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 performing mass spectrometry, we here identify the polarity protein Scribble as a scaffold for the RhoGAP protein DLC3 (also known as StarD8) at cell-cell adhesions. This mutually dependent interaction is mediated by the PDZ domains of Scribble and a PDZ ligand (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 new function for Scribble in Rho regulation that entails positioning of DLC3 GAP activity at cell junctions in polarized epithelial cells.