Identification of targets of c-Src tyrosine kinase by chemical complementation and phosphoproteomics

Isabel Martinez Ferrando, Raghothama Chaerkady, Jun Zhong, Henrik Molina, Harrys K C Jacob, Katie Herbst-Robinson, Beverley M. Dancy, Vikram Katju, Ron Bose, Jin Zhang, Akhilesh Pandey, Philip A. Cole

Research output: Contribution to journalArticle

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-C3GRap1 pathway, these results exemplify a strategy for obtaining a comprehensive understanding of the functions of nonreceptor tyrosine kinases with high specificity and kinetic resolution.

Original languageEnglish (US)
Pages (from-to)355-369
Number of pages15
JournalMolecular and Cellular Proteomics
Volume11
Issue number8
DOIs
StatePublished - Aug 2012

Fingerprint

Phosphopeptides
Phosphorylation
Tyrosine
Chemical activation
Integrins
Protein-Tyrosine Kinases
Substrates
Adhesion
src Genes
Isotope Labeling
Focal Adhesion Protein-Tyrosine Kinases
Phosphotyrosine
Focal Adhesions
GTP Phosphohydrolases
Phosphoproteins
Cell adhesion
Guanine
Cell growth
Cell culture
Cell Adhesion

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Analytical Chemistry

Cite this

Ferrando, I. M., Chaerkady, R., Zhong, J., Molina, H., Jacob, H. K. C., Herbst-Robinson, K., ... Cole, P. A. (2012). Identification of targets of c-Src tyrosine kinase by chemical complementation and phosphoproteomics. Molecular and Cellular Proteomics, 11(8), 355-369. https://doi.org/10.1074/mcp.M111.015750

Identification of targets of c-Src tyrosine kinase by chemical complementation and phosphoproteomics. / Ferrando, Isabel Martinez; Chaerkady, Raghothama; Zhong, Jun; Molina, Henrik; Jacob, Harrys K C; Herbst-Robinson, Katie; Dancy, Beverley M.; Katju, Vikram; Bose, Ron; Zhang, Jin; Pandey, Akhilesh; Cole, Philip A.

In: Molecular and Cellular Proteomics, Vol. 11, No. 8, 08.2012, p. 355-369.

Research output: Contribution to journalArticle

Ferrando, IM, Chaerkady, R, Zhong, J, Molina, H, Jacob, HKC, Herbst-Robinson, K, Dancy, BM, Katju, V, Bose, R, Zhang, J, Pandey, A & Cole, PA 2012, 'Identification of targets of c-Src tyrosine kinase by chemical complementation and phosphoproteomics', Molecular and Cellular Proteomics, vol. 11, no. 8, pp. 355-369. https://doi.org/10.1074/mcp.M111.015750
Ferrando, Isabel Martinez ; Chaerkady, Raghothama ; Zhong, Jun ; Molina, Henrik ; Jacob, Harrys K C ; Herbst-Robinson, Katie ; Dancy, Beverley M. ; Katju, Vikram ; Bose, Ron ; Zhang, Jin ; Pandey, Akhilesh ; Cole, Philip A. / Identification of targets of c-Src tyrosine kinase by chemical complementation and phosphoproteomics. In: Molecular and Cellular Proteomics. 2012 ; Vol. 11, No. 8. pp. 355-369.
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