Interaction of heat-shock protein 70 with p53 translated in vitro: evidence for interaction with dimeric p53 and for a role in the regulation of p53 conformation.

In intact cells, hsp70 proteins selectively complex with mutant p53. We report here that rabbit reticulocyte lysate contains hsp70 which selectively complexes with the mutant p53 translated in vitro. Hsp70 complexes with dimers and possibly monomers of p53 in a manner that requires the terminal 28 amino acids of p53. Using murine p53Val135, which is temperature-sensitive for phenotype, we demonstrate that p53-hsp70 complexes can occur after post-translational switching from wild-type to mutant p53 phenotype. Moreover, the temperature-induced switch of full-length p53Val135 from wild-type to mutant phenotype is ATP-independent, whereas the switch from mutant to wild-type form requires ATP hydrolysis and involves hsp70. These results imply that hsp70 is involved in the regulation of p53 conformation.     

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.     

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.     

Expression of wild-type p53 is not compatible with continued growth of p53-negative tumor cells.

Inactivation of the cellular p53 gene is a common feature of Friend virus-induced murine erythroleukemia cell lines and may represent a necessary step in the progression of this disease. As well, frequent loss or mutation of p53 alleles in diverse human tumors is consistent with the view of p53 as a tumor suppressor gene. To examine the significance of p53 gene inactivation in tumorigenesis, we have attempted to express transfected wild-type p53 in three p53-negative tumor cell lines: murine DP16-1 Friend erythroleukemia cells, human K562 cells, and SKOV-3 cells. We found that aberrant p53 proteins, which differ from wild-type p53 by a single amino acid substitution, were expressed stably in these cells, whereas wild-type p53 expression was not tolerated. The inability of p53-negative tumor cell lines to support long-term expression of wild-type p53 protein is consistent with the view that p53 is a tumor suppressor gene.     

Cotranslation of activated mutant p53 with wild type drives the wild-type p53 protein into the mutant conformation

Activating mutations of p53 promote tumor progression. The mutant protein adopts a characteristic conformation, which lacks the growth suppressor function of wild-type p53. We show that mutant p53 can drive cotranslated wild-type p53 into the mutant conformation: a similar effect in vivo would block wild-type suppressor function with dominant negative effect. The cotranslational effect of mutant p53 on wild-type conformation depends upon interaction between nascent polypeptides and oligomerization of the full-length proteins. We also show that oligomers of p53 proteins can be induced to change conformation in a cooperative manner. Cell growth stimulation induces a similar conformational change in p53, and our present results indicate that this may involve allosteric regulation.     

Mutational analysis of the p53 core domain L1 loop

The p53 tumor suppressor gene acquires missense mutations in over 50% of human cancers, and most of these mutations occur within the central core DNA binding domain. One structurally defined region of the core, the L1 loop (residues 112-124), is a mutational "cold spot" in which relatively few tumor-derived mutations have been identified. To further understand the L1 loop, we subjected this region to both alanine- and arginine-scanning mutagenesis and tested mutants for DNA binding in vitro. Select mutants were then analyzed for transactivation and cell cycle analysis in either transiently transfected cells or cells stably expressing wild-type and mutant proteins at regulatable physiological levels. We focused most extensively on two p53 L1 loop mutants, T123A and K120A. The T123A mutant p53 displayed significantly better DNA binding in vitro as well as stronger transactivation and apoptotic activity in vivo than wild-type p53, particularly toward its pro-apoptotic target AIP1. By contrast, K120A mutant p53, although capable of strong binding in vitro and wild-type levels of transactivation and apoptosis when transfected into cells, showed impaired activity when expressed at normal cellular levels. Our experiments indicate a weaker affinity for DNA in vivo by K120A p53 as the main reason for its defects in transactivation and apoptosis. Overall, our findings demonstrate an important, yet highly modular role for the L1 loop in the recognition of specific DNA sequences, target transactivation, and apoptotic signaling by p53.     

Conformational effects of selected cancer-related amino acid substitutions in the p53 protein.

The tumor suppressor gene p53 has been identified as the most frequent target of genetic alterations in human cancers. Cancer-related mutations in the human p53 protein tend to cluster in four of the five highly conserved domains of the protein, and, in particular, in the central region of domain IV from residues 241 to 253. Using conformational energy analysis based on ECEPP (Empirical Conformational Energies for Polypeptides Program), we have determined the preferred three dimensional structures for this tridecapeptide sequence for the human wild-type p53 protein and four cancer-related mutant p53 proteins (Ala 245, Ile 246, Trp 248, Ser 249). The results show that the mutant peptides adopt conformations that are distinctly different from that of the wild-type peptide. These results are consistent with experimental conformational studies demonstrating altered detectability of antigenic epitopes in murine wild-type and mutant p53 proteins. These results suggest that the oncogenic effects of human mutant p53 proteins may be mediated by distinct local conformational changes in the protein.     

