O-GlcNAc Regulation of the KLHL and WNK Kinase Protein Families

Abstract

O-GlcNAcylation is a monosaccharide post-translational modification (PTM) that modifies cytoplasmic, mitochondrial, and nuclear proteins at serine and threonine residues. Its regulatory roles have been found in many cellular processes including metabolism, transcription and translation. It is also dysregulated in many human diseases, such as cancer, diabetes, neurodegenerative diseases, and cardiovascular diseases.Previously, we discovered O-GlcNAc regulation on the kelch-like (KLHL) family of proteins. KLHL proteins function as adaptors in E3 ubiquitin ligase complexes and are responsible for targeting substrates for ubiquitination and (usually) proteasomal degradation. In humans, 42 KLHL proteins have been identified, and they all have similar domain structures, comprised of an N-terminal BTB domain, a BACK domain, and C-terminal Kelch repeats.Our prior results indicated that O-GlcNAcylation regulates substrate degradation and protein-protein interactions on two members of the KLHL family, KEAP1 (KLHL19) and gigaxonin (KLHL16). We and others have also identified O-GlcNAcylation on the substrates of KEAP1 (NRF2) and gigaxonin (intermediate filaments). With the similar domain structures and ubiquitination functions of KLHL proteins, we hypothesize that O-GlcNAc regulation of KLHL proteins and their substrates may be generalized to other KLHL family members. Therefore, I tested this hypothesis by examining the O-GlcNAc status of other KLHL proteins we have not yet studied and focused on the KLHL3/with-no-lysine (WNK) pathway. KLHL3 regulates ion homeostasis in the kidney by regulating WNK kinase stability. Our biochemical and proteomic data suggest that KLHL3 and all four WNK isoforms in human are O-GlcNAcylated, and we mapped O-GlcNAc sites on KLHL3 and WNK4 by mass spectrometry (MS). We further explored the biological functions of O-GlcNAcylation on KLHL3 and WNK4 and identified the involvement of WNK4 O-GlcNAcylation in osmolarity and ferroptosis regulation. WNK kinases are well-known osmoregulators, and our data suggest that WNK4 O-GlcNAcylation is affected by osmotic stress. WNK4 localization is also influenced by changes in osmolarity, and normal O-GlcNAc cycling is required for WNK4 relocalization under osmotic stress. We also identified a novel function of WNK4 in ferroptosis. We first observed protective effects of WNK4 in ferroptosis in a cell survival assay, and then examined the involvement of WNK4 O-GlcNAcylation in ferroptosis in a brain slice model for neurodegeneration. These results provide evidence that O-GlcNAc regulates the functions of WNK4 in different cellular contexts. In addition to KLHL3, I also investigated whether KLHL2, KLHL40, KLHL1, and KLHL38 are O-GlcNAcylated. I detected O-GlcNAcylation on KLHL2 and KLHL40, and further exploration is needed to elucidate whether O-GlcNAc regulates their functions.Taken together, in my thesis work, I investigated whether O-GlcNAc regulation of KLHL proteins and their substrates can be generalized to other members of the KLHL family, beyond KEAP1 and gigaxonin. Data on KLHL3 and WNK kinase O-GlcNAcylation suggest that O-GlcNAc may mediate the response of WNK4 in different physiological and pathological contexts, specifically osmolarity and ferroptosis. Future work will focus on deciphering the detailed molecular mechanisms of O-GlcNAc regulation on WNK4 and extending our knowledge of O-GlcNAcylation to more KLHL proteins and their substrates.