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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

Authors

  • Sato K
  • Kimoto M
  • Kakumoto M
  • Horiuchi D
  • Iwasaki T
  • Tokmakov AA
  • Fukami Y

Journal

Genes to cells : devoted to molecular & cellular mechanisms

Publication Data

September 7, 2000

Associated Grants

None

Mesh Terms

  • 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