Protein-protein interactions involved in Rap1-mediated signal transduction

Abstract

Signal transduction pathways play important roles in cell differentiation, proliferation, and survival. Specific protein-protein interactions mediate the assemblies of protein complexes in response to different signals therefore regulate the proper transmission of cellular signals. Inappropriate protein-protein interactions within signalling pathways can lead to many diseases, including cancer. Proteomics-based approaches, which enable the quantitative investigation of protein-protein interactions involved in signalling networks, provide us with techniques to define the molecules controlling various signalling pathways. The small GTPase Rap1 belongs to the Ras family and shares high similarity with Ras protein. Rap1 cycles between an inactive GDP-bound state and an active GTP-bound state. This cycle is regulated by guanine nucleotide exchange factors (GEFs), such as Epac and GTPase activating proteins (GAPs), such as Rap1GAP. The most well-established processes where Rap1 is involved in include integrin-mediated adhesion and cadherin-mediated cell-cell adhesion. The GEFs are regulated by second messengers and are part of a protein-protein network, which regulates their temporal and spatial activities. The objective of the work described in this thesis was to investigate how Rap1 signaling pathway is regulated and to identify and characterize signaling proteins that direct the Rap1 pathway. In chapter 2, we studied the relation between AF6 and Rap1. We show that the overexpression of AF6 inhibits Rap1 induced adhesion. In addition, knocking-down the endogenous AF6 increases adhesion. These results show that in Jurkat T cells, AF6 functions to buffer GTP-Rap in resting cells and negatively regulates Rap1 function. In chapter 3 we analyze three Rap-like pseudogenes (mRap1A-retro1, mRap1A-retro2 and hRap1B-retro) in mouse and human genome. We show that all three retrogenes are expressed and encode functional proteins. These proteins appeared to stay more in a GTP-bound state compared to wild type Rap1. More interestingly, they exhibit clear differences in their ability to induce cell adhesion and spreading. To gain more insight in the protein interaction network, which controls the spatial and temporal organisation of Rap specific GEFs we performed yeast two-hybrid screens using Epac and PDZ-GEF as baits. This is described in chapter 4 addendum. A general summary of the results is given and candidate proteins were discussed. In chapter 4 and chapter 5 we characterized in detail the interaction between ERM (Ezrin-Radixin-Moesin) proteins and Epac1. We show that both Ezrin and Radixin interact with Epac1 in an activation-dependent manner. The Ezrin/Radixin binding region was identified in the N-terminus of Epac1. Furthermore, we demonstrate that this region is also required for the localization of Epac1 at microvilli in fully polarized cells. We show that Ezrin couples the activation of the ?-adrenergic receptor to Rap1 signalling via the recruitment of Epac1. In chapter 5 a novel Radixin mutant was characterized. This mutant Radixin in which both I577 and F580 are substituted for aspartic acids fulfils the classical criteria of being in an active state. Interestingly, active Radixin facilitates Rap1 activation and Rap1 mediated adhesion