Abstract
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.
Highlights
From the ‡Department of Pharmacology and Molecular Sciences and the §McKusick-Nathans Institute of Genetic Medicine and Department of Biological Chemistry, and the ¶¶Department of Pathology and Oncology, Johns Hopkins School of Medicine, Baltimore, Maryland 21205 and the ¶Institute of Bioinformatics, International Technology Park, Bangalore, 560 066 India
By combining the specific and controlled activation of c-Src by chemical rescue with the ability of quantitative SILAC phosphoproteomics to provide the phosphorylation fingerprint of cells in different conditions, we have identified a number of potential novel c-Src targets and discuss the potential ramifications of these phosphorylation events in cell signaling
To further ensure that imidazole had no major effects on cellular tyrosine phosphorylation outside of chemical rescue under these conditions, we investigated its effect on SYF and wild-type MEF cells
Summary
Cell Culture and SILAC—Src-, Yes-, and Fyn-triple knockout mouse embryonic fibroblast cells (hereafter referred to as SYF cells) (ATCC) stably transfected with either R390A c-Src or D388N c-Src constructs as previously described [3] were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) qualified fetal bovine serum (Invitrogen), 1% (v/v) of penicillin-streptomycin-glutamine (Invitrogen) and 300 g/ml of hygromycin B (Roche Applied Science) and incubated at 37 °C in the presence of 5% CO2. C-Src R390A stably transfected SYF cells were cultured in Arg-/Lys-free Dulbecco’s modified Eagle’s medium supplemented with light (12C6 Arg/12C6 Lys) amino acids from Sigma or heavy (13C6 Arg/13C6 Lys) amino acids from Cambridge Isotope Laboratories, Inc Both media contained 10% qualified fetal bovine serum (Invitrogen), 1% penicillin-streptomycin-glutamine (Invitrogen) and 300 g/ml of hygromycin B (Roche Applied Science). LC-MS analysis of sample aliquots were performed to observe the equal mixing of heavy and light cell lysate proteins using a Micromass quadrupole time of flight mass spectrometer Ultima API (Waters) (supplemental Fig. 1C). The phosphotyrosine antibody-linked beads (Cell Signaling Technology) (250 g in 40 l) were washed with IAP buffer (1.4 ml of IAP buffer, mixed by invert five times) once and incubated with the cleared peptide solution for 30 min at 4 °C with gentle shaking. The raw data associated with this manuscript may be downloaded from ProteomeCommons.org Tranche, http://proteomecommons. org/tranche/, using the following hash code: 04W8a1fuϩ GSc7GMzdx0PJMaHuNϩTEK8OpXcdzz07rrnT9h7USvqudUouF1v SinkQgXwrpϩSA6WuF5IBCwMuF4oCtFDgAAAAAAAAJSgϭϭ
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
