The cellular proto-oncogene c-Src is a nonreceptor 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 the substrates of c-Src remains a major challenge, because 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 29 potential novel c-Src 
substrate proteins. Tyrosine phosphopeptide mapping allowed the identification 
of 382 nonredundant tyrosine phosphopeptides on 213 phosphoproteins. Stable 
isotope labeling of amino acids in cell culture-based quantitation allowed the 
detection of 97 nonredundant 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 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, C3G, one of the identified potential c-Src substrates that 
plays a role in focal adhesion signaling. 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 nonreceptor tyrosine kinases with high specificity and kinetic 
resolution.