Moyamoya disease (MMD) is a cerebrovascular disease characterized by progressive occlusion of the internal carotid arteries. Genetic studies originally identified RNF213 as an MMD susceptibility gene that encodes a large 591 kDa protein with a functional RING domain and dual AAA+ ATPase domains. As the functions of RNF213 and its relationship to MMD onset are unknown, we set out to characterize the ubiquitin ligase activity of RNF213, and the effects of MMD patient mutations on these activities and on other cellular processes. In vitro ubiquitination assays, using the RNF213 RING domain, identified Ubc13/Uev1A as a key ubiquitin conjugating enzyme that together generate K63-linked polyubiquitin chains. However, nearly all MMD patient mutations in the RING domain greatly reduced this activity. When full-length proteins were overexpressed in HEK293T cells, patient mutations that abolished the ubiquitin ligase activities conversely enhanced nuclear factor κB (NFκB) activation and induced apoptosis accompanied with Caspase-3 activation. These induced activities were dependent on the RNF213 AAA+ domain. Our results suggest that the NFκB- and apoptosis-inducing functions of RNF213 may be negatively regulated by its ubiquitin ligase activity and that disruption of this regulation could contribute towards MMD onset.

RNF213 encodes a unique protein with AAA+ ATPase and E3 ubiquitin ligase activities that are critical for its diverse roles, which range from involvement in human vasculopathies, such as Moyamoya disease, to ubiquitination of viral and bacterial pathogens. Nevertheless, its primary functions in human signaling remain unclear due to the limited identification of direct substrates. Here, we investigated the interaction between RNF213 and caveolin-1 (Cav-1), a small scaffolding protein vital for caveolae formation and the regulation of a plethora of cellular processes. Cav-1 specifically binds within the two functional AAA+ domains of RNF213 in an ATP-dependent manner, highlighting the influence of cellular energy status on this interaction. Consequently, RNF213 ubiquitinates Cav-1 at several N-terminal lysine residues through K48 and K63 linkages, although several Moyamoya disease-associated RNF213 mutations greatly reduce this polyubiquitination. Moreover, RNF213 activity inhibits phosphorylation of a key regulatory residue of Cav-1, as RNF213 knockdown under oxidative stress markedly enhances Cav-1 Tyr14 phosphorylation and modifies nitric oxide bioavailability in endothelial cells. Collectively, our results indicate that RNF213 functions as a molecular switch modulating Cav-1 signaling based on RNF213 functionality and cellular conditions. These findings offer new insights into vascular pathogenesis and the vast signal pathways along the RNF213–Cav-1 axis.

In this study, we reveal a novel relationship between RNF213, an E3 ubiquitin ligase associated with Moyamoya disease (MMD) and the ubiquitination of both endogenous and pathogenic substrates, and EGFR, the epithelial growth factor receptor involved in cell growth, angiogenesis, and cancer. RNF213 knockdown or knockout in HeLa and A549 cells markedly reduces EGFR phosphorylation at key tyrosine sites following EGF and TGFα stimulation. In RNF213 knockout cells, HER2 phosphorylation, typically activated through heterodimerization with EGFR, and Src recruitment and/or phosphorylation are also diminished. Mutations in the RNF213 RING, RZ finger, or AAA+ domains, including the prevalent R4810K mutation in MMD, consistently reduce EGFR phosphorylation. In vivo, EGF injections increase EGFR and HER2 phosphorylation in WT but not in RNF213 knockout mice. Despite the reduced phosphorylation levels of these tyrosine kinases in knockout cells, the activation of downstream signals such as AKT, ERK1/2, and STAT3 remains unaffected, although phosphorylation of PLCγ, a key mediator of Ca²⁺ release, is selectively reduced by RNF213 knockout. These findings demonstrate that RNF213 modulates EGFR‐related pathways and specific downstream signal pathways, possibly affecting physiologic and pathogenic angiogenesis, and may have implications for unraveling the etiology of MMD and for developing cancer therapies that target RNF213.