Phosphotyrosine Profiles from Nontransformed versus Src-Transformed Cells Obtained through Extensive Application of a Shotgun Proteomics Strategy
We analyzed the global pTyr state of a population of MEFs that express oncogenic mouse Src-F529 in which the negative regulatory Tyr-529 was changed to phenylalanine. These cells have greatly elevated cellular pTyr levels and properties classically associated with Src-transformation including anchorage-independent growth, altered morphology, formation of podosome rosettes, and invasive/metastatic behavior.13,20
To assess the impact of Src-F529, a parallel analysis was performed on a counterpart population of nontransformed cells. The shotgun pTyr proteomics strategy12
was applied in large scale through 18 rounds of analysis for each cell type, including 10 biological replicates with peptides generated by trypsin and 8 biological replicates with cleavage by chymotrypsin. Peptides were enriched using either or two different anti-pTyr antibodies, PY100 or 4G10, with each antibody used for half of the replicates. Resulting peptides were analyzed by LC-MS/MS with spectra matching against the mouse subset of the UniRef100 database. shows representative annotated spectra for four pTyr-containing peptides, representing four different proteins (additional representative annotated spectra are presented in Supplemental Figure S1
). After applying filtering criteria, a total of 867 unique peptides representing 563 distinct pTyr sites on 374 different proteins were retained from the analysis of Src-transformed cells, while 514 unique peptides representing 275 pTyr sites on 167 proteins were retained from the nontransformed cells (). Searching the spectra against the sequence-reversed database, while employing the same criteria for data retention, gave an estimate of false-discovery rates as less than 1.5%.
Figure 1 Representative MS/MS spectra for pTyr-containing peptides. The spectra shown are from four different pTyr-containing tryptic peptides that were readily detected in both the Src-transformed and nontransformed cell populations: (A) doubly-charged tryptic (more ...)
Figure 2 Overview of the pTyr profiles. (A) Totals of pTyr sites and represented proteins from the nontransformed vs Src-transformed cell populations, considering either all retained sites (All) or only sites with at least 4 independent identifications (≥4 (more ...)
All retained peptides for the nontransformed and Src-transformed cells are listed in Supplemental Table S1
and Supplemental Table S2
, respectively, and organized according to a functional classification of the represented proteins. These tables also include information on the amino acid position of the pTyr site, tallies of independent spectral counts, highest cross-correlation (xcorr) values for each peptide and charge state with associated precursor ion mass and second-ranking peptides with delta correlation (dCn) values >0.2. Inspection of these tables indicates that the chymotryptic digests revealed several sites not detected in tryptic digests and provided critical confirmation for others. The two anti-pTyr antibodies generated similar data, with little distinction in their abilities to recover pTyr peptides.
Multiple distinct peptides were identified for 111 (40%) of the nontransformed cell pTyr sites and for 203 (36%) of the sites identified from the Src-transformed cells. Of the sites represented by single phosphopeptides, the large majority were retained on the basis of multiple independent identifications with at least two xcorr values meeting the retention threshold of ≥3.3 for charge of 3, ≥2.2 for charge of 2, and ≥1.5 for charge of 1. In addition, we chose to include in the final data set 32 pTyr sites from nontransformed cells and 90 sites from Src-transformed cells that were identified only once from our 36 combined rounds of analysis but which have been reported in the PhosphoSite online resource of in vivo phosphorylation sites. This latter group includes some well-established pTyr sites, but is also more likely to include misidentified sites. Plots of identification frequencies (combined spectral counts) for all pTyr sites retained approximate the Poisson distribution (). The numbers of distinct sites (and proteins) are reduced by about half when only sites with four or more independent identifications are considered ().
