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

Stella-Rita C Ibeawuchi; Larry N Agbor; Frederick W Quelle; Curt D Sigmund

doi:10.1074/jbc.M115.645358

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

Journal article

Open access

Peer reviewed

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

Stella-Rita C Ibeawuchi, Larry N Agbor, Frederick W Quelle and Curt D Sigmund

The Journal of biological chemistry, Vol.290(31), pp.19208-19217

07/31/2015

DOI: 10.1074/jbc.M115.645358

PMCID: PMC4521042

PMID: 26100637

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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.

Cullin Proteins - genetics

Gene Expression

Ubiquitination

Humans

Protein Multimerization

HEK293 Cells

Protein Binding

rhoA GTP-Binding Protein - metabolism

Hypertension - enzymology

Hypertension - genetics

Carrier Proteins

Details

Title: Subtitle: Hypertension-causing Mutations in Cullin3 Protein Impair RhoA Protein Ubiquitination and Augment the Association with Substrate Adaptors
Creators: Stella-Rita C Ibeawuchi - From the Department of Pharmacology, University of Iowa, Iowa City, Iowa 52242
Larry N Agbor - From the Department of Pharmacology, University of Iowa, Iowa City, Iowa 52242
Frederick W Quelle - From the Department of Pharmacology, University of Iowa, Iowa City, Iowa 52242
Curt D Sigmund - From the Department of Pharmacology, University of Iowa, Iowa City, Iowa 52242 curt-sigmund@uiowa.edu
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.290(31), pp.19208-19217
DOI: 10.1074/jbc.M115.645358
PMID: 26100637
PMCID: PMC4521042
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: United States
Grant note: R01 HL125603 / NHLBI NIH HHS HL048058 / NHLBI NIH HHS T32 GM067795 / NIGMS NIH HHS HL084207 / NHLBI NIH HHS R01 HL048058 / NHLBI NIH HHS P01 HL062984 / NHLBI NIH HHS P01 HL084207 / NHLBI NIH HHS HL062984 / NHLBI NIH HHS R37 HL048058 / NHLBI NIH HHS
Language: English
Date published: 07/31/2015
Academic Unit: Molecular Physiology and Biophysics; Neuroscience and Pharmacology; Internal Medicine
Record Identifier: 9984040283102771

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