title: Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling.
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.
authors.affiliation: Evotec (München) GmbH, Martinsried, Germany.
authors.forename: Sebastian K
authors.initials: SK
authors.lastname: Wandinger
authors.affiliation: VIB Center for the Biology of Disease, Leuven, Belgium.|KU Leuven, Center for Human Genetics, Leuven, Belgium.
authors.forename: Idoya
authors.initials: I
authors.lastname: Lahortiga
authors.affiliation: VIB Center for the Biology of Disease, Leuven, Belgium.|KU Leuven, Center for Human Genetics, Leuven, Belgium.
authors.forename: Kris
authors.initials: K
authors.lastname: Jacobs
authors.affiliation: Evotec (München) GmbH, Martinsried, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Klammer
authors.affiliation: Evotec (München) GmbH, Martinsried, Germany.
authors.forename: Nicole
authors.initials: N
authors.lastname: Jordan
authors.affiliation: Evotec (München) GmbH, Martinsried, Germany.
authors.forename: Sarah
authors.initials: S
authors.lastname: Elschenbroich
authors.affiliation: Oncology Drug Discovery, Janssen Research & Development, Janssen Pharmaceutica NV, Beerse, Belgium.
authors.forename: Marc
authors.initials: M
authors.lastname: Parade
authors.affiliation: Oncology Drug Discovery, Janssen Research & Development, Janssen Pharmaceutica NV, Beerse, Belgium.
authors.forename: Edgar
authors.initials: E
authors.lastname: Jacoby
authors.affiliation: Oncology Drug Discovery, Janssen Research & Development, Janssen Pharmaceutica NV, Beerse, Belgium.
authors.forename: Joannes T M
authors.initials: JT
authors.lastname: Linders
authors.affiliation: Oncology Drug Discovery, Janssen Research & Development, Janssen Pharmaceutica NV, Beerse, Belgium.
authors.forename: Dirk
authors.initials: D
authors.lastname: Brehmer
authors.affiliation: VIB Center for the Biology of Disease, Leuven, Belgium.|KU Leuven, Center for Human Genetics, Leuven, Belgium.
authors.forename: Jan
authors.initials: J
authors.lastname: Cools
authors.affiliation: Evotec (München) GmbH, Martinsried, Germany.
authors.forename: Henrik
authors.initials: H
authors.lastname: Daub
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002460:Cell Line; D005786:Gene Expression Regulation; D053263:Gene Regulatory Networks; D005818:Genetic Engineering; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D020890:Neuregulin-1 / Q000378:metabolism / Q000494:pharmacology; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D025941:Protein Interaction Mapping; D020543:Proteome / Q000235:genetics* / Q000378:metabolism; D020893:Receptor, ErbB-3 / Q000235:genetics* / Q000378:metabolism; D066247:Receptor, ErbB-4 / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D020890:Neuregulin-1; C490581:Nrg1 protein, mouse; D010750:Phosphoproteins; D020543:Proteome; C000607800:ErbB3 protein, mouse; C587690:Erbb4 protein, mouse; D020893:Receptor, ErbB-3; D066247:Receptor, ErbB-4