title: Kelch-like 3 and Cullin 3 regulate electrolyte homeostasis via ubiquitination and degradation of WNK4.
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.
authors.affiliation: Department of Genetics, Howard Hughes Medical Institute, and W. M. Keck Facility, Yale University School of Medicine, New Haven, CT 06510, USA.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Shibata
authors.forename: Junhui
authors.initials: J
authors.lastname: Zhang
authors.forename: Jeremy
authors.initials: J
authors.lastname: Puthumana
authors.forename: Kathryn L
authors.initials: KL
authors.lastname: Stone
authors.forename: Richard P
authors.initials: RP
authors.lastname: Lifton
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism*; D002522:Chlorocebus aethiops; D044842:Cullin Proteins / Q000378:metabolism*; D004573:Electrolytes; D005786:Gene Expression Regulation; D006706:Homeostasis; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008840:Microfilament Proteins; D015778:Minor Histocompatibility Antigens; D009154:Mutation; D024661:Potassium Channels, Inwardly Rectifying / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D011546:Pseudohypoaldosteronism / Q000235:genetics; D025801:Ubiquitin / Q000378:metabolism; D054875:Ubiquitination / Q000502:physiology*; D000076204:WNK Lysine-Deficient Protein Kinase 1
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C503645:CUL3 protein, human; D002352:Carrier Proteins; D044842:Cullin Proteins; D004573:Electrolytes; D047908:Intracellular Signaling Peptides and Proteins; C086989:KCNJ1 protein, human; C489837:KLHL3 protein, human; D008840:Microfilament Proteins; D015778:Minor Histocompatibility Antigens; D024661:Potassium Channels, Inwardly Rectifying; D025801:Ubiquitin; D017346:Protein Serine-Threonine Kinases; D000076204:WNK Lysine-Deficient Protein Kinase 1; C498743:WNK1 protein, human; C433806:WNK4 protein, human
keywords: Gordon syndrome; Kir1.1; proteomics