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Adaptor protein Shc undergoes translocation and mediates up‐regulation of the tyrosine kinase c‐Src in EGF‐stimulated A431 cells

2000; Wiley; Volume: 5; Issue: 9 Linguagem: Inglês

10.1046/j.1365-2443.2000.00358.x

1365-2443

Kenichi Sato, Miwa Kimoto, Miki Kakumoto, Dai Horiuchi, Tetsushi Iwasaki, Alexander A. Tokmakov, Yasuo Fukami,

Glycosylation and Glycoproteins Research

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.