Rap1 signaling in monolayer integrity: a bilateral approach

Abstract

The small G protein Rap1 is an important regulator of cell-cell adhesion and cycles between an active GTP-bound and an inactive GDP-bound state. This cycling is regulated by guanine nucleotide exchange factors (GEFs) which facilitate the exchange of GDP for GTP, and GTPase activating proteins (GAPs) that promote Rap1 inactivation. Active Rap1 promotes cell-cell adhesion, hereby contributing to the integrity of a cell monolayer. In endothelial cells the integrity of a cell monolayer is reflected by its barrier function. Activation of Rap1 induces the tightening and/or maturation of the cell junctions and promotes the formation of cortical/junctional actin. Together these two processes contribute to Rap1’s stabilizing effect on the endothelial barrier function. Here we have studied Rap1 signaling in the context of monolayer integrity using a bilateral approach. We have investigated regulators and effectors of Rap1 in the endothelial barrier function and have implicated Rap1 signaling in the polarization of endothelial cells during wound healing. In addition, we have investigated signaling pathways downstream of Rap by identifying global phosphorylation changes that occur upon activation of the Rap exchange factor Epac1. In the regulation of endothelial barrier function we show that different RapGEFs regulate Rap1 activity under different circumstances. The exchange factors PDZ-GEF1 and 2 are responsible for Rap1 activity in the control of basal (unstimulated) barrier function, whereas Rap1 activation by Epac1 mediates the cAMP induced barrier function. The depletion of the PDZ-GEFs or Rap1 induces an irregular and immature organization of the adherens junctions together with an increase in junction motility. Activation of Epac1 using the Epac-selective cAMP analogue 007-AM increases the formation of junctional actin, inhibits junction motility and restores the barrier function of PDZ-GEF depleted, but not Rap1 depleted cells. In addition, we have implicated the Epac1-Rap1 signaling pathway in the directional polarization of endothelial cells during wound healing. The accelerated cell polarization is associated with an earlier start in directional cell migration, but not with an increase in migration speed. We propose that the activation of Epac1 and Rap1 accelerates cell polarization and contributes to directional cell migration in wound healing by the earlier onset of cell migration, rather than an increased cell migration speed. Lastly, in order to identify novel proteins/pathways downstream of Rap1 we employed quantitative phosphoproteomics to detect global phosphorylation changes after activation of Epac1 and Rap using 007-AM. We applied this experimental set-up on three different cell systems: (1) epithelial HeLa cell-line stably overexpressing Epac1, (2) primary endothelial cells endogenously expressing Epac1 (HUVECs) and (3) HUVECs depleted of PDZ-GEF 1 and 2 to maximize the 007-AM induced activation of Rap1. Gene Ontology (GO) term analysis reveals that the majority of the proteins that present with 007-AM regulated changes in phosphorylation are associated with cell-cell adhesion, cell-substratum adhesion and the microtubule and actin cytoskeleton. This widespread screen provides us with various novel target proteins for further study of the Epac1-Rap signaling pathway.