Regulation of the transcription factor AP-1 in benign and malignant mouse keratinocyte cells.
The mouse benign keratinocyte cell line 308 was previously shown to have less AP-1 DNA binding and transactivation ability than its malignant variant 10Gy5. Because elevated AP-1 activity in 10Gy5 appears to be critical for its malignant phenotype, we were interested in examining the molecular mechanisms that regulate activator protein 1 (AP-1) in this system. In both 308 and 10Gy5 cells, c-fos, fra-2, c-jun, jun B, and jun D were capable of binding to an AP-1 DNA binding site as determined by antibody clearance gel mobility shift assays. By western analysis, jun B steady-state nuclear and cytoplasmic protein levels were reduced in 10Gy5 cells as compared with 308 cells and jun B steady-state mRNA levels were similar in the two cell lines. The rate of jun B protein synthesis was decreased in 10Gy5 cells in comparison with 308 cells. Gel mobility shift experiments indicated that AP-1 inhibitory proteins were not present in the cytoplasm of 308 cells. Oxidation-reduction posttranslational modification was not a major mechanism of AP-1 regulation in these cells as shown by 12-O-tetradecanoylphorbol-13-acetate-responsive element (TRE) gel mobility shift assay of nuclear protein treated with a reducing agent and by western analysis for ref-1 protein. Overall phosphorylation of AP-1 proteins in 308 and 10Gy5 cells was examined by 32P orthophosphate labeling and immunoprecipitation. A difference in jun B protein overall phosphorylation was observed in the two cell lines. Our experiments suggest that decreased jun B protein levels may be a mechanism that results in elevated AP-1 activity in malignant 10Gy5 cells.
Joseloff
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Department of Radiation Oncology, University of Arizona Health Sciences Center, Tucson 85724, USA.
Bowden
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GT
D000818:Animals; D000906:Antibodies / Q000378:metabolism; D015152:Blotting, Northern; D015153:Blotting, Western; D002471:Cell Transformation, Neoplastic / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015603:Keratinocytes / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D010084:Oxidation-Reduction; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011233:Precipitin Tests; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000906:Antibodies; D010761:Phosphorus Radioisotopes; D016755:Proto-Oncogene Proteins c-jun; D018808:Transcription Factor AP-1; D004247:DNA; D008715:Methionine