Hypertension-causing Mutations in Cullin3 Protein Impair RhoA Protein Ubiquitination and Augment the Association with Substrate Adaptors

Abstract

Background: Cullin3 ubiquitin ligase regulates protein turnover by promoting the ubiquitination of substrates. Results: Ubiquitination of RhoA is impaired by mutations in Cullin3. Conclusion: Disease-causing Cullin3 mutations impair the turnover of RhoA protein and may sequester substrates adaptors. Significance: Mutations in Cullin3 cause reduced ubiquitination and elevation of RhoA levels, which may enhance RhoA and Rho kinase signaling in a variety of cell types and could potentially contribute to hypertension.

Mutant Cullin 3 causes familial hyperkalemic hypertension via dominant effects

“…We previously proposed that CUL3-Δ9 is an overactive form of CUL3 that inappropriately degrades KLHL3 (28). The Sigmund group reported that WT CUL3/CUL3-Δ9 heterodimers are less stable than CUL3 WT/WT homodimers, and that CUL3-Δ9 may also sequester adapters (29). Finally, the Kurz group confirmed our finding with respect to inappropriate degradation of KLHL3 but also reported that CUL3-Δ9 displays autoubiquitination, leading to its own proteasomal degradation (30).…”

“…The mechanism by which CUL3-Δ9 leads to FHHt is controversial, but this is primarily related to the fact that most studies have been performed in vitro (28)(29)(30)34). A constitutive deletion of exon 9 in mice suggested that FHHt is due to haploinsufficiency resulting from degradation of CUL3-Δ9 following autoubiquitination (30).…”

“…Attempts to use an antibody recently shown to be specific using kidney lysates from KLHL3-KO mice (27) have thus far been unsuccessful (data not shown), but we detected no differences in KLHL3 abundance along DCT1 and DCT2/CNT by IF using this antibody (Supplemental Figure 11). Another model for CUL3-Δ9-mediated FHHt proposes that CUL3-Δ9 exerts dominant-negative effects, disrupting WT CUL3 homodimers, and possibly also causing sequestration of substrate adaptors, thus preventing substrate ubiquitination (29). Combined with the reduction of abundance of WT CUL3, WT CUL3 homodimer disruption and adapter sequestration would also be expected to further reduce activity of the WT CRL.…”

“…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.…”

Cullin-3 mutation causes arterial stiffness and hypertension through a vascular smooth muscle mechanism

“…This results in an in-frame deletion of 57 amino acids internal to CUL3. Evidence that the mutant protein (termed CUL3Δ9) has dominant-negative activity includes that (a) CUL3Δ9 supports impaired ubiquitin ligase activity toward normal substrates, (b) CUL3Δ9 exhibits enhanced binding to substrate adaptors and promotes their degradation, and (c) CUL3Δ9 may form inhibitory and unstable heterodimers with wildtype CUL3 (CUL3WT) (4)(5)(6).…”