Rap-afadin axis in control of Rho signaling and endothelial barrier recovery

• Published in: Molecular biology of the cell Vol. 24; no. 17; pp. 2678 - 2688
• Main Authors: Birukova, Anna A, Tian, Xinyong, Tian, Yufeng, Higginbotham, Katherine, Birukov, Konstantin G
• Format: Journal Article
• Language: English
• Published: United States The American Society for Cell Biology 01-09-2013
• Subjects: Cytoskeleton - genetics; Cytoskeleton - metabolism; Down-Regulation; Endothelial Cells - cytology; Endothelial Cells - metabolism; Gene Knockdown Techniques; Guanine Nucleotide Exchange Factors - genetics; Guanine Nucleotide Exchange Factors - metabolism; Humans; Microfilament Proteins - metabolism; Phosphorylation; rap1 GTP-Binding Proteins - metabolism; rho GTP-Binding Proteins - genetics; rho GTP-Binding Proteins - metabolism; Signal Transduction - drug effects; Signal Transduction - genetics; Thrombin - pharmacology
• ISSN: 1059-1524; 1939-4586
• DOI: 10.1091/mbc.e13-02-0098

Activation of the Rho GTPase pathway determines endothelial cell (EC) hyperpermeability after injurious stimuli. To date, feedback mechanisms of Rho down-regulation critical for barrier restoration remain poorly understood. We tested a hypothesis that Rho down-regulation and barrier recovery of agonist-stimulated ECs is mediated by the Ras family GTPase Rap1. Thrombin-induced EC permeability driven by rapid activation of the Rho GTPase pathway was followed by Src kinase-dependent phosphorylation of the Rap1-specific guanine nucleotide exchange factor (GEF) C3G, activation of Rap1, and initiation of EC barrier recovery. Knockdown experiments showed that Rap1 activation was essential for down-regulation of Rho signaling and actin stress fiber dissolution. Rap1 activation also enhanced interaction between adherens junction (AJ) proteins VE-cadherin and p120-catenin and stimulated AJ reannealing mediated by the Rap1 effector afadin. This mechanism also included Rap1-dependent membrane translocation of the Rac1-specific GEF Tiam1 and activation of Rac1-dependent peripheral cytoskeletal dynamics, leading to resealing of intercellular gaps. These data demonstrate that activation of the Rap1-afadin axis is a physiological mechanism driving restoration of barrier integrity in agonist-stimulated EC monolayers via negative-feedback regulation of Rho signaling, stimulation of actin peripheral dynamics, and reestablishment of cell-cell adhesive complexes.