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

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/β 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.

Renal COP9 Signalosome Deficiency Alters CUL3-KLHL3-WNK Signaling Pathway

“…33,52 Thus, hyperneddylation can disrupt regulated degradation of substrates by abnormally targeting its substrate adaptor. The results here are consistent with previous work in cultured cells using CSN inhibitors; KLHL3 was degraded 36 and Keap1 abundance increased. 53 Furthermore, CUL3D9 showed a similar effect on the substrate adaptors, both in vitro 36,37 and in vivo.…”

“…First, to mimic the KS-Jab1 2/2 mice we inhibited endogenous JAB1 expression by using siRNA in cells overexpressing NCC ( Figure 5C). As shown previously, 36 a reduction in JAB1 protein caused an increased in cullin neddylation compared with control siRNA. NCC abundance was dramatically reduced in cells transfected with Jab1 siRNA compared with cells containing control siRNA.…”

“…[25][26][27][28][29] Inhibition of any subunit, either in vitro [30][31][32][33][34] or in vivo, 33,35 leads to decreased expression of substrate adaptor proteins. Two groups have shown that the CUL3 FHHt mutant (CUL3D9) has impaired binding to the CSN 11,36 ; our group found that CUL3D9 also targets KLHL3 for degradation. The loss of interaction with the CSN could account for enhanced KLHL3 degradation, perhaps explaining the disease phenotype, as we postulated previously.…”

“…In cultured cells, the protein produced by the disease-causing mutant CUL3D9 does not associate normally with the CSN, 11,36 is hyperneddylated, 11,36 and degrades KLHL3 anomalously. 37 On the basis of these findings, we postulated that the resulting low KLHL3 abundance impairs WNK kinase degradation, contributing to the phenotype of FHHt.…”

“…37 We recently proposed, on the basis of work primarily in cells, that CUL3D9 exhibits both gain-and loss-of-function features, with regard to specific substrates and substrate adaptors. 36 As CSN's catalytic activity is contained within the JAB1 subunit, 21 and CUL3D9 is hyperneddylated in cultured cells, 11,37 we postulated that hyperneddylation contributes to substrate accumulation and substrate adaptor instability. Here, we generated mice in which JAB1 could be deleted from kidney-tubule cells (KS-Jab1 2/2 ) to test the importance of the CSN-binding deficiency in disease pathogenesis, in vivo.…”