Phosphoproteomic analysis of Her2/neu signaling and inhibition

Ron Bose, Henrik Molina, A. Scott Patterson, John K. Bitok, Balamurugan Periaswamy, Joel S. Baderh, Akhilesh Pandey, Philip A. Cole

Research output: Contribution to journalArticle

174 Scopus citations

Abstract

Her2/neu (Her2) is a tyrosine kinase belonging to the EGF receptor (EGFR)/ErbB family and is overexpressed in 20-30% of human breast cancers. We sought to characterize Her2 signal transduction pathways further by using MS-based quantitative proteomics. Stably transfected cell lines overexpressing Her2 or empty vector were generated, and the effect of an EGFR and Her2 selective tyrosine kinase inhibitor, PD168393, on these cells was characterized. Quantitative measurements were obtained on 462 proteins by using the SILAC (stable isotope labeling with amino acids in cell culture) method to monitor three conditions simultaneously. Of these proteins, 198 showed a significant increase in tyrosine phosphorylation in Her2-overexpressing cells, and 81 showed a significant decrease in phosphorylation. Treatment of Her2-overexpressing cells with PD168393 showed rapid reversibility of the majority of the Her2-triggered phosphorylation events. Phosphoproteins that were identified included many known Her2 signaling molecules as well as known EGFR signaling proteins that had not been previously linked to Her2, such as Stat1, Dok1, and δ-catenin. Importantly, several previously uncharacterized Her2 signaling proteins were identified, including Axl tyrosine kinase, the adaptor protein Fyb, and the calcium-binding protein Pdcd-6/Alg-2. We also identified a phosphorylation site in Her2, Y877, which is located in the activation loop of the kinase domain, is distinct from the known C-terminal tail autophosphorylation sites, and may have important implications for regulation of Her2 signaling. Network modeling, which combined phosphoproteomic results with literature-curated protein-protein interaction data, was used to suggest roles for some of the previously unidentified Her2 signaling proteins.

Original languageEnglish
Pages (from-to)9773-9778
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume103
Issue number26
DOIs
StatePublished - Jun 27 2006
Externally publishedYes

Keywords

  • Breast cancer
  • Cellular networks
  • Proteomics signal transduction
  • Tyrosine kinase inhibitors

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    Bose, R., Molina, H., Scott Patterson, A., Bitok, J. K., Periaswamy, B., Baderh, J. S., Pandey, A., & Cole, P. A. (2006). Phosphoproteomic analysis of Her2/neu signaling and inhibition. Proceedings of the National Academy of Sciences of the United States of America, 103(26), 9773-9778. https://doi.org/10.1073/pnas.0603948103