Identification of the p53 protein domain involved in formation of the simian virus 40 large T-antigen-p53 protein complex.

An expression vector utilizing the enhancer and promoter region of the simian virus 40 (SV40) DNA regulating a murine p53 cDNA clone was constructed. The vector produced murine p53 protein in monkey cells identified by five different monoclonal antibodies, three of which were specific for the murine form of p53. The murine p53 produced in monkey cells formed an oligomeric protein complex with the SV40 large tumor antigen. A large number of deletion mutations, in-frame linker insertion mutations, and linker insertion mutations resulting in a frameshift mutation were constructed in the cDNA coding portion of the p53 protein expression vector. The wild-type and mutant p53 cDNA vectors were expressed in monkey cells producing the SV40 large T antigen. The conformation and levels of p53 protein and its ability to form protein complexes with the SV40 T antigen were determined by using five different monoclonal antibodies with quite distinct epitope recognition sites. Insertion mutations between amino acid residues 123 and 215 (of a total of 390 amino acids) eliminated the ability of murine p53 to bind to the SV40 large T antigen. Deletion (at amino acids 11 through 33) and insertion mutations (amino acids 222 through 344) located on either side of this T-antigen-binding protein domain produced a murine p53 protein that bound to the SV40 large T antigen. The same five insertion mutations that failed to bind with the SV40 large T antigen also failed to react with a specific monoclonal antibody, PAb246. In contrast, six additional deletion and insertion mutations that produced p53 protein that did bind with T antigen were each recognized by PAb246. The proposed epitope for PAb246 has been mapped adjacent (amino acids 88 through 109) to the T-antigen-binding domain (amino acids 123 through 215) localized by the mutations mapped in this study. Finally, some insertion mutations that produced a protein that failed to bind to the SV40 T antigen appeared to have an enhanced ability to complex with a 68-kilodalton cellular protein in monkey cells.     

A large-tumor-antigen-specific monoclonal antibody inhibits DNA replication of simian virus 40 minichromosomes in an in vitro elongation system.

In productively infected cells, a fraction of large-tumor antigen (T antigen) is tightly bound to replicating simian virus 40 (SV40) minichromosomes and does not dissociate at salt concentrations of greater than 1 M NaCl. We present electronmicrograms demonstrating the presence of T antigen on the replicated sections of replicating SV40 minichromosomes. We also show that the fraction of tightly bound T antigen is recognized by antibodies from mouse tumor serum and, more specifically, by a particular T-antigen-specific monoclonal antibody, PAb 1630. A second T-antigen-specific monoclonal antibody, PAb 101, does not react with the T-antigen fraction remaining on replicating SV40 chromatin at high salt concentrations. We used an in vitro replication system which allows, via semiconservative DNA replication, the completion of in vivo-initiated replicative intermediate DNA molecules. We show that monoclonal antibody PAb 1630, but not monoclonal antibody PAb 101, inhibits viral DNA replication. We discuss the possibility that SV40 T antigen may play a role in chain elongation during SV40 chromatin replication.     

Complex formation between the lymphotropic papovavirus large tumor antigen and the tumor suppressor protein p53

The simian B-lymphotropic papovavirus (LPV) encodes a large tumor antigen (T antigen) which is 45% identical to both the simian virus 40 (SV40) and the polyomavirus (PyV) large T antigens. In transgenic mice, the transforming properties of the LPV T antigen are similar to those of the SV40 T antigen. However, little is known about its biochemical activities. Since SV40 T antigen forms a complex with and stabilizes the host cell tumor suppressor protein p53 while the PyV large T antigen does not, we characterized the LPV T antigen for its ability to complex p53. We demonstrate an association between LPV T antigen and p53 in both a tumor-derived cell line and BALB/c 3T3 cells transformed in culture. A third protein of approximately 68 kDa which was found associated with the LPV T antigen-p53 complex in tumor-derived cells appears to be heat shock protein 70 (hsp70). The half-life of p53 in all LPV T-antigen-transformed cells was extended significantly; i.e., it was 3 to 7 h compared with 19 minutes in BALB/c 3T3 cells. The half-life of the LPV T antigen itself was 5 to 9 h depending on the cell line origin. That p53 was stabilized because of association with LPV T antigen and not because of mutation was demonstrated with the p53 conformation-dependent monoclonal antibody PAb246. This antibody distinguishes between wild-type p53 (PAb246+) and mutant, oncogenic p53 (PAb246-). Sequential immunoprecipitation showed all detectable p53 to be of the PAb246+ class in each LPV-transformed cell line, suggesting that the stable p53 was indeed wild type.     

p53 mutations are not selected for in simian virus 40 T-antigen-induced tumors from transgenic mice.

