Phosphoproteomic analysis of Her2/neu signaling and inhibition

Abstract

Her2͞neu (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 ␦-catenin. Importantly, several previously uncharacterized Her2 signaling proteins were identified, including Axl tyrosine kinase, the adaptor protein Fyb, and the calcium-binding protein Pdcd-6͞Alg-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.breast cancer ͉ cellular networks ͉ proteomics ͉ signal transduction ͉ tyrosine kinase inhibitors

Axl is an essential epithelial-to-mesenchymal transition-induced regulator of breast cancer metastasis and patient survival

“…RTKs are a well-established cancer therapeutic class, and small molecule inhibitors of Axl tyrosine kinase activity have been reported (27,28). Interestingly, Her2 is reported to trigger Axl activation, suggesting crosstalk with this clinically important pathway (29). The strong independent correlation of Axl expression with reduced overall survival and persistent Axl expression in subsequent metastasis suggests that the 40% of patients with Axl-expressing primary tumors could benefit from Axl-targeting therapy.…”

Time Series Proteome Profiling To Study Endoplasmic Reticulum Stress Response

“…Additionally, SILAC can allow accurate temporal proteome profiling 15 and monitoring of protein phosphorylation to study signaling pathways. [16][17][18][19][20] In this study, we implemented the SILAC strategy in combination with SDS-PAGE and LC-MS/MS to monitor temporal changes in the expression and dynamics of proteins within the endoplasmic reticulum (ER) compartment of human primary fibroblast cells exposed to ER stress inducers tunicamycin (Tun) and thapsigargin (Thp). To demonstrate the feasibility of temporal profiling using this method, we have chosen a model system using Tun and Thp, as these ER stress agents have been documented to induce temporal changes in a number of ER resident proteins within minutes to hours.…”

Oncogenic Tyrosine Kinases Target Dok-1 for Ubiquitin-Mediated Proteasomal Degradation To Promote Cell Transformation

“…Dok-1, also known as p62 dok , is the prototypical family member. It was first identified as a substrate of OTKs p210 bcr-abl and v-Abl (13, 93) and found to be a substrate of many endogenous PTKs (9,18,19,23,34,51,53,59,60,64,78,91,92,97); hence, it was termed Dok, for downstream of tyrosine kinases. Since the identification of Dok-1, six additional family members, Dok-2 to Dok-7, have been identified in human and mice, all of which are substrates of various receptor or cytoplasmic PTKs, with some of them having partially overlapping functions while others having distinct functions (15,16,20,30,40,49,61,66).…”