title: Hypertension-causing Mutations in Cullin3 Protein Impair RhoA Protein Ubiquitination and Augment the Association with Substrate Adaptors.
abstract: 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 (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 (HEK293T(Cul3KO)). When expressed in these cells, Cul3Δ9 supported reduced ubiquitin ligase activity toward RhoA compared with equivalent levels of wild-type Cul3 (Cul3WT). Consistent with its reduced activity, binding of Cul3Δ9 to the E3 ubiquitin ligase Rbx1 and neddylation of Cul3Δ9 were impaired significantly compared with Cul3WT. Conversely, Cul3Δ9 bound to substrate adaptor proteins more efficiently than Cul3WT. Cul3Δ9 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 Cul3Δ9 in HEK293T(Cul3KO) 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.
authors.affiliation: From the Department of Pharmacology, University of Iowa, Iowa City, Iowa 52242.
authors.forename: Stella-Rita C
authors.initials: SR
authors.lastname: Ibeawuchi
authors.affiliation: From the Department of Pharmacology, University of Iowa, Iowa City, Iowa 52242.
authors.forename: Larry N
authors.initials: LN
authors.lastname: Agbor
authors.affiliation: From the Department of Pharmacology, University of Iowa, Iowa City, Iowa 52242.
authors.forename: Frederick W
authors.initials: FW
authors.lastname: Quelle
authors.affiliation: From the Department of Pharmacology, University of Iowa, Iowa City, Iowa 52242 curt-sigmund@uiowa.edu.
authors.forename: Curt D
authors.initials: CD
authors.lastname: Sigmund
mesh_terms: D002352:Carrier Proteins; D044842:Cullin Proteins / Q000235:genetics*; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D006973:Hypertension / Q000201:enzymology / Q000235:genetics*; D011485:Protein Binding; D055503:Protein Multimerization; D054875:Ubiquitination; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: C503645:CUL3 protein, human; D002352:Carrier Proteins; D044842:Cullin Proteins; C479529:RBX1 protein, human; C066737:RHOA protein, human; D020742:rhoA GTP-Binding Protein
keywords: E3 ubiquitin ligase; Ras homolog gene family, member A (RhoA); hypertension; mutation; protein turnover; scaffold protein