Many diverse tumors contain cells that select for mutations at the p53 gene locus. This appears to be the case because the p53 gene product can act as a negative regulator of cell division or a tumor suppressor. These mutations then eliminate this activity of the p53 gene product. The simian virus 40 (SV40) large T antigen binds to p53 and acts as an oncogene to promote cellular transformation and initiate tumors. If the binding of T antigen to the p53 protein inactivated its tumor suppressor activity, there would be no selection pressure for p53 mutants to appear in tumors. To test this idea, transgenic mice that carried and expressed the SV40 large T-antigen gene were created. Expression of the T antigen was directed to the liver, using the albumin promoter, and the choroid plexus, using the SV40 enhancer-promoter. A large number of papillomas (indicated in parentheses) of the choroid plexus (14), hepatocellular carcinomas (5), liver adenomas (10), and tumors of clear-cell foci (5) were examined for mutant and wild-type p53 genes and gene products. In all cases, the tumor extracts contained readily detectable T-antigen-p53 protein complexes. A monoclonal antibody specifically recognizing the wild-type p53 protein (PAb246) reacted with p53 in every tumor extract. A monoclonal antibody specifically recognizing mutant forms of the p53 protein (PAb240) failed to detect p53 antigens in these extracts. Finally, p53 partial cDNAs were sequenced across the regions of common mutations in this gene, and in every case only the wild-type sequence was detected. These results strongly support the hypothesis that T antigen inactivates the wild-type p53 tumor-suppressing activity and there is no need to select for mutations at the p53 locus.     

Monoclonal antibody analysis of p53 expression in normal and transformed cells.

The cellular phosphoprotein p53 binds tightly and specifically to simian virus 40 T antigen and the 58,000-molecular-weight adenovirus E1b protein. Many human and murine tumor cell lines contain elevated levels of the p53 protein even in the absence of these associated viral proteins. Recently the cloned p53 gene, linked to strong viral promoters, has been shown to complement activated ras genes in transformation of primary rodent cell cultures. Overexpression of the p53 gene alone rescues some primary rodent cell cultures from senescence. We isolated three new monoclonal antibodies to the p53 protein, designated PAb242, PAb246, and PAb248, and mapped the epitopes they recognized on p53 in comparison with other previously isolated antibodies. At least five sterically separate epitopes were defined on murine p53. One of the antibodies, PAb246, recognizes an epitope on p53 that is unstable in the absence of bound simian virus 40 T antigen. This effect is demonstrable in vivo and in newly developed in vitro assays of T-p53 complex formation. Using the panel of anti-p53 antibodies and sensitive immunocytochemical methods, we found that p53 has a predominantly nuclear location in established but not transformed cells as well as in the vast majority of transformed cell lines. Several monoclonal antibodies to p53 showed cross-reactions with non-p53 components in immunocytochemical staining.     

Activating mutations in p53 produce a common conformational effect. A monoclonal antibody specific for the mutant form.

Point mutations in the p53 gene are the most frequently identified genetic change in human cancer. They convert murine p53 from a tumour suppressor gene into a dominant transforming oncogene able to immortalize primary cells and bring about full transformation in combination with an activated ras gene. In both the human and murine systems the mutations lie in regions of p53 conserved from man to Xenopus. We have developed a monoclonal antibody to p53 designated PAb240 which does not immunoprecipitate wild type p53. A series of different p53 mutants all react more strongly with PAb240 than with PAb246. The PAb240 reactive form of p53 cannot bind to SV40 large T antigen but does bind to HSP70. In contrast, the PAb246 form binds to T antigen but not to HSP70. PAb240 recognizes all forms of p53 when they are denatured. It reacts with all mammalian p53 and chicken p53 in immunoblots. We propose that immunoprecipitation of p53 by PAb240 is diagnostic of mutation in both murine and human systems and suggest that the different point mutations which convert p53 from a recessive to a dominant oncogene exert a common conformational effect on the protein. This conformational change abolishes T antigen binding and promotes self-oligomerization. These results are consistent with a dominant negative model where mutant p53 protein binds to and neutralizes the activity of p53 in the wild type conformation.     

