Identiﬁcation of c-Src Kinase Substrates We have used the stable isotope labeling with amino acids in cell culture (SILAC) approach which enables identiﬁcation of tyrosine kinase substrates based on a unique signature in mass spectrometry experiments.10–12 The main objective of this work was to identify novel c-Src substrates by overexpression of a constitutively active form of c-Src followed by enrichment of tyrosine-phosphorylated proteins. We have identiﬁed 26 novel c-Src tyrosine kinase substrates in addition to 10 others, which were either known Src family kinase substrates or proteins known to associate with Src family kinases. We have experimentally conﬁrmed 4 novel substrates, NICE-4, RNA binding motif 10, FUSE-binding protein 1 and TRK-fused gene, to be direct substrates of c-Src using in vitro kinase assays. We were also able to implicate EWS1, RNA binding motif 10 and Bcl-2 associated transcription factor in PDGF signaling using a chemical inhibitor of c-Src. Our peptide microarray approach led to identiﬁcation of a number of peptides that are phos- phorylated by c-Src. To our knowledge, this is the ﬁrst reported integrated proteomics strategy that couples cell culture, mass spectrometry and peptide microarrays to identify tyrosine kinase substrates. Experimental Procedures Chemicals and Antibodies. Stable isotope containing amino acids, 12C6-arginine, 13C6-arginine and 13C -15N4-arginine, were 6 purchased from Cambridge Isotope Labs (Andover, MA). Complete protease inhibitor cocktail tablets were purchased from Roche (Indianapolis, IN), sodium orthovanadate and anti- Flag M2 monoclonal antibody from Sigma-Aldrich Co. (St. Louis, MO), SU6656 from EMD Biosciences, Inc. (San Diego, CA), anti-phosphotyrosine antibodies (4G10) agarose-conjugate and streptavidin-agarose beads from Upstate Biotechnology (Lake Placid, NY), antiphosphotyrosine-RC20 biotin conjugate from BD Biosciences (San Jose, CA) and PDGF-BB from Invitrogen (Carlsbad, CA). Sequencing grade trypsin was pur- chased from Promega (Madison, WI). Antibodies against cort- actin were purchased from Upstate USA, Inc. (Chicago, IL), p130CAS and EWS1 from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), BTF from Bethyl, Inc. (Montgomery, TX), and RBM10 was from Abcam, Inc. (Cambridge, MA). Phospho-Src (Tyr416) antibody and PhosphoScan Kit (P-Tyr-100) were purchased from cell signaling technology (Boston, MA). Cell Culture and Stable Isotope Labeling with Amino Acid in Cell Culture (SILAC). Human embryonic kidney 293T cells were grown in Dulbecco’s modiﬁed Eagle’s medium (DMEM) containing ‘light’, ‘medium’ or ‘heavy’ arginine supple- mented with 10% dialyzed fetal bovine serum (FBS) plus antibiotics. The 293T cells were adapted to growing in isotope rich-medium supplemented with dialyzed serum prior to initiating these experiments. In each experiment, 20 10-cm dishes were used per condition and the cells were transfected with 15 µ g of DNA using the standard calcium phosphate method (Invitrogen, Carlsbad, CA). Six hours after transfection, the cells were serum-starved for 10 or 20 h. After starvation, the cells were lysed in modiﬁed RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.25% sodium deoxycholate, and 1 mM sodium orthovanadate in the presence of protease inhibitors). Upon cell lysis, proteins lysates were either subjected to afﬁnity puriﬁcation of tyrosine phos- phorylated proteins13 or peptides containing phosphotyrosine were enriched directly from trypsin-digested cell lysates14 using speciﬁc antibodies against phosphotyrosine and identiﬁed by tandem mass spectrometry. research articles Immunoprecipitation and Western Blotting. Light, medium and heavy cell lysates were precleared with protein A-agarose, mixed, and incubated with 400 µ g of 4G10 monoclonal anti- bodies coupled with agarose beads, 75 µ g of biotin-conjugated RC20 antibody, and streptavidin-agarose beads overnight at 4 °C. Precipitated immune complexes were then washed three times with lysis buffer. Agarose beads were boiled and resolved by 10% SDS-PAGE. The gel was silver-stained for visualizing protein bands. Western blotting experiments were performed using anti-phosphotyrosine antibody (4G10) and reprobing was carried out using anti-Flag antibody. Cloning and Transfection. NICE-4 protein (NP_055662), RNA Binding Motif protein 10 isoform 1 (NP_05667), Far upstream element-binding protein (NP_003893), and TRK- Fused gene (NP_006061) were subcloned into a Flag epitope- tagged mammalian expression vector, pCMVtag4A. 293T cells were grown in 10 cm dishes. One dish transfected with wild- type c-Src and pCMVtag4A vector as control; one was cotrans- fected with wild-type c-Src and Flag-tagged cDNAs. The expressed proteins were immunoprecipitated using anti-Flag antibody, followed by SDS-PAGE and Western blotting. The blots were probed with anti-phosphotyrosine antibody followed by stripping and reprobing with anti-Flag antibodies. In Vitro Kinase Assays Using GST-Fusion Proteins. Fusion proteins were made using TNT-coupled rabbit reticulocyte lysate system (Promega, Madison, WI) with the cDNAs cloned in GST expression vector, PGEX4T1. The in vitro translated GST-tagged proteins were puriﬁed with 10 µ g of GST beads for 12 h at 4 °C. After incubation, the beads were washed two times in lysis buffer and two times in kinase buffer (20 mM Hepes, pH 7.4, 5 mM MgCl2, 2 mM MnCl2, 50 µ M sodium vanadate, 50 µ M DTT). Immune complexes were incubated for 30 min at 30 °C in 5 µ L of ATP mixture (10 µ M cold ATP and 10 µ Ci of [γ-32P] ATP) and c-Src Kinase. Protein samples were then eluted by boiling in sample buffer and resolved by SDS-PAGE. The gel was dried and exposed to X-ray ﬁlm to visualize the 32P- labeled protein bands. PDGF Stimulation and Inhibition of Src Kinase. NIH3T3 cells were grown in DMEM containing 10% FBS supplemented with antibiotics. For all PDGF stimulation experiments, cells were stimulated with 100 ng/mL PDGF-BB for 5 min. For inhibition of c-Src kinase, cells were treated with 2 µ M c-Src kinase inhibitor, SU6656, for 1 h prior to stimulation of cells with PDGF-BB for 5 min. In-Gel Trypsin Digestion and In-Solution Trypsin Diges- tion. The silver-stained protein bands were excised and in-gel trypsin digestion was performed as described previously.15 Brieﬂy, the gel slices were excised and incubated with trypsin overnight at 37 °C to allow digestion of proteins after a reduction and alkylation step. After in-gel digestion, the tryptic peptides were extracted. The supernatants from the in-gel and in-solution trypsin digestion containing the peptide mixture were partially dried down in a vacufuge to approximately 10 µ L. For in-solution digestion and enrichment of phosphopep- tides, phosphoscan kit was used according to manufacturer’s prescribed conditions. Liquid Chromatography-Mass Spectrometry. The extracted peptide mixture was centrifuged for 2 min at 12 000g and 4 °C and resolved by reversed-phase liquid chromatography on Agilent 1100 Series LC system (Agilent Technologies, Palo Alto, CA) equipped with a well plate sampler, a vacuum degasser, and a capillary pump. Each fraction from the digested peptide mixture was analyzed by automated nanoﬂow LC-MS/MS. An Journal of Proteome Research Vol. 7, No. 9, 2008 3901