An advantage of using an ion trap mass spectrometer for these studies is that the adjustable injection time can increase the sensitivity and improve the quality of the MS/MS spectrum, thus, enhancing the capacity of spectral counting to evaluate differences in the two cell populations. In comparing the nontransformed versus Src-transformed pTyr profiles, however, it became clear that a limitation of the shotgun approach in this particular application was the data-dependent acquisition step during MS/MS, which tends to select only the most intense peptide ions for analysis. While ions once detected are excluded from fragmentation to enhance the identification of lower level ions, large differences in sample complexity can still impact on the comparison. The pTyr peptide preparations from the Src-transformed cells are necessarily much more complex than the preparations from nontransformed cells (producing many more intense signals), such that peptides representing sites phosphorylated to similar extents in both populations will be relatively less abundant in the Src-transformed preparation and therefore less likely selected for MS/MS analysis. For this reason, observed differences between the two cell populations in a site’s identification frequency cannot be used as an absolute quantitative measure of the relative differences in phosphorylation stoichiometry. Nevertheless, the site identification frequency can be taken as an indicator of peptide relative abundance in each of the pTyr-enriched preparations and thus a useful statistic for assessing the global impact of oncogenic Src. Later in this report, we present an independent quantitative analysis using the SILAC approach.
The pTyr Profiles Obtained from Nontransformed versus Src-Transformed Cells Have Distinct Characteristics
Of the 563 sites detected from Src-transformed cells, only 123 were also detected in the nontransformed cells (, left). The nontransformed cells also have a specific profile, with 152 sites detected exclusively in this population. Thus, from the two populations combined, 715 total pTyr sites (representing 458 different proteins) were identified. Many of the sites (the majority from Src-transformed cells) have not previously been described in the published literature and/or remain functionally uncharacterized. The largely distinct natures of the two profiles are maintained when the data are limited to more frequently identified sites, exemplified by those found ≥4 times (, right).
The rather distinct characteristics of the two profiles are further evident from the functional classification of represented proteins (, and see Supplemental Table S1
and Supplemental Table S2
). From nontransformed cells, about half of the sites are on proteins classified under the broad “Signaling” category and another 25% on proteins in the “Adhesion/Cytoskeleton” category. Considering only those sites with ≥4 identifications resulted in further enrichment in these categories (, left). By comparison, the Src-transformed profile was more broadly distributed, with the Signaling and Adhesion/Cytoskeleton categories together comprising less than half of the total and with large representations of proteins classified under RNA Synthesis and Processing, Protein Synthesis and Processing, and Others of miscellaneous or unknown function (, right). For Src-transformed cells, limiting the analysis to sites identified ≥4 times has little effect on these distributions (). The distinct characteristics of the two profiles are also readily apparent from inspection of the most frequently identified sites (). Most frequently detected in the nontransformed cells is pTyr-15 on CDK1, a target of a dual-specificity kinase that maintains this cyclin-dependent kinase in an inactive state during interphase.21
From Src-transformed cells, most frequently detected is pTyr-24 on the glycolytic enzyme enolase, one of the first documented v-Src substrates.22
Figure 3 Protein functional classes represented in the profiles. The pie-charts show distributions of pTyr sites from nontransformed (left) and Src-transformed (right) cells according to a broad functional classification of represented proteins. Color-coding is (more ...)
Ten Most Frequently Identified Sites
To further evaluate differences between the two profiles, a statistical permutation test based on the Poisson distribution was used to display sites detected with significantly higher frequency (p < 0.01) in one cell type versus the other. With this test, 70 sites on 56 proteins were judged as being comparatively “highly enriched” in the nontransformed cell preparations, while 51 sites on 42 proteins were highly enriched in Src-transformed cell preparations ( and , respectively). For nontransformed cells, such sites fall almost entirely in the Signaling and Adhesion/Cytoskeleton categories, but for the Src-transformed cells, these categories were reduced through considering only the highly enriched sites (). The frequently identified () and highly enriched ( and ) sites provide some focus for the following discussions of prominent features of the two pTyr profiles.
