Species-specific phosphorylation of mouse and rat p53 in simian virus 40-transformed cells.

We have analyzed in detail the phosphorylation of p53 from normal (3T3) and simian virus 40 (SV40)-transformed (SV3T3) BALB/c mouse cells and from normal (F111) and SV40-transformed [FR(wt648)] rat cells by two-dimensional tryptic peptide mapping and phosphoamino acid analyses. To accommodate the different half-lives of p53 in normal (half-life, 15 min) and transformed (half-life, 20 h) cells and possible differences in the rates of turnover of phosphate at specific sites, cells were labeled for 2 h (short-term labeling) or 18 h (long-term labeling). Depending on the labeling conditions, either close similarities or marked differences were observed in the phosphorylation patterns of p53 from normal and transformed cells. After the 2-h labeling, the phosphorylation patterns of p53 from normal and transformed mouse cells were quite similar. In contrast, p53 from normal and transformed rat cells exhibited dramatic quantitative and qualitative differences under these labeling conditions. The reverse was found after an 18-h label leading to steady-state phosphorylation of p53 in transformed cells: while p53 in transformed mouse cells revealed a marked quantitative increase in phosphorylation compared with p53 from normal cells, the corresponding patterns of p53 from normal and transformed rat cells were similar. Our data thus indicate species-specific differences in the phosphorylation of mouse and rat p53 in SV40-transformed cells, reflected by (i) different turnover rates at specific sites in mouse and rat p53 and (ii) phosphorylation of nonhomologous serine and threonine residues in rat p53, as revealed by indirect assignment of phosphorylation sites to the phosphopeptides of rat p53. Analyses of p53 from the SV40 tsA58 mutant-transformed F111 cell lines FR(tsA58)A (N type) and FR(tsA58)57 (A type) yielded no conclusive evidence for a direct correlation between phosphorylation of p53, the metabolic stabilization of p53, and expression of the transformed phenotype.     

Targeting of p300/CREB binding protein coactivators by simian virus 40 is mediated through p53

The primary transforming functions of simian virus 40 large T antigen (SV40 LT) are conferred primarily through the binding and inactivation of p53 and the retinoblastoma family members. Normal p53 function requires an association with the CREB binding protein (CBP)/p300 coactivators, and a ternary complex containing SV40 LT, p53, and CBP/p300 has been identified previously. In this report, we have evaluated a secondary function of p53 bound to the SV40 LT complex in mediating the binding of human CBP/p300. We demonstrate that p53 associated with SV40 LT was posttranslationally modified in a manner consistent with the binding of CBP/p300. Furthermore, expression of SV40 LT induced the proportion of p53 phosphorylated on S15. An essential function for p53 in bridging the interaction between SV40 LT and CBP/p300 was identified through the reconstitution of the SV40 LT-CBP/p300 complex upon p53 reexpression in p53-null cells. In addition, the SV40 LT-CBP/p300 complex was disrupted through RNA interference-mediated depletion of endogenous p53. We also demonstrate that SV40 LT was acetylated in a p300- and p53-dependent manner, at least in part through the CH3 domain of p300. Therefore, the binding of p53 serves to modify SV40 LT by targeting CBP and p300 binding to direct the acetylation of SV40 LT.     

Evidence for free and metabolically stable p53 protein in nuclear subfractions of simian virus 40-transformed cells.

To determine functional subcellular loci of p53, a cellular protein associated with cellular transformation, we analyzed the nucleoplasmic, chromatin, and nuclear matrix fractions from normal mouse 3T3 cells, from methylcholanthren-transformed mouse (MethA) cells, and from various simian virus 40 (SV40)-transformed cells for the presence of p53. In 3T3 and MethA cells, p53 was present in all nuclear subfractions, suggesting an association of p53 with different structural components of the nucleus. In 3T3 cells, p53 was rapidly turned over, whereas in MethA cells, p53 was metabolically stable. In SV40-transformed cells, p53 complexed to large tumor antigen (large T) was found in the nucleoplasmic and nuclear matrix fractions, as described previously (M. Staufenbiel and W. Deppert, Cell 33:173-181, 1983). In addition, however, metabolically stable p53 not complexed to large T (free p53) was also found in the chromatin and nuclear matrix fractions of these cells. This free p53 did not arise by dissociation of large T-p53 complexes, suggesting that stabilization of p53 in SV40-transformed cells can also occur by means other than formation of a complex with large T.     

Antigenic structure of simian virus 40 large tumor antigen and association with cellular protein p53 on the surfaces of simian virus 40-infected and -transformed cells.

