Concerted regulation of wild-type p53 nuclear accumulation and activation by S100B and calcium-dependent protein kinase C

The calcium ionophore ionomycin cooperates with the S100B protein to rescue a p53-dependent G(1) checkpoint control in S100B-expressing mouse embryo f ibroblasts and rat embryo fibroblasts (REF cells) which express the tempera ture-sensitive p53Val135 mutant (C. Scotto, J, C, Deloulme, D. Rousseau, E, Chambaz, and J, Baudier, Mol, Cell. Biol, 18:4272-4281, 1998). We investig ated in this study the contributions of S100B and calcium-dependent PKC (cP KC) signalling pathways to the activation of wild-type p53. We first confir med that S100B expression in mouse embryo fibroblasts enhanced specific nuc lear accumulation of wild-type p53. We next demonstrated that wild-type p53 nuclear translocation and accumulation is dependent on cPKC activity. Muta tion of the five putative cPKC phosphorylation sites on murine p53 into ala nine or aspartic residues had no significant effect on p53 nuclear localiza tion, suggesting that the cPKC effect on p53 nuclear translocation is indir ect. A concerted regulation by S100B and cPKC of wild-type p53 nuclear tran slocation and activation was confirmed with REF cells expressing S100B (S10 0B-REF cells) overexpressing the temperature-sensitive p53Val135 mutant. St imulation of S100B-REF cells with the PKC activator phorbol ester phorbol m yristate acetate (PMA) promoted specific nuclear translocation of the wild- type p53Val135 species in cells positioned in early G(1) phase of the cell cycle. PMA also substituted for ionomycin in the mediating of p53-dependent G(1) arrest at the nonpermissive temperature (37.5 degrees C). PMA-depende nt growth arrest was linked to the cell apoptosis response to UV irradiatio n. In contrast, growth arrest mediated by a temperature shift to 32 degrees C protected S100B-REF cells from apoptosis. Our results suggest a model in which calcium signalling, linked with cPKC activation, cooperates with S10 0B to promote wild-type p53 nuclear translocation in early G(1) phase and a ctivation of a p53-dependent G(1) checkpoint control.