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 antin eoplastic activity were screened as inhibitors of Shc/Grb2 interaction in i n vitro assay systems. To investigate the effects of actinomycin D on Shc/G rb2 interaction in cell-based experiments, we used SAA (normal hEGFR-overex pressed NIH3T3) cells and B104-1-1 (neu*-transformed NIH3T3) cells, because a large number of the Shc/Grb2 complexes were detected. Associated protein completes containing Shc were immunoprecipitated from actinomycin D-treate d cell lysates with polyclonal anti-She antibody. Then the association with Grb2 was assessed by immunoblotting with monoclonal anti-Grb2 antibody. Th e result of the immunoblotting experiment revealed that actinomycin D inhib ited Shc/Grb2 interaction in a dose-dependent manner in both B104-1-1 and E GF-stimulated SAA cells. The inhibition of Shc/Grb2 interaction by actinomy cin 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 p athway. These results suggest that actinomycin D could be a non-phosphoryla ted natural and cellular membrane-permeable SH2 domain antagonist. (C) 1999 Federation of European Biochemical Societies.