Abstract 297: Naturally Occurring Cullin-3 Mutations Decrease Substrate Ubiquitination and Acts Dominantly by Sequestering Substrate Adaptors for Cul3

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Cullin-Ring ubiquitin Ligases (CRL) regulate protein turnover by promoting ubiquitination of substrate proteins targeting them for proteosomal degradation. Impaired Cullin-3 (Cul3) activity in vascular smooth muscle causes increased RhoA protein and Rho kinase signaling leading to impaired vasodilation and augmented contraction. PHAII patients with de novo mutations in Cul3 which cause skipping of exon 9 in Cul3 (Cul3Δ9) have early onset hypertension, but the mechanistic basis remains unclear. We hypothesize that Cul3Δ9 results in reduced RhoA ubiquitination and degradation, increasing RhoA activity. We cloned a mutant Cul3Δ9 cDNA by “splicing by overhang extension” PCR. Endogenous RhoA protein was increased in HEK293T cells expressing Flag-Cul3Δ9 compared with cells expressing wildtype Flag-Cul3 (Cul3WT). 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 epitope-tagged RhoA by Cul3Δ9 was significantly impaired compared with Cul3WT both in vivo and in vitro. Thus Cul3Δ9 exhibits impaired ubiquitination of Cul3 substrates. Interestingly, co-IP studies revealed that the substrate adaptor 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 Cul3 ubiquitin ligase complexes is required for efficient ubiquitination of some substrates. Thus Cul3Δ9 may act dominantly by interfering with Cul3WT by sequestering substrate adaptors from the CRL3 complex or by directly inhibiting the Cul3 dimer. We conclude that Cul3Δ9 exhibits impaired ubiquitin ligase activity and interferes with activity of Cul3WT. In smooth muscle, this would increase the RhoA pool that can be activated 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 contributing to their hypertension.