Dual Gain and Loss of Cullin 3 Function Mediates Familial Hyperkalemic Hypertension

Abstract

Familial hyperkalemic hypertension is caused by mutations in with‐no‐lysine (WNK) kinases, or in proteins that mediate their degradation, kelch‐like 3 (KLHL3) and cullin 3 (Cul3). While 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 enhanced binding and degradation of KLHL3 have all been postulated. Here, we systematically evaluated the effects of Cul3Δ403–459 using cultured kidney cells. First, using co‐immunoprecipitation, we identified that the catalytically active CSN subunit JAB1 does not associate with the deleted Cul3 4HB domain, but instead with the adjacent α/β1 domain, indicating that resulting secondary structural effects underlie impaired binding. Inhibition of deneddylation, with JAB1 siRNA, increased Cul3 neddylation, but did not mimic the effects of Cul3Δ403–459 with respect to KLHL3 and WNK4, indicating that hyperneddylation is not essential. Next, we prevented Cul3 neddylation by introducing a point mutation at the neddylation site (K712R). The neddylation‐deficient Cul3Δ403–459 K712R construct was still able to significantly degrade KLHL3, indicating that this degradation has both ubiquitin ligase‐dependent and ‐independent components. Pharmacological inhibition of the degradative pathways showed that proteasomal degradation of KLHL3 was enhanced by Cul3Δ403–459; however, autophagic degradation was also upregulated by the 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.Support or Funding InformationNIH R01 DK51496, NIH T32 DK067864, VA 1I01BX002228‐01A1, AHA 16POST3064003, NIH F32 DK112531, National Natural Science Foundation of China 81570634 and 81770706This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.