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 PHAIIcausing 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 diseasecausing mutations in both KLHL3 and WNK4.proteomics | Gordon syndrome | Kir1.1 H ypertension affects 1 billion people worldwide and is a principal reversible risk factor for cardiovascular disease. Identification of the causes of rare Mendelian forms of hypertension has demonstrated the key role of increased renal salt reabsorption in the pathogenesis of this common disease (1, 2).Among Mendelian hypertensive syndromes, pseudohypoaldosteronism type II (PHAII, also known as familial hypertensive hyperkalemia, Gordon syndrome, OMIM no. 145260) is particularly interesting because it has revealed previously unrecognized physiology involved in orchestrating the activities of different electrolyte flux pathways (3). The kidney is exposed to elevated levels of the steroid hormone aldosterone in two distinct physiologic conditions. Intravascular volume depletion activates the renin-angiotensin system, leading to increased angiotensin II (AII) levels. AII binds to its receptor in adrenal glomerulosa, leading to aldosterone secretion. In this setting, aldosterone signaling leads to a marked increase in renal Na-Cl reabsorption, defending intravascular volume. In the setting of hyperkalemia, high plasma K + levels depolarize glomerulosa cells, directly producing aldosterone secretion. In this case, aldosterone signaling supports increased electrogenic Na + reabsorption, providing the electrical driving force for K + secretion, restoring normal plasma K + levels. The kidney must be able to distinguish between these tw...

Angiotensin II signaling via protein kinase C phosphorylates Kelch-like 3, preventing WNK4 degradation

“…Interestingly, S433 is recurrently mutated in autosomal dominant PHAII (4,26). Mutation of S433 abrogates WNK4 binding and degradation, increasing WNK4 levels (22,23). This observation suggested that S433 phosphorylation might modulate KLHL3 binding and degradation of WNK4.…”

“…We quantitated WNK4 levels when expressed with either KLHL3 WT or KLHL3 S433E (23). As reported (23), KLHL3 WT reduced WNK4 levels by 57% (P = 0.002; Fig.…”

“…FLAG-KLHL3 WT or -KLHL3 S433E was incubated with WNK4-HA, and IP was performed with anti-FLAG, followed by Western blotting with anti-HA to detect WNK4. These experiments were performed in vitro to ensure that equal amounts of protein were input into all experiments, eliminating the confounding effect of KLHL3 causing WNK4 degradation in vivo (23). Whereas KLHL3 WT robustly bound WNK4, KLHL3 S433E showed almost no binding ( Fig.…”

“…Multiple lines of evidence suggest that WNK4 lies downstream of AII signaling (14,27,28), suggesting that AII regulates WNK4 levels via phosphorylation of KLHL3 S433 (23,29). Angiotensin type 1 receptor (AT1R) activation by AII induces release of the G protein alpha subunit, G q , which leads to activation of protein kinase C (PKC) (30).…”

“…[16][17][18][19][20][21], and WNK4 function can be modulated by phosphorylation (21). CUL3/KLHL3 has been shown to target WNK4 and WNK1 for ubiquitination and degradation, and disease-causing mutations impair this binding and degradation (22)(23)(24). In particular, dominant mutations in the Kelch domain of KLHL3 prevent binding to WNKs; reciprocally, disease-causing point mutations in WNK4 also prevent WNK4-KLHL3 binding.…”