PMID:23576762

Citation	Shibata, S, Zhang, J, Puthumana, J, Stone, KL and Lifton, RP (2013) Kelch-like 3 and Cullin 3 regulate electrolyte homeostasis via ubiquitination and degradation of WNK4. Proc. Natl. Acad. Sci. U.S.A. 110:7838-43
Abstract	Pseudohypoaldosteronism type II (PHAII) is a rare Mendelian syndrome featuring hypertension and hyperkalemia resulting from constitutive renal salt reabsorption and impaired K(+) secretion. Recently, mutations in Kelch-like 3 (KLHL3) and Cullin 3 (CUL3), components of an E3 ubiquitin ligase complex, were found to cause PHAII, suggesting that loss of this complex's ability to target specific substrates for ubiquitination leads to PHAII. By MS and coimmunoprecipitation, we show that KLHL3 normally binds to WNK1 and WNK4, members of WNK (with no lysine) kinase family that have previously been found mutated in PHAII. We show that this binding leads to ubiquitination, including polyubiquitination, of at least 15 specific sites in WNK4, resulting in reduced WNK4 levels. Dominant disease-causing mutations in KLHL3 and WNK4 both impair WNK4 binding, ubiquitination, and degradation. WNK4 normally induces clearance of the renal outer medullary K(+) channel (ROMK) from the cell surface. We show that WT but not mutant KLHL3 inhibits WNK4-induced reduction of ROMK level. We show that PHAII-causing mutations in WNK4 lead to a marked increase in WNK4 protein levels in the kidney in vivo. These findings demonstrate that CUL3-RING (really interesting new gene) ligases that contain KLHL3 target ubiquitination of WNK4 and thereby regulate WNK4 levels, which in turn regulate levels of ROMK. These findings reveal a specific role of CUL3 and KLHL3 in electrolyte homeostasis and provide a molecular explanation for the effects of disease-causing mutations in both KLHL3 and WNK4.
Links	PubMed PMC3651502 Online version:10.1073/pnas.1304592110
Keywords	Animals; COS Cells; Carrier Proteins/metabolism; Cercopithecus aethiops; Cullin Proteins/metabolism; Electrolytes; Gene Expression Regulation; Homeostasis; Humans; Intracellular Signaling Peptides and Proteins/metabolism; Mutation; Potassium Channels, Inwardly Rectifying/metabolism; Protein Binding; Protein-Serine-Threonine Kinases/metabolism; Proteomics/methods; Pseudohypoaldosteronism/genetics; Ubiquitin/metabolism; Ubiquitination/physiology
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
MOUSE:KLHL3	involved_in	GO:0010467: gene expression	ECO:0000314: direct assay evidence used in manual assertion		P		Seeded From UniProt	complete
MOUSE:KLHL3	involved_in	GO:0000209: protein polyubiquitination	ECO:0000314: direct assay evidence used in manual assertion		P		Seeded From UniProt	complete
MOUSE:KLHL3	involved_in	GO:0050801: ion homeostasis	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete
MOUSE:KLHL3	involved_in	GO:0010467: gene expression	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete
MOUSE:KLHL3	involved_in	GO:0030163: protein catabolic process	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete
MOUSE:KLHL3	involved_in	GO:0000209: protein polyubiquitination	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete
MOUSE:KLHL3	enables	GO:0005515: protein binding	ECO:0000353: physical interaction evidence used in manual assertion	UniProtKB:P83741	F		Seeded From UniProt	complete
MOUSE:KLHL3	enables	GO:0005515: protein binding	ECO:0000353: physical interaction evidence used in manual assertion	UniProtKB:Q80UE6	F		Seeded From UniProt	complete
MOUSE:WNK1	enables	GO:0005515: protein binding	ECO:0000353: physical interaction evidence used in manual assertion	UniProtKB:E0CZ16	F		Seeded From UniProt	complete
MOUSE:WNK4	involved_in	GO:0050801: ion homeostasis	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete
MOUSE:WNK4	involved_in	GO:0010467: gene expression	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete
MOUSE:WNK4	enables	GO:0005515: protein binding	ECO:0000353: physical interaction evidence used in manual assertion	UniProtKB:E0CZ16	F		Seeded From UniProt	complete
HUMAN:WNK4	involved_in	GO:0050801: ion homeostasis	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete
HUMAN:WNK4	involved_in	GO:0070294: renal sodium ion absorption	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete
HUMAN:WNK4	involved_in	GO:0072156: distal tubule morphogenesis	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete
HUMAN:WNK4	enables	GO:0005515: protein binding	ECO:0000353: physical interaction evidence used in manual assertion	UniProtKB:Q9UH77	F		Seeded From UniProt	complete
HUMAN:KLHL3	part_of	GO:0031463: Cul3-RING ubiquitin ligase complex	ECO:0000314: direct assay evidence used in manual assertion		C		Seeded From UniProt	complete
HUMAN:KLHL3	involved_in	GO:0070936: protein K48-linked ubiquitination	ECO:0000314: direct assay evidence used in manual assertion		P		Seeded From UniProt	complete
HUMAN:KLHL3	involved_in	GO:0006511: ubiquitin-dependent protein catabolic process	ECO:0000314: direct assay evidence used in manual assertion		P		Seeded From UniProt	complete
HUMAN:KLHL3	involved_in	GO:0050801: ion homeostasis	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete
HUMAN:KLHL3	involved_in	GO:0070294: renal sodium ion absorption	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete
HUMAN:KLHL3	involved_in	GO:0072156: distal tubule morphogenesis	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete
HUMAN:KLHL3	enables	GO:0005515: protein binding	ECO:0000353: physical interaction evidence used in manual assertion	UniProtKB:Q96J92	F		Seeded From UniProt	complete
HUMAN:CUL3	part_of	GO:0031463: Cul3-RING ubiquitin ligase complex	ECO:0000314: direct assay evidence used in manual assertion		C		Seeded From UniProt	complete

References

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