Adaptor protein Shc undergoes translocation and mediates up-regulation of the tyrosine kinase c-Src in EGF-stimulated A431 cells.
BACKGROUND: Shc is the adaptor protein that exists in three isoforms, P46, P52 and P66, and acts as a bridge between activated cell surface receptors and downstream signalling molecules which act in extracellular signal-regulated cell events such as cell cycle progression. In our previous studies, Shc was shown to be a substrate of the tyrosine kinase c-Src in vitro and in vivo. RESULTS: Using green fluorescent protein-fusion Shc (GFP-Shc), we have shown that following epidermal growth factor (EGF) stimulation of A431 cells, all Shc isoforms were rapidly recruited from the cytoplasm to the plasma membrane (within 5 min) and then redistributed to the cytoplasmic vesicle structures (in the next 10-20 min). Indirect immunofluorescent study demonstrated that all Shc isoforms co-localize with EGF receptor (EGFR) and activated c-Src in both plasma membranes and cytoplasmic vesicle structures. Our previous study has shown that EGF induces the indirect association of EGFR and c-Src and activation of c-Src in A431 cells. An immunoprecipitation study demonstrated that the EGFR-Src association and c-Src activation are augmented in cells expressing GFP-Shc P52 or P66, but not P46. In addition, P52 and P66, but not P46, are in association with EGFR-Src complex. We also found that EGFR and Shc can be dissociated from c-Src by the addition of a synthetic peptide that corresponds to the autophosphorylation site of c-Src. Interestingly, the peptide-induced dissociation of the complex was not affected by the tyrosine phosphorylation state of the peptide. CONCLUSION: These results demonstrated a dynamic subcellular movement of Shc in response to EGF, and suggested a hitherto unknown scheme whereby Shc can work not only as a substrate of c-Src but also as a mediator of the EGF-induced activation of c-Src in an isoform-specific manner.
Pubmed ID: 10971656 RIS Download
Adaptor Proteins, Signal Transducing | Adaptor Proteins, Vesicular Transport | Amino Acid Sequence | Animals | Cell Line | Cytoplasmic Vesicles | Epidermal Growth Factor | Green Fluorescent Proteins | Humans | Immunoblotting | Luminescent Proteins | Mice | Microscopy, Confocal | Mitogen-Activated Protein Kinases | Molecular Sequence Data | Protein Isoforms | Proteins | Proto-Oncogene Proteins pp60(c-src) | Receptor, Epidermal Growth Factor | Recombinant Fusion Proteins | Shc Signaling Adaptor Proteins | Up-Regulation