| First Author | Ibeawuchi SR | Year | 2015 |
| Journal | J Biol Chem | Volume | 290 |
| Issue | 31 | Pages | 19208-17 |
| PubMed ID | 26100637 | Mgi Jnum | J:224601 |
| Mgi Id | MGI:5688412 | Doi | 10.1074/jbc.M115.645358 |
| Citation | Ibeawuchi SR, et al. (2015) Hypertension-causing Mutations in Cullin3 Protein Impair RhoA Protein Ubiquitination and Augment the Association with Substrate Adaptors. J Biol Chem 290(31):19208-17 |
| abstractText | 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 (Cul3Delta9) 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, Cul3Delta9 supported reduced ubiquitin ligase activity toward RhoA compared with equivalent levels of wild-type Cul3 (Cul3WT). Consistent with its reduced activity, binding of Cul3Delta9 to the E3 ubiquitin ligase Rbx1 and neddylation of Cul3Delta9 were impaired significantly compared with Cul3WT. Conversely, Cul3Delta9 bound to substrate adaptor proteins more efficiently than Cul3WT. Cul3Delta9 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 Cul3Delta9 in HEK293T(Cul3KO) cells resulted in a decrease in the active form of Cul3WT. We conclude that Cul3Delta9-associated ubiquitin ligase activity toward RhoA is impaired and suggest that Cul3Delta9 mutations may act dominantly by sequestering substrate adaptors and disrupting Cul3WT complexes. |
