Endothelial cells serve as a barrier between blood and tissues. Maintenance of the endothelial cell barrier depends on the integrity of intercellular junctions, which is regulated by a polarity complex that includes the ζ isoform of atypical protein kinase C (PKCζ) and partitioning defective 3 (PAR3). We revealed that the E3 ubiquitin ligase PDZ domain-containing ring finger 3 (PDZRN3) regulated endothelial intercellular junction integrity. Endothelial cell-specific overexpression of Pdzrn3 led to early embryonic lethality with severe hemorrhaging and altered organization of endothelial intercellular junctions. Conversely, endothelial-specific loss of Pdzrn3 prevented vascular leakage in a mouse model of transient ischemic stroke, an effect that was mimicked by pharmacological inhibition of PKCζ. PDZRN3 regulated Wnt signaling and associated with a complex containing PAR3, PKCζ, and the multi-PDZ domain protein MUPP1 (Discs Lost-multi-PDZ domain protein 1) and targeted MUPP1 for proteasomal degradation in transfected cells. Transient ischemic stroke increased the ubiquitination of MUPP1, and deficiency of MUPP1 in endothelial cells was associated with decreased localization of PKCζ and PAR3 at intercellular junctions. In endothelial cells, Pdzrn3 overexpression increased permeability through a PKCζ-dependent pathway. In contrast, Pdzrn3 depletion enhanced PKCζ accumulation at cell-cell contacts and reinforced the cortical actin cytoskeleton under stress conditions. These findings reveal how PDZRN3 regulates vascular permeability through a PKCζ-containing complex.< Réduire