Multiple p53 protein isoforms and formation of oligomeric complexes with heat shock proteins Hsp70 and Hsp90 in the human mammary tumor, T47D, cell line.
At least eleven isoforms of p53 protein were observed in a human mammary tumor cell line. T47D. Comparative 33P and 35S incorporation analysis showed an equal distribution of P53 isoforms within cytoplasmic and nuclear compartments, although phosphorylation was unequal among isoforms and the most basic p53 species was unphosphorylated. Using a combination of immunoprecipitation with monoclonal antibodies for p53 and heat shock proteins Hsp70 & Hsp90, and two-dimensional gel electrophoretic analysis, T47D p53 protein oligomers were observed with several species of Hsp70 and Hsp90. The p53/Hsp70/Hsp90 aggregate dissociates after nuclear translocation. Immunoprecipitation of Hsp70 and Hsp90 using monoclonal antibodies showed formation of a heteroligomer between Hsp70 and Hsp90 in cytoplasm but not nucleus. This suggests these Hsp proteins can form a complex in the cytoplasm but undergo a conformational change after nuclear translocation such that Hsp/Hsp binding sites are no longer recognized. These data indicate T47D cells have multiple p53 precursor molecules probably at different stages of phosphorylation, and which may be sequestered from proteases by binding to Hsp proteins. Hsp proteins also can heterocomplex in the cytoplasm, also possibly as protection against protease degradation until bound to p53. After translocation, p53 is freed from Hsp proteins for binding to DNA where Hsp70 and Hsp90 are no longer able to form a nuclear complex probably rendering Hsp's labile to proteolysis.
Selkirk
J K
JK
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Merrick
B A
BA
Stackhouse
B L
BL
He
C
C
D000911:Antibodies, Monoclonal; D001943:Breast Neoplasms / Q000378:metabolism* / Q000648:ultrastructure; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007163:Immunosorbent Techniques; D046911:Macromolecular Substances; D010766:Phosphorylation; D011487:Protein Conformation; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D046911:Macromolecular Substances; D016159:Tumor Suppressor Protein p53