Abstract
Mutations in the ubiquitin ligase scaffold protein Cullin 3 (CUL3) cause the disease familial hyperkalemic hypertension (FHHt). In the kidney, mutant CUL3 (CUL3-Δ9) increases abundance of With-No-Lysine [K] Kinase 4 (WNK4), with excessive activation of the downstream Sterile 20 (STE20)/SPS-1-related proline/alanine-rich kinase (SPAK) increasing phosphorylation of the Na+-Cl- cotransporter (NCC). CUL3-Δ9 promotes its own degradation via autoubiquitination, leading to the hypothesis that Cul3 haploinsufficiency causes FHHt. To directly test this, we generated Cul3 heterozygous mice (CUL3-Het), and Cul3 heterozygotes also expressing CUL3-Δ9 (CUL3-Het/Δ9), using an inducible renal epithelial-specific system. Endogenous CUL3 was reduced to 50% in both models, and consistent with autoubiquitination, CUL3-Δ9 protein was undetectable in CUL3-Het/Δ9 kidneys unless primary renal epithelia cells were cultured. Abundances of WNK4 and phosphorylated NCC did not differ between control and CUL3-Het mice, but they were elevated in CUL3-Het/Δ9 mice, which also displayed higher plasma [K+] and blood pressure. Abundance of phosphorylated Na+-K+-2Cl- cotransporter (NKCC2) was also increased, which may contribute to the severity of CUL3-Δ9-mediated FHHt. WNK4 and SPAK localized to puncta in NCC-positive segments but not in NKCC2-positive segments, suggesting differential effects of CUL3-Δ9. These results indicate that Cul3 haploinsufficiency does not cause FHHt, but dominant effects of CUL3-Δ9 are required.
Highlights
Identifying the genes responsible for rare inherited diseases has led to profound insights into both normal and abnormal physiological processes
The discovery that mutations in the With-no-lysine [K] kinases (WNKs) Wnk1 and Wnk4 cause familial hyperkalemic hypertension (FHHt) [1, 2] and, more recently, that mutations in Cullin 3 (Cul3) (OMIM 614496, PHA2E) [3,4,5] and Kelch-like 3 (Klhl3) [4, 5] — components of an E3 ubiquitin ligase complex — cause the disease has revealed a canonical pathway for the regulation of the Na+Cl– cotransporter (NCC)
The mechanism by which Cullin 3 (CUL3)-Δ9 leads to FHHt is controversial, but this is primarily related to the fact that most studies have been performed in vitro [28,29,30, 34]
Summary
Identifying the genes responsible for rare inherited diseases has led to profound insights into both normal and abnormal physiological processes. Rather than being directly phosphorylated by WNKs, phosphorylation is performed primarily by Sterile 20 (STE20)/SPS-1–related proline/alanine-rich kinase (SPAK) [8, 9], with Oxidative stress-response kinase-1 (OSR1) playing a minor role [6]. These kinases are activated by WNK-mediated phosphorylation. FHHt-causing mutations in Wnk or Wnk increase abundances of WNK1 and WNK4 protein, increasing phosphorylation and activation of SPAK/OSR1 [1, 2, 10] and, NCC phosphorylation.
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