Controlling epidermal growth factor (EGF)-stimulated Ras activation in intact cells by a cell-permeable peptide mimicking phosphorylated EGF receptor.
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.
Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2363, USA. Liny@ctrvax.vanderbilt.edu
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D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002463:Cell Membrane Permeability; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D051380:GRB2 Adaptor Protein; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010748:Phosphopeptides / Q000737:chemistry / Q000494:pharmacology*; D011506:Proteins / Q000378:metabolism*; D015398:Signal Transduction; D014158:Transcription, Genetic; D018631:ras Proteins / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; C495949:Grb2 protein, mouse; D010446:Peptide Fragments; D010748:Phosphopeptides; D011506:Proteins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D018631:ras Proteins