Phosphoproteomic analysis of Her2/neu signaling and inhibition

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 delta-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.

Fig. 1.

Effect of Her2 and PD168393 on protein tyrosine phosphorylation. (A) Western blot with anti-phosphotyrosine antibody on lysates from 3T3-Her2 or empty vector cells. (B) 3T3-Her2 cells treated for 1 h with PD168393, gefitinib, or DMSO vehicle were blotted as in A. (C) 3T3-Her2 cells treated with 0.5 μM PD168393 for the indicated times were blotted as in A.

Fig. 2.

Schematic for SILAC and representative MS spectra. (A) Schematic for SILAC. (B) The MS and MS/MS spectra of a tryptic peptide derived from Her2. Identified y- and b-ions are indicated. R** represents heavy Arg. (C) MS spectra showing the four major patterns of phosphorylation observed. Peptide sequences are VGQAQDILR (Dok1), VAGQSSPSGIQSR (Fyb), FFEILSPVYR (FAK), and HDGAFLIR (Grb2).

Fig. 3.

Quantification of phosphorylation by SILAC. (A) Summary of fold change with Her2 for all 462 proteins, with several individual proteins highlighted. Proteins with a ratio >1.5 (upper red line) are considered as increased in their tyrosine phosphorylation level, and those with ratios <0.66 (lower red line) are considered as decreased in their tyrosine phosphorylation level. (B) Effect of PD168393 on proteins that show increased phosphorylation, no change, or decreased phosphorylation in 3T3-Her2 cells. The number of proteins in each category is shown. Areas without numbers have fewer than five proteins.

Fig. 4.

Confirmation of protein phosphorylation by immunoprecipitation and Western blot. 3T3 transfectants (A) or BT-474 cells (B) were treated with PD168393 for 1 h and then lysed as described in ref. .

Fig. 5.

Identification of the Y877 phosphorylation site in the Her2 kinase domain. (A) MS/MS spectra of the peptide LLDIDETE(pY)HADGGKVPIK, which bears the phosphotyrosine residue at Y877 of Her2. The charge state of the parent ion is +3, thus yielding both +1 (upper line) and +2 (lower line) charged daughter ions on fragmentation. The mass difference corresponding to the phosphotyrosine residue, y₁₁ minus y₁₀ ion, is seen in both +1 and +2 charged fragment series. (B) clustalw 1.82 alignment of the kinase domains of human Her2, EGFR, Src, and insulin receptor. The phosphopeptide identified by MS in A is underlined. Black and gray arrows mark the conserved tyrosine residue and EGFR L858, respectively. The activation loop is indicated by horizontal arrows. (C) 3T3 transfectants or BT-474 cells were treated as in Fig. 4, and Western blotting was performed with phospho-Y877-specific Her2 antibody or anti-Her2 antibody.

Fig. 6.

Network modeling. (A) A focused view of a module of the EGFR/ErbB signaling pathway linking two proteins identified in this study, Pdcd6 and Pabpc1. Gene symbols used are from HPRD. Yellow and purple boxes represent known and previously unrecognized Her2 signaling proteins identified in this study, respectively. White boxes represent connecting proteins not identified by MS but known to be involved in EGFR signaling. (B) Bayesian network model showing a directed graph of influence on protein phosphorylation. Blue hexagons, tan ovals, and white ovals represent ErbB receptor tyrosine kinases, other protein kinases, and other proteins, respectively. Edges in green, yellow, and red are conserved in >400, >300, and >200 of the 500 highest scoring networks, respectively. Based on the underlying assumptions of Bayesian networks, loops are not permitted, and red edges represent alternate routes taken by the simulation on different runs.