The role of Rap1 in cell adhesion and migration

Abstract

cAMP is a pivotal second messenger that regulates a wide range of cellular processes. Besides protein kinase A, cAMP also activates the guanine nucleotide exchange factor (GEF) Epac that can subsequently activate the small GTPase Rap1. Rap1 is an important regulator of E-cadherin mediated cell-cell junctions and integrin-mediated cell adhesion tot the extracellular matrix (ECM). In cells containing both cell-ECM adhesions and cell-cell junctions, such as epithelial cells, crosstalk between and regulation of both types of adhesion are required for efficient cell migration. As Rap1 is important for both types of adhesion, we set out to investigate the role of Rap1 in growth factor-induced cell migration. For proper cell migration to occur, a cell has to polarize, form initial adhesions to the ECM, modify its actin cytoskeleton and induce focal adhesion (FA) assembly at the front of the cell to generate contractile forces. Focal adhesions are large protein complexes that link the actin cytoskeleton to the ECM to provide structural integrity, sense the environment and initiate signaling. The coordinated formation and turnover of FAs is crucial for optimal cell migration. Here, we describe that the activation of Rap1 can inhibit growth factor-induced cell migration of epithelial cells. We show that Rap1 inhibits cell migration in the absence of cell-cell junctions and that this inhibition correlates with a decrease in focal adhesion and protrusion dynamics. To identify the mechanism responsible for the effect of Rap1 on focal adhesion dynamics, we investigated several major pathways responsible for the regulation FA dynamics. These pathways were not affected by 007 stimulation. We hypothesize that Rap1 might be involved in modulating the strength of the integrin-cytoskeleton connection. The stability of FAs is regulated by tension generated in the acto-myosin cytoskeleton and the connectivity of this tension to the integrins. Although we could not find any evidence for direct regulation of tension by Rap1, the generation of tension is required for the Rap1-induced stabilization of FAs. This suggests that Rap1 may be involved in regulating the dynamic connection of the actin cytoskeleton and the integrins. This may be regulated by receptor-mediated signaling, including cAMP-Epac-mediated signaling, but also by outside-in signaling. Furthermore, we used an siRNA approach to determine the molecular mechanism of Rap1-mediated regulation of focal adhesions. We used a small customized siRNA library to identify proteins involved in the effect of Rap1 activation of cell spreading and FA formation. In this screen, we show that these effects are mediated by Rap1A and identify a number of proteins that partially modulate the Rap1 effect. In conclusion, we show that the regulation of cell adhesion by Rap1 likely comprises multiple different effector proteins and pathways, controlling separate aspects of this process, such as integrin activation, spreading, protrusion and the control of actin cytoskeleton dynamics. Our growing understanding of cell adhesion processes and the emergence of refined imaging techniques should contribute to the elucidation of the molecular mechanism of Rap1 function in cell adhesion.