Abstract

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 muLC-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 Cgamma (PLCgamma), 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.

Highlights

  • Overexpression and enhanced activation of the epidermal growth factor (EGF) receptor are frequent events in human cancers that correlate with poor prognosis

  • Constitutive EGF receptor signaling has been associated with altered signaling resulting in cell transformation and HN5 cells, expressing ϳ5 ϫ 106 receptors per cell [20], show high-constitutive EGF receptor kinase activity due to autocrine TGF-␣ expression

  • Due to the involvement of Grb2 in EGF receptor signaling, we examined the cross-identification of proteins between the two studies

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Summary

Introduction

Overexpression and enhanced activation of the epidermal growth factor (EGF) receptor are frequent events in human cancers that correlate with poor prognosis. The abbreviations used are: EGF, epidermal growth factor; HNSCC, head and neck squamous carcinoma; EGFR, epidermal growth factor receptor; IGF-1, insulin-like growth factor-1; FAK, focal adhesion kinase; HB-EGF, heparin-binding epidermal growth factor; SH2, Src homology 2 domain; PTB, phosphotyrosine binding domain; LPA, lysophosphatidic acid; TGF␣, transforming growth factor ␣; IC50, half maximal inhibitory concentration; pY, phosphotyrosine; SCX, strong cation exchange; PLC␥, phospholipase C␥; PI3-kinase, phosphatidyl inositol-3 kinase; GO, Gene Ontology database; OSI774, erlotinib (Tarceva). The translocation and internalization of the receptor into early endosomes place the receptor in an important subcellular localization for the transduction of signals through the Ras-Raf-Mek-Erk pathway important in the mitogenic effects of EGF [8] Both EGF receptor protein interactions as well as the cellular location of receptor complexes determine the downstream signals produced. The EGF receptor and proteins controlling cell adhesion assembly and disassembly have been shown to physically interact and cross-regulate in a complex manner dependent on receptor activity and cell adhesion factors [12, 13]

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