Spatial regulation of Rap signaling

Abstract

By cycling between an inactive GDP-bound and active GTP-bound state, small G-proteins of the Rap family act as molecular switches that relay upstream signals to diverse cellular processes. This GDP/GTP-cycle and consequently downstream signaling by Rap is under tight regulation by its GEFs and GAPs. The biological consequence of Rap activity depends on its cellular position and local environment. As specific anchoring mechanisms may recruit the individual RapGEFs and RapGAPs to distinct cellular locations, they can control discrete cellular pools of Rap in order to establish different downstream signaling events. In this thesis, we have explored several aspects of this spatial regulation of Rap signaling. We have focused on the regulation of the RapGEF Epac1, which is activated by the second messenger cAMP that induces a conformational change to release the protein from auto-inhibition. We have revealed that binding of cAMP in addition induces the relocalization of Epac1 from the cytosol to the plasma membrane (PM). This allows activation of Rap at this compartment, which links the activation of Epac1 to PM-localized processes including Rap-mediated adhesion of cells to the extracellular matrix. As Epac1 is not statically confined to the PM but translocates here upon binding of cAMP, this allows further dynamic regulation of its cellular distribution. Indeed, we describe an alternative targeting mechanism for Epac1, mediated by binding to proteins of the Ezrin, Radixin, Moesin (ERM) family in their open conformation. Numerous signaling pathways can trigger the open conformation of the ERM proteins, which are subsequently targeted to the PM and thus provide an additional anchoring mechanism for Epac1 at this compartment. Epac1 in addition interacts with the nucleoporin RanBP2, which targets Epac1 to the nuclear pore. This interaction is mediated by the domain of Epac1 that catalyses the nucleotide exchange of Rap. As a result, binding of RanBP2 inhibits the activity of Epac1 towards Rap in vitro and in vivo. Diverse pathways may impinge on this interaction to augment the available pool of Epac1 that signals to Rap, which involves phosphorylation of RanBP2. RanBP2 thus functions to maintain a regulated, inactive cellular pool of Epac1. Finally, we have demonstrated how localized Rap activity regulates a specific signaling pathway during epithelial cell polarization. In intestinal epithelial cells, the kinase Lkb1 controls the establishment of apico-basal polarity and the subsequent formation of apical brush borders that allows the efficient exchange of nutrients with the intestinal lumen. Rap2A is activated downstream of Lkb1, which occurs specifically at the apical membrane. Rap2A controls brush border formation by targeting its effector TNIK to the apical compartment. TNIK in turn allows the recruitment of Mst4, which mediates activation of the actin-linking protein Ezrin to induce brush border formation. Thus, we have identified Rap2A and TNIK as novel components of the Lbk1 signaling pathway in a specific aspect of epithelial polarity. In summary, we have unveiled multiple aspects of the spatial regulation of Rap, which will help to broaden our understanding of the Rap signaling network