Sites Detected with Significantly Higher Frequency in the Nontransformed Cell Populationa
Sites Detected with Significantly Higher Frequency in the Src-Transformed Cell Populationa
Prominent Features of the pTyr Profile Obtained from Nontransformed Cells
Sites on protein kinases account for 30 of the 70 highly enriched sites in the nontransformed cell profile. Included are sites on 12 traditional tyrosine kinases and 10 other proten kinases in the phylogenetic “CMGC” group.23
Highly enriched sites on CMGC kinases CDK1, GSK3, ERK1, ERK2, p38α, DYRK1A, and PRP4 are also among those most frequently identified from these cells. GSK3β pTyr-216 (not distinguishable from GSK3α), an important site of activation loop autophosphorylation during protein folding,24
is the second most frequently identified in nontransformed cells. The activation loop sites identified on the related dual-specificity kinases DYRK1A and PRP4 are likely also sites of autophosphorylation during maturation, as demonstrated for Drosophila DYRK family members.25
The sites on ERK1, ERK2 and p38α are also activation loop sites, targeted by other dual-specificity kinases, that reflect the active states of these MAPKs. Other CMGC kinases represented by nontransformed cell highly enriched sites are ERK5 and dual-specificity kinases HIPK1 and HIPK3.
Among traditional tyrosine kinases, focal adhesion kinase (FAK) stands out in the nontransformed cell profile with 3 highly enriched sites: pTyr-397, pTyr-576, and pTyr-577. FAK Tyr-397 is a site of autophosphorylation in response to cell-ECM adhesion, and Src is recruited to this site to phosphorylate FAK activation loop tyrosines 576 and 577.26
Activation of the FAK/Src complex influences adhesion/cytoskeletal dynamics and is important for efficient cell motility.6,26
Further reflecting the prominence of cell-ECM adhesion signaling in the nontransformed cell profile are highly enriched sites on two major substrates of the FAK/Src complex, paxillin (pTyr-88 and pTyr-118) and p130Cas (pTyr-238 and pTyr-253), that serve to dock downstream signaling effectors.26
FAK pTyr-576, paxillin pTyr-118, and p130Cas pTyr-253 are among the most frequently identified in these cells. P130Cas expression is known to enhance Src-mediated phosphorylation of certain adhesion/cytoskeletal proteins including FAK and paxillin.20
The ~3-fold higher level of p130Cas expression in the nontransformed versus the Src-transformed cells13
could therefore have accounted for some of the enrichment of cell/ECM adhesion proteins in the nontransformed cell profile. Other nontransformed cell highly enriched sites are on proteins implicated in regulating the adhesion-actin cytoskeletal network, including p190RhoGAP pTyr-94327
and protein-tyrosine phosphatase PTPα pTyr-825.28
Highly enriched sites on other cell-ECM adhesion proteins (tensin, vinculin), cell–cell adhesion proteins (p120catenin, plakophilin-4, PARD3), and others (CrkL, RhoGAP-12, BCAR3, N-WASP) may also impact adhesion–cytoskeletal dynamics. In addition to FAK, nonreceptor tyrosine kinases are further represented by highly enriched regulatory sites on members of the Src family and on ACK that is activated in response to both cell-ECM adhesion and growth factors.29
Receptor tyrosine kinases figure prominently in the nontransformed cell profile. Sites were detected on 7 members of the Eph receptor family including highly enriched sites on EphA2 and EphB3. Bidirectional signaling from Eph receptors and their transmembrane ephrin ligands regulate cytoskeletal, adhesive, and motile properties of interacting cells.30
One Eph receptor ligand, ephrin B2 (most peptides common to ephrin B1), was also part of the nontransformed cell profile with multiple sites identified including one highly enriched site. Also highly enriched are activation loop sites on receptor tyrosine kinases Met, Axl, DDR2, and IGF1R. The docking protein Dok1, a downstream target of various receptor and nonreceptor tyrosine kinases, is prominent in the nontransformed cell profile with five sites identified. Dok1 pTyr-361, the only site ranking among the 10 most frequently identified in both populations, is important for recruitment of the adaptor Nck to stimulate F-actin reorganization and cell motility.31,32
Other downstream effectors for receptor tyrosine kinases that are represented by nontransformed cell highly enriched sites are adaptor/docking proteins LAP2, Shb, Shc1, Gab1, and insulin receptor substrate 2. Other notable signaling/regulatory proteins represented by nontransformed cell highly enriched sites are caveolin-2, calmodulin, and STAT3.
