Immunological evidence for the association of p53 with a heat shock protein, hsc70, in p53-plus-ras-transformed cell lines.
A rabbit antiserum was prepared against the C-terminal peptide of 21 amino acids from the human heat shock protein hsp70. These antibodies were shown to be specific for this highly inducible heat shock protein (72 kilodaltons [kDa] in rat cells), and for a moderately inducible, constitutively expressed heat shock protein, hsc70 (74 kDa). In six independently derived rat cell lines transformed by a murine cDNA-genomic hybrid clone of p53 plus an activated Ha-ras gene, elevated levels of p53 were detected by immunoprecipitation by using murine-specific anti-p53 monoclonal antibodies. In all cases, the hsc70, but not the hsp70, protein was coimmunoprecipitated with the murine p53 protein. Similarly, antiserum to heat shock protein coimmunoprecipitated p53. Western blot (immunoblot) analysis demonstrated that the hsc70 and p53 proteins did not share detectable antigenic epitopes. The results provide clear immunological evidence for the specific association of a single heat shock protein, hsc70, with p53 in p53-plus-ras-transformed cell lines. A p53 cDNA clone, p11-4, failed to produce clonable cell lines from foci of primary rat cells transfected with p11-4 plus Ha-ras. A mutant p53 cDNA clone derived from p11-4, SVKH215, yielded a 2- to 35-fold increase in the number of foci produced after transfection of rat cells with SVKH215 plus Ha-ras. When cloned, 87.5% of these foci produced transformed cell lines. SVKH215 encodes a mutant p53 protein that binds preferentially to the heat shock proteins of 70 kDa compared with binding by the parental p11-4 p53 gene product. These data suggest that the p53-hsc70 protein complex could have functional significance in these transformed cells.
Hinds
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PW
Department of Molecular Biology, Princeton University, New Jersey 08544.
Finlay
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Frey
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Levine
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D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis; D006367:HeLa Cells / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000235:genetics*; D006801:Humans; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics*; D009698:Nucleoproteins / Q000235:genetics*; D009857:Oncogenes; D010750:Phosphoproteins / Q000235:genetics*; D051381:Rats; D016159:Tumor Suppressor Protein p53
D006360:Heat-Shock Proteins; D009363:Neoplasm Proteins; D009698:Nucleoproteins; D010750:Phosphoproteins; D016159:Tumor Suppressor Protein p53; D004247:DNA