Mutant Cullin 3 causes familial hyperkalemic hypertension via dominant effects
Mohammed Z. Ferdaus, … , Curt D. Sigmund, James A. McCormick
Mohammed Z. Ferdaus, … , Curt D. Sigmund, James A. McCormick
Published December 21, 2017
Citation Information: JCI Insight. 2017;2(24):e96700. https://doi.org/10.1172/jci.insight.96700.
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Research Article Nephrology

Mutant Cullin 3 causes familial hyperkalemic hypertension via dominant effects

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.

Authors

Mohammed Z. Ferdaus, Lauren N. Miller, Larry N. Agbor, Turgay Saritas, Jeffrey D. Singer, Curt D. Sigmund, James A. McCormick

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Figure 7

Model of normal Cullin RING Ligase (CRL) function and proposed mechanisms of CUL3-Δ9–mediated familial hyperkalemic hypertension (FHHt).

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Model of normal Cullin RING Ligase (CRL) function and proposed mechanism...
(A) The active WT CRL is a complex composed of 2 Cullin 3–containing (CUL3-containing) complexes. CUL3 acts as a scaffold for the complex. One CUL3 monomer is covalently linked to Neural precursor cell expressed developmentally downregulated protein 8 (Nedd8), which may facilitate dimerization with a non-Nedd8–conjugated CUL3 monomer and also activates the CRL (35). Each CUL3 monomer can interact with many different substrate adaptors, but Kelch-like 3 (KLHL3 is shown here since it is relevant to FHHt. WNK kinases interact with KLHL3, which brings them close to the RING E2 ubiquitin ligase that covalently attaches ubiquitin (Ub) to With-No-Lysine [K] Kinases (WNKs), targeting them for proteasomal degradation. (B) Three mechanisms have been proposed to explain how CUL3-Δ9 causes FHHt. In the haploinsufficiency model, CUL3-Δ9 triggers degradation of itself, but not of WT CUL3, by the proteasome (30). Fewer functional CRLs are available to degrade WNKs, which accumulate, leading to inappropriate Na+-Cl– cotransporter (NCC) phosphorylation and activation by Sterile 20 (STE20)/SPS-1–related proline/alanine-rich kinase (SPAK)/oxidative stress–response kinase-1 (OSR1). Our data do not support this model. In the KLHL3 degradation model, CUL3-Δ9 inappropriately degrades KLHL3, decreasing the number of active CRLs and causing accumulation of WNKs (28). Since KLHL3 is highly expressed along the distal convoluted tubule, the site of NCC expression, this leads to FHHt, which is primarily a disease of NCC dysregulation. This model assumes that CUL3-Δ9 exerts most of its effects on KLHL3 and not on other CUL3 adaptors. Western blotting (30) and immunofluorescence for KLHL3 (Supplemental Figure 10) in CUL3-Δ9–expressing mouse models do not support this model. In the sequestration model, CUL3/CUL3-Δ9 heterodimers are less stable than CUL3/CUL3 homodimers, and CUL3-Δ9 may also sequester adapters (29), leading to a lower number of active CRLs and resulting in WNK accumulation. Our data do not refute this model, but more extensive disruption of CRL activity might be expected to cause multiple defects, not just FHHt.