The protein kinase C activator, phorbol ester, cooperates with the wild-type p53 species of Ras-transformed embryo fibroblasts growth arrest.
Clone 112 cells, a rat embryo fibroblast cell line cotransfected by an activated ras gene and a temperature-sensitive mutant p53 gene (p53val135) grow well at 37 degrees C but cease DNA synthesis and cell division when shifted to 32 degrees C (Michalovitz, D., Halevy, O., and Oren, M. (1990) Cell 62, 671-680). Characterization of the p53 protein in exponentially growing clone 112 cells at 37 degrees C revealed that both wild-type (reactive with the monoclonal antibody PAb 246) and mutant (reactive with PAb 240) p53 conformational forms are co-expressed. These results indicate that in clone 112 cells the growth suppressor activity of the wild-type p53 species is inactivated at 37 degrees C. We show that clone 112 cells grown at 37 degrees C elicits specific growth inhibition response to stimulation by the tumor promoter phorbol ester, phorbol 12-myristate 13-acetate (PMA). At 37 degrees C, PMA induced nuclear accumulation of the p53 protein, a behavior that is also observed in growth-arrested cells at 32 degrees C. Furthermore, when cells are growth arrested at 32 degrees C, PMA prevented the cells from re-entering the cell cycle when they are shifted back to 37 degrees C. All these observations suggest that PMA can cooperate with the wild-type p53 in cell growth arrest. At 37 and 32 degrees C, PMA stimulation of clone 112 cells resulted in specific enhancement of phosphorylation of the wild-type p53 species but not of the mutant form. We also demonstrate that the growth arrest of clone 112 cells at 37 degrees C is correlated with stimulation of the nuclear wild-type p53-DNA binding activities. The PMA-mediated increase in p53 DNA binding activity coincides with the loss of the PAb 421 epitope on the p53.DNA complex. PAb 421 non-reactivity with p53 has been shown by others to occur in growth-arrested cells and upon phosphorylation of p53 by protein kinase C. We also provide evidence that, in vitro, the protein kinase C mode of phosphorylation stimulates DNA binding activities of purified recombinant wild-type p53 and that in a mutant conformation p53 is not a substrate for protein kinase C. We propose that wild-type p53 and protein kinase C, the cellular receptor of phorbol ester, could participate in the negative feedback controls associated with the phosphoinositide-derived signals common to a number of mitogenic stimulations.
Département de Biologie Moléculaire et Structural, INSERM Unité 309, Commissariat à l'Energie Atomique, Grenoble, France.
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D000818:Animals; D001692:Biological Transport; D002455:Cell Division / Q000187:drug effects; D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000378:metabolism; D002999:Clone Cells; D004268:DNA-Binding Proteins / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology; D004789:Enzyme Activation; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D011905:Genes, ras; D006358:Hot Temperature; D009154:Mutation; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate