Zhang Y, Wolf-Yadlin A, Ross PL, Pappin DJ, Rush J, Lauffenburger DA et al.. Time-resolved mass spectrometry of tyrosine phosphorylation sites in the epidermal growth factor receptor signaling network reveals dynamic modules. Mol Cell Proteomics 4: 1240-1250

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.

Download full-text


Available from: Alejandro Wolf-Yadlin, Aug 27, 2015
  • Source
    • "such as quantitative tyrosine phosphorylation profiles [96]. All above-mentioned clustering methods require calculation of feature-feature or feature-centroid distances, being mostly on "
    [Show abstract] [Hide abstract]
    ABSTRACT: The investigation of post-translational modifications (PTMs) represents one of the main research focuses for the study of protein function and cell signaling. Mass spectrometry instrumentation with increasing sensitivity, improved protocols for PTM enrichment and recently established pipelines for high-throughput experiments allow large-scale identification and quantification of several PTM types. This review addresses the concurrently emerging challenges for the computational analysis of the resulting data and presents PTM-centered approaches for spectra identification, statistical analysis, multivariate analysis and data interpretation. We furthermore discuss the potential of future developments that will help to gain deep insight into the PTM-ome and its biological role in cells. Copyright © 2015. Published by Elsevier B.V.
    Journal of Proteomics 07/2015; DOI:10.1016/j.jprot.2015.07.016 · 3.93 Impact Factor
  • Source
    • "This differential attenuation of prosurvival and proapoptotic signals may underlie the acute sensitivity of these lung adenocarcinoma cells to mutant EGFR inhibition [11]. Recent advances in quantitative MS and profiling of PTMs, such as phosphorylation has enabled global proteome-wide analysis of normal EGFR signaling pathway [12] [13] [14] [15] [16]. More specifically, a global phosphoproteomic approach to identify tyrosine phosphorylated peptides has identified several oncogenic kinases such as EGFR, MET, PDGFR␣, DDR1, and novel ALK and ROS fusions in nonsmall cell lung cancer (NSCLC) cell lines and tumor specimens [17]. "
    [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
  • Source
    • "Phosphoproteomics MCF10A vector control and shCul5 cells were grown to 90% confluency in normal growth medium on three 15 cm plates. Cell lysis and protein digestion was performed as previously described (Zhang et al., 2005), except that a 20:1 substrate:trypsin ratio was used for digestion. Lyophilized peptides were resuspended in 0.5 ml of 100 mM Tris-HCl, pH 7.4, with 100 mM NaCl, 0.1% NP-40 and immunoprecipitated overnight at 4˚C with 2.5 mg of anti-pY antibody-conjugated beads (PY- 99, Santa Cruz). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Phosphorylation-dependent protein ubiquitylation and degradation provides an irreversible mechanism to terminate protein kinase signaling. Here we report that mammary epithelial cells require Cullin 5 RING E3 ubiquitin ligase complexes (Cul5-CRLs) to prevent transformation by a Src-Cas signaling pathway. Removing Cul5 stimulates growth factor-independent growth and migration, membrane dynamics, and colony dysmorphogenesis, dependent on the endogenous tyrosine kinase, Src. Src is activated in Cul5-deficient cells, but Src activation alone is not sufficient to cause transformation. We found that Cul5 and Src together stimulate degradation of the Src substrate, p130Cas (Crk-associated substrate). Phosphorylation stimulates Cas binding to the Cul5-CRL adaptor protein SOCS6 and consequent proteasome-dependent degradation. Cas is necessary for the transformation of Cul5-deficient cells. Either SOCS6 knockdown or a degradation-resistant Cas mutant stimulates membrane ruffling but not other aspects of transformation. Our results show that endogenous Cul5 suppresses epithelial cell transformation by several pathways, including inhibiting Src-Cas-induced ruffling through SOCS6.
    Journal of Cell Science 11/2013; 127(3). DOI:10.1242/jcs.127829 · 5.33 Impact Factor
Show more