research articles Amanchy et al. Figure 2. Tyrosine phosphorylation proﬁle of proteins on transfection with inactive and active forms of c-Src. 293T cells were transfected with inactive and active forms of c-Src, cells were lysed, and tyrosine-phosphorylated proteins were immunoprecipitated from the cell lysates as described in Experimental Procedures. Cell lysates and immunoprecipitates were then run on a 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were probed with anti-phosphotyrosine antibodies and reprobed with phospho (Y416)- Src antibody. Table 1. Known Src Family Kinase Substrates and Interactors Identiﬁed in This Study by Overexpression of c-Src Kinase in 293T Cells Followed by SILAC fold increase (heavy/light)a ( SD Reference 1 2 3 4 5 6 7 8 9 10 NCBI accession no. NP_005408 NP_005222 NP_005234 NP_112552 NP_001830 NP_005745 NP_112533 NP_002129 NP_006833 NP_005511 protein Known c-Src Substrates c-Src Cortactin Ewing sarcoma breakpoint region 1 Heterogeneous nuclear ribonucleoprotein K Calponin 3 RasGAP SH3-domain binding protein Known Src Family Kinase Interactors Heterogeneous nuclear ribonucleoprotein A2/B1 Heterogeneous nuclear ribonucleoprotein D Splicing factor 3B subunit 2 Heterogeneous nuclear ribonucleoprotein H1 30 ( 9.1 4.9 ( 2.6 4.6 3.7 ( 0.2 3.4 2.2 10.2 ( 5.1 4.3 5.6 ( 0.9 3.0 35 28 27 30, 31 29 32–34 45 45 46 45 a Heavy refers to Arg ‘10’ and light refers to Arg ‘0’ containing peptides. (Figure 4B) identiﬁed in this study were reported earlier. We also identiﬁed 4 novel tyrosine phosphorylation sites on each on EWS1 (Figure 4C), FUSE-binding protein 1 (Figure 4D), calponin-3 (Figure 4E) and FIP1-like1 (Figure 4F). We note that, although EWS1 was a known c-Src substrate, no tyrosine phosphorylation sites were previously localized. One of the drawbacks of our experiments is that lysine was not available as 3 different isotopic forms at the time we initiated our experiments. By using 3 isotopes of lysine along with arginine, we would have obtained a better peptide coverage for each protein and likely identiﬁed additional proteins as substrates of c-Src. Nevertheless, we performed a lysine and arginine labeled SILAC experiment in a similar manner but followed by enrichment of phosphopeptides using antibodies against phosphotyrosine to see if we could identify phosphorylated peptides, but unfortunately, we could only identify 8 tyrosine phosphorylation sites (data not shown). We 3904 Journal of Proteome Research Vol. 7, No. 9, 2008 do not know the reasons for such a low yield. However, a protein IP serves our purpose of identifying the proteins that are substrates of Src even though the site is still not identiﬁed. This is the reason we coupled our approach with peptide microarrays to aid in identiﬁcation of phosphopeptides. Validation of a Subset of Novel c-Src Substrates. Further validation of the proteins identiﬁed by SILAC to prove that they are bona ﬁde substrates usually involves the use of antibodies against these proteins. The validation of all of the protein candidates is not always possible, especially for novel proteins, as it depends on the availability of good antibodies. As commercial antibodies were not available for many of the proteins identiﬁed, we chose to investigate a subset of proteins, if they were direct substrates of c-Src using in vitro kinase assays. We selected NICE-4, RBM10, FBP1 and TRK-fused gene for this purpose, as their cDNAs were readily available. We used rabbit reticulocytes to perform in vitro transcription and