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

Cullin/Ring ubiquitin ligases regulate protein turnover by promoting the ubiquitination of substrate proteins, targeting them for proteasomal degradation. It has been shown previously that mutations in Cullin3 (Cul3) causing deletion of 57 amino acids encoded by exon 9 (Cul3 Delta 9) cause hypertension. Moreover, RhoA activity contributes to vascular constriction and hypertension. We show that ubiquitination and degradation of RhoA is dependent on Cul3 in HEK293T cells in which Cul3 expression is ablated by either siRNA or by CRISPR-Cas9 genome editing. The latter was used to generate a Cul3-null cell line (HEK293T(Cul3KO)). When expressed in these cells, Cul3 Delta 9 supported reduced ubiquitin ligase activity toward RhoA compared with equivalent levels of wild-type Cul3 (Cul3WT). Consistent with its reduced activity, binding of Cul3 Delta 9 to the E3 ubiquitin ligase Rbx1 and neddylation of Cul3 Delta 9 were impaired significantly compared with Cul3WT. Conversely, Cul3 Delta 9 bound to substrate adaptor proteins more efficiently than Cul3WT. Cul3 Delta 9 also forms unstable dimers with Cul3WT, disrupting dimers of Cul3WT complexes that are required for efficient ubiquitination of some substrates. Indeed, coexpression of Cul3WT and Cul3 Delta 9 in HEK293T(Cul3KO) cells resulted in a decrease in the active form of Cul3WT. We conclude that Cul3 Delta 9-associated ubiquitin ligase activity toward RhoA is impaired and suggest that Cul3 Delta 9 mutations may act dominantly by sequestering substrate adaptors and disrupting Cul3WT complexes.