Dual gain and loss of cullin 3 function mediates familial hyperkalemic hypertension

Ryan J. Cornelius, Chong Zhang, Kayla J. Erspamer, Larry N. Agbor, Curt D. Sigmund, Jeffrey D. Singer, Chao-Ling Yang, David Ellison

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Familial hyperkalemic hypertension is caused by mutations in with-no-lysine kinases (WNKs) or in proteins that mediate their degradation, kelch-like 3 (KLHL3) and cullin 3 (CUL3). Although the mechanisms by which WNK and KLHL3 mutations cause the disease are now clear, the effects of the disease-causing CUL3Δ403-459 mutation remain controversial. Possible mechanisms, including hyperneddylation, altered ubiquitin ligase activity, decreased association with the COP9 signalosome (CSN), and increased association with and degradation of KLHL3 have all been postulated. Here, we systematically evaluated the effects of Cul3Δ403-459 using cultured kidney cells. We first identified that the catalytically active CSN subunit jun activation domain-binding protein-1 (JAB1) does not associate with the deleted Cul3 4-helix bundle domain but instead with the adjacent α/β1 domain, suggesting that altered protein folding underlies the impaired binding. Inhibition of deneddylation with JAB1 siRNA increased Cul3 neddylation and decreased KLHL3 abundance, similar to the Cul3 mutant. We next determined that KLHL3 degradation has both ubiquitin ligase-dependent and -independent components. Proteasomal KLHL3 degradation was enhanced by Cul3Δ403-459; however, autophagic degradation was also upregulated by this Cul3 mutant. Finally, to evaluate whether deficient substrate adaptor was responsible for the disease, we restored KLHL3 to wild-type (WT) Cul3 levels. In the absence of WT Cul3, WNK4 was not degraded, demonstrating that Cul3Δ403-459 itself cannot degrade WNK4; conversely, when WT Cul3 was present, as in diseased humans, WNK4 degradation was restored. In conclusion, deletion of exon 9 from Cul3 generates a protein that is itself ubiquitin-ligase defective but also capable of enhanced autophagocytic KLHL3 degradation, thereby exerting dominant-negative effects on the WT allele.

Original languageEnglish (US)
Pages (from-to)F1006-F1018
JournalAmerican journal of physiology. Renal physiology
Volume315
Issue number4
DOIs
StatePublished - Oct 1 2018

Fingerprint

Cullin Proteins
Ligases
Ubiquitin
Hypertension
Mutation
Lysine
Carrier Proteins
Phosphotransferases
Protein Folding
Small Interfering RNA
Exons
Cultured Cells
Proteins
Alleles
Kidney

Keywords

  • cullin-RING ubiquitin ligase
  • deneddylation
  • JAB1
  • neddylation

ASJC Scopus subject areas

  • Physiology
  • Urology

Cite this

Dual gain and loss of cullin 3 function mediates familial hyperkalemic hypertension. / Cornelius, Ryan J.; Zhang, Chong; Erspamer, Kayla J.; Agbor, Larry N.; Sigmund, Curt D.; Singer, Jeffrey D.; Yang, Chao-Ling; Ellison, David.

In: American journal of physiology. Renal physiology, Vol. 315, No. 4, 01.10.2018, p. F1006-F1018.

Research output: Contribution to journalArticle

Cornelius, Ryan J. ; Zhang, Chong ; Erspamer, Kayla J. ; Agbor, Larry N. ; Sigmund, Curt D. ; Singer, Jeffrey D. ; Yang, Chao-Ling ; Ellison, David. / Dual gain and loss of cullin 3 function mediates familial hyperkalemic hypertension. In: American journal of physiology. Renal physiology. 2018 ; Vol. 315, No. 4. pp. F1006-F1018.
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abstract = "Familial hyperkalemic hypertension is caused by mutations in with-no-lysine kinases (WNKs) or in proteins that mediate their degradation, kelch-like 3 (KLHL3) and cullin 3 (CUL3). Although the mechanisms by which WNK and KLHL3 mutations cause the disease are now clear, the effects of the disease-causing CUL3Δ403-459 mutation remain controversial. Possible mechanisms, including hyperneddylation, altered ubiquitin ligase activity, decreased association with the COP9 signalosome (CSN), and increased association with and degradation of KLHL3 have all been postulated. Here, we systematically evaluated the effects of Cul3Δ403-459 using cultured kidney cells. We first identified that the catalytically active CSN subunit jun activation domain-binding protein-1 (JAB1) does not associate with the deleted Cul3 4-helix bundle domain but instead with the adjacent α/β1 domain, suggesting that altered protein folding underlies the impaired binding. Inhibition of deneddylation with JAB1 siRNA increased Cul3 neddylation and decreased KLHL3 abundance, similar to the Cul3 mutant. We next determined that KLHL3 degradation has both ubiquitin ligase-dependent and -independent components. Proteasomal KLHL3 degradation was enhanced by Cul3Δ403-459; however, autophagic degradation was also upregulated by this Cul3 mutant. Finally, to evaluate whether deficient substrate adaptor was responsible for the disease, we restored KLHL3 to wild-type (WT) Cul3 levels. In the absence of WT Cul3, WNK4 was not degraded, demonstrating that Cul3Δ403-459 itself cannot degrade WNK4; conversely, when WT Cul3 was present, as in diseased humans, WNK4 degradation was restored. In conclusion, deletion of exon 9 from Cul3 generates a protein that is itself ubiquitin-ligase defective but also capable of enhanced autophagocytic KLHL3 degradation, thereby exerting dominant-negative effects on the WT allele.",
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AU - Sigmund, Curt D.

AU - Singer, Jeffrey D.

AU - Yang, Chao-Ling

AU - Ellison, David

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