Intracellular location and kinetics of complex formation between simian virus 40 T antigen and cellular protein p53

Abstract

The intracellular location and kinetics at which the simian virus 40 T antigen and the cellular protein p53 associate with one another were determined for simian virus 40-transformed mouse (215) and rat (14B) cells. Cells were labeled under pulse-chase conditions and fractionated into nuclear and cytoplasmic components, and the proteins were immunoprecipitated with monoclonal antibodies (pAb 416, 101, and 122). We found that newly made T antigen and p53 migrated to the nucleus of these cells independently; that is, in uncomplexed form. Newly made p53 was transported to the nucleus more rapidly than T antigen in both cell lines and formed a complex with a mature form of T antigen recognizable by pAb 101. This association was very rapid in both cell lines (t 1/2, 5 to 15 min). In contrast, the time course of complex formation between newly made T antigen and the p53 in the nucleus varied with the ratio of T antigen to p53 of the cell line studied. In 215 cells, where the ratio was 3.6, the kinetics were quite slow (t 1/2, 30 min), whereas in 14B cells, where the ratio was 1.7, they were quite rapid (t 1/2, 5 min). We suggest that a competition between newly made and uncomplexed T antigen for the p53 in the nucleus is the major determinant of the rate of complex formation for newly made T antigen. Our studies indicate that this macromolecular interaction is extremely dynamic.