Phosphotyrosine Signaling Networks in Epidermal Growth Factor Receptor Overexpressing Squamous Carcinoma Cells

• Published in: Molecular & cellular proteomics Vol. 4; no. 4; pp. 356 - 376
• Main Authors: Thelemann, April, Petti, Filippo, Griffin, Graeme, Iwata, Ken, Hunt, Tony, Settinari, Tina, Fenyo, David, Gibson, Neil, Haley, John D
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 01-04-2005
• Subjects: Carcinoma, Squamous Cell - metabolism; Carcinoma, Squamous Cell - pathology; Cell Adhesion; Cell Line, Tumor; Chromatography, Affinity; Chromatography, Liquid; Enzyme Activation; ErbB Receptors - antagonists & inhibitors; ErbB Receptors - drug effects; ErbB Receptors - metabolism; Erlotinib Hydrochloride; Head and Neck Neoplasms - metabolism; Head and Neck Neoplasms - pathology; Humans; Immunoblotting; Isotopes; Mass Spectrometry; Models, Biological; Peptide Mapping; Phosphorylation - drug effects; Phosphotyrosine - metabolism; Proteins - analysis; Quinazolines - pharmacology; Signal Transduction - drug effects; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; src-Family Kinases - metabolism
• ISSN: 1535-9476; 1535-9484; 1535-9484
• DOI: 10.1074/mcp.M400118-MCP200

Overexpression and enhanced activation of the epidermal growth factor (EGF) receptor are frequent events in human cancers
that correlate with poor prognosis. Anti-phosphotyrosine and anti-EGFr affinity chromatography, isotope-coded μLC-MS/MS, and
immunoblot methods were combined to describe and measure signaling networks associated with EGF receptor activation and pharmacological
inhibition. The squamous carcinoma cell line HN5, which overexpresses EGF receptor and displays sustained receptor kinase
activation, was used as a model system, where pharmacological inhibition of EGF receptor kinase by erlotinib markedly reduced
auto and substrate phosphorylation, Src family phosphorylation at EGFR Y845, while increasing total EGF receptor protein.
Diverse sets of known and poorly described functional protein classes were unequivocally identified by affinity selection,
comprising either proteins tyrosine phosphorylated or complexed therewith, predominantly through EGF receptor and Src family
kinases, principally 1) immediate EGF receptor signaling complexes (18%); 2) complexes involved in adhesion and cell-cell
contacts (34%); and 3) receptor internalization and degradation signals. Novel and known phosphorylation sites could be located
despite the complexity of the peptide mixtures. In addition to interactions with multiple signaling adaptors Grb2, SHC, SCK,
and NSP2, EGF receptors in HN5 cells were shown to form direct or indirect physical interactions with additional kinases including
ACK1, focal adhesion kinase (FAK), Pyk2, Yes, EphA2, and EphB4. Pharmacological inhibition of EGF receptor kinase activity
by erlotinib resulted in reduced phosphorylation of downstream signaling, for example through Cbl/Cbl-B, phospholipase Cγ
(PLCγ), Erk1/2, PI-3 kinase, and STAT3/5. Focal adhesion proteins, FAK, Pyk2, paxillin, ARF/GIT1, and plakophillin were down-regulated
by transient EGF stimulation suggesting a complex balance between growth factor induced kinase and phosphatase activities
in the control of cell adhesion complexes. The functional interactions between IGF-1 receptor, lysophosphatidic acid (LPA)
signaling, and EGF receptor were observed, both direct and/or indirectly on phospho-Akt, phospho-Erk1/2, and phospho-ribosomal
S6.