Conformational phenotype of p53 is linked to nuclear translocation

Abstract

P53 is inactivated in tumors by mechanisms other than mutations in the p53 gene itself. To gain insight into the mechanisms by which this inactivation occurs, we chemically mutagenized A1-5 cells expressing high levels of temperature sensitive p53 val135 (tsp53) and selected for clones that were capable of growth at the permissive temperature for p53 activation. We expanded 22 clones (ALTR cells for A1-5 Low Temperature Resistant) that could grow at the permissive temperature. Most exhibited cytoplasmic sequestration as the mechanism by which p53 was inactivated. We show here that this cytoplasmically sequestered tsp53 protein is maintained in a mutant conformation. Only in clones with nuclear localized p53 is it also expressed in the wild-type conformation suggesting that subcellular localization of tsp53 is important in determining the conformation of the protein. Consistent with this, we show that the changes in conformation of p53 in A1-5 and SK-N-SH cells induced by ionizing radiation also correlate with nuclear translocation of p53. We suggest that nuclear translocation of p53 can result in a change in the conformation from mutant to wild-type but that these may be two separable events. Oncogene (2000) 19, 4042 ± 4049.

Genetic analysis of p53 nuclear importation

“…Previously, we showed that ALTR cells with cytoplasmically sequestered tsp53 at 321C also exhibited a predominantly mutant conformation of tsp53 and only in cells with nuclear-localized p53 was the tsp53 present in a wildtype conformation, suggesting that there is a correlation between the localization of p53 protein and its conformation (Gaitonde et al, 2000). This correlation suggests that there is a link between protein conformation and nuclear localization.…”

“…The immunoprecipitation and indirect immunofluorescence were performed as described previously (Gaitonde et al, 2000).…”

DNA binding and selective gene induction by different forms of the p53 protein

“…Next cells were lysed and processed according to Gaitonde et al (2000). The antibodies used included: goat anti-PC4 polycolonal antibody (N-17) (Santa Cruz, Santa Cruz, CA, USA) and conformation specific antip53 antibodies: PAb 421, PAb 242, PAb 246 and PAb 240.…”

“…After quantitation using scintillation counter an equal amount of protein from each sample was subjected to immunoprecipitation by protein A-sepharose beads conjugated with the conformation specific anti-p53 antibodies: PAb 421, PAb 242, PAb 246 or PAb 240 (Courtesy of Dr David Lane). The beads were processed as described by Gaitonde et al (2000). Samples were electrophoresed on a 10% denaturing polyacrylamide gel.…”

“…After incubating for 20 h at appropriate temperatures the cells were processed and analysed according to the protocol by Gaitonde et al (2000). ChIP assays ChIPs using the p53 antibodies was performed according Watts et al (2004).…”