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Query: UNIPROT:P04637 (p53)
 77,613 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To study the effect of SV40 T-antigen in mammary epithelial cells, a rat beta-casein promoter-driven SV40 early-region construct was stably introduced into the clonal mouse mammary epithelial cell line HC11. With the expression of the viral T-antigens under the control of a hormone-inducible promoter, it was possible to dissociate the effects of different levels of T-antigen expression on cell growth, morphology, and gene expression. Following hormonal induction, a rapid but transient induction of T-antigen was observed, followed by a delayed induction of H4 histone mRNA. In T-antigen-positive HC11 cells cultured in the absence of EGF, the expression of basal levels of T-antigen (in the absence of hormonal induction) led to a decreased doubling time and an increased cell density. In the presence of EGF, T-antigen expression resulted additionally in an altered cell morphology. Despite the effects of T-antigen on cell growth and gene expression, the cells were unable to form colonies in soft agar and were nontumorigenic when transplanted into cleared mammary fat pads. They were, however, weakly tumorigenic in nude mice. Relatively high levels of p53 protein synthesis were observed in both the transfected HC11 cells and the parental COMMA-D cells, as compared to 3T3E fibroblasts and another mammary epithelial cell line. The HC11 and COMMA-D cells synthesized approximately equal levels of wild-type and mutated p53 proteins as defined by their reactivities with monoclonal antibodies PAb246 and PAb240, respectively. Interactions between excess p53 and T-antigen may, in part, explain the failure of these cells to display a completely transformed phenotype.
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PMID:Differential effects of the simian virus 40 early genes on mammary epithelial cell growth, morphology, and gene expression. 132 45

A series of human diploid fibroblast cell clones were developed by DNA transfection with either wild-type SV40 T-antigen (SV40 T) or T-antigen mutants defective in its various functional domains. Cell clones expressing the wild-type SV40 T were significantly radioresistant as compared with clones transfected with the neo gene only (D0 = 192 +/- 13 vs 127 +/- 19). This radioresistance persisted in post-crisis, immortalized cell lines. A series of mutants with point or deletion mutations within each functionally active domain of SV40 T were also examined for their ability to alter radiosensitivity and induce morphological transformation. Cell clones transfected with T-antigen mutants defective in nuclear localization or origin binding showed increased radioresistance similar to clones transfected with wild-type T-antigen, and expressed morphological changes characteristic of SV40 T-transfected cells. A retinoblastoma susceptibility gene (RB) binding defective mutant showed moderately increased radioresistance (D0 = 174 +/- 10). However, cell clones transfected with three different p53 binding defective mutants showed no change in radiosensitivity (D0 = 132 +/- 5) as compared with neo gene transfected controls. Transfection with T-antigen mutants defective in either the RB or p53 binding domain yielded no morphological alterations characteristic of transformation. These data suggest that the SV40 T/p53 complex may be of importance in the radioresistance phenotype.
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PMID:Transformation and radiosensitivity of human diploid skin fibroblasts transfected with SV40 T-antigen mutants defective in RB and P53 binding domains. 135 59

Deletion mutants of SV40 T-antigen were stably expressed in BALB/c 3T3E cells and characterized in immunoprecipitation assays using T-antigen-specific monoclonal antibodies. The epitope recognized by antibody PAb602 was narrowed to T-antigen residues 230-362. Coprecipitation results were compatible with a p53-binding region between T-antigen amino acids 347 and 517. Amino terminal deletions (1-108 or 98-229) of T-antigen appeared to have pronounced effects on the conformation of distal regions of the molecule, based both on antibody binding and on association with p53.
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PMID:Epitope mapping and conformational analysis of SV40 T-antigen deletion mutants. 137 93

The observed interaction between p53 and the oncoproteins encoded by several DNA tumor viruses suggests that these viruses mediate their transforming activities at least in part by altering the normal growth regulatory function of p53. In this study we examined the effect of viral oncoprotein expression on the transcriptional transactivation function of wild-type p53 in human cells. Plasmids expressing human p53 were cotransfected with either SV40 large T-antigen or human papillomavirus (HPV) type 16 E6 expression plasmids and assayed for transactivation function using a reporter gene driven by a p53-responsive promoter containing multiple copies of the consensus p53 DNA binding motif, TGCCT. Both large T-antigen and E6 were able to inhibit transactivation by wild-type p53. Furthermore, SV40 T-antigen mutants that are defective for p53 binding were not able to inhibit transactivation and HPV E6 proteins that were either mutant or derived from non-oncogenic HPV types and unable to bind p53, had no effect on p53 transactivation. These results demonstrate the physiological relevance of the interaction of SV40 T-antigen and HPV E6 oncoproteins with p53 in vivo and suggest that the transforming functions of these viral oncoproteins may be linked to their ability to inhibit p53-mediated transcriptional activation.
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PMID:The transcriptional transactivation function of wild-type p53 is inhibited by SV40 large T-antigen and by HPV-16 E6 oncoprotein. 146 23

