Identiﬁcation of c-Src Kinase Substrates Figure 1. (A) Schematic for the integrated proteomic approach for the identiﬁcation of c-Src kinase substrates. Human embry- onic kidney (HEK) 293T cells growing in Arg ‘0’ containing medium were transiently transfected with a kinase-dead Src (K298M) and 293T cells growing in Arg ‘6’ and Arg ‘10’ were transiently transfected with constitutively active Src kinase (Y527F). Arg ‘0’ refers to 12C6-arginine, Arg ‘6’ refers to 13C - 6 arginine and Arg ‘10’ refers to 13C -15N4-arginine, isotopic labeled 6 forms of arginine used to differentially label 293T cells for identiﬁcation of Src substrates. method also allows one to distinguish contaminating proteins in immunoprecipitates that arise due to nonspeciﬁc binding. Because of the complexity of cell lysates, and because kinase substrates generally exhibit low stoichiometry of tyrosine phosphorylation, speciﬁc identiﬁcation of tyrosine kinase substrates can be facilitated by prior enrichment of tyrosine- phosphorylated proteins with anti-phosphotyrosine antibodies. We applied SILAC for the identiﬁcation of the c-Src kinase substrates in human embryonic kidney cells by overexpression of a constitutively active form of c-Src followed by afﬁnity puriﬁcation of tyrosine-phosphorylated proteins. We transiently overexpressed either a kinase inactive c-Src (K298M) as a control or a constitutively active c-Src (Y527F) kinase in 293T cells. Phosphorylation of the C-terminal tyrosine by C-terminal Src Kinase (CSK) allows inactivation of c-Src.20–22 Hence, mutation of this tyrosine residue to phenylalanine allows c-Src kinase to be constitutively active by preventing its folding and by allowing the kinase domain access to its substrates.23,24 Inhibition of c-Src activity is often achieved by coexpression of the c-Src-inactivating C-terminal Src Kinase (CSK), the kinase-inactive Src mutant Src K298 M,25 or by treatment of the cells with c-Src inhibitors. Three populations of human embryonic kidney cells were grown in DMEM containing 12C6-arginine (light), 13C6-arginine (medium) and 13C -15N4-arginine (heavy), respectively (Figure 6 1). The cells grown in light medium were transfected with a kinase inactive c-Src as a negative control.26 Cells grown in medium and heavy isotope containing media were transfected with a constitutively active form of c-Src (Y527F) and harvested at 12 or 24 h post-transfection, respectively. We found an increased tyrosine phosphorylation upon transfection of con- research articles stitutively active form of c-Src for 24 h (Figure 2). We have also observed that the Y416 in c-Src gets phosphorylated more in this state which points to the increased c-Src tyrosine kinase activity (Figure 2). In addition, the trend of increasing tyrosine phosphorylation could also serve as one more surrogate signature of substrates as one would expect the phosphoryla- tion level to increase during this time course. Mixing of light, medium and heavy isotope labeled cell lysates allowed us to compare the proﬁle of proteins in a single MS experiment. In MS/MS spectra, fragmentation patterns generated by light, medium and heavy peptide pairs are identical except for the expected mass shift of the fragment ions. The ratio of the intensity of the heavier versus the light peptides provides information about the degree of phospho- rylation of a protein and hence its enrichment upon expression of an active c-Src kinase. Thus, the greater the extent of phosphorylation of a protein by c-Src kinase, the higher should be its abundance in anti-phosphotyrosine antibody immuno- precipitates. Peptide sets with a little or no increase in intensity indicate that the protein is not different in abundance in the different states being compared. Such proteins were not investigated further as they are likely nonspeciﬁcally bound proteins. An increase in heavy/light intensity ratio, indicating an increase in total phosphotyrosine content upon Src kinase expression and activity, was found in peptides derived from 36 proteins (Tables 1 and 2). Of these, 10 proteins were either known Src family kinase substrates or proteins known to interact with Src family kinases (Table 1), whereas the remain- ing 26 proteins have not previously been described as sub- strates of c-Src or Src family members (Table 2) in higher eukaryotes. The known substrates identiﬁed in this screen included EWS1 (Ewing sarcoma breakpoint region 1),27 cort- actin,28 calponin-3,29 hnRNP-K (Heterogeneous nuclear ribo- nucleoprotein K),30,31 G3BP (RasGAP SH3-domain binding protein)32–34 and c-Src itself.35 The protein with maximum increase in tyrosine phosphorylation upon c-Src overexpression was c-Src itself. Other known and novel Src family substrates displayed >2-fold increase in intensity of phosphorylation. Apart from signaling and cytoskeletal proteins, we also identi- ﬁed DNA and RNA binding proteins in our analysis. Some of the reasons it is not possible in a kinase-substrate identiﬁca- tion screen of this type to possibly identify every known substrate are (i) previously described substrates might not be expressed in the cell line that we have used; (ii) although the substrates might be expressed, they might not be abundant enough to be enriched and detected in our experiments; (iii) the phosphorylation might not occur or occur at a lower level in the cells that we have used; and (iv) the time course and kinetics of phosphorylation in the system that we have em- ployed might be different from the systems previously used in the literature to describe substrates. Thus, although it is not possible to identify all of the known substrates of Src, identi- ﬁcation of 6 known substrates of Src along with validation of some novel ones is indicative of the success of this type of phosphoproteomic screen. We performed relative quantitation of tyrosine phosphory- lation for each protein as a measure of increase in intensity ratios from light isotope to heavier isotope containing peptides (Supplementary Table 1). Figure 3 shows representative MS spectra for six of the proteins identiﬁed from our screen. In addition to identiﬁcation of proteins with increased phospho- rylation and quantitation, we also mapped 6 phosphorylation sites; Y334 in cortactin10 (Figure 4A) and Y72 in hnRNP-K30 Journal of Proteome Research Vol. 7, No. 9, 2008 3903