Spatial Regulation of Cyclic AMP-Epac1 Signaling in Cell Adhesion by ERM Proteins

Abstract

Epac1 is a guanine nucleotide exchange factor for the small G protein Rap and is involved in membrane-localized processes such as integrin-mediated cell adhesion and cell-cell junction formation. Cyclic AMP (cAMP) directly activates Epac1 by release of autoinhibition and in addition induces its translocation to the plasma membrane. Here, we show an additional mechanism of Epac1 recruitment, mediated by activated ezrin-radixin-moesin (ERM) proteins. Epac1 directly binds with its N-terminal 49 amino acids to ERM proteins in their open conformation. Receptor-induced activation of ERM proteins results in increased binding of Epac1 and consequently the clustered localization of Epac1 at the plasma membrane. Deletion of the N terminus of Epac1, as well as disruption of the Epac1-ERM interaction by an interfering radixin mutant or small interfering RNA (siRNA)-mediated depletion of the ERM proteins, impairs Epac1-mediated cell adhesion. We conclude that ERM proteins are involved in the spatial regulation of Epac1 and cooperate with cAMP- and Rap-mediated signaling to regulate adhesion to the extracellular matrix.

We thank John de Koning, Hybrigenics S.A. (Paris, France) and UMC Utrecht, for the yeast two-hybrid screen, Kjetil Taskén (Oslo) for performing a peptide scan with the N-terminal 49 amino acids of Epac1 and ezrin, Hans Clevers and Jean Paul ten Klooster (Utrecht, Netherlands) for the LS174T-W4 cells and help with experiments with these cells, Daan Visser (Amsterdam, Netherlands) for technical assistance, and the members of our laboratories for support and stimulating discussions.

This study is supported by Chemical Sciences (M.G.), Earth and Life Sciences (H.R.), a Netherlands Organization for Scientific Research (NWO) material investment grant (K.J.), and the Netherlands Genomics Initiative of the NWO (J.L.B.).