histocompatibility Antigens
  • Mutation*
  • Phenotype
  • Potassium Channels, Inwardly Rectifying / metabolism
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Protein Serine-Threonine Kinases / genetics
  • Protein Serine-Threonine Kinases / metabolism*
  • Proteolysis
  • Pseudohypoaldosteronism / genetics*
  • Pseudohypoaldosteronism / metabolism*
  • Sodium Channels / metabolism
  • Ubiquitination
  • WNK Lysine-Deficient Protein Kinase 1


  • Adaptor Proteins, Signal Transducing
  • KLHL3 protein, mouse
  • Kcnj1 protein, mouse
  • Microfilament Proteins
  • Minor Histocompatibility Antigens
  • Potassium Channels, Inwardly Rectifying
  • Sodium Channels
  • Prkwnk4 protein, mouse
  • Protein Serine-Threonine Kinases
  • WNK Lysine-Deficient Protein Kinase 1
  • Wnk1 protein, mouse