Abstract
The four members of the epidermal growth factor receptor (EGFR/ERBB) family form homo- and heterodimers which mediate ligand-specific regulation of many key cellular processes in normal and cancer tissues. While signaling through the EGFR has been extensively studied on the molecular level, signal transduction through ERBB3/ERBB4 heterodimers is less well understood. Here, we generated isogenic mouse Ba/F3 cells that express full-length and functional membrane-integrated ERBB3 and ERBB4 or ERBB4 alone, to serve as a defined cellular model for biological and phosphoproteomics analysis of ERBB3/ERBB4 signaling. ERBB3 co-expression significantly enhanced Ba/F3 cell proliferation upon neuregulin-1 (NRG1) treatment. For comprehensive signaling studies we performed quantitative mass spectrometry (MS) experiments to compare the basal ERBB3/ERBB4 cell phosphoproteome to NRG1 treatment of ERBB3/ERBB4 and ERBB4 cells. We employed a workflow comprising differential isotope labeling with mTRAQ reagents followed by chromatographic peptide separation and final phosphopeptide enrichment prior to MS analysis. Overall, we identified 9686 phosphorylation sites which could be confidently localized to specific residues. Statistical analysis of three replicate experiments revealed 492 phosphorylation sites which were significantly changed in NRG1-treated ERBB3/ERBB4 cells. Bioinformatics data analysis recapitulated regulation of mitogen-activated protein kinase and Akt pathways, but also indicated signaling links to cytoskeletal functions and nuclear biology. Comparative assessment of NRG1-stimulated ERBB4 Ba/F3 cells revealed that ERBB3 did not trigger defined signaling pathways but more broadly enhanced phosphoproteome regulation in cells expressing both receptors. In conclusion, our data provide the first global picture of ERBB3/ERBB4 signaling and provide numerous potential starting points for further mechanistic studies.
Highlights
The HER family of receptor tyrosine kinases (RTKs), known as ERBB receptors or epidermal growth factor receptor (EGFR) family, consists of the four members EGFR/ERBB1, ERBB2, ERBB3 and ERBB4, referred to as HER1, HER2, HER3 and HER4 for the human orthologs
By using a quantitative mass spectrometry (MS) approach we investigated the phosphoproteome regulation mediated by NRG1 treatment in cells expressing both ERBB3 and ERBB4 or ERBB4 alone, to systematically analyze and compare signal transduction processes mediated through these receptor tyrosine kinases
While the Ba/F3 cell line model enables the defined reconstitution of signaling through human EGFR family members, some care must be taken when interpreting results due to receptor coupling to murine signal transduction proteins which may be expressed at different levels than their counterparts in human cancer cell lines
Summary
The HER family of receptor tyrosine kinases (RTKs), known as ERBB receptors or epidermal growth factor receptor (EGFR) family, consists of the four members EGFR/ERBB1, ERBB2, ERBB3 and ERBB4, referred to as HER1, HER2, HER3 and HER4 for the human orthologs. The ERBB receptors are activated by multiple ligands including EGF, transforming growth factor alpha and neuregulins, leading to heterodimerization or homodimerization of the receptors [3]. EGFR mutations and ERBB2 overexpression are well known mechanisms that lead to constitutive activation of ERBB signaling pathways in lung and breast carcinoma [1, 8]. In breast cancer cell lines that overexpress ERBB2, increased levels of ERBB3 drive continued oncogenic signaling and, resistance to the ERBB2 inhibitory activity of the kinase inhibitors gefitinib and erlotinib [10]. Acquired resistance to the monoclonal antibody cetuximab, which targets the EGFR, might partially result from ERBB3-dependent signaling and activation of the phosphatidylinositol 3-kinase (PI3K)–Akt pathway [11]. The activation of an early feedback survival loop involving ERBB3 has been recently reported to occur in melanoma cells after treatment with RAF/MEK inhibitors [12]
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