Identification of c-Src tyrosine kinase substrates using mass spectrometry and peptide microarrays

Ramars Amanchy, Jun Zhong, Henrik Molina, Raghothama Chaerkady, Akiko Iwahori, Dario Eluan Kalume, Mads Grønborg, Jos Joore, Leslie Cope, Akhilesh Pandey

Research output: Contribution to journalArticle

45 Scopus citations

Abstract

c-Src tyrosine kinase plays a critical role in signal transduction downstream of growth factor receptors, integrins and G protein-coupled receptors. We used stable isotope labeling with amino acids in cell culture (SILAC) approach to identify additional substrates of c-Src tyrosine kinase in human embryonic kidney 293T cells. We have identified 10 known substrates and interactors of c-Src and Src family kinases along with 26 novel substrates. We have experimentally validated 4 of the novel proteins (NICE-4, RNA binding motif 10, FUSE-binding protein 1 and TRK-fused gene) as direct substrates of c-Src using in vitro kinase assays and cotransfection experiments. Significantly, using a c-Src specific inhibitor, we were also able to implicate 3 novel substrates (RNA binding motif 10, EWS1 and Bcl-2 associated transcription factor) in PDGF signaling. Finally, to identify the exact tyrosine residues that are phosphorylated by c-Src on the novel c-Src substrates, we designed custom peptide microarrays containing all possible tyrosine-containing peptides (312 unique peptides) and their mutant counterparts containing a Tyr → Phe substitution from 14 of the identified substrates. Using this platform, we identified 34 peptides that are phosphorylated by c-Src. We have demonstrated that SILAC-based quantitative proteomics approach is suitable for identification of substrates of nonreceptor tyrosine kinases and can be coupled with peptide microarrays for high-throughput identification of substrate phosphopeptides.

Original languageEnglish (US)
Pages (from-to)3900-3910
Number of pages11
JournalJournal of Proteome Research
Volume7
Issue number9
DOIs
StatePublished - Sep 1 2008

Keywords

  • PDGF
  • Phosphorylation
  • Quantitative mass spectrometry
  • SILAC
  • Systems biology

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

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    Amanchy, R., Zhong, J., Molina, H., Chaerkady, R., Iwahori, A., Kalume, D. E., Grønborg, M., Joore, J., Cope, L., & Pandey, A. (2008). Identification of c-Src tyrosine kinase substrates using mass spectrometry and peptide microarrays. Journal of Proteome Research, 7(9), 3900-3910. https://doi.org/10.1021/pr800198w