Hypertension Causing Mutations in Cullin3 Decrease RhoA Ubiquitination and Sequester Substrate Adaptors

Abstract

Cullin-Ring ubiquitin Ligases (CRL) regulate protein turnover by promoting ubiquitination of substrates. Impaired Cullin3 (Cul3) activity in vascular smooth muscle causes increased RhoA/Rho kinase signaling leading to augmented contraction. PHAII patients with de novo Cul3 mutations which cause skipping of exon 9 (Cul3Δ9) have early onset hypertension, but the mechanistic basis remains unclear. We hypothesize that Cul3Δ9 reduces RhoA ubiquitination and degradation. There was no defect in neddylation of Cul3Δ9. However, binding of Cul3Δ9 to Rbx1, which is necessary for delivering ubiquitin to the substrate was impaired. Consistent with this, ubiquitination of RhoA by Cul3Δ9 was significantly impaired compared with Cul3WT. Thus Cul3Δ9 exhibits impaired ubiquitination of Cul3 substrates. Interestingly, substrate recognition proteins Bacurd1, RhoBTB1 and KLHL3 bound more efficiently to Cul3Δ9 than Cul3WT. Moreover, we observed a significant increase in Cul3Δ9:Cul3WT heterodimers compared to Cul3Δ9:Cul3Δ9 homodimers. This is important because dimerization of CRL complexes is required for efficient ubiquitination activity. Thus Cul3Δ9 may act dominantly by sequestering substrate adaptors from the CRL3 complex or by directly interfering with Cul3WT. We conclude that Cul3Δ9 exhibits impaired ubiquitin ligase activity. In smooth muscle, this would increase the pool of activatable RhoA in response to contractile agonists. When combined with our previous data, we suggest that Cul3-mediated regulation of RhoA protein turnover controls both vascular function and arterial blood pressure. Patients carrying Cul3Δ9 mutations may exhibit elevated smooth muscle RhoA/Rho kinase activity significantly contributing to their hypertension.