Cerebral Cavernous Malformation 2 Protein Promotes Smad Ubiquitin Regulatory Factor 1-mediated RhoA Degradation in Endothelial Cells

Mutation of CCM2 predisposes individuals to cerebral cavernous malformations, vascular abnormalities that cause seizures and hemorrhagic stroke. CCM2 has been proposed to regulate the activity of RhoA for maintenance of vascular integrity. Herein, we define a novel mechanism where the CCM2 phosphotyrosine binding (PTB) domain binds the ubiquitin ligase (E3) Smurf1, controlling RhoA degradation. Brain endothelial cells with knockdown of CCM2 have increased RhoA protein and display impaired directed cell migration. CCM2 binding of Smurf1 increases Smurf1-mediated degradation of RhoA. CCM2 does not significantly alter the catalytic activity of Smurf1, nor is CCM2 a Smurf1 substrate. Rather the CCM2-Smurf1 interaction functions to localize Smurf1 for RhoA degradation. These findings provide a molecular mechanism for the pathogenesis of cerebral cavernous malformations (CCM) resulting from loss of CCM2-mediated localization of Smurf1, which controls RhoA degradation required for maintenance of normal endothelial cell physiology.We previously characterized a scaffold-like protein named osmosensing scaffold for MEKK3 (OSM) for its ability to bind actin and localize to Rac-containing membrane ruffles and its obligate requirement for p38 activation in response to hyperosmotic stress (1). Subsequently, the gene encoding OSM, CCM2, was found to be mutated in the human disease cerebral cavernous malformations (CCM)² (2). Cerebral cavernous malformations are vascular lesions of the central nervous system characterized as clusters of dilated, thin walled blood vessels. CCM lesions are fragile and prone to vascular leakiness and rupture, leading to hemorrhages that cause seizure and stroke (3, 4).Recently, CCM2 knockdown endothelial cells were shown to have increased activation of RhoA (5), although the mechanism was not defined. Herein, we demonstrate a molecular mechanism for activation of this pathway. Through a novel CCM2 PTB domain interaction with the Smurf1 homologous to the E6-AP C terminus (HECT) domain, we now show that CCM2 binds the E3 ligase Smurf1 for the control of RhoA degradation.