Identification of new Sam68-interacting protein in cancer cells by using yeast two-hybrid screening and mass spectrometry analysis

Sam68 is a member of the Signal Transduction and Activation of RNA metabolism (STAR) family of RNA-binding proteins and it is implicated in many cellular processes, such as RNA metabolism, transcription, signal transduction, cell cycle regulation and apoptosis. Sam68 function is modulated by several post-translational modifications and it is thought to integrate signal transduction pathways with the regulation of RNA metabolism. Sam68 contains several motifs that can explain its multifunctional role. First, Sam68 shares the GSG domain (Gpr33-Sam68-GLD-1) with proteins of STAR family, which is required for RNA binding and homodimerization. Second, Sam68 contains six consensus proline-rich motifs (P0– P5) that are involved in the protein-protein interaction with SH3- and WW-domain containing protein. Third, a C-terminal tyrosine-rich domain (YY) can modulate binding to SH2-containing proteins and contains a nuclear localization signal (NLS). Fourth, Sam68 contains RGG boxes, which are potential sites for protein arginine methylation. Sam68 function can be modulate by several post-translational modification and/or by interaction with several signalling protein, both mediated by cellular cues. In fact, it has been shown that (i) Sam68 interacts with the SH2 and SH3 domains of several signalling proteins acting as a scaffold molecule in response to different stimuli; or that (ii) the tyrosine phosphorylation of Sam68, mediated by interacting with Src-related kinases, reduce its affinity for RNA and regulate alternative splicing of the Bcl-x pre-mRNA, whereas serine/threonine phosphorylation (iii) by the Erk1/2 mitogen-activated protein kinases (MAPK), affects alternative splicing of the CD44 receptor pre-mRNA. Furthermore, methylation, tyrosine phosphorylation and the binding to polysomes can regulate the intracellular localization of Sam68. Nevertheless, the exact function of this protein is still largely unknown. We have recently shown that Sam68 is up-regulated in prostate cancer (PCa) and that its function is required to promote proliferation and survival of PCa cells. To gain insight into the function of Sam68 in PCa cells, we performed a 2-hybrid screen and a co-immunoprecipitation/mass spectrometry experiment to identify proteins that interact with Sam68 in these cells. We found several proteins that specifically interacted with Sam68 in PCa cells. Functional annotation of these interacting proteins revealed an enrichment in ribosomal proteins and proteins involved in several RNA processes, such as regulation or termination of the transcription and alternative splicing. Several of these interactions have been confirmed by co-immunoprecipitation and pull-down experiments. Functional analyses of these interactions are currently being performed.