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.


CONCLUSIONS
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.
Sato
K
K
Laboratory of Molecular Biology, Biosignal Research Center, and; Department of Biology, Faculty of Science, Kobe University, Kobe 657-8501, Japan. kksato@kobe-u.ac.jp
Kimoto
M
M
Kakumoto
M
M
Horiuchi
D
D
Iwasaki
T
T
Tokmakov
A A
AA
Fukami
Y
Y
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D022162:Cytoplasmic Vesicles / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D020033:Protein Isoforms; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D015854:Up-Regulation
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D008164:Luminescent Proteins; D020033:Protein Isoforms; D011506:Proteins; D011993:Recombinant Fusion Proteins; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; C528034:Shc1 protein, mouse; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D049452:Green Fluorescent Proteins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D016392:Proto-Oncogene Proteins pp60(c-src); D020928:Mitogen-Activated Protein Kinases