Comparisons of tyrosine phosphorylated proteins in cells expressing lung cancer-specific alleles of EGFR and KRAS

Udayan Guha, Raghothama Chaerkady, Arivusudar Marimuthu, A. Scott Patterson, Manoj K. Kashyap, H. C. Harsha, Mitsuo Sato, Joel S. Bader, Alex E. Lash, John D. Minna, Akhilesh Pandey, Harold E. Varmus

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Abstract

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 (β-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.

Original languageEnglish (US)
Pages (from-to)14112-14117
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume105
Issue number37
DOIs
StatePublished - Sep 16 2008

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Tyrosine
Lung Neoplasms
Alleles
Epithelial Cells
Phosphorylation
Proteins
gamma Catenin
Isotope Labeling
Catenins
Intercellular Junctions
Bayes Theorem
Receptor Protein-Tyrosine Kinases
Cadherins
Mass Spectrometry
Phosphotransferases
Nucleotides
Cell Culture Techniques
Amino Acids
Cell Line

Keywords

  • Proteomics
  • Tyrosine phosphorylation

ASJC Scopus subject areas

  • General

Cite this

Comparisons of tyrosine phosphorylated proteins in cells expressing lung cancer-specific alleles of EGFR and KRAS. / Guha, Udayan; Chaerkady, Raghothama; Marimuthu, Arivusudar; Patterson, A. Scott; Kashyap, Manoj K.; Harsha, H. C.; Sato, Mitsuo; Bader, Joel S.; Lash, Alex E.; Minna, John D.; Pandey, Akhilesh; Varmus, Harold E.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 105, No. 37, 16.09.2008, p. 14112-14117.

Research output: Contribution to journalArticle

Guha, U, Chaerkady, R, Marimuthu, A, Patterson, AS, Kashyap, MK, Harsha, HC, Sato, M, Bader, JS, Lash, AE, Minna, JD, Pandey, A & Varmus, HE 2008, 'Comparisons of tyrosine phosphorylated proteins in cells expressing lung cancer-specific alleles of EGFR and KRAS', Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 37, pp. 14112-14117. https://doi.org/10.1073/pnas.0806158105
Guha, Udayan ; Chaerkady, Raghothama ; Marimuthu, Arivusudar ; Patterson, A. Scott ; Kashyap, Manoj K. ; Harsha, H. C. ; Sato, Mitsuo ; Bader, Joel S. ; Lash, Alex E. ; Minna, John D. ; Pandey, Akhilesh ; Varmus, Harold E. / Comparisons of tyrosine phosphorylated proteins in cells expressing lung cancer-specific alleles of EGFR and KRAS. In: Proceedings of the National Academy of Sciences of the United States of America. 2008 ; Vol. 105, No. 37. pp. 14112-14117.
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abstract = "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 (β-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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