Epidermal growth factor and other mitogens induce binding of a protein complex to the c-fos serum response element in human astrocytoma and other cells.
Rapid stimulation of c-fos transcription by many agonists requires the serum response element (SRE), which binds at least two distinct nuclear proteins, p67SRF and p62TCF. Using nuclear protein extracts from 1321-N1 human astrocytoma cells, we investigated ligand-induced changes in binding of these proteins to SRE probes. In these cells c-fos mRNA expression can be induced by epidermal growth factor (EGF) through protein kinase C-independent pathways and by phorbol esters through protein kinase C. We detected two DNA-protein complexes that formed specifically with the SRE (bands 1 and 2). Band 2 formation was increased 4-6 min after stimulation with EGF as well as serum and phorbol esters; this peaked at 10-30 min and returned to basal levels by 60 min. Induction of band 2 formation preceded the onset and peak accumulation of c-fos mRNA (15 and 30 min after EGF stimulation, respectively) and its return to basal levels (by 1-2 h). Band 2 formation was also increased A431 cells stimulated with EGF and in HeLa and Swiss-3T3 cells stimulated with serum. We found that band 1 contained p67SRF bound to the SRE; band 2 contained p67SRF and a second protein. Gel shift analyses using [35S]methionine-labeled p67SRF and nonradioactive DNA probes suggested that hormone treatment most likely modified the second protein component of band 2. Transient transfection of 1321-N1 cells with plasmids containing point mutations that prevented band 2 formation in vitro also abolished induction of c-fos transcription in vivo as assayed by RNase protection analysis. Thus, hormone-stimulated formation of the protein-DNA complex represented by band 2 may be involved in the activation of c-fos transcription.
Malik
R K
RK
Department of Pediatrics, Duke University Medical Center, Durham, North Carolina 27710.
Roe
M W
MW
Blackshear
P J
PJ
D001254:Astrocytoma; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008934:Mitogens / Q000494:pharmacology*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016760:Proto-Oncogene Proteins c-fos; D011519:Proto-Oncogenes; D026362:Serum Response Factor; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D008934:Mitogens; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D016760:Proto-Oncogene Proteins c-fos; D026362:Serum Response Factor; D004815:Epidermal Growth Factor; D004247:DNA; D011493:Protein Kinase C