Article

Time-resolved mass spectrometry of tyrosine phosphorylation sites in the epidermal growth factor receptor signaling network reveals dynamic modules.

Biological Engineering Division, Massachusetts Institute of Technnology, Cambridge, Massachusetts 02139, USA.
Molecular &amp Cellular Proteomics (Impact Factor: 7.25). 10/2005; 4(9):1240-50. DOI: 10.1074/mcp.M500089-MCP200
Source: PubMed

ABSTRACT Ligand binding to cell surface receptors initiates a cascade of signaling events regulated by dynamic phosphorylation events on a multitude of pathway proteins. Quantitative features, including intensity, timing, and duration of phosphorylation of particular residues, may play a role in determining cellular response, but experimental data required for analysis of these features have not previously been available. To understand the dynamic operation of signaling cascades, we have developed a method enabling the simultaneous quantification of tyrosine phosphorylation of specific residues on dozens of key proteins in a time-resolved manner, downstream of epidermal growth factor receptor (EGFR) activation. Tryptic peptides from four different EGFR stimulation time points were labeled with four isoforms of the iTRAQ reagent to enable downstream quantification. After mixing of the labeled samples, tyrosine-phosphorylated peptides were immunoprecipitated with an anti-phosphotyrosine antibody and further enriched by IMAC before LC/MS/MS analysis. Database searching and manual confirmation of peptide phosphorylation site assignments led to the identification of 78 tyrosine phosphorylation sites on 58 proteins from a single analysis. Replicate analyses of a separate biological sample provided both validation of this first data set and identification of 26 additional tyrosine phosphorylation sites and 18 additional proteins. iTRAQ fragment ion ratios provided time course phosphorylation profiles for each site. The data set of quantitative temporal phosphorylation profiles was further characterized by self-organizing maps, which resulted in identification of several cohorts of tyrosine residues exhibiting self-similar temporal phosphorylation profiles, operationally defining dynamic modules in the EGFR signaling network consistent with particular cellular processes. The presence of novel proteins and associated tyrosine phosphorylation sites within these modules indicates additional components of this network and potentially localizes the topological action of these proteins. Additional analysis and modeling of the data generated in this study are likely to yield more sophisticated models of receptor tyrosine kinase-initiated signal transduction, trafficking, and regulation.

0 Bookmarks
 · 
100 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Mutations in the Epidermal growth factor receptor (EGFR) kinase domain occur in 10–30% of lung adenocarcinoma and are associated with tyrosine kinase inhibitor (TKI) sensitivity. We sought to identify the immediate direct and indirect phosphorylation targets of mutant EGFRs in lung adenocarcinoma. We undertook stable isotope labeling of amino acids in cell culture (SILAC) strategy, phosphopeptide enrichment, and quantitative mass spectrometry to identify dynamic changes of phosphorylation downstream of mutant EGFRs in lung adenocarcinoma cells harboring EGFRL858R and EGFRL858R/T790M, the TKI-sensitive and -resistant mutations, respectively. Top canonical pathways that were inhibited upon erlotinib treatment in sensitive cells, but not in the resistant cells include EGFR, Insulin receptor, HGF, MAPK, mTOR, p70S6K and JAK/STAT signaling. We identified phosphosites in proteins of the autophagy network, such as ULK1 (S623) that is constitutively phosphorylated in these lung adenocarcinoma cells; phosphorylation is inhibited upon erlotinib treatment in sensitive cells, but not in resistant cells. Finally, kinase-substrate prediction analysis from our data indicated that substrates of basophilic kinase families, AGC, CAMK and STE were significantly enriched and those of proline directed kinase families, CMGC and CK were significantly depleted among substrates that exhibited increased phosphorylation upon EGF stimulation and reduced phosphorylation upon TKI inhibition. This is the first study to date to examine global phosphorylation changes upon erlotinib treatment of lung adenocarcinoma cells and results from this study provide new insights into signaling downstream of mutant EGFRs in lung adenocarcinoma.This article is protected by copyright. All rights reserved
    Proteomics 11/2014; 15(2-3). DOI:10.1002/pmic.201400315 · 3.97 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Receptor tyrosine kinases (RTKs) bind to a subset of growth factors on the surface of cells and elicit responses with broad roles in developmental and postnatal cellular processes. Receptors in this subclass consist of an extracellular ligand-binding domain, a single transmembrane domain, and an intracellular domain harboring a catalytic tyrosine kinase and regulatory sequences that are phosphorylated either by the receptor itself or by various interacting proteins. Once activated, RTKs bind signaling molecules and recruit effector proteins to mediate downstream cellular responses through various intracellular signaling pathways. In this chapter, we highlight the role of a subset of RTK families in regulating the activity of neural crest cells (NCCs) and the development of their derivatives in mammalian systems. NCCs are migratory, multipotent cells that can be subdivided into four axial populations, cranial, cardiac, vagal, and trunk. These cells migrate throughout the vertebrate embryo along defined pathways and give rise to unique cell types and structures. Interestingly, individual RTK families often have specific functions in a subpopulation of NCCs that contribute to the diversity of these cells and their derivatives in the mammalian embryo. We additionally discuss current methods used to investigate RTK signaling, including genetic, biochemical, large-scale proteomic, and biosensor approaches, which can be applied to study intracellular signaling pathways active downstream of this receptor subclass during NCC development. © 2015 Elsevier Inc. All rights reserved.
    Current Topics in Developmental Biology 01/2015; 111:135-82. DOI:10.1016/bs.ctdb.2014.11.005 · 4.21 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We sought to explore the molecular mechanism of type 2 diabetes (T2D) and identify potential drug targets and candidate agents for T2D treatment. The differentially expressed genes (DEGs) were assessed between human pancreatic islets with T2D and normal islets. The dysfunctional pathways, the potential transcription factor, and microRNA targets were analyzed by bioinformatics methods. Moreover, a group of bioactive small molecules were identified based on the connectivity map database. The pathways of Eicosanoid Synthesis, TGF-beta signaling pathway, Prostaglandin Synthesis and Regulation, and Integrated Pancreatic Cancer Pathway were found to be significantly dysregulated in the progression of T2D. The genes of ZADH2 (zinc binding alcohol dehydrogenase domain containing 2), BTBD3 (BTB (POZ) domain containing 3), Cul3-based ligases, LTBP1 (latent-transforming growth factor beta binding protein 1), PDGFRA (alpha-type platelet-derived growth factor receptor), and FST (follistatin) were determined to be significant nodes regulated by potential transcription factors and microRNAs. Besides, two small molecules (sanguinarine and DL-thiorphan) were identified to be capable of reverse T2D. In the present study, a systematic understanding for the mechanism underlying T2D development was provided with biological informatics methods. The significant nodes and bioactive small molecules may be drug targets and candidate agents for T2D treatment.
    Journal of Diabetes Research 01/2014; 2014:763936. DOI:10.1155/2014/763936 · 3.54 Impact Factor

Full-text (2 Sources)

Download
305 Downloads
Available from
Jun 3, 2014