Variation in antigenic determinants of p53 transformation-related protein obtained from various species

p53 is a cellular-encoded transformation-related protein. It is synthesized at elevated levels in tumor cells but has also been detected at low concentrations in several types of nontransformed cells. The p53 of tumor cells is immunogenic and elicits specific antibody production. The antigenic determinants of the p53 protein were studied by specific binding to anti-p53 monoclonal antibodies obtained from the RA3-2C2, PAb122, and PAb421 established hybridoma cell lines, and their conservation was followed in various animal species. We found that whereas mouse p53 efficiently immunoprecipitated with all three anti-p53 monoclonal antibodies, human and rat p53 bound PAb122 and PAb421 but lacked a determinant binding RA3-2C2. The hamster p53 molecule represented a third category, which immunoprecipitated with polyclonal anti-p53 antibodies but failed to bind all three monoclonal antibodies analyzed here. Using these monoclonal antibodies, we detected no variations between p53 found in transformed and p53 found in nontransformed cells, within a given species. The results also showed that RA3-2C2, which recognizes a mouse-specific determinant, binds a site located at a proteolytic digestion fragment of the p53 molecule that differs from that containing PAb122 and PAb421 recognition site(s). p53 is a single protein that can be immunoprecipitated through different antigenic determinants that vary between species.     

Activating mutations for transformation by p53 produce a gene product that forms an hsc70-p53 complex with an altered half-life.

The 11-4 p53 cDNA clone failed to transform primary rat fibroblasts when cotransfected with the ras oncogene. Two linker insertion mutations at amino acid 158 or 215 (of 390 amino acids) activated this p53 cDNA for transformation with ras. These mutant cDNAs produced a p53 protein that lacked an epitope, recognized by monoclonal antibody PAb246 (localized at amino acids 88 to 110 in the protein) and preferentially bound to a heat shock protein, hsc70. In rat cells transformed by a genomic p53 clone plus ras, two populations of p53 proteins were detected, PAb246+ and PAb246-, which did or did not bind to this monoclonal antibody, respectively. The PAb246- p53 preferentially associated with hsc70, and this protein had a half-life 4- to 20-fold longer than free p53 (PAb246+). These data suggest a possible functional role for hsc70 in the transformation process. cDNAs for p53 derived from methylcholanthrene-transformed cells transform rat cells in cooperation with the ras oncogene and produce a protein that bound with the heat shock proteins. Recombinant clones produced between a Meth A cDNA and 11-4 were tested for the ability to transform rat cells. A single amino acid substitution at residue 132 was sufficient to activate the 11-4 p53 cDNA for transformation. These studies have identified a region between amino acids 132 and 215 in the p53 protein which, when mutated, can activate the p53 cDNA. These results also call into question what the correct p53 wild-type sequence is and whether a wild-type p53 gene can transform cells in culture.     

Correlation of DNA content, p53, T antigen, and V antigen in simian virus 40-infected human diploid cells

Human diploid fibroblasts (HDF) have a finite life span in cell culture which can be extended when transformed with simian virus 40 (SV40). Flow cytometric analysis of SV40-HDF transformation allowed DNA content changes to be correlated with the appearance, quantity, and distribution of T antigen, p53, and V antigen, three proteins associated with this process. These studies demonstrated a shift in the DNA content to tetraploidy, which was correlated with the age of the SV40-HDF but not the time of infection. A significant increase of the epitope recognized by PAb122 to host p53 and the epitope PAb101 to SV40 T antigen occurred at the same time the tetraploid population appeared. However, an antigen reactive with SV40 V antibody was present at high levels in most of the population early after infection, but the levels declined with time. The percentage of PAb101-T antigen-positive cells increased more rapidly in cells infected at a late passage, and this was concomitant with the shift in DNA content to tetraploid. Analysis of the mean fluorescence of total, gated populations (G1, G2, and greater than G2) demonstrated that a threshold level of p53 and T antigen was reached in each compartment of the cell cycle. As the transformed phenotype appeared, a population of cells was continually released into the supernatant, and although these cells had a DNA pattern similar to the monolayer cells, the T antigen and p53 levels were 3-5 times higher in the tetraploid G2 cells. These studies correlated the expression of proteins associated with viral transformation in HDF which vary with time and shift in DNA content.     

Characterization of mutant p53-hsp72/73 protein-protein complexes by transient expression in monkey COS cells.