In summary, the pTyr profile of the nontransformed cell population strongly reflects the receptor- and adhesion-associated signaling pathways the act to drive proliferative and motile cell behavior.
Prominent Features of the Src-Transformed Cell pTyr Profile
A major distinction of the Src-transformed profile is an absence of protein kinases, other than Src itself, in the lists of sites either most frequently identified or highly enriched. Two sites on Src, pTyr-92 and pTyr-438, are highly enriched in the transformed cells, and remarkably, both are essentially uncharacterized. Tyr-92 is a conserved residue in the SH3 domain that has an important role in ligand binding.33
Tyr-438 is in a region of the kinase domain implicated in substrate selection, and has been previously described as an autophosphorylation site in studies on a recombinant kinase domain.34
A third novel site on Src, detected at low frequency, is Tyr-231 in the SH2 domain. These findings suggest that oncogenic Src has the capacity to undergo more extensive autophosphorylation than previously appreciated, with implications for regulation of substrate selection. While no other sites on protein kinases made the lists of highly enriched or most frequently identified, these signaling enzymes are nevertheless well-represented in the Src-transformed profile with 30 additional sites representing 20 protein kinases. Included are the sites on FAK, EphA2, CDK1, GSK3, and PRP4 that were frequently detected in nontransformed cells.
It is notable that sites on several other protein kinases that were very frequently detected in the nontransformed cells are absent in the Src-transformed profile, including those on Met, ACK, ERK1, ERK2, and p38α. The limitation of shotgun proteomics imposed by data-dependent acquisition is clearly a factor in the comparatively low-frequency identification in Src-transformed cells of such pTyr sites that are not known or not likely to be directly targeted by the oncogenic Src kinase. As an illustration of this point, the ERK1/ERK2 activation loop sites are still quite readily detected in the Src-transformed cells and nontransformed cells through immunoblotting with phosphospecific antibodies (), although the signal is reduced in comparison to the nontransformed cells. A more revealing example of the sensitivity limitation is the FAK activation loop. Immunoblotting shows that FAK activation loop phosphorylation is elevated in the Src-transformed cells (), as expected for this recognized Src target, although the acivtion loop sites were detected by the shotgun proteomics approach with significantly higher frequency in the nontransformed cells.
Immunoblot analyses of representative pTyr sites. Total cell lysates (30 µg protein/lane) were analyzed using phosphospecific antibodies as indicated.
