Identification of Mutated in colorectal cancer (MCC) protein as a novel partner of PDZRN3 during Blood brain barrier development

Abstract

Blood brain barrier (BBB) disruption is a critical component of the physiopathology of neurological disorder. Wnt signalling is crucial for BBB development and its stability. We have reported that an endothelial excessive activation of the ubiquitin ligase PDZRN3, a partner in Wnt signalling, destabilizes the BBB. However, the molecular pathway is still poorly understood. Using a differential screening strategy by BioID, MCC was discovered as a potential PDZRN3 interacting partner. This study focused on understanding how MCC/PDZRN3 interaction regulates Wnt signalling in vitro and in vivo. We then investigated the role of MCC in endothelial function. PDZRN3 and MCC protein partners were identified by a proteomic screening strategy (bioID). MCC functional role in Wnt signalling was studied in brain endothelial cells (HBMEC) using siRNA strategy, and specific chemical inhibitors. MCC in vivo expression was analysed in extracted blood vessels from wild type and transgenic mouse brains. MCC is a direct target of the kinase CKIɛ, a key regulator of the canonical Wnt pathway. We demonstrated that MCC is constantly phosphorylated by this kinase. We then reported that PDZRN3 prevents CKIɛ-induced phosphorylation of MCC. During post-natal mouse BBB development, MCC undergoes a switch from un- to hyper-phosphorylated state, correlated with a decrease in PDZRN3 expression level. Interestingly, in mouse mutants, specific ectopic Pdzrn3 over-expression in brain endothelial cells impairs MCC phosphorylation switch. We then investigated MCC involvement in endothelial function. MCC knock down impairs HBMEC directed migration under flow condition and planar MTOC polarization. Finally, using a bioID screening, we have identified several centrosomal proteins, such as CEP131, as potential MCC partners. Centrosome being a key organelle in cell polarization, we propose that MCC may regulate its organisation during HBMEC planar migration. By forming a stable complex with MCC, PDZRN3 prevents CKIɛ-induced phosphorylation of MCC. MCC would then regulate centrosome polarisation required for endothelial planar migration, making MCC an actor of BBB dynamic maturation.