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

The 42-member Kelch-like (KLHL) protein family are adaptors for ubiquitin E3 ligase complexes, governing the stability of a wide range of substrates. KLHL proteins are critical for maintaining proteostasis in a variety of tissues and are mutated in human diseases, including cancer, neurodegeneration, and familial hyperkalemic hypertension. However, the regulation of KLHL proteins remains incompletely understood. Previously, we reported that two KLHL family members, KEAP1 and gigaxonin, are regulated by O-linked β-N-acetylglucosamine (O-GlcNAc), an intracellular form of glycosylation. Interestingly, some ubiquitination targets of KEAP1 and gigaxonin are themselves also O-GlcNAcylated, suggesting that multi-level control by this post-translational modification may influence many KLHL pathways. To test this hypothesis, we examined KLHL3, which ubiquitinates with-no-lysine (WNK) kinases to modulate downstream ion channel activity. Our biochemical and glycoproteomic data demonstrate that human KLHL3 and all four WNK kinases (WNK1-4) are O-GlcNAcylated. Moreover, our results suggest that O-GlcNAcylation affects WNK4 function in both osmolarity control and ferroptosis, with potential implications ranging from blood pressure regulation to neuronal health and survival. This work demonstrates the functional regulation of the KLHL3/WNK axis by O-GlcNAcylation and supports a broader model of O-GlcNAc serving as a general regulator of KLHL signaling and proteostasis.