Protein-protein interactions are implied in glucocorticoid receptor mutant 465*-mediated cell death.
Previously we have shown that ICR-27, a clone of glucocorticoid-resistant human leukemic T cells, showed rapid cell loss upon transient transfection with plasmids expressing certain fragments of the human glucocorticoid receptor lacking the ligand binding domain. An extreme example was the frameshift GR mutant 465*, mutated after amino acid 465. This generated a novel 21-amino acid "tail," beginning within the second zinc finger of the human glucocorticoid receptor DNA binding domain, a region required for ICR-27 cell death caused by hologlucocorticoid receptor plus hormone. The cell loss mediated by 465* was faster but quantitatively equivalent to that caused by hologlucocorticoid receptor plus hormone. We are therefore investigating the mechanism of action of 465*. We overexpressed 465* with or without a glutathione S-transferase tag fused to its N terminus and tested its ability to affect glucocorticoid response element (GRE)-driven reactions in vitro. Partially purified 465* showed little binding to a consensus GRE, caused virtually no stimulation of transcription from a GRE, and failed to inhibit GR-driven transcription. However, GST-465* "trapped" several proteins from ICR-27 cell extracts, including c-Jun; recombinant c-Jun also was bound in vitro. In co-transfection assays of CV-1 cells, 465* expression reduced phorbol ester (12-O-tetradecanoylphorbol-13-acetate) transcriptional activation from a promoter containing multiple AP-1 sites. Further studies proved the repressive activity of 465* was c-Jun-specific and not due to squelching artifacts. The data suggest that interaction of 465* with other proteins, such as c-Jun, might be responsible for its cell killing function.
Chen
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H
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555-0645, USA.
Srinivasan
G
G
Thompson
E B
EB
D000818:Animals; D017209:Apoptosis; D006801:Humans; D016296:Mutagenesis; D011485:Protein Binding; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000235:genetics* / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D016755:Proto-Oncogene Proteins c-jun; D011965:Receptors, Glucocorticoid; D011994:Recombinant Proteins; D018808:Transcription Factor AP-1