We present evidence for the possible involvement of both the RB and p53 proteins in the regulation of cellular senescence. Human fibroblasts immortalized with an inducible SV40 T-antigen become senescent following the de-induction of T-antigen. Plasmids expressing an alternative source of intact T-antigen restore proliferation but T-antigen deletion mutants lacking either the RB or p53 binding domains are unable to do so. Similarly, combinations of adenovirus E1A + E1B or human papillomavirus E6 + E7 genes are able to replace T-antigen functions and permit cell proliferation, whereas the individual genes do not. These results are discussed in terms of a two-stage model for the escape from in vitro cellular senescence.
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PMID:A role for both RB and p53 in the regulation of human cellular senescence. 165 50

T antigen is able to transactivate gene expression from the simian virus 40 (SV40) late promoter and from several other viral and cellular promoters. Neither the mechanisms of transactivation by T antigen nor the regions of T antigen required for this activity have been determined. To address the latter point, we have measured the ability of a set of SV40 large T antigen mutants to stimulate gene expression in CV-1 monkey kidney cells from the SV40 late promoter and Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter. Transactivation, although reduced, was retained by an N-terminal 138-amino-acid fragment of T antigen. Mutants with alterations at various locations within the N-terminal 85 amino acids transactivated the RSV LTR promoter less well than did wild-type T antigen. Most of these were also partially defective in their ability to transactivate the SV40 late promoter. Two mutants with lesions in the DNA-binding domain that were unable to bind to SV40 DNA were completely defective for transactivation of both promoter, while a third mutant with a lesion in the DNA-binding domain which retained origin-binding activity transactivated both promoters as well as did wild-type T antigen. Only a low level of transactivation was seen with mutant T antigens which had lesions in or near the zinc finger region (amino acids 300 to 350). Mutations which caused defects in ATPase activity, host range/helper function, binding to p53, binding to the retinoblastoma susceptibility protein, or nuclear localization had little or no effect on transactivation. These results suggest that N-terminal portion of T antigen possesses an activation activity. The data are consistent with the idea that the overall conformation of T antigen is important for transactivation and that mutations in other regions that reduce or eliminate transactivation do so by altering the conformation or orientation of the N-terminal region so that its ability to interact with various targets is diminished or abolished.
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PMID:Mapping the transcriptional transactivation function of simian virus 40 large T antigen. 185 53

We used 21 monoclonal antibodies (PAbs 100 to 117, 405, 419, and KT3) specific for different determinants in simian virus 40 (SV40) large T antigen (T) and one antibody specific for p53 that coprecipitates T complexed with p53 (T-p53) to analyze T in SV40-infected CV1 cells. We measured the ATPase specific activity, extent of adenylylation, and p53 content of T precipitated by antibodies directed against the N-terminal region I (0.65 to 0.62 map units), the midregion III (0.43 to 0.28 map units) containing both the ATPase- and nucleotide-binding sites, and the C-terminal region IV (0.28 to 0.17 map units) of T. Lytic T appeared to exist in three different forms with respect to p53 binding and ATPase activity. The most ATPase-active form of T was that precipitated by PAb 122. This T-p53 complex contained only 6% of the total T but contributed 35% of the ATPase activity, on average. Free p53 isolated from 3T6, Ann-1, or L929 cells had no apparent ATPase activity. A second form of T precipitated by several antibodies had little associated p53 but appreciable ATPase activity, accounting for 15 to 20% of total T and 60 to 70% of the ATPase activity. The rest of T constituted the third form and was also depleted in p53 but had a decreased ATPase specific activity. Thus, the remaining 75 to 80% of T had 15 to 20% of the ATPase specific activity. Antibodies specific for region III precipitated T with both altered ATPase activity and altered amounts of bound p53. PAbs 104 and 114 reacted with ATPase-active T but inhibited ADP hydrolysis, suggesting that they were inactivating antibodies. T that was preferentially adenylylated in vitro corresponded to T that was also preferentially ATPase active. T bound to p53 was adenylylated to a higher specific activity than total T. In addition, p53 itself was significantly adenylylated under these conditions. The results suggest that ATPase activity and p53 binding are structurally and functionally related and that p53 alters biochemical activities of T and plays a role in productive infection.
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PMID:Characterization of simian virus 40 large T antigen by using different monoclonal antibodies: T-p53 complexes are preferentially ATPase active and adenylylated. 244 96

