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Submitted on September 2, 2004
OSI Pharmaceuticals Inc., Farmingdale, NY 11735
Corresponding Author: jhaley{at}osip.com
Over-expression and enhanced activation of the epidermal growth factor (EGF) receptor are frequent events in human cancers, which correlate with poor prognosis. Anti-phosphotyrosine and anti-EGFr affinity chromatography, isotope-coded uLC-MSMS mass spectrometry 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, 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, PLCgamma, Erk1/2, PI-3 kinase and STAT3/5. Focal adhesion proteins, FAK, Pyk2, paxillin, ARF/GIT1, plakophillin were downregulated 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.
Revised on January 15, 2005
Accepted on January 17, 2005
Phosphotyrosine signaling networks in epidermal growth factor receptor overexpressing squamous carcinoma cells
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