comprehensive analysis of interactions between the src-associated protein in mitosis of 68 kda and the human src-homology 3 proteome综合分析src-associated蛋白质之间的相互作用的有丝分裂68 kda和人类src-homology 3蛋白质组.pdfVIP

Comprehensive Analysis of Interactions between the Src- Associated Protein in Mitosis of 68 kDa and the Human Src-Homology 3 Proteome 1 1 2 1 Benedikt Asbach , Christine Ludwig , Kalle Saksela , Ralf Wagner * 1 Institute of Medical Microbiology and Hygiene, University of Regensburg, Regensburg, Germany, 2 Department of Virology, Haartman Institute, University of Helsinki, Helsinki, Finland Abstract The protein Sam68 is involved in many cellular processes such as cell-cycle regulation, RNA metabolism, or signal transduction. Sam68 comprises a central RNA-binding domain flanked by unstructured tails containing docking sites for signalling proteins including seven proline-rich sequences (denoted P0 to P6) as potential SH3-domain binding motifs. To comprehensively assess Sam68-SH3-interactions, we applied a phage-display screening of a library containing all approx. 300 human SH3 domains. Thereby we identified five new (from intersectin 2, the osteoclast stimulating factor OSF, nephrocystin, sorting nexin 9, and CIN85) and seven already known high-confidence Sam68-ligands (mainly from the Src- kinase family), as well as several lower-affinity binders. Interaction of the high-affinity Sam68-binders was confirmed in independent assays in vitro (phage-ELISA, GST-pull-down) and in vivo (FACS-based FRET-analysis with CFP- and YFP-tagged proteins). Fine-mapping analyses with peptides established P0, P3, P4, and P5 as exclusive docking-sites for SH3 domains, which showed varying preferences for these motifs. Mutational analyses identified individual residues within the proline-rich motifs being crucial for the interactions. Based on these data, we generated a Sam68-mutant incapable of interacting with SH3 domains