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

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

J Proteome Res

McKusick-Nathans Institute of Genetic Medicine and the Departments of Biological Chemistry, Pathology and Oncology, Johns Hopkins University, Baltimore, Maryland 21205, USA.

Published: September 2008

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.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2646669PMC
http://dx.doi.org/10.1021/pr800198wDOI Listing

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