Regulation of the transcription factor AP-1 in benign and malignant mouse keratinocyte cells

Mol Carcinog. 1997 Jan;18(1):26-36. doi: 10.1002/(sici)1098-2744(199701)18:1<26::aid-mc4>;2-p.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Antibodies / metabolism
  • Blotting, Northern
  • Blotting, Western
  • Cell Transformation, Neoplastic / metabolism*
  • DNA / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Keratinocytes / metabolism*
  • Methionine / metabolism
  • Mice
  • Mice, Inbred BALB C
  • Oxidation-Reduction
  • Phosphorus Radioisotopes
  • Phosphorylation
  • Precipitin Tests
  • Proto-Oncogene Proteins c-jun / metabolism
  • Transcription Factor AP-1 / metabolism*
  • Tumor Cells, Cultured


  • Antibodies
  • Phosphorus Radioisotopes
  • Proto-Oncogene Proteins c-jun
  • Transcription Factor AP-1
  • DNA
  • Methionine