Mutational status of the p53 gene modulates the basal level of jun N-terminal kinase and its inducibility by ultraviolet irradiation in A1-5 rat fibroblasts

Exposure of mammalian cells to ultraviolet (UV) light and other DNA-damagin g agents triggers the UV response which is characterized by induction of a large number of genes including c-fos, c-jun, and the genes For DNA repair enzymes and cell-cycle regulatory proteins such as p21(WAF1) and p53. Upon DNA damage, the p53 tumor suppressor protein transmits signals to restrict cell-cycle progression, thereby allowing time For DNA repair to occur. Cell s also respond to genotoxic stress by activation of the jun N-terminal kina se (JNK)/stress-activated protein kinase pathway. In this report we investi gated the effects of modulation of the level of wild-type and mutant p53 pr otein on basal and UV-inducible JNK activity We used the A1-5 rat fibroblas t cell line, which contains a p53 gene coding for a temperature-sensitive p 53 protein, which allows vs to regulate the relative level of wild-type and mutant p53 protein produced in a cell. We measured the relative levels of JNK activity in sham-irradiated and UV-irradiated cells by using the immune complex kinase assay and then computed the fold induction of JNK after UV exposure. We demonstrated that cells expressing p53 protein in the wild-typ e conformation (when grown at 32 degrees C) exhibited a very low level of J NK activity that was induced 14- to 16-fold by UVC irradiation. When cells were grown at 37 degrees C or 39 degrees C to express predominantly mutant p53 protein, basal JNK activity was significantly higher than at 32 degrees C. UVC irradiation of cells expressing mutant p53 protein resulted in JNK activation, although the overall fold-induction was only two-fold because J NK1 activity was already high in the sham-treated controls. UVB irradiation also induced JNK1 activity, although we again observed a relatively high l evel of basal JNK activity in sham irradiated cells expressing mutant p53 p rotein compared with cells expressing wild-type p53. Control experiments co nfirmed that JNK1 basal activity was not affected by temperature alone. Wes tern blot analysis of cell extracts indicated that expression of p21(WAF) p rotein was significantly higher in cells expressing wild-type p53 protein a nd was associated with low basal levels of JNK1 activity. In contrast, cell s expressing mutant p53 protein and very low levels of p21(WAF1) protein we re found to have a higher level of basal JNK1 activity. We also observed a reduced ability to induce JNK1 after UV irradiation of several other cell l ines with p53-mutant or p53-null genotypes. Our results provide evidence fo r a novel connection between p53 status and the basal level of JNK1, a crit ical enzyme in the stress-activated protein kinase family. In addition, the se studies suggest that the presence of mutant p53 protein in a cell not on ly affects basal activity of JNK1 but also affects the ability of a cell to respond to UV-induced stress by transmitting signals via induction or acti vation of the JNK1 cascade. (C) 1999 Wiley-Liss, Inc.