Abstract
Exposure of mammalian cells to genotoxic stress results in activation of the c‐jun amino‐terminal kinase (JNK)‐stress‐activated protein kinase (SAPK) pathway and induction of DNA repair enzymes and cell cycle–regulatory proteins such as p53 and p21waf1. The p53 tumor suppressor protein transmits signals that activate p21waf1 gene expression. The p21waf1 protein then restricts cell‐cycle progression, thereby allowing time for DNA repair to occur. In this study, we investigated the effects of modulation of the level of wild‐type and mutant p53 protein on basal JNK1 activity in the A1‐5 rat fibroblast cell line. This cell line contains a p53 gene coding for a temperature‐sensitive p53 protein, which allows us to regulate the relative level of wild‐type and mutant p53 protein produced in cells. Using the immune complex kinase assay to measure JNK1 activity, we demonstrated that cells expressing the wild‐type–conformation p53 protein (when grown at 32.5°C) exhibited a very low level of JNK1 activity. When cells were grown at 37°C or 39°C to express predominantly mutant p53 protein, basal level of JNK1 activity was significantly higher than at 32.5°C. We also demonstrated protein‐protein interactions between the p53, p21waf1, and JNK1 proteins in this cell line. Both wild‐type p53 protein (expressed at 32.5°C) and mutant p53^val135^ protein (expressed at 37°C and 39°C) were present in immunocomplexes of JNK1 protein. Under conditions where wild‐type p53 protein was present to induce p21waf1 expression (at 32.5°C), a higher level of p21waf1 protein was also detected in the JNK1 immunocomplexes than in those at 37°C and 39°C. We next investigated the effect that co‐association of p53 protein and p21waf1 protein would have on JNK1 activity. We measured basal levels of JNK1 activity in cells expressing wild‐type p53 and p21waf1, or in __p21waf1‐__null cells, and demonstrated that cells expressing both p53 and p21waf1 proteins exhibited an approximately threefold lower basal level of JNK1 activity when compared with p21waf1‐null cells. To confirm that p21waf1 protein expression in cells resulted in reduced JNK1 activity, we transfected p21waf1−/− cells with a p21waf1 expression vector. We observed that JNK1 activity was inhibited after exogenous p21waf1 protein was expressed in these cells. Our results provide evidence for modulation of the JNK1 pathway by p53 and p21waf1 proteins and support the hypothesis that modulation of JNK1 activity occurred through protein‐protein interactions between JNK1, p53, and p21waf1 proteins. © 2002 Wiley‐Liss, Inc.