Phosphoproteomic analysis of Her2/neu signaling and inhibition

doi:10.1073/pnas.0603948103

Phosphoproteomic analysis of Her2/neu signaling and inhibition

Departments of *Pharmacology,
^†Oncology, and
**Biomedical Engineering,
^‡McKusick–Nathans Institute for Genetic Medicine,
^§Institute in Multiscale Modeling of Biological Interactions, and
^‖High-Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205; and
^¶Institute of Bioinformatics, International Tech Park, Bangalore 560066, India

Communicated by Paul Talalay, Johns Hopkins University School of Medicine, Baltimore, MD, May 12, 2006 (received for review March 3, 2006)

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.

Footnotes

^††To whom correspondence may be addressed. E-mail: pandey{at}jhmi.edu or pcole{at}jhmi.edu
Author contributions: R.B., J.S.B., A.P., and P.A.C. designed research; R.B., H.M., A.S.P., and J.K.B. performed research; R.B., H.M., and A.P. contributed new reagents/analytic tools; R.B., H.M., A.S.P., B.P., and J.S.B. analyzed data; and R.B., A.S.P., and P.A.C. wrote the paper.
Conflict of interest statement: No conflicts declared.
Abbreviations:

Abbreviations:

SILAC,

stable isotope labeling with amino acids in cell culture;

TKI,

tyrosine kinase inhibitor;

PPI,

protein–protein interaction;

HPRD,

Human Protein Reference Database;

EGFR,

EGF receptor;

PLCγ1,

phospholipase C γ1;

PI3K,

phosphatidylinositol 3-kinase;

FAK,

focal adhesion kinase.