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. 2008 Aug;7(8):3447-60.
doi: 10.1021/pr800187n. Epub 2008 Jun 19.

Global impact of oncogenic Src on a phosphotyrosine proteome

Weifeng Luo  1 , Robbert J Slebos, Salisha Hill, Ming Li, Jan Brábek, Ramars Amanchy, Raghothama Chaerkady, Akhilesh Pandey, Amy-Joan L Ham, Steven K Hanks
Affiliations

Global impact of oncogenic Src on a phosphotyrosine proteome

Weifeng Luo et al. J Proteome Res. 2008 Aug.

Abstract

Elevated activity of Src, the first characterized protein-tyrosine kinase, is associated with progression of many human cancers, and Src has attracted interest as a therapeutic target. Src is known to act in various receptor signaling systems to impact cell behavior, yet it remains likely that the spectrum of Src protein substrates relevant to cancer is incompletely understood. To better understand the cellular impact of deregulated Src kinase activity, we extensively applied a mass spectrometry shotgun phosphotyrosine (pTyr) proteomics strategy to obtain global pTyr profiles of Src-transformed mouse fibroblasts as well as their nontransformed counterparts. A total of 867 peptides representing 563 distinct pTyr sites on 374 different proteins were identified from the Src-transformed cells, while 514 peptides representing 275 pTyr sites on 167 proteins were identified from nontransformed cells. Distinct characteristics of the two profiles were revealed by spectral counting, indicative of pTyr site relative abundance, and by complementary quantitative analysis using stable isotope labeling with amino acids in cell culture (SILAC). While both pTyr profiles are replete with sites on signaling and adhesion/cytoskeletal regulatory proteins, the Src-transformed profile is more diverse with enrichment in sites on metabolic enzymes and RNA and protein synthesis and processing machinery. Forty-three pTyr sites (32 proteins) are predicted as major biologically relevant Src targets on the basis of frequent identification in both cell populations. This select group, of particular interest as diagnostic biomarkers, includes well-established Src sites on signaling/adhesion/cytoskeletal proteins, but also uncharacterized sites of potential relevance to the transformed cell phenotype.

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Figures

Figure 1
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 peptide from tyrosine phosphatase SHP-2 pTyr-62, (B) doubly-charged tryptic peptide from p130Cas pTyr-253, (C) triply-charged tryptic peptide from cofilin-1 pTyr-139, and (D) doubly-charged tryptic peptide from VASP pTyr-39. The precursor m/z values for each spectrum are indicated on each panel. In the peptide sequences, pY indicates phosphotyrosine, while “cmC” in the cofilin-1 peptide indicates a carboxyamidomethylated cysteine.
Figure 2
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 IDs). (B) Identification frequencies of retained pTyr sites reflects a Poisson distribution. (C) Venn diagrams depicting pTyr sites identified uniquely in each cell population and common to both. Left, all retained sites (715 total). Right, sites with ≥4 identifications (374 total).
Figure 3
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 according to the functional categories shown at bottom, with numbers indicating distinct sites within each. (A) All retained sites. (B) Sites with ≥4 identifications. (C) Sites "highly-enriched" in one cell population vs the other based on peptide frequency-based analysis.
Figure 4
Figure 4
Immunoblot analyses of representative pTyr sites. Total cell lysates (30 µg protein/lane) were analyzed using phosphospecific antibodies as indicated.
Figure 5
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 cells). MS/MS spectra shows the annotation of fragment ions (b and y ions) of the more intense heavy peptide peak, confirming the sequence and phosphorylation sites (pY). Asterisk (*) represents the 13C6-labeled amino acid (R/K). Top, MS and corresponding MS/MS spectra of peptide LIEDNEpYTAR from Src (pTyr-418). Middle, MS and corresponding MS/MS spectra of peptide NEEENIpYSVPHDSTQGK from p190RhoGAP (pTyr-943). Bottom, MS and corresponding MS/MS spectra of peptide HLLAPGPQDIpYDVPPVR from p130Cas (pTyr-253).
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