Hypertension Causing Mutations in Cullin3 Impair RhoA Ubiquitination and Augment Association with Substrate Adaptors [Protein Synthesis and Degradation]

June 22nd, 2015 by Ibeawuchi, S.-R. C., Agbor, L. N., Quelle, F. W., Sigmund, C. D.

Cullin-Ring ubiquitin Ligases (CRL) regulate protein turnover by promoting ubiquitination of substrate proteins, targeting them for proteasomal degradation. It was previously shown that mutations in Cullin3 (Cul3) causing deletion of 57 amino acids encoded by exon 9 (Cul3Δ9) 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 (HEK293TCul3KO). When expressed in these cells, Cul3Δ9 supported reduced ubiquitin ligase activity toward RhoA compared to equivalent levels of wildtype Cul3 (Cul3WT). Consistent with its reduced activity, binding of Cul3Δ9 to the E3 ubiquitin ligase, Rbx1, and neddylation of Cul3Δ9 was significantly impaired compared to Cul3WT. Conversely, Cul3Δ9 bound to substrate adaptor proteins more efficiently than Cul3WT. Cul3Δ9 also forms unstable dimers with Cul3WT, thus disrupting dimers of Cul3WT complexes that are required for efficient ubiquitination of some substrates. Indeed, co-expression of Cul3WT and Cul3Δ9 in HEK293TCul3KO cells resulted in a decrease in the active form of Cul3WT. We conclude that Cul3Δ9-associated ubiquitin ligase activity toward RhoA is impaired, and suggest that Cul3Δ9 mutations may act dominantly by sequestering substrate adaptors and disrupting Cul3WT complexes.
  • Posted in Journal of Biological Chemistry, Publications
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