This work was supported by NIH grants R01-DK-51496 (to D. Ellison and C. Yang) and T32-DK-067864 (to D. H. Ellison), as well as by Merit Review Grant no. 1I01BX002228–01A1 from the Department of Veterans Affairs (to D. Ellison), and by American Heart Association grant no. 16POST3064003 and NIH grant no. F32-DK-112531 (to R. Cornelius). C. Zhang was supported by the National Natural Science Foundation of China grant. nos. 81570634 and 81770706. C. Sigmund was supported by NIH grant no. R01-HL-125603.
American Journal of Physiology
Hypertension -- Etiology, Ubiquitin -- Pathophysiology, Ligases, Protein kinases -- Mutation
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.
Cornelius, Ryan J.; Zhang, Chang; Erspamer, Kayla J.; Agbor, Larry N.; Sigmund, Curt D.; Singer, Jeffrey; Yang, Chao-Ling; and Ellison, David H., "Dual Gain and Loss of Cullin 3 Function Mediates Familial Hyperkalemic Hypertension" (2018). Biology Faculty Publications and Presentations. 228.
This is the author’s version of a work that was accepted for publication in American Journal of Physiology-Renal Physiology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication.
A definitive version was subsequently published in American Journal of Physiology-Renal Physiology, Vol. 315, Issue 4, September 2018 and is available online: https://doi.org/10.1152/ajprenal.00602.2017