Abstract
Epidermal growth factor (EGF)-stimulated Ras activation involves specific interactions between the EGF receptor (EGFR), the adaptor proteins Grb2 and Shc, and the nucleotide exchange factor Sos-1. Study and control of these protein-protein interactions in vivo can be greatly promoted by introducing intracellular reagents that mimic EGFR functions. Here, we showed that a synthetic phosphopeptide encompassing the autophosphorylation site 1068 of EGFR formed a complex with endogenous Grb2 after this peptide was delivered into intact cells by a cell-permeable peptide import technique. Consequently, this intracellular peptide inhibited EGF-induced EGFR/Grb2 associations but not EGFR/Shc or Shc/Grb2 associations. Peptide-mediated disruption of the EGF/Grb2/Sos-1 cascade led to reduced Ras activation and mitogen-activated protein kinase activation. These results indicate that the binding of Grb2 to the phosphorylated Tyr-1068 of EGFR is crucial to the EGF-induced Ras/mitogen-activated protein kinase signaling pathway. The application of cell-permeable peptides to this study demonstrates a useful biochemical tool to probe and control various intracellular processes involved in signal transduction and gene transcription.
Highlights
Mitogenic signaling stimulated by epidermal growth factor (EGF)1 requires the intrinsic tyrosine kinase activity of its transmembrane receptor
We showed in this report that this outside-in peptide formed an intracellular peptide-protein complex with endogenous Grb2, resulting in a substantial inhibition of EGF-stimulated EGF receptor (EGFR)/Grb2 association and Ras/MAP kinase activation
We first examined whether cell-permeable SP1068 peptide can be taken up efficiently by living NIH 3T3 cells overexpressing human EGF receptors (SAA cells [21])
Summary
Mitogenic signaling stimulated by epidermal growth factor (EGF) requires the intrinsic tyrosine kinase activity of its transmembrane receptor (for review see Refs. 1 and 2). Mitogenic signaling stimulated by epidermal growth factor (EGF) requires the intrinsic tyrosine kinase activity of its transmembrane receptor Several autophosphorylation sites have been identified in the carboxyl-terminal region of the EGF receptor. These sites and their flanking regions constitute specific motifs that can be recognized by the Src homology 2 (SH2) domains or the phosphotyrosine binding/phosphotyrosine interaction domains of many intracellular signaling proteins We showed in this report that this outside-in peptide formed an intracellular peptide-protein complex with endogenous Grb, resulting in a substantial inhibition of EGF-stimulated EGFR/Grb association and Ras/MAP kinase activation. Our results demonstrate that intracellular tyrosine kinase signaling pathways can be studied and regulated in living cells by cell-permeable peptides carrying specific functional motifs, such as phosphotyrosine-containing sequences
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