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.
Kim
H K
HK
Korea Research Institute of Bioscience and Biotechnology, KIST, Taejon, South Korea.
Nam
J Y
JY
Han
M Y
MY
Lee
E K
EK
Choi
J D
JD
Bok
S H
SH
Kwon
B M
BM
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D003609:Dactinomycin / Q000494:pharmacology*; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D051380:GRB2 Adaptor Protein; D008024:Ligands; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D008958:Models, Molecular; D008968:Molecular Conformation; D011485:Protein Binding / Q000187:drug effects; D011506:Proteins / Q000378:metabolism*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction / Q000187:drug effects; D014170:Transformation, Genetic; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000900:Anti-Bacterial Agents; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D003609:Dactinomycin; D066246:ErbB Receptors; D051380:GRB2 Adaptor Protein; D008024:Ligands; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D011506:Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; D011994:Recombinant Proteins; D055765:Shc Signaling Adaptor Proteins; C064740:epidermal growth factor receptor-neu receptor