Role of the paxillin/poly(A)-binding protein 1 complex in mRNA trafficking during cell migration

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During cell migration the adaptor protein, paxillin, is recruited to integrin complexes at the cell's leading edge and is thought to be involved in positioning multiprotein complexes that are key to the maintenance of polarity during cell migration. One of the mechanisms by which cells generate and maintain polarity is by trafficking and localising mRNA transcripts to particular subcellular locations, and it is therefore of interest that we have recently identified poly(A)-binding protein 1 (PABP1) as a novel ligand for paxillin. PABP1 associates with paxillin with high affinity, and the resulting complex shuttles in and out of the nucleus and localises to the leading edge of migrating cells. In preliminary studies, mutants of PABP1 that cannot bind paxillin are inefficiently exported from the nucleus and are thus unable to reach the leading edge of migrating cells. Furthermore, cells expressing these mutants are unable to migrate directionally, raising the possibility that PABP1 must associate with paxillin to maintain cell polarity during migration.Recent NMR and crystallographic studies of the complex formed between paxillin and focal adhesion kinase highlight the dangers of undertaking mutagenesis studies without detailed information on the structure of the intermolecular complex. We therefore propose a detailed structural analysis of the paxillin/PABP1 complex using biochemical and biophysical technologies. We have demonstrated the feasibility of this approach by collecting preliminary NMR spectra showing a domain/domain interaction between paxillin and PABP1. We propose to pursue these approaches to obtain detailed information of the intermolecular contacts between paxillin and PABP1, and how these relate to the association of PABP1 with mRNA and components of the translation apparatus. Using this information we will construct mutants of PABP1 that have reduced affinity for paxillin, and synthesise peptide antagonists of paxillin/PABP1 interaction. These will be designed to have minimal effect on the folding of PABP1, its association with mRNA and ability to enhance poly(A)-dependent translation. These mutant PABP1s and cell permeant peptides will allow determination of the requirement for paxillin in the export of mRNA from the nucleus and its transport to the leading edge of migrating cells. We will also measure various parameters of cell movement on 2D and 3D matrices following disruption of the paxillin/PABP1 complex and evaluate the requirement for this aspect of mRNA trafficking during directional migration. We anticipate that this parallel application of biochemical, biophysical and cell biological approaches will yield valuable information concerning the molecular mechanisms by which mRNA is trafficked in migrating cells and how this relates to the development of cell polarity.