Several mutant, but not wild-type, p53 proteins form complexes with hsp72/73 heat shock-related proteins in simian virus 40-transformed monkey COS cells. We carried out a detailed biochemical and structural mapping analysis of p53 and report here that p53-hsp72/73 complex formation showed considerable structural specificity. Such complexes were remarkably stable, but unlike analogous complexes formed between p53 and simian virus 40 T antigen, they did not form in in vitro association assays. p53-hsp72/73 complex formation in vivo appears to be dependent on aspects of mutant p53 protein conformation. However, absence of the conformation-sensitive epitope recognized by monoclonal antibody PAb 246 was not reliably diagnostic of such complexes, nor was p53-hsp72173 binding reliably diagnostic of oncogenic activation.     

Monoclonal antibodies displaying a novel species specificity for the primate transformation-related protein, p53

SV40 large T antigen associates with a cellular phosphoprotein, p53, in virus-transformed cells. We have raised three new monoclonal antibodies, PAb1101, PAb1102 and PAb1103, to this cellular protein, derived from SV40-transformed human fibroblasts. These define at least two non-overlapping determinants on human p53 that are in different areas of the molecule from those recognised by previously available antibodies. Unlike those antibodies, PAb1102 and PAb1103 do not react with rodent p53. PAb1101 reacts far more weakly with rodent p53 than with primate p53. All three antibodies show a preference for binding to the large T-associated form of p53, an effect that is particularly marked with PAb1102. The novel specificity of these antibodies allows further probing of the nature and function of the large T/p53 complex in human cells.     

Isolation of a full-length mouse cDNA clone coding for an immunologically distinct p53 molecule.

Transfection of a cloned p53 gene into a p53 nonproducer Abelson murine leukemia virus-transformed cell line, L12, reconstituted p53 expression. The protein expressed in these cells was indistinguishable from that naturally expressed in p53 producer tumor cells. Conversely, p53 protein expressed in L12-derived clones that were established by transfection with a full-length p53 cDNA clone (pM8) exhibited a discrete immunological form. Immunoprecipitation of p53 with a panel of monoclonal anti-p53 antibodies showed that L12-derived clones that were transfected with the genomic p53 clone contained the same antigenic determinants as those found in the p53 protein expressed in tumor cells. These p53 proteins bound all monoclonal antibody types as well as the polyclonal anti-p53 tested. However, L12-derived clones established by transfection of the p53 cDNA clone (pM8) expressed a p53 protein that bound the RA3-2C2 and PAb200.47 anti-p53 monoclonal antibodies as well as polyclonal anti-p53 serum but totally lacked the antigenic receptor for the PAb122 and PAb421 monoclonal antibodies. The p53 proteins expressed by either genomic or cDNA p53 clones exhibited the same apparent molecular sizes and identical partial peptide maps. We suggest that transfection of the p53 gene induced expression of the entire group of the possible mRNA species, whereas cloned p53 cDNA (pM8) represented a single mRNA molecule that codes for a discrete species of p53 protein.     

Monoclonal antibodies against simian virus 40 nuclear large T tumour antigen: epitope mapping, papova virus cross-reaction and cell surface staining.

Thirty six cloned hybridomas have been isolated which produce monoclonal antibodies directed against simian virus 40 (SV40) large T tumour antigen. They have been shown to recognize at least six different epitopes along the T antigen polypeptide according to their reaction with the various truncated forms of T antigen expressed by adenovirus-SV 40 hybrid viruses. Sixteen antibodies cross-react with cells infected by the closely related human BK virus. Only two antibodies, PAb1604 and PAb1614, directed against different epitopes of the SV40 T antigen, cross-react with polyoma large T tumour antigen which has a more limited amino acid sequence homology. This cross-reaction is rarely seen with polyclonal antibodies. Monoclonal antibody PAb1620 gave nuclear immunofluorescence only with murine cells transformed by SV40 and was found to react with a complex of T-antigen and 53 000-dalton host-coded protein. All the monoclonal antibodies react with nuclear T antigen and all but four antibodies stained the surface of SV40-transformed cells. These were four of the five antibodies directed against the central third of the T antigen. Thus the monoclonal antibodies show that cell surface T antigen differs from nuclear T antigen, either in accessibility or structure.     

