Negative cooperativity underlies dynamic assembly of the Par complex regulators Cdc42 and Par-3

Abstract

The Par complex polarizes diverse animal cells through the concerted action of multiple regulators. Binding to Par-3 couples the complex to cortical flows that construct the Par membrane domain. Once localized properly, the complex is thought to transition from Par-3 to the Rho GTPase Cdc42 to activate the complex. While this transition is a critical step in Par-mediated polarity, little is known about how it occurs. Through a biochemical reconstitution approach utilizing purified, intact Par complex and qualitative binding assays, we found that Par-3 and Cdc42 exhibit strong negative cooperativity for the Par complex. We show that the interactions between the second and third PDZ protein interaction domains of Par-3 and the aPKC Kinase-PBM (PDZ binding motif) mediate the displacement of Cdc42 from the Par complex. Our results indicate that Par-3, Cdc42, Par-6, and aPKC are the minimal components that are sufficient for this transition to occur and that no external factors are required. Our findings provide the mechanistic framework for understanding a critical step in the regulation of Par complex polarization and activity.