Cellular localization and cell cycle regulation by a temperature-sensitive p53 protein.

Abstract

Primary rat embryo fibroblasts were transformed by a p53 mutant (alanine to valine change at amino acid 135) plus ras. This p53^^'*^^ mutant is temperature sensitive for a conformational change detected by the binding of a monoclonal antibody, PAb246, which recognizes the wild-type protein or the great majority of p53 vail 35 at 32.5°C. At 37°C, both mutant and wild-type p53 conformational forms co-exist in the cells, while at 39.5°C, the majority of the p53^"'^^^ in the cell is in a mutant conformation not recognized by PAb246 antibody. At 39.5°C, the mutant p53 is localized in the cytoplasm of the cell. At 32.5°C, the p53 protein enters the nucleus and stops the growth of these cells. At 37°C where there is a mixture of mutant and wild-type p53, the wild-type p53 protein is in a complex with hsc70 and mutant p53 protein in the cytoplasm of the cell during Gj. This wild-type protein enters the nucleus as the cells enter the S-phase of the cell cycle. At 32.5°C, the cells stop replication and arrest at the G^/S border. After 48 hr at 32.5°C, 91% of the cells are in the Gj fraction of the cell cycle. The S-phase cells appear to be immune to the p53 negative regulation of growth until they enter the next G^ period. These data strongly suggest that mutant p53 proteins in transformed cells act to sequester the wild-type p53 protein in an hsc70-p53 complex, which resides in the cytoplasm during the stage of the cell cycle, G^, when nuclear wild-type p53 would normally act to regulate cell growth and progression through the cycle. In this way, mutant p53 proteins can act in a trans-dominant fashion to overcome growth regulation by the wild-type p53 allele and protein in a cell.

Human smooth muscle α-actin gene is a transcriptional target of the p53 tumor suppressor protein

“…Actin ®laments were visualized using rhodamine-conjugated phalloidin. Photographs shown are two examples of cells maintained at the two temperatures ®broblast cells (A1-5) that express a temperaturesensitive murine mutant p53 protein (Martinez et al, 1991). The temperature sensitive p53 gene contains a point mutation that results in an alanine to valine substitution at amino acid residue 135 (Martinez et al, 1991).…”

“…Photographs shown are two examples of cells maintained at the two temperatures ®broblast cells (A1-5) that express a temperaturesensitive murine mutant p53 protein (Martinez et al, 1991). The temperature sensitive p53 gene contains a point mutation that results in an alanine to valine substitution at amino acid residue 135 (Martinez et al, 1991). At high temperature (398C), *80% of the protein is in the mutant conformation as determined by reactivity with conformation-dependent monoclonal antibodies and the cells grow exponentially (Martinez et al, 1991).…”

“…The temperature sensitive p53 gene contains a point mutation that results in an alanine to valine substitution at amino acid residue 135 (Martinez et al, 1991). At high temperature (398C), *80% of the protein is in the mutant conformation as determined by reactivity with conformation-dependent monoclonal antibodies and the cells grow exponentially (Martinez et al, 1991). In contrast, at low temperature, (328C), *80% of the p53 is in the wild-type conformation and the cells are growth arrested in the G1 phase of the cell cycle (Martinez et al, 1991).…”

Dissociation of p53-mediated suppression of homologous recombination from G1/S cell cycle checkpoint control

“…(ii) 10.1/VAS5 cells expressed the same mutant, but with the last 26 amino acids (normally-spliced form) exchanged with 17 di erent residues (alternatively-spliced, Val135AS) leading to loss of non-speci®c DNA binding activity (Kulesz-Martin et al, 1994;Bayle et al, 1995). Considering suggestions that at 378C p53-Val135 could display features of both, mutant and wild-type conformation, at least in rat ®broblast lines (Martinez et al, 1991;Stewart et al, 1995), we sought to con®rm the absence of damage-inducible p53 activation at this temperature.…”

“…The data presented here imply that p53 can maintain genome integrity by suppression of spontaneous HR independently of the G1/S checkpoint and probably also in the absence of transcriptional activation. Furthermore, p53 has been suggested to act in a post-replicative mismatch repair pathway (Mummenbrauer et al, 1996;Huang, 1998), it co-localizes with DNA synthesis and the DNA replication apparatus (Huang, 1998), interacts with viral replication (Deppert, 1994), migrates into the nucleus with S phase (Shaulsky et al, 1990;Martinez et al, 1991), and interacts with a variety of proteins involved in DNA repair (for further review, see Janus et al, 1999a). Deppert and colleagues (Janus et al, 1999b) recently made the intriguing observation that p53's exonuclease and sequencespeci®c DNA binding activities appeared to be mutually exclusive functions.…”