The polarity protein Scribble positions DLC3 at adherens junctions to regulate Rho signaling

The spatial regulation of cellular Rho signaling by GAP proteins is still poorly understood. By mass spectrometry we here identify the polarity protein Scribble as a scaffold for the RhoGAP protein DLC3 at cell-cell adhesions. This mutually dependent interaction is mediated by the PDZ domains of Scribble and a PDZL motif in DLC3. Both Scribble depletion and PDZL deletion abrogated DLC3 junctional localization. Using a RhoA biosensor and a targeted GAP domain, we demonstrate that DLC3 activity locally regulates RhoA-ROCK signaling at and Scribble localization to adherens junctions and is required for their functional integrity. In a 3D model of cyst development, we furthermore show that DLC3 depletion impairs polarized morphogenesis, phenocopying the effects observed upon Scribble knockdown. We thus propose a novel function for Scribble in Rho regulation that entails positioning of DLC3 GAP activity at cell junctions in polarized epithelial cells.