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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    • "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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