c-Src Chemical Rescue: Contribution to the c-Src Phosphoproteome and the Elucidation of the Mechanism of Activation of C3G

The cellular proto-oncogene c-Src is a non-receptor tyrosine kinase involved in cell growth and cytoskeletal regulation. Despite being dysregulated in a variety of human cancers, its precise functions are not fully understood. Identification of c-Src's substrates remains a major challenge, as there is no simple way to directly stimulate its activity. Here we combine the chemical rescue of mutant c-Src and global quantitative phosphoproteomics to obtain the first high-resolution snapshot of the range of tyrosine phosphorylation events that occur in the cell immediately after specific c-Src stimulation. After enrichment by anti-phosphotyrosine antibodies, we identified 28 potential novel c-Src substrate proteins. Tyrosine phosphopeptide mapping allowed the identification of 382 non-redundant tyrosine phosphopeptides on 213 phosphoproteins. SILAC-based quantitation allowed the detection of 97 non-redundant tyrosine phosphopeptides whose level of phosphorylation is increased by c-Src. A large number of previously uncharacterized c-Src putative protein targets and phosphorylation sites are presented here, a majority of which play key roles in signaling and cytoskeletal networks, particularly in cell adhesion. Integrin signaling and focal adhesion kinase (FAK) signaling pathway are two of the most altered pathways upon c-Src activation through chemical rescue. In this context, our study revealed the temporal connection between c-Src activation and the GTPase Rap1, known to stimulate integrin-dependent adhesion. Chemical rescue of c-Src provided a tool to dissect the spatiotemporal mechanism of activation of the Rap1 guanine exchange factor (Rap1GEF), C3G, one of the identified potential c-Src substrates that plays a role in focal adhesion signaling. Phosphorylation of C3G on tyrosine 514 is known to increase activation of C3G. Translocation of C3G to the membrane by the adaptor protein Crk brings C3G to close proximity to its effector Rap1. Chemical rescue of c-Src allowed clarification of the timing and connection between both events as well as the identification of Crk phosphorylation as the possible event that lead to the observed C3G-Crk disassembly. In addition to unveiling the role of c-Src in the cell and, specifically, in the Crk-C3G-Rap1 pathway, these results exemplify a strategy for obtaining a comprehensive understanding of the functions of non-receptor tyrosine kinases with high specificity and kinetic resolution.