Other Signaling and Adhesion/Cytoskeleton proteins make notable contributions to the Src-transformed cell profile. Annexins, which are calcium-dependent phospholipid-binding proteins involved in structural organization and intracellular signaling, are well-represented by annexins A1, A2, A5, A6, and A11. Annexin A2 pTyr-274 and annexin A6 pTyr-29 are highly enriched sites. Four additional sites were detected on annexin A2, the first identified v-Src substrate previously known as p36 calpactin I/lipocortin II,35
including pTyr-23 also found in nontransformed cells. Other sites detected multiple times in Src-transformed cells, but absent in the nontransformed profile, are on signaling proteins phospholipase Cγ1, Rab GDIβ, RACK1 (receptor of activated protein kinase C), striatin-3, and Tks4 (related to Tks5/FISH). Cell-ECM adhesion-associated proteins including p130Cas, LPP (lipoma preferred partner), paxillin, talin, tensin, vinculin, and VASP were readily detected, with several previously unrecognized sites among the many identified on these proteins. On vinculin, the first cytoskeletal substrate identified for v-Src,36
pTyr-99 is highly enriched. Similarly, cell–cell adhesion proteins contributed to the Src-transformed profile including p120catenin, PARD3, plakophilin-4, ZO-1, and ZO-2, again with several novel sites detected. Actin regulators are notably represented by sites on cofilin, cortactin, and by a highly enriched site on actin interacting protein AIP1. Intriguing novel sites were found on three components (Arp2, Arp3, and p21Arc) of the Arp2/3 complex that acts to nucleate new actin filaments. Arp2 pTyr-91 and p21Arc pTyr-46 are among the highly enriched sites. Other readily detected sites include actin cross-linkers α-actinin and filamin B, and cytokinesis regulators anillin and septin-2. Sites on α-actinin, filamin B, and plectin (a cross-linker of microtubules to intermediate and actin filaments) are among those highly enriched.
The Src-transformed profile includes many metabolic enzymes including several involved in glycolysis. In addition to enolase with two of the three most frequently identified sites, glycolytic enzymes are represented by GAPDH, phosphofructokinase, lactate dehydrogenase, phosphoglycerate mutase, and pyruvate kinase isoform M2. Glycolytic enzyme pTyr sites are of interest in light of findings implicating tyrosine phosphorylation in the switch from oxidative phosphorylation to aerobic glycolysis (the “Warburg effect”) that is important for cancer cell metabolism and tumor growth. Of particular note in this context are recent studies showing that the Warburg effect can result as a consequence of pyruvate kinase M2 binding to pTyr-binding peptides, including sites identified on enolase (pY43) and lactate dehydrogenase (pY238), which inhibits pyruvate kinase M2 enzymatic activity to divert glucose metabolites to anabolic processes.37,38
GAPDH and the rate-limiting phosphofructokinase have previously been characterized as substrates for the EGF receptor39
prompting speculation that these phosphorylation events could contribute to the altered energy metabolism in cancer cells. Sites on over a dozen other diverse metabolic/biosynthetic enzymes were detected in the Src-transformed cells, including several additional highly enriched sites.
Proteins involved in RNA and protein synthesis and processing are a major aspect of the Src-transformed cell profile. Included are many RNA helicases, hnRNPs, splicing factors, tRNA synthetases, ribosomal proteins, translation initiation factors, chaperones, and components of the ubiquitin–proteasome system. Proteins in these categories also account for a large fraction of the Src-transformed cell frequently identified and highly enriched sites. The second most frequently detected site in the Src-transformed cells is pTyr-485 on hnRNP Q, almost all from chymotryptic peptides. The DEAD-box RNA helicase, DDX3X, emerges as a prominent likely target of oncogenic Src, with 7 distinct sites detected including one of the most frequently identified. Tyrosine phosphorylation of proteins that function in RNA processing may account for past observations of oncogenic Src activity causing partially spliced transcripts to accumulate in the nucleus.40
Also among the most frequently identified in Src-transformed cells are two sites on proteasome subunit PSMA2.
To summarize, a distinguishing aspect of the Src-transformed cell profile is the large representation of proteins having basic “housekeeping” functions including metabolic enzymes and macromolecular synthetic machinery. For pTyr sites on such abundant cellular proteins that were detected only in the Src-transformed cells, it must be considered that these may not be normal targets of endogenous tyrosine kinases but rather represent superfluous targets of the catalytically potent oncogenic Src “gone wild”. Nevertheless, such sites could have an impact on the neoplastic properties of these cells.
