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.

Keywords: Gordon syndrome; Kir1.1; proteomics.

Fig. 1.

Association of WNK kinases with CUL3–KLHL3 complex. (A and B) KLHL3 binds to endogenous CUL3 and WNK1. Products of IP of FLAG-KLHL3 from COS-7 cell lysates were analyzed by LC-MS/MS. Representative MS/MS spectra from peptides mapping to CUL3 (Macaca mulatta, amino acids 717–731) (A) or WNK1 (Macaca mulatta, amino acids 1855–1868) (B) are shown (all peptides in Tables S1 and S2). (C) IP of cell lysates with anti-FLAG (KLHL3) was followed by Western blotting (WB). CUL3 is pulled down only when FLAG-KLHL3 is expressed. (D) Cell lysates expressing indicated proteins were immunoprecipitated with anti-HA, followed by Western blotting. When WNK4-HA is coexpressed with KLHL3, both KLHL3 and CUL3 are immunoprecipitated, whereas neither CUL3 nor KLHL3 is detected in IP products in the absence of KLHL3. (E) Cell lysates expressing the indicated proteins were immunoprecipitated using FLAG antibody to IP FLAG-KLHL3, followed by Western analysis with antibody to HA to detect WNK4-HA (Upper) or FLAG to detect KLHL3 (Lower). IP of FLAG-KLHL3 pulls down WNK4.

Fig. 2.

PHAII mutations in WNK4 or KLHL3 abrogate WNK4 binding to KLHL3. Cell lysates from cells expressing FLAG-KLHL3^WT or FLAG-KLHL3^R528H were incubated with lysates from cells expressing WNK4^WT-HA, WNK4^E559K-HA, or WNK4^Q562E-HA followed by anti-FLAG (KLHL3) immunoprecipitation and Western blotting (WB). PHAII mutations in KLHL3 and WNK4 eliminate or reduce binding of WNK4 to KLHL3.

Fig. 3.

Identification of ubiquitin conjugation sites in WNK4 by MS. (A) Cell lysates expressing the indicated proteins were immunoprecipitated in denaturing condition with anti-HA, followed by Western blotting (WB) with anti-ubiquitin antibodies. WNK4 is ubiquitinated in the presence but not the absence of KLHL3. (B) MS/MS spectrum of purified ubiquitinated WNK4 showing assignment of the peptide containing ubiquitinated K325 in the WNK4 kinase domain. The precursor ion, 635.88²⁺, was selected and produced the fragment ion spectrum shown. Specific y and b fragment ions allowed the identification of K325 with di-glycine modification (shown as GG) that is derived from the C terminus of ubiquitin. (C) Ubiquitination sites of WNK4 in COS-7 cells expressing KLHL3. Sites are numbered according to their position in mouse sequence. The domains of WNK4 are indicated and lysines that are ubiquitinated are shown. Peptides and Mascot scores are shown in Table 1. CC, coiled-coil domain.

Fig. 4.

KLHL3^WT, but not KLHL3^R528H reduces WNK4 levels. (A) Western blots of cell lysates show markedly reduced levels of WNK4 when KLHL3^WT but not KLHL3^R528H is coexpressed. An empty vector was used as control to equalize the total amount of DNA in transfections lacking KLHL3. (Lower) Bar graphs show quantitation of the results of four independent experiments in each condition. **P < 0.01. (B) COS-7 cell lysates expressing the indicated proteins were immunoprecipitated with anti-HA, followed by Western blotting (WB) with anti-ubiquitin antibodies. Polyubiquitination of WNK4 was markedly reduced in the presence of KLHL3^R528H. In addition, WNK4 level was reduced in cells expressing KLHL3^WT compared with KLHL3^R528H. Representative results of triplicate experiments are shown. (C) Western blots of lysates of cells expressing FLAG-KLHL3^WT and either WNK4^WT-HA, WNK4^E559K-HA, or WNK4^Q562E-HA are shown. Mutant WNK4s show significantly highly levels compared with WT. Bar graph shows the results of quantitation (n = 4). Data are expressed as mean ± SEM.

Fig. 5.

KLHL3 abrogates WNK4’s inhibition of ROMK expression. The indicated proteins were expressed, and levels of EGFP-ROMK in the membrane fraction were analyzed by Western blotting (Upper). Cadherin was used as a loading control (Lower). KLHL3^WT inhibits WNK4-dependent reduction of ROMK level, and effect lost with KLHL3^R528H. Results from biological replicates are shown and bar graphs show the results of quantitation (n = 4). Data are expressed as means ± SEM; *P < 0.05, **P < 0.01.

Fig. 6.

Renal expression of WNK4 in WT, Tg(WNK4^WT), and Tg(WNK4^PHAII) mice. Kidneys from 3- to 5-mo-old mice of indicated genotypes (n = 3 of each) were fixed by in vivo perfusion and sectioned and stained with anti-WNK4. (Upper) High-power and (Lower) low-power images. Intensity of WNK4 staining (arrows) was consistently increased in Tg(WNK4^PHAII) mice compared with WT littermates or Tg(WNK4^WT). (Scale bars, 100 μm.)