Disordered Motifs and Domains in Cell Control - October 11-15, 2014

Disordered Motifs and Domains in Cell Control

Poster Session I

19-POS Board 19 Re-Assembling Gab Family Protein-based Signalling Complexes Katharina Mandel 1 , Marc Lewitzky 1 , Philip C. Simister 1 , Ingrid Tessmer 2 , Jörg Bürger 3 , Christian M. T. Spahn 3 , Thorsten Mielke 4 , Stephan M. Feller 1 1 University of Oxford, Oxford, United Kingdom, 2 University of Würzburg, Würzburg, Germany, 3 Charité – Universitätsmedizin Berlin, Berlin, Germany, 4 Max Planck Institute for Molecular Genetics, Berlin, Germany. Gab family proteins are large multi-site docking proteins serving as dynamic assembly platforms for signal processing protein complexes. Gab proteins contain a well-folded N-terminal pleckstrin homology (PH) domain, followed by a long tail region predicted to be largely disordered. However, the molecular details of how Gab facilitates the computation of specific biological responses remain largely unstudied. Upon receptor activation multiple tyrosine residues within the tail of Gab become phosphorylated and serve as binding sites for signalling proteins including SHP2, Crk, PLCγ and PI3 kinase. Two highly-conserved RxxK epitopes with helical secondary structure serve as critically important binding sites for the adaptor protein Grb2 [1], connecting the Gab proteins to a plethora of functionally distinct receptors. Our study aims to investigate the structure and dynamics of Gab protein-based signalling complexes by attempting a step-wise assembly of recombinantly expressed key proteins. Initially, we are focusing on the caspase-generated Gab1 fragment p35Gab1 [2], which contains binding sites for Grb2, c-Met, and PI3 kinase. We co-purified p35Gab1 and Grb2 and studied the properties of the p35Gab1–Grb2 complex by biophysical techniques. In preliminary atomic force microscopy (AFM) and electron microscopy (EM) experiments, we detected clear signals for discrete, defined asymmetrical particles, which have dimensions consistent with that of Grb2 bound to additional protein material, and thus are likely to represent the p35Gab1–Grb2 complex. These initial findings suggest to us that it may be feasible to build even larger complexes containing Gab proteins bound to several interaction partners and to study their structural, biophysical and biochemical properties. Further structural analyses of step-wise assembled signalling protein complexes at higher resolution are planned to provide a first glimpse into the virtually unknown molecular architecture of these signalling network hub protein assemblies. [1] Harkiolaki et al. (2009) Structure [2] Le Goff et al. (2012) JBC

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