Characterization of ubiquitination dependent dynamics in growth factor receptor signaling by quantitative proteomics

摘要: Protein ubiquitination is a dynamic reversible post-translational modification that plays key role in the regulation of numerous cellular processes including signal transduction, endocytosis, cell cycle control, DNA repair and gene transcription. The conjugation small protein ubiquitin or chains molecules various types lengths to targeted proteins known alter proteins' lifespan, localization function modulate interactions. Despite its central importance aspects life there are only limited number reports investigating on proteomic scale, mainly due inherited complexity heterogeneity ubiquitination. We describe here quantitative proteomics strategy based specificity binding domains (UBDs) Stable Isotope Labeling by Amino acids Cell culture (SILAC) for selectively decoding ubiquitination-driven involved signaling networks. applied this approach characterize temporal dynamics events accompanying epidermal growth factor receptor (EGFR) transduction. used recombinant UBDs derived from endocytic adaptor specific enrichment ubiquitinated complexes EGFR network subsequent analyses high accuracy mass spectrometry. show suitable profiling occurring individual as well ubiquitination-dependent pathways. In addition detailed seven time-point profile over 30 minutes ligand stimulation, our data determined prominent involvement Lysine-63 branching EGF signaling. Furthermore, we found two centrosomal proteins, PCM1 Azi1, form multi-protein complex with E3 ligases MIB1 WWP2 downstream EGFR, thereby revealing possible cross-talk between centrosomal-dependent rearrangements microtubules. This general can be utilized study other systems modifications.