We have observed the tumor suppressor protein p53 to be present in cytoplasm and nucleus in a family of at least eleven isoforms that differ in charge but not molecular weight suggesting these isoforms are the product of post-translational modification. We have also shown by two dimensional gel electrophoretic analysis that the isoforms are increasingly acidic, presumably by addition of phosphorus, suggesting an array of precursor molecules at various stages of completion for rapid utilization when signaled for transduction to the nucleus. In addition, we have found at least 5 HSP70 and 3 HSP90 species complexed with the p53 protein in the cytoplasm which are transported to the nucleus bound to p53, Once inside the nucleus these proteins are released indicating a conformational change occurring in either or both families of proteins. We plan to isolate the p53 complex both from cell free and modified cellular systems. We have developed a baculovirus system for the production of large quantities of wild-type human p53 protein, as well as an immunoaffinity column using the hybridoma P122 for purification of p53. Our cell-free approach is to radiolabel Saos-2 or Cos-1 cell proteins with 35S-methionine, with and without heat shock, then incubate the lysate with pure unlabeled p53 in the presence of protease inhibitors followed by gentle immunoprecipitation of the p53 complex with the monoclonal antibody Ab-1. Analysis by two-dimensional gel electrophoresis and fluorography will separate which proteins are associated with p53 in addition to the known HSP species which will be used as biomarkers. Cellular studies will utilize human cell lines constructed from the N-terminal and C-terminal truncated human p53 genes as well mouse constructs with mutated p53 nuclear localization signals to allow for cytoplasmic accumulation of complexed proteins due to inhibition of apoptosis since the complex will not translocate to the nucleus.