P-Tyr Sites Identified Frequently in Both Nontransformed and Src-Transformed Cells
Considering once again the limitations of data-dependent acquisition in profiling the Src-transformed cells, pTyr sites that were frequently identified in both cell populations are likely to include the major (most abundant) biologically relevant Src sites. That is, such sites are likely to be prominently phosphorylated in normal cells but also undergo elevated phosphorylation in the presence of oncogenic Src. Forty-seven sites, representing 36 proteins, met the arbitrary cutoff of ≥4 identifications in each cell type. Four of these sites, on CMGC kinases CDK1, GSK3β, DYRK1A, and PRP4, are among those most frequently identified in the nontransformed cells () while being identified with greatly reduced frequencies in Src-transformed cells, which is consistent with these sites not being direct Src targets.
The remaining 43 sites representing 32 proteins () we consider as candidate Src substrates (see Supplemental Figure S1
for representative annotated spectra of peptides containing these sites, and Supplemental Text
File for brief overviews of these proteins focused on their tyrosine phosphorylation). For 23 of these proteins (red highlighting in ), the existing evidence supports them as being bona fide
Src substrates. Src is on this list by virtue of its activation loop autophosphorylation site, and also represented are other established Src sites on FAK, p130Cas, paxillin, p120catenin, SHIP2, p190RhoGAP, caveolin-1, cortactin, Nck1, Shc1, Dok1 (pTyr-361), annexin A2, and enolase.
Sites Detected Frequently in Both Nontransformed and Src-Transformed Cell Populationsa
Also identified ≥4 times from both populations are sites on proteins previously characterized as known or likely Src substrates but for which published evidence is lacking for the specific site(s) being directly targeted by Src, namely, talin-1, GIT1, ZO-1, cofilin-1, Cbl, eEF1A, CDV3A, PI3K p85α, SHP2, and two additional sites on Dok1 (). Of particular interest in this group are sites on the protein-tyrosine phosphatase SHP2 (pTyr-62) and the p85α regulatory subunit of PI3K (pTyr-467) as they lie in regions involved in regulating enzyme activity. SHP2 exhibits constitutive tyrosine-phosphorylation in Src-transformed cells and has been implicated in morphological transformation.41,42
Structural studies showed SHP2 Tyr-62 to lie in a region of the N-terminal SH2 domain that inserts into the catalytic cleft to autoinhibit phosphatase activity.43
PI3K activity is also known to be elevated in Src-transformed cells and implicated in transformation.44,45
Tyr-467 of the p85α subunit is part of a coiled-coiled domain in the inter-SH2 region that interacts with the p110 catalytic subunit to inhibit activity.46
Immunoblot analysis using a phosphospecific antibody raised against the p85 pTyr-467 provided further evidence for the elevated phosphorylation of this site in Src-transformed cells ().
Many other sites found ≥4 times from both populations are on proteins not previously implicated as Src substrates. This group includes a predicted protein kinase SgK269 (two sites), a zyxin-family member LPP (lipoma preferred partner, three sites) known to localize to cell/ECM adhesions, actin-associated proteins septin-2 and VASP, small G-protein regulators intersectin-2 and RIN1, a predicted Rho-family GAP that is most closely related to oligophrenin 1, ANKS1/Odin, and the glycolytic enzyme GAPDH (). VASP Tyr-39 is notable among this group of sites as it is a conserved residue in the EVH1 domain important for subcellular targeting.47,48
RIN1 Tyr-35 has been previously characterized as a site of phosphorylation by the Abl tyrosine kinase.49
Immunoblotting with a phosphospecific antibody against RIN Tyr-35 further supports this site as being elevated in the Src-transformed cells (). As Src can act as a positive regulator of Abl by phosphorylating the Abl kinase domain activation loop Tyr-39350
(in our study, identified 4 times in nontransformed cells and 3 times in Src-transformed cells), it is possible that increased Abl activity contributes to the observed RIN1 Tyr-35 phosphorylation. Other sites presented here as candidate novel Src sites could similarly be targets of other kinases that are activated downstream of Src.
