HSP BINDING AND MITOCHONDRIAL LOCALIZATION OF P53 PROTEIN IN HUMAN HT1080 AND MOUSE C3H10T1 2 CELL-LINES/

In normal cells, the tumor suppressor actions of p53 protein are media ted by specific DNA binding and protein-protein interactions within th e nucleus. Mutant p53 proteins, however, often assume an aberrant conf ormation devoid of tumor suppressor activity and newly capable of bind ing to the cognate or inducible HSP70. Recent reports from our laborat ory and others show that additional unknown proteins may also complex with mutant p53. In this study, we characterize p53:HSP complexes and their subcellular location in the transformed cell lines, human HT1080 and murine C3H10T1/2, which both contain aberrant p53 conformers. Imm unoprecipitation and SDS-PAGE of p53 from whole cell lysates revealed the additional presence of a broad 70 kDa band and a 90 kDa band in bo th lines, while p53 isolated from nuclear lysates was free from other proteins. 2D-PAGE was used to isolate and identify HSP members from cy toplasmic and nuclear lysates by immunoprecipitation, Western blotting and protein sequencing. Anti-p53 immune complexes from cytoplasmic ly sates contained not only HSC70 but also GRP75, GRP78 and a weakly basi c 90 kDa protein, which may be related to HSP90. The inducible form of HSP70 was not complexed to p53 protein, even though expressed in thes e cells. Analysis of anti-HSP70, anti-GRP75 and anti-HSP90 immune comp lexes suggests that HSP members exist as preformed complexes in the cy toplasm, but not the nucleus. The presence of the mitochondrial and en doplasmic reticular chaperones, GRP75 and GRP78. in p53:HSP complexes suggested that p53 might be found in these cytoplasmic organelles whic h was confirmed in mitochondria by biochemical and immunoelectron micr oscopic evidence. These studies suggest that newly identified members of p53:HSP complexes represent components of a chaperone program which affects the subcellular distribution of p53 protein in these transfor med lines.