We have compared the ATPase, DNA-binding, and helicase activities of free simian virus 40 (SV40) large T antigen (To) and T antigen complexed with cellular p53 (T+p53). Each activity is essential for productive viral infection. The T+p53 and To fractions were prepared by sequential immunosorption of infected monkey cells with monoclonal antibodies specific for p53 and T antigen. The immune-complexed T fractions were then assayed in parallel. For ATP hydrolysis, the Vmax for T+p53 was 143 nmol of ADP per min per mg of protein, or 18-fold greater than for To. ATP had no effect on the stability of the T+p53 complex. The T+p53 complex was significantly more active than To in hydrolyzing dATP, dGTP, GTP, and UTP. Of the nucleotide substrates tested, the greatest relative increase (T+p53/To) was in hydrolyzing dGTP and GTP. In DNase footprinting assays performed under replication conditions, the T+p53 complex protected regions I, II, and III of origin DNA while equivalent amounts of To protected only regions I and II. Region III is known to contribute to the efficiency of DNA replication and contains the SP1-binding sites of the early viral promoter. The T+p53 fraction was also a more efficient helicase than To, especially with a GC-rich primer and template. Thus, the T+p53 complex has enhanced ATPase, GTPase, DNA-binding, and helicase activities. These findings imply that complex formation between cellular monkey p53 and SV40 T antigen modulates a number of essential activities of T in SV40 productive infection.
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PMID:The p53 complex from monkey cells modulates the biochemical activities of simian virus 40 large T antigen. 252 75

We have analyzed the biochemical properties of a nonviable simian virus 40 (SV40) mutant encoding a large T antigen (T) bearing an amino acid substitution (Pro-584-Leu) in its hydrophobic region. Mutant 5080 has an altered cell type specificity for transformation (transforming mouse C3H10T1/2 but not rat REF52 cells), is defective for viral DNA replication, and encodes a T that is unable to form a complex with the cellular p53 protein (K. Peden, A. Srinivasan, J. Farber, and J. Pipas, Virology 168:13-21, 1989). In this article, we show that 5080-transformed C3H10T1/2 cell lines express an altered T that is synthesized at a significantly higher rate but with a shorter half-life than normal T from wild-type SV40-transformed cells. 5080 T did not oligomerize beyond 5 to 10S in size compared with normal T, which oligomerized predominantly to 14 to 20S species. In addition, the 5080 T complex had significantly decreased ATPase activity and had a 10-fold-lower level of in vivo phosphorylation compared with that of normal T. Two-dimensional phosphopeptide analysis indicated several changes in the specific 32P labeling pattern, with altered phosphorylation occurring at both termini of the mutant protein compared with the wild-type T. Loss of p53 binding is therefore concomitant with changes in ATPase activity, oligomerization, stability, and in vivo phosphorylation of T and can be correlated with defective replication and restricted transformation functions. That so many biochemical changes are associated with a single substitution in the hydrophobic region of T is consistent with its importance in regulating higher-order structural and functional relationships in SV40 T.
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PMID:Properties of a simian virus 40 mutant T antigen substituted in the hydrophobic region: defective ATPase and oligomerization activities and altered phosphorylation accompany an inability to complex with cellular p53. 254 11

SV40 T-antigen (T-ag) is localized in both the nucleus (nT-ag) and plasma membrane (pmT-ag) of cells and provides multiple functions necessary for cell transformation. The pmT-ag population is structurally very similar to the nT-ag. Transport to the cell surface is by an unknown mechanism that does not involve the secretory pathway. The disposition of T-ag in the membrane exposes both the amino and the carboxyl terminus on the exterior of the cell. Nuclear-transport-defective mutants of T-ag can transform established cells in culture, but not primary cells, suggesting that non-nuclear forms of T-ag may mediate some transformation-related process(es). A non-cytolytic protein extraction technique utilizing 1-butanol solubilized from SV40-transformed cells a multimeric complex composed of pmT-ag and at least five cellular proteins ranging in size from 35,000 (35K) to 60K M. Both amino- and carboxylterminal T-ag-specific monoclonal antibodies co-precipitated T-ag and the 35-60K Mr proteins, but antibodies against the internal portion of T-ag precipitated only uncomplexed T-ag. The growth state of the cells markedly influenced the expression of the T-ag-containing surface complexes; more complexes were recovered from actively dividing cells than from confluent cell cultures, and suspension cells yielded more complexes than cells on a substratum. The complex exhibited a highly dynamic association with the cell membrane, as demonstrated by pulse-chase analysis. The characteristics of growth-dependent expression and rapid turnover rate suggest a functional role for the membrane complex. The identities of the cellular proteins in the complex with pmT-ag are unknown, although one member (56K) is recognized by p53-specific monoclonal antibodies.
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PMID:SV40 T-antigen as a dual oncogene: structure and function of the plasma membrane-associated population. 255 88


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