In vivo effects of abolishing the single canonical sumoylation site in the C-terminal region of Drosophila p53

Using yeast two-hybrid screens we determined that Drosophila (Dm)p53 interacts with proteins involved in sumoylation (UBA2, UBC9 and PIAS) through different regions of its C-terminal domain. A K302R point mutation within a single canonical sumoylation site of Dmp53 did not abolish the observed interactions. These observations prompted us to analyze whether Dmp53 sumoylation at this site has any functional role in vivo. Genetic assays showed that deleting one copy of genes involved in sumoylation (lwr, Su(var)2-10 or smt3 heterozygosity) enhanced slightly the mutator phenotype of Dmp53. We compared the in vivo effects of wild type and K302R Dmp53 overproduced from transgenes and determined that similar levels of expression of the mutant and wild type proteins resulted in similar phenotype, and the two proteins showed similar cellular localization. The half life and the trans-activator activity of K302R mutant and wild type Dmp53 were also comparable. Lastly, by analyzing wild type and K302R Dmp53 expressed at different levels in animals and in S2 cells we detected no differences between the mobility of the mutant and wild-type protein. From these data we conclude that under normal developmental conditions the loss of SUMO modification at K302 does not affect Dmp53 function significantly.     

Expression of wild-type and mutant p53 proteins by recombinant vaccinia viruses.

To facilitate the purification of wild type p53 protein, we established a recombinant p53 vaccinia viral expression system. Using this efficient eukaryotic expression vector, we found that the expressed p53 proteins retained their specific structural characteristics. A comparison between wild type and mutant p53 proteins showed the conservation of the typical subcellular localization and the expression of specific antigenic determinants. Furthermore, wild type p53 exhibited a typical binding with large T antigen, whereas no binding was detected with mutant p53. Both wild type and mutant p53 proteins were highly stable and constituted 5-7% of total protein expressed in the infected cells. These expression recombinant viruses offer a simple, valuable system for the purification of wild type and mutant p53 proteins that are expressed abundantly in eukaryotic cells.     

Activation of the cryptic DNA binding function of mutant forms of p53.

Wild type p53 assembles into a latent multiprotein complex which can be activated for sequence-specific DNA binding in vitro by proteins targeting the carboxy-terminal domain. Using an optimized system coupling the post-translational modification of wild type p53 to activation of sequence specific DNA binding, we examined the affects of common mutations on the cryptic DNA binding function of p53. Two mutant forms of p53 were shown to be efficiently converted from the latent state by PAb421 and DnaK, but were defective in activation by casein kinase II, indicating that mutant p53 may not be receptive to allosteric regulation by casein kinase II phosphorylation. A reactive sulfhydryl group is absolutely required for DNA binding by wild type and mutant forms of p53 once converted to the activated state. Together, these data show that some mutant forms of p53 harbour the wild-type machinery required to engage in sequence-specific DNA binding and define a signalling pathway whose inactivation may directly result in a loss of p53 function.     

Wild-type p53 restores cell cycle control and inhibits gene amplification in cells with mutant p53 alleles.

Loss of cell cycle control and acquisition of chromosomal rearrangements such as gene amplification often occur during tumor progression, suggesting that they may be correlated. We show here that the wild-type p53 allele is lost when fibroblasts from patients with the Li-Fraumeni syndrome (LFS) are passaged in vitro. Normal and LFS cells containing wild-type p53 arrested in G1 when challenged with the uridine biosynthesis inhibitor PALA and did not undergo PALA-selected gene amplification. The converse occurred in cells lacking wild-type p53 expression. Expression of wild-type p53 in transformants of immortal and tumor cells containing mutant p53 alleles restored G1 control and reduced the frequency of gene amplification to undetectable levels. These studies reveal that p53 contributes to a metabolically regulated G1 check-point, and they provide a model for understanding how abnormal cell cycle progression leads to the genetic rearrangements involved in tumor progression.     

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.     

Co-chaperones Bag-1, Hop and Hsp40 regulate Hsc70 and Hsp90 interactions with wild-type or mutant p53.

Using highly purified proteins, we have identified intermediate reactions that lead to the assembly of molecular chaperone complexes with wild-type or mutant p53R175H protein. Hsp90 possesses higher affinity for wild-type p53 than for the conformational mutant p53R175H. The presence of Hsp90 in a complex with wild-type p53 inhibits the binding of Hsp40 and Hsc70 to p53, consequently preventing the formation of wild-type p53-multiple chaperone complexes. The conformational mutant p53R175H can form a stable heterocomplex with Hsp90 only in the presence of Hsc70, Hsp40, Hop and ATP. The anti-apoptotic factor Bag-1 can dissociate Hsp90 from a pre- assembled complex wild-type p53 protein, but it cannot dissociate a pre-assembled p53R175H-Hsp40- Hsc70-Hop-Hsp90 heterocomplex. The results presented here provide possible molecular mechanisms that can help to explain the observed in vivo role of molecular chaperones in the stabilization and cellular localization of wild-type and mutant p53 protein.