Identiﬁcation of c-Src Kinase Substrates Table 2. List of Novel Potential Src Substrates Identiﬁed Using SILAC by Overexpression of Src Kinase in 293T Cells research articles accession no. NP_005454 NP_113680 NP_003760 NP_004490 NP_005667 NP_003893 NP_055662 NP_919223 NP_003676 NP_004951 NP_005849 NP_055554 NP_877952 NP_473357 NP_005110 NP_008937 XP_028253 NP_079222 NP_060082 NP_005057 NP_005780 NP_006061 NP_003746 NP_663760 NP_067038 NP_057123 protein Heterogeneous nuclear ribonucleoprotein D-like RNA binding motif protein 4B Splicing factor, arginine/serine-rich 9 Heterogeneous nuclear ribonucleoprotein AB RNA binding motif protein 10 FUSE binding protein NICE-4 Heterogeneous nuclear ribonucleoprotein A3 FUSE binding protein 2 FUS/TLS oncogene A-kinase anchor protein 8 Bcl-2-associated transcription factor 1 Arsenate resistance protein ARS2 FUS interacting protein (serine/arginine-rich) 1 Thyroid hormone receptor associated protein 3 Cleavage and polyadenylation speciﬁc factor 5 Similar to Zinc ﬁnger CCCH-type domain-containing protein 6 NEFA-interacting nuclear protein Zinc ﬁnger, CCHC domain containing 8 Splicing factor proline/glutamine-rich Proteasome activator subunit 3 TRK-fused gene Eukaryotic translation initiation factor 3, subunit 4 Ataxin 2 related protein Chromosome 20 open reading frame 77 Homeobox prox 1 fold increase (heavy/light)a ( SD 10.0 ( 5.2 9.4 ( 3.7 9.3 ( 3.6 8.0 7.2 ( 0.6 6.9 ( 0.8 6.6 ( 2.1 6.1 ( 2.5 5.7 ( 2.2 5.0 ( 0.9 5.5 5.5 5.4 5.1 5.1 4.8 ( 3.1 3.8 ( 1.9 3.8 ( 0.8 3.7 3.1 ( 1.0 2.6 ( 0.4 2.4 2.2 2.2 2.0 ( 0.3 2.0 ( 0.3 a Heavy refers to Arg ‘10’ and light refers to Arg ‘0’ containing peptides. translation reactions. These proteins were puriﬁed and incu- bated along with c-Src kinase to investigate if it could phos- phorylate these proteins. After incubation with c-Src kinase, the proteins were resolved by SDS-PAGE and autoradiographs were obtained. We found that all of the tested proteins were tyrosine phosphorylated upon incubation with c-Src kinase (Figure 5A). TRK-fused gene from Xenopus laevis has been shown to interact with SH3 domains of various proteins including v-Src but did not bind to neuronal speciﬁc Src in vitro.36 We veriﬁed if the above c-Src substrates were also substrates of c-Src in vivo by cotransfecting these proteins with wild-type c-Src in 293T cells. We also subcloned these cDNAs into a Flag epitope-tagged vector, pCMVtag4A, and cotransfected 293T cells with wild-type c-Src kinase or with an empty vector. The proteins were immunoprecipitated using anti-Flag antibodies, resolved by SDS-PAGE, and immunoblotted with anti-phos- photyrosine antibodies. Upon cotransfection with c-Src kinase, we again observed increased tyrosine phosphorylation of all the tested proteins suggesting that these proteins were also in vivo substrates of c-Src (Figure 5B). We have previously identiﬁed NICE-4 as a tyrosine-phosphorylated protein in a global phosphoproteomic study of HeLa cells.10 RBM10, FBP1 and TRK-fused gene are novel tyrosine-phosphorylated proteins and further investigations need to be carried out to determine how they transduce signals downstream of c-Src kinase and if tyrosine phosphorylation regulates this process. Involvement of a Subset of Novel Substrates in Platelet- Derived Growth Factor Signaling. Since kinase activity of c-Src is required for modulating cellular responses to PDGF receptor stimulation,37 we chose to study the role of a subset of novel substrates in PDGF signaling. The role of c-Src has been well- studied in PDGF signaling.38 We investigated the involvement of EWS1, BTF and RBM10 in PDGF receptor signaling. Cortactin and p130CAS were used as positive controls. For this experi- ment, NIH3T3 cells, which express endogenous PDGF recep- tors, were treated with PDGF-BB in the presence or absence of a c-Src kinase inhibitor, SU6656 (2-oxo-3-(4,5,6,7-tetrahy- dro-1 H-indol-2-ylmethylene)-2,3-dihydro-1H-indole-5-sulfonic acid dimethylamide).38 By activating PDGF signaling in NIH3T3 cells, we investigated the ability of these proteins to get tyrosine-phosphorylated upon ligand-induced stimulation of the PDGF receptor. We found that all of the novel proteins were tyrosine-phosphorylated upon stimulation of PDGF receptor (Figure 5C), as was the case with the two known substrates. Using SU6656, a potent inhibitor of c-Src kinase, we have shown the involvement of three novel proteins RBM10, EWS1 and BTF as c-Src substrates in PDGF signaling (Figure 5C). c-Src has been shown to have been involved in the regulation of nuclear proteins and transcription factors downstream of PDGF signaling and plays an important role in controlling DNA synthesis.39 We have also examined the involvement of RasGAP SH3-domain binding protein and Thyroid hormone receptor associated protein 3 in PDGF signaling using SU6656 but could not detect any tyrosine phosphorylation of these proteins in any state (data not shown). Further experiments need to be done in order to examine the precise role of these proteins and the importance of their phosphorylation in PDGF signaling. One important caveat of these experiments using SU6656 for inhibition of Src kinase is that it could bind and inhibit other related kinases. When better inhibitors become available, this would be a great approach to identify the kinase-substrate relationships. Ewing sarcoma breakpoint region 1 (EWS1) is an RNA binding protein and has been shown to be involved in gene translocations and often appear as fusion of EWS with ETS family transcription factor genes and known to cause Ewing sarcoma tumors.40 One of the fusion proteins EWS-Fli1 has been implicated in insulin-like growth factor 1 (IGF-1)41 as well as PDGF-BB42 induced proliferation of Ewing sarcoma cells. The implication of EWS1 as a downstream substrate of c-Src in PDGF-signaling might shed light into the mechanism of induction of proliferation by these genes in signaling and tumors. Bcl-2-associated transcription factor 1 (BTF) is an Journal of Proteome Research Vol. 7, No. 9, 2008 3905