Global Impact of Oncogenic Src on a Phosphotyrosine Proteome

Department of Cell and Developmental Biology, Cancer Biology, Biostatistics, and Biochemistry, and The Proteomics Laboratory of the Mass Spectrometry Research Center, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
Journal of Proteome Research (Impact Factor: 4.25). 07/2008; 7(8):3447-60. DOI: 10.1021/pr800187n
Source: PubMed


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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    • "Further indication of the capacity of pY proteomics to reveal distinct signaling activities in HCT-116 cells are pY sites readily detected on tyrosine kinases EGFR (pY1197), macrophage-stimulating protein receptor (MST1/RON (pY1238)), and the JAK-family member TYK2 (pY292). These tyrosine kinase sites were prominent in the HCT-116 cell profile with 9, 15, and 13 spectral IDs, respectively, but none were detected in the previous MEF analysis [28]. An intriguing observation is that equivalent pY sites in the SH2 domains of two different Src-family kinases (FYN pY213 and LYN pY193) are among the most frequently identified HCT-116 cell sites not found MEFs (Table 2). "
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    ABSTRACT: Sensitive and specific biomarkers of protein kinase inhibition can be leveraged to accelerate drug development studies in oncology by associating early molecular responses with target inhibition. In this study, we utilized unbiased shotgun phosphotyrosine (pY) proteomics to discover novel biomarkers of response to dasatinib, a small molecule Src-selective inhibitor, in preclinical models of colorectal cancer (CRC). We performed unbiased mass spectrometry shotgun pY proteomics to reveal the pY proteome of cultured HCT-116 colonic carcinoma cells, and then extended this analysis to HCT-116 xenograft tumors to identify pY biomarkers of dasatinib-responsiveness in vivo. Major dasatinib-responsive pY sites in xenograft tumors included sites on delta-type protein kinase C (PKCδ), CUB-domain-containing protein 1 (CDCP1), Type-II SH2-domain-containing inositol 5-phosphatase (SHIP2), and receptor protein-tyrosine phosphatase alpha (RPTPα). The pY313 site PKCδ was further supported as a relevant biomarker of dasatinib-mediated Src inhibition in HCT-116 xenografts by immunohistochemistry and immunoblotting with a phosphospecific antibody. Reduction of PKCδ pY313 was further correlated with dasatinib-mediated inhibition of Src and diminished growth as spheroids of a panel of human CRC cell lines. These studies reveal PKCδ pY313 as a promising readout of Src inhibition in CRC and potentially other solid tumors and may reflect responsiveness to dasatinib in a subset of colorectal cancers.
    Preview · Article · Nov 2013 · PLoS ONE
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    • "The first amino acid of the motif (Ala) is hidden behind the plane of the figure. The phosphorylation on Tyr 7 (Y7) in the ALYD(Y/F) motif was documented in mouse Src [45] and in human Abl [13]. The figure was created using PyMol. "
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    ABSTRACT: SH3 domains are eukaryotic protein domains that participate in a plethora of cellular processes including signal transduction, proliferation, and cellular movement. Several studies indicate that tyrosine phosphorylation could play a significant role in the regulation of SH3 domains. To explore the incidence of the tyrosine phosphorylation within SH3 domains we queried the PhosphoSite Plus database of phosphorylation sites. Over 100 tyrosine phosphorylations occurring on 20 different SH3 domain positions were identified. The tyrosine corresponding to c-Src Tyr-90 was by far the most frequently identified SH3 domain phosphorylation site. A comparison of sequences around this tyrosine led to delineation of a preferred sequence motif ALYD(Y/F). This motif is present in about 15% of human SH3 domains and is structurally well conserved. We further observed that tyrosine phosphorylation is more abundant than serine or threonine phosphorylation within SH3 domains and other adaptor domains, such as SH2 or WW domains. Tyrosine phosphorylation could represent an important regulatory mechanism of adaptor domains. While tyrosine phosphorylation typically promotes signaling protein interactions via SH2 or PTB domains, its role in SH3 domains is the opposite - it blocks or prevents interactions. The regulatory function of tyrosine phosphorylation is most likely achieved by the phosphate moiety and its charge interfering with binding of polyproline helices of SH3 domain interacting partners.
    Full-text · Article · May 2012 · PLoS ONE
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    • "(C) Y140 is close to actin binding residue E142 (green) but points to the back and thus away from the actin binding interface (green). (D) Ingenuity Pathway Analysis of kinases potentially involved in phosphorylation of Y140 cofilin 1: Src, the Ephrin receptors, PYK/PTK2B and FCeR1 based on proteomics experiments where treatments resulted in increased cofilin 1 phosphorylation [32] [33] [34] [35]. These kinases have direct binding (full lines), direct activation by phosphorylation (full arrows) or indirect interactions (striped arrows) with each other or with a complex containing Abl, Cbl, GAB2 and 14-3-3 proteins (YWHAG, YWHAZ) [36] [37]. "
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    ABSTRACT: Evolutionary conservation for structure function relations is commonly accepted. Here we hypothesize that closely related single domain paralogous proteins, having similar expression profiles and redundant biochemical core functions, additionally evolved to allow and maintain isoform specific differential regulation by single conserved amino acid substitutions. To substantiate this, we considered two families of closely related actin binding proteins combined with data mining of phosphorylated residues in human and mouse proteins. We show that such residues are identical in other orthologs whereas paralogs have a different, but also conserved, non-phosphorylatable residue at the equivalent positions.
    Full-text · Article · Feb 2012 · FEBS letters
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