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Evidence for functional regulation of the KLHL3/WNK pathway by O-GlcNAcylation

Jimin Hu1, Duc T Huynh1, Denise E Dunn2

  • 1Department of Biochemistry, Duke University School of Medicine, Box 3711 DUMC, Durham, NC 27710, United States.

Glycobiology
|August 12, 2025
PubMed

Insights

O-linked β-N-acetylglucosamine (O-GlcNAc) modifies Kelch-like (KLHL) proteins, including KLHL3, impacting WNK kinase function. This glycosylation regulates cellular processes like osmolarity control and ferroptosis, crucial for health.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cellular Signaling

Background:

  • Kelch-like (KLHL) proteins are crucial adaptors for ubiquitin E3 ligase complexes, regulating substrate stability and proteostasis.
  • Dysregulation of KLHL proteins is implicated in diseases such as cancer, neurodegeneration, and hypertension.
  • Previous studies indicated O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation regulates KEAP1 and gigaxonin, other KLHL family members.

Purpose of the Study:

  • To investigate the role of O-GlcNAc in the regulation of the KLHL3/WNK signaling axis.
  • To determine if KLHL3 and its substrates, the with-no-lysine (WNK) kinases, are O-GlcNAcylated.
  • To explore the functional consequences of O-GlcNAc modification on WNK kinase activity and cellular processes.

Main Methods:

  • Biochemical assays to assess protein interactions and modifications.
  • Quantitative glycoproteomics to identify O-GlcNAcylated proteins.
  • Functional assays to evaluate the impact of O-GlcNAc on WNK4 activity in osmolarity control and ferroptosis.

Main Results:

  • Human KLHL3 and all four WNK kinases (WNK1-4) were found to be O-GlcNAcylated.
  • O-GlcNAcylation was demonstrated to affect WNK4 function in regulating osmolarity control.
  • Evidence suggests O-GlcNAc modification influences WNK4's role in ferroptosis.

Conclusions:

  • O-GlcNAcylation functionally regulates the KLHL3/WNK signaling pathway.
  • This study supports a broader model where O-GlcNAc acts as a general regulator of KLHL protein signaling and cellular proteostasis.
  • Findings have potential implications for blood pressure regulation, neuronal health, and survival.
Keywords:
KLHLO-GlcNAcWNKsignaling

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