Phosphorylation des récepteurs nucléaires de l'acide rétinoïque et interaction avec des adaptateurs à domaines SH3: Etudes structurales et fonctionnelles – VINRAR

This project aims at deciphering the moledular, dynamic and structural determinants of how a disordered domain such as the N-terminal domain (NTD) of nuclear retinoic acid (RA) receptors (RARs) can regulate their function. RARs are ligand-dependent transcriptional transregulators which control a multitude of physiological phenomena (embryogenesis, homeostasis, reproduction, cell growth, cell differentiation and apoptosis). Molecular and structural studies provided a huge amount of information on how the ligand constitutes the 'on switch' signal leading to communication with the general transcriptional machinery via conformational changes affecting the well folded Ligand Binding Domain (LBD). However, it becomes increasingly clear that these features, though necessary, are not sufficient to explain the fine-tuning of RA signaling and that the NTD of RARs is also functionally important. Up to now, the importance of this domain was controversial, as no structural data were available for the NTD and prediction algorithms suggested a naturally disordered structure. However, the group of C.Rochette-Egly revealed that the particularity of the NTD of RARs lies in the presence of a proline rich motif (PRM) with a serine residue, which becomes phosphorylated in response to RA. Moreover this motif provides a surface for adaptor proteins characterized by SH3 or SH3-like domains such as Vinexin ' and Profilin IIA, which are also actin-binding proteins. Consequently, the aim of the present project will be to decipher the biophysical-biochemical determinants of how the PRM of RARs can regulate their function. The originality and novel aspect of the present project lies in the multidisciplinary strategy in the fields of molecular, cellular and structural biology. First, we will analyze the molecular, structural and thermodynamical aspects of the interaction of the proline-rich rich motif of RARs with the SH3 and SH3-like domains of vinexin ' and profilin IIA. As the serine residue located in the PRM of RARs becomes phosphorylated in response to RA, we will focus on the impact of phosphorylation of this domain on its interaction with vinexin ' and profilin IIA. We will also analyze the consequences of phosphorylation on the structure and dynamics of the NTD and adjacent structured DBD. Such a cross-talk between two domains will be possible by combining NMR analysis and peptide ligation promoted by inteins, to produce NTD-DBD fusion proteins Finally, we will investigate how vinexin ' and Profilin IIA control RAR-mediated transcription. This part will involve chromatin immunoprecipitation experiments and the characterization of the other nuclear proteins that interact with vinexin ' and Profilin IIA within the RAR complexes. This part is quite novative as to our knowledge, it is the first study highlighting a new functional relationship between RARs and cytoskeletal proteins. Indeed vinexin ' and profilin IIA belong to a growing number of cytoskeleton proteins that are also present in the nucleus and interact with several activators of transcription. In the particular case of vinexin ', which contains three SH3 domains, it will also include the thermodynamic and structural analysis of potential cross talk between the individual SH3 domains. In this project, the classical cellular and functional techniques (gene expression, phosphorylation analysis, immunoprecipitation) will be combined with modeling approaches (numerical simulations) and appropriate biophysical techniques (NMR, Biacore, FRET, SAXS) focusing on the thermodynamical and structural aspects of the interactions and of the perturbations induced by the phosphorylation signal. Such criteria are fulfilled by the partnership between the teams headed by C.Rochette-Egly, B.Kieffer and A. Dejaegere who will contribute studies of the molecular and functional correlation aspects, the biophysical aspects (NMR) and the modeling, respectively. This multidisciplinary strategy should provide the first thermodynamic and structural data for the NTD of RARs. It should also shed light on how this domain participates to the molecular communication between RARs and the basal transcription machinery.