Comparisons of tyrosine phosphorylated proteins in cells expressing lung cancer-specific alleles of EGFR and KRAS. Proc Natl Acad Sci USA

Program in Cancer Biology and Genetics, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 10/2008; 105(37):14112-7. DOI: 10.1073/pnas.0806158105
Source: PubMed


We have used unbiased phosphoproteomic approaches, based on quantitative mass spectrometry using stable isotope labeling with amino acids in cell culture (SILAC), to identify tyrosine phosphorylated proteins in isogenic human bronchial epithelial cells (HBECs) and human lung adenocarcinoma cell lines, expressing either of the two mutant alleles of EGFR (L858R and Del E746-A750), or a mutant KRAS allele, which are common in human lung adenocarcinomas. Tyrosine phosphorylation of signaling molecules was greater in HBECs expressing the mutant EGFRs than in cells expressing WT EGFR or mutant KRAS. Receptor tyrosine kinases (such as EGFR, ERBB2, MET, and IGF1R), and Mig-6, an inhibitor of EGFR signaling, were more phosphorylated in HBECs expressing mutant EGFR than in cells expressing WT EGFR or mutant RAS. Phosphorylation of some proteins differed in the two EGFR mutant-expressing cells; for example, some cell junction proteins (beta-catenin, plakoglobin, and E-cadherin) were more phosphorylated in HBECs expressing L858R EGFR than in cells expressing Del EGFR. There were also differences in degree of phosphorylation at individual tyrosine sites within a protein; for example, a previously uncharacterized phosphorylation site in the nucleotide-binding loop of the kinase domains of EGFR (Y727), ERBB2 (Y735), or ERBB4 (Y733), is phosphorylated significantly more in HBECs expressing the deletion mutant than in cells expressing the wild type or L858R EGFR. Signaling molecules not previously implicated in ERBB signaling, such as polymerase transcript release factor (PTRF), were also phosphorylated in cells expressing mutant EGFR. Bayesian network analysis of these and other datasets revealed that PTRF might be a potentially important component of the ERBB signaling network.

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Available from: Joel S Bader, Dec 16, 2014
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    • "Tyrosine phosphorylation of Y394 in Mig-6 has been reported by Guha et al (2008), which is consistent with our MS data for endo Mig-6. However, serine/threonine phosphorylation of Mig-6 has not been reported at all. "
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    ABSTRACT: The tumour suppressor gene product Mig-6 acts as an inhibitor of epidermal growth factor (EGF) signalling. However, its posttranslational modifications and regulatory mechanisms have not been elucidated. Here, we investigated the phosphorylation of human Mig-6 and found that Chk1 phosphorylated Mig-6 in vivo as well as in vitro. Moreover, EGF stimulation promoted phosphorylation of Mig-6 without DNA damage and the phosphorylation was inhibited by depletion of Chk1. EGF also increased Ser280-phosphorylated Chk1, a cytoplasmic-tethering form, via PI3K pathway. Mass spectrometric analyses suggested that Ser 251 of Mig-6 was a major phosphorylation site by Chk1 in vitro and in vivo. Substitution of Ser 251 to alanine increased inhibitory activity of Mig-6 against EGF receptor (EGFR) activation. Moreover, EGF-dependent activation of EGFR and cell growth were inhibited by Chk1 depletion, and were rescued by co-depletion of Mig-6. Our results suggest that Chk1 phosphorylates Mig-6 on Ser 251, resulting in the inhibition of Mig-6, and that Chk1 acts as a positive regulator of EGF signalling. This is a novel function of Chk1.
    The EMBO Journal 04/2012; 31(10):2365-77. DOI:10.1038/emboj.2012.88 · 10.43 Impact Factor
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    • "Bai and colleagues have reported recently that PTRF protein was down-regulated in breast cancer cell lines and breast tumor tissue, and that down-regulation of PTRF in breast cancer cells was associated with the promoter methylation [49]. PTRF/cavin-1 phosphorylated species have been described in cells that over-express EGFR, which suggests a function in this signaling pathway [50]. Our label-free proteomics results indicate that PTRF expression is lost in NSCLC samples. "
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    ABSTRACT: With the completion of the human genome sequence, biomedical sciences have entered in the "omics" era, mainly due to high-throughput genomics techniques and the recent application of mass spectrometry to proteomics analyses. However, there is still a time lag between these technological advances and their application in the clinical setting. Our work is designed to build bridges between high-performance proteomics and clinical routine. Protein extracts were obtained from fresh frozen normal lung and non-small cell lung cancer samples. We applied a phosphopeptide enrichment followed by LC-MS/MS. Subsequent label-free quantification and bioinformatics analyses were performed. We assessed protein patterns on these samples, showing dozens of differential markers between normal and tumor tissue. Gene ontology and interactome analyses identified signaling pathways altered on tumor tissue. We have identified two proteins, PTRF/cavin-1 and MIF, which are differentially expressed between normal lung and non-small cell lung cancer. These potential biomarkers were validated using western blot and immunohistochemistry. The application of discovery-based proteomics analyses in clinical samples allowed us to identify new potential biomarkers and therapeutic targets in non-small cell lung cancer.
    PLoS ONE 03/2012; 7(3):e33752. DOI:10.1371/journal.pone.0033752 · 3.23 Impact Factor
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    • "activated proteins or phosphorylated sequences into subclasses to reflect the kinetics of their recruitment in the cascade (Zhang et al., 2005; Schmelzle et al., 2006; Wolf-Yadlin et al., 2006) or their correlation with the activation level of the governing RTK (Petti et al., 2005; Huang et al., 2007). Alternatively, Bayesian network modeling was used to analyze several datasets that target the same RTK, but obtained in different conditions on different cell types, and to suggest probable logical organizations of proteins repeatedly identified in several studies (Bose et al., 2006; Guha et al., 2008). The presented studies also often evidenced interconnections between several signaling pathways, which could explain in particular why some cancer types are resistant to drugs that specifically inhibit one RTK. "
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    ABSTRACT: Proteomes are intricate. Typically, thousands of proteins interact through physical association and post-translational modifications (PTMs) to give rise to the emergent functions of cells. Understanding these functions requires one to study proteomes as "systems" rather than collections of individual protein molecules. The abstraction of the interacting proteome to "protein networks" has recently gained much attention, as networks are effective representations, that lose specific molecular details, but provide the ability to see the proteome as a whole. Mostly two aspects of the proteome have been represented by network models: proteome-wide physical protein-protein-binding interactions organized into Protein Interaction Networks (PINs), and proteome-wide PTM relations organized into Protein Signaling Networks (PSNs). Mass spectrometry (MS) techniques have been shown to be essential to reveal both of these aspects on a proteome-wide scale. Techniques such as affinity purification followed by MS have been used to elucidate protein-protein interactions, and MS-based quantitative phosphoproteomics is critical to understand the structure and dynamics of signaling through the proteome. We here review the current state-of-the-art MS-based analytical pipelines for the purpose to characterize proteome-scale networks.
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