Temperature-dependent switching between "wild-type" and "mutant" forms of p53-Val135

The p53 gene is a suppressor of abnormal cell growth but is also subject to oncogenic activation by mutation. The mutant allele p53-Val135, has recently been discovered to be temperature-sensitive and functions as an oncogene at 37 degrees C and as a tumor suppressor at 32.5 degrees C. In order to investigate the molecular mechanism underlying the temperature sensitivity of p53-Val135 rabbit reticulocyte lysate was used to translate the p53 mRNAs in vitro at 37 degrees C and at 30 degrees C. The immunoreactivity and T antigen binding of wild-type protein p53-Ala135 were unaffected by temperature and were similar to wild-type p53 expressed in vivo. In contrast, the mutant p53-Val135 protein was markedly affected by temperature. At 37 degrees C p53-Val135 showed reduced T antigen binding and did not react with monoclonal antibodies PAb246 and PAb1620. At 30 degrees C, p53-Val135 behaved as the wild-type p53. Temperature also exerted a post-translational effect on p53-Val135 with complete conversion from wild-type to mutant phenotype within two minutes of temperature shift from 30 degrees C to 37 degrees C. There was incomplete conversion from mutant to wild-type phenotype when the temperature was shifted down from 37 degrees C to 30 degrees C. We propose that the temperature dependent forms of p53-Val135 represent conformational variants of the p53 protein with opposing functions in cell growth control.     

Intracellular location and kinetics of complex formation between simian virus 40 T antigen and cellular protein p53

The intracellular location and kinetics at which the simian virus 40 T antigen and the cellular protein p53 associate with one another were determined for simian virus 40-transformed mouse (215) and rat (14B) cells. Cells were labeled under pulse-chase conditions and fractionated into nuclear and cytoplasmic components, and the proteins were immunoprecipitated with monoclonal antibodies (pAb 416, 101, and 122). We found that newly made T antigen and p53 migrated to the nucleus of these cells independently; that is, in uncomplexed form. Newly made p53 was transported to the nucleus more rapidly than T antigen in both cell lines and formed a complex with a mature form of T antigen recognizable by pAb 101. This association was very rapid in both cell lines (t 1/2, 5 to 15 min). In contrast, the time course of complex formation between newly made T antigen and the p53 in the nucleus varied with the ratio of T antigen to p53 of the cell line studied. In 215 cells, where the ratio was 3.6, the kinetics were quite slow (t 1/2, 30 min), whereas in 14B cells, where the ratio was 1.7, they were quite rapid (t 1/2, 5 min). We suggest that a competition between newly made and uncomplexed T antigen for the p53 in the nucleus is the major determinant of the rate of complex formation for newly made T antigen. Our studies indicate that this macromolecular interaction is extremely dynamic.     

Mutant conformation of p53. Precise epitope mapping using a filamentous phage epitope library.

Many naturally occurring point mutations in the p53 gene lead to a proportion of the encoded protein molecules adopting a distinct, "mutant" conformation characterized by exposure of a normally cryptic epitope recognized by the monoclonal antibody PAb240. Here the PAb240 epitope is defined using a filamentous phage epitope library. The hexapeptides displayed by the PAb240-binding phage isolated from the library were all highly related and allowed both direct localization of the epitope and prediction of a specific interaction between PAb240 and Xenopus TFIIIA. This study demonstrates for the first time the power of phage epitope libraries in the precise definition of previously unmapped epitopes. Identification of the PAb240 epitope precisely defines a region of the p53 molecule structurally altered by the mutation-induced conformational shift.     

Mutation of the serine 312 phosphorylation site does not alter the ability of mouse p53 to inhibit simian virus 40 DNA replication in vivo.

Two mutations were introduced into the wild-type mouse p53 gene by oligonucleotide-directed mutagenesis. These mutations substituted alanine or aspartic acid for serine at position 312, which is constitutively phosphorylated. Phosphopeptide mapping of the mutant proteins, expressed in COS cells, confirmed the loss of phosphorylation at position 312. There were no changes in the ability of the mutant p53s to express the conformation-dependent epitope for monoclonal antibody PAb246 or to participate in complexes with the simian virus 40 (SV40) large T antigen. Replication of a plasmid containing the SV40 origin of replication was inhibited in COS cells by wild-type p53 and both of the phosphorylation site mutants with equal efficiency. A transforming mutant of p53, encoding valine at position 135, did not inhibit SV40 DNA replication in COS cells.     

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.

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.