ABSTRACT
To identify novel tyrosine kinase substrates that have never been implicated in cancer, we studied the phosphoproteomic changes in the MCF10AT model of breast cancer progression using a combination of phosphotyrosyl affinity enrichment, iTRAQ technology, and LC-MS/MS. Using complementary MALDI- and ESI-based mass spectrometry, 57 unique proteins comprising tyrosine kinases, phosphatases, and other signaling proteins were detected to undergo differential phosphorylation during disease progression. Seven of these proteins (SPAG9, Toll-interacting protein (TOLLIP), WBP2, NSFL1C, SLC4A7, CYFIP1, and RPS2) were validated to be novel tyrosine kinase substrates. SPAG9, TOLLIP, WBP2, and NSFL1C were further proven to be authentic targets of epidermal growth factor signaling and Iressa (gefitinib). A closer examination revealed that the expression of SLC4A7, a bicarbonate transporter, was down-regulated in 64% of the 25 matched normal and tumor clinical samples. The expression of TOLLIP in clinical breast cancers was heterogeneous with 25% showing higher expression in tumor compared with normal tissues and 35% showing the reverse trend. Preliminary studies on SPAG9, on the other hand, did not show differential expression between normal and diseased states. This is the first time SLC4A7 and TOLLIP have been discovered as novel tyrosine kinase substrates that are also associated with human cancer development. Future molecular and functional studies will provide novel insights into the roles of TOLLIP and SLC4A7 in the molecular etiology of breast cancer.
Subject(s)
Breast Neoplasms/pathology , Protein-Tyrosine Kinases/metabolism , Breast Neoplasms/enzymology , Cell Line, Tumor , Chromatography, Liquid , Humans , Immunohistochemistry , Phosphorylation , Spectrometry, Mass, Electrospray Ionization , Substrate Specificity , Tandem Mass SpectrometryABSTRACT
With the completion of the human genome project, analysis of enriched phosphotyrosyl proteins from epidermal growth factor (EGF)-induced phosphotyrosine proteome permits the identification of novel downstream substrates of the EGF receptor (EGFR). Using cICAT-based LC-MS/MS method, we identified and relatively quantified the tyrosine phosphorylation levels of 21 proteins between control and EGF-treated A431 human cervical cancer cells. Of these, Endofin, DCBLD2, and KIAA0582 were validated to be novel tyrosine-phosphorylation targets of EGF signaling and Iressa, a highly selective inhibitor of EGFR. In addition, EGFR activity was shown to be necessary for EGF-induced localization of Endofin, an FYVE domain-containing protein regulated by phosphoinositol lipid and engaged in endosome-mediated receptor modulation. Although several groups have conducted phosphoproteomics of EGF signaling in recent years, our study is the first to identify and validate Endofin, DCBLD2, and KIAA0582 as part of a complex EGF phosphotyrosine signaling network. These novel data will provide new insights into the complex EGF signaling and may have implications on target-directed cancer therapeutics.
Subject(s)
Epidermal Growth Factor/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Neoplasms/metabolism , Phosphoproteins/metabolism , Phosphotyrosine/metabolism , Quinazolines/pharmacology , Serine Endopeptidases/metabolism , Signal Transduction , Amino Acid Sequence , Cell Line, Tumor , Gefitinib , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Membrane Proteins/chemistry , Microtubule-Associated Proteins , Molecular Sequence Data , Neoplasms/pathology , Phosphoproteins/chemistry , Proteomics , Serine Endopeptidases/chemistry , Signal Transduction/drug effects , Tandem Mass SpectrometryABSTRACT
The Gastric Cancer (Biomarkers) Knowledgebase (GCBKB) (http://biomarkers.bii.a-star.edu.sg/background/gastricCancerBiomarkersKb.php) is a curated and fully integrated knowledgebase that provides data relating to putative biomarkers that may be used in the diagnosis and prognosis of gastric cancer. It is freely available to all users. The data contained in the knowledgebase was derived from a large literature source and the putative biomarkers therein have been annotated with data from the public domain. The knowledgebase is maintained by a curation team who update the data from a defined source. As well as mining data from the literature, the knowledgebase will also be populated with unpublished experimental data from investigators working in the gastric cancer biomarker discovery field. Users can perform searches to identify potential markers defined by experiment type, tissue type and disease state. Search results may be saved, manipulated and retrieved at a later date. As far as the authors are aware this is the first open access database dedicated to the discovery and investigation of gastric cancer biomarkers.
