Actinomycin D as a novel SH2 domain ligand inhibits Shc/Grb2 interaction in B104-1-1 (neu*-transformed NIH3T3) and SAA (hEGFR-overexpressed NIH3T3) cells.

Actinomycins, a family of bicyclic chromopeptide lactones with strong antineoplastic activity, were screened as inhibitors of Shc/Grb2 interaction in in vitro assay systems. To investigate the effects of actinomycin D on Shc/Grb2 interaction in cell-based experiments, we used SAA (normal hEGFR-overexpressed NIH3T3) cells and B104-1-1 (neu*-transformed NIH3T3) cells, because a large number of the Shc/Grb2 complexes were detected. Associated protein complexes containing Shc were immunoprecipitated from actinomycin D-treated cell lysates with polyclonal anti-Shc antibody. Then the association with Grb2 was assessed by immunoblotting with monoclonal anti-Grb2 antibody. The result of the immunoblotting experiment revealed that actinomycin D inhibited Shc/Grb2 interaction in a dose-dependent manner in both B104-1-1 and EGF-stimulated SAA cells. The inhibition of Shc/Grb2 interaction by actinomycin D in B104-1-1 cells also reduced tyrosine phosphorylation of MAP kinase (Erk1/Erk2), one of the major components in the Ras-MAP kinase signaling pathway. These results suggest that actinomycin D could be a non-phosphorylated natural and cellular membrane-permeable SH2 domain antagonist.