title: Purification of complexes of nuclear oncogene p53 with rat and Escherichia coli heat shock proteins: in vitro dissociation of hsc70 and dnaK from murine p53 by ATP.
abstract: Oligomeric protein complexes containing the nuclear oncogene p53 and the simian virus 40 large tumor antigen (D. I. H. Linzer and A. J. Levine, Cell 17:43-51, 1979), the adenovirus E1B 55-kilodalton (kDa) tumor antigen, and the heat shock protein hsc70 (P. Hinds, C. Finlay, A. Frey, and A. J. Levine, Mol. Cell. Biol. 7:2863-2869, 1987) have all been previously described. To begin isolating, purifying, and testing these complexes for functional activities, we have developed a rapid immunoaffinity column purification. p53-protein complexes are eluted from the immunoaffinity column by using a molar excess of a peptide comprising the epitope recognized by the p53 monoclonal antibody. This mild and specific elution condition allows p53-protein interactions to be maintained. The hsc70-p53 complex from rat cells is heterogeneous in size, with some forms of this complex associated with a 110-kDa protein. The maximum apparent molecular mass of such complexes is 660,000 daltons. Incubation with micromolar levels of ATP dissociates this complex in vitro into p53 and hsc70 110-kDa components. Nonhydrolyzable substrates of ATP fail to promote this dissociation of the complex. Murine p53 synthesized in Escherichia coli has been purified 660-fold on the same antibody affinity column and was found to be associated with an E. coli protein of 70 kDa. Immunoblot analysis with specific antisera demonstrated that this E. coli protein was the heat shock protein dnaK, which has extensive sequence homology with the rat hsc70 protein. Incubation of the immunopurified p53-dnaK complex with ATP resulted in the dissociation of the p53-dnaK complex as it did with the p53-hsc70 complex. This remarkable conservation of p53-heat shock protein interactions and the specificity of dissociation reactions suggest a functionally important role for heat shock proteins in their interactions with oncogene proteins.
authors.affiliation: Department of Molecular Biology, Princeton University, New Jersey 08544.
authors.forename: C F
authors.initials: CF
authors.lastname: Clarke
authors.forename: K
authors.initials: K
authors.lastname: Cheng
authors.forename: A B
authors.initials: AB
authors.lastname: Frey
authors.forename: R
authors.initials: R
authors.lastname: Stein
authors.forename: P W
authors.initials: PW
authors.lastname: Hinds
authors.forename: A J
authors.initials: AJ
authors.lastname: Levine
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification*; D002461:Cell Line, Transformed; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000235:genetics / Q000302:isolation & purification*; D007118:Immunoassay; D009363:Neoplasm Proteins / Q000235:genetics / Q000302:isolation & purification*; D009687:Nuclear Proteins / Q000235:genetics / Q000302:isolation & purification*; D009857:Oncogenes; D010750:Phosphoproteins / Q000235:genetics / Q000302:isolation & purification*; D010957:Plasmids; D051381:Rats; D016159:Tumor Suppressor Protein p53
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D016159:Tumor Suppressor Protein p53; D000255:Adenosine Triphosphate; C063842:dnaK protein, E coli