Identification of c-Src tyrosine kinase substrates using mass spectrometry and peptide microarrays.
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.
Amanchy
Ramars
R
McKusick-Nathans Institute of Genetic Medicine and the Departments of Biological Chemistry, Pathology and Oncology, Johns Hopkins University, Baltimore, Maryland 21205, USA.
Zhong
Jun
J
Molina
Henrik
H
Chaerkady
Raghothama
R
Iwahori
Akiko
A
Kalume
Dario Eluan
DE
Grønborg
Mads
M
Joore
Jos
J
Cope
Leslie
L
Pandey
Akhilesh
A
D000595:Amino Acid Sequence; D002460:Cell Line; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D010766:Phosphorylation; D040081:Protein Array Analysis; D013379:Substrate Specificity; D019061:src-Family Kinases / Q000378:metabolism*
D010455:Peptides; D019061:src-Family Kinases