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PMID: 3285177 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

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.

Molecular and cellular biology ·Vol. 8 ·No. 3 ·1988-03-00 ·Pages 1206-15

Clarke CF, Cheng K, Frey AB, Stein R, Hinds PW, Levine AJ

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.

MeSH Terms
Adenosine Triphosphate/pharmacology Animals Bacterial Proteins/genetics,isolation & purification Cell Line, Transformed Chromatography, Affinity Chromatography, Gel Densitometry Electrophoresis, Polyacrylamide Gel Escherichia coli Escherichia coli Proteins HSP70 Heat-Shock Proteins Heat-Shock Proteins/genetics,isolation & purification Immunoassay Neoplasm Proteins/genetics,isolation & purification Nuclear Proteins/genetics,isolation & purification Oncogenes Phosphoproteins/genetics,isolation & purification Plasmids Rats Tumor Suppressor Protein p53
Chemicals
Bacterial Proteins Escherichia coli Proteins HSP70 Heat-Shock Proteins Heat-Shock Proteins Neoplasm Proteins Nuclear Proteins Phosphoproteins Tumor Suppressor Protein p53 Adenosine Triphosphate dnaK protein, E coli
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Clarke C F
Department of Molecular Biology, Princeton University, New Jersey 08544.
Cheng K
Frey A B
Stein R
Hinds P W
Levine A J
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/mcb.8.3.1206-1215.1988
Published
1988-03-00
Pages
1206-15
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC363265
Subset
IM
Grants
NCI NIH HHS · CA41086-02 · United States
External Links
PubMed source DOI: 10.1128/mcb.8.3.1206-1215.1988 PMC Full Text View journal details
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