SILAC Analysis of the Impact of Oncogenic Src on the pTyr Proteome
While the spectral-counting analysis revealed much about the striking impact of oncogenic Src on the pTyr proteome, the much greater complexity of the peptide preparations from the Src-transformed cells precluded the use of this approach as a means to provide accurate information on differences between the two cell populations in phosphorylation site stoichiometry. Thus, to complement the spectral counting data, we carried out an independent quantitative comparison using the SILAC method.51,52
For the SILAC analysis, proteins in the Src-transformed cell population were labeled through six passages in media containing heavy 13
-arginine and 13
-lysine, while the nontransformed cells were maintained in standard growth medium. Then equal numbers of cells from each population were combined prior to cell lysis and subsequent tryptic peptide enrichment through pTyr immunoaffinity purification. Corresponding light and heavy SILAC phopeptides, that co-elute in the reversed-phase step, appear as doublets in MS with the ratio of peak intensities indicating relative differences in phosphorylation stoichiometry (, and Supplemental Figure S2
). A single round of SILAC analysis was carried out, providing quantitative measures for 48 peptides representing 42 different pTyr sites on 38 different proteins (Supplemental Table S3
). Of these 42 sites, 10 were represented by peptides that were clearly detected only as heavy peaks representative of the Src-transformed cell sample.
Figure 5 Examples of quantitation by SILAC. The panels show the MS and MS/MS spectra of three different pTyr-containing peptides. MS spectra show the fold changes in the level of heavy labeled peptide (from Src-transformed cells) versus light peptide (from nontransformed (more ...)
The SILAC results provided general support for the conclusions made from the label-free spectral count analysis. All but two of the sites identified by SILAC were indicated to have elevated phosphorylation in the Src-transformed cells, with the two exceptions being CDK1 pTyr-15, that was elevated in the nontransformed cells, and GSK3 pTyr-216 that was essentially unchanged (). In the label-free analysis, these two CMGC kinase sites were among those identified with significantly higher frequency in the nontransformed cells.
SILAC Quantification of Sites Frequently Detected in Both Cell Populationsa
Notable among the 40 pTyr sites indicated by SILAC to have elevated phosphorylation in the Src-transformed cells are 11 sites () predicted from the label-free analysis as major biologically relevant Src substrates on the basis of their frequent identification in both cell populations. Included are five established Src sites (Src Tyr-418, p190RhoGAP Tyr-943, p130Cas Tyr-253, p120catenin Tyr-96, and enolase Tyr-43) and six of the sites not previously established as Src targets (talin-1 Tyr-26, talin-1 Tyr-70, eEF1A Tyr-29, VASP Tyr-39, RIN-1 Tyr-35, and GAPDH Tyr-315). Thus SILAC provided further evidence in support of these sites being direct targets of oncogenic Src. Of these 11 sites, only enolase Tyr-43 was identified with significantly higher frequency in the Src-transformed cells in the label-free analysis, while the two sites with the lowest heavy/light peak intensity ratios among this group, p190RhoGAP Tyr-943 (heavy/light ) 2.8) and p130Cas Tyr-253 (heavy/light = 4.0), were identified with significantly higher frequency in the nontransformed cells.
A majority of the remaining pTyr sites identified by SILAC as elevated in the Src-transformed cells were on housekeeping proteins including metabolic enzymes and macromolecular synthetic machinery. Thirteen sites identified by SILAC, including enolase Tyr-43 and Src-Tyr-438, were also found in the label-free analysis with significantly higher frequency in the Src-transformed cells and these tended to have higher heavy/light peak intensity ratios. Five sites emerged from the SILAC analysis (on alpha-1 catenin, tight junction protein ZO-1, elongation complex protein 3, DNA damage-binding protein 1, and U1 snRNP 70 kDa; see Supplemental Table S3
for details) that had not been found in the label-free analysis, which is not surprising in light the different instrumentation and set up used in the two approaches.