The antigenic structure of simian virus 40 (SV40) large tumor antigen (T-ag) in the plasma membranes of SV40-transformed mouse cells and SV40-infected monkey cells was characterized as a step toward defining possible biological function(s). Wild-type SV40, as well as a deletion mutant of SV40 (dl1263) which codes for a truncated T-ag with an altered carboxy terminus, was used to infect permissive cells. Members of a series of monoclonal antibodies directed against antigenic determinants on either the amino or the carboxy terminus of the T-ag polypeptide were able to precipitate surface T-ag (as well as nuclear T-ag) from both SV40-transformed and SV40-infected cells. Cellular protein p53 was coprecipitated with T-ag by all T-ag-reactive reagents from the surface and nucleus of SV40-transformed cells. In contrast, T-ag, but not T-ag-p53 complex, was recovered from the surface of SV40-infected cells. These results confirm that nuclear T-ag and surface T-ag are highly related molecules and that a complex of SV40 T-ag and p53 is present at the surface of SV40-transformed cells. Detectable levels of such a complex do not appear to be present on SV40-infected cells. Both the carboxy and amino termini of T-ag are exposed on the surfaces of SV40-transformed and -infected cells. The possible relevance of the presence of a T-ag-p53 complex on the surface of SV40-transformed cells and its absence from SV40-infected cells is considered.     

Complex of simian virus 40 large-T antigen and host 53,000-molecular-weight protein in monkey cells.

Mouse cells transformed by simian virus 40 (SV40) have been shown to contain a complex of the virus-coded large-T antigen with a host 53,000-molecular-weight (53K) protein. Initial attempts to detect a similar complex in lytically infected cells were unsuccessful, and it therefore seemed that the complex might be peculiar to transformed or abortively transformed nonpermissive cells. Immunoprecipitation of [32P]phosphate-labeled extracts of SV40-infected CV-1 African green monkey kidney cells with antibodies specific for large-T or the 53K protein revealed that the large-T-53K protein complex was formed during lytic infections. Only a minor fraction of the large-T present was associated with 53K protein, and large-T and the 53K host protein cosedimented during centrifugation through sucrose gradients. We used monospecific sera and monoclonal antibodies to study the rate of synthesis and phosphorylation of the 53K protein during lytic infections. Infection of CV-1 cells with SV40 increased the rate of synthesis of the 53K protein fivefold over that in mock-infected cells. At the same time, the rate of phosphorylation of the 53K protein increased more than 30-fold compared with control cultures. Monkey cells transformed by UV-irradiated SV40 (Gluzman et al., J. Virol. 22:256-266, 1977) also contained the large-T-53K protein complex. The formation of the complex is therefore not a peculiarity of SV40-transformed rodent cells but is a common feature of SV40 infections.     

Simian virus 40 large T antigen induces or activates a protein kinase which phosphorylates the transformation-associated protein p53.

The cellular phosphoprotein p53 is presumably involved in simian virus 40 (SV40)-induced transformation. We have monitored changes in the state of phosphorylation of p53 from normal versus SV40-infected or -transformed cells. In normal cells, p 53 was hardly phosphorylated. Upon infection or transformation, a quantitative and qualitative increase in p53 phosphorylation was observed as revealed by two-dimensional phosphopeptide analysis. This increase was dependent on a functional large T antigen. In rat cells, enhanced phosphorylation of p53 resulted in conversion to a second, electrophoretically distinct form. In cells transformed with transformation-defective mutants, phosphorylation of p53 was reduced and conversion to form 2 was inefficient. These data suggest (i) that SV40 large T antigen induces or activates a protein kinase, one substrate of which is p53, (ii) that transformation-defective mutants are impaired in kinase induction, and (iii) that either a certain phosphorylation state of p53 or the SV40-induced kinase is critical for efficient transformation.     

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.

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.     

Dephosphorylation of simian virus 40 large-T antigen and p53 protein by protein phosphatase 2A: inhibition by small-t antigen.

Simian virus 40 (SV40) large-T antigen and the cellular protein p53 were phosphorylated in vivo by growing cells in the presence of 32Pi. The large-T/p53 complex was isolated by immunoprecipitation and used as a substrate for protein phosphatase 2A (PP2A) consisting of the catalytic subunit (C) and the two regulatory subunits, A and B. Three different purified forms of PP2A, including free C, the AC form, and the ABC form, could readily dephosphorylate both proteins. With both large-T and p53, the C subunit was most active, followed by the AC form, which was more active than the ABC form. The activity of all three forms of PP2A toward these proteins was strongly stimulated by manganese ions and to a lesser extent by magnesium ions. The presence of complexed p53 did not affect the dephosphorylation of large-T antigen by PP2A. The dephosphorylation of individual phosphorylation sites of large-T and p53 were determined by two-dimensional peptide mapping. Individual sites within large-T and p53 were dephosphorylated at different rates by all three forms of PP2A. The phosphates at Ser-120 and Ser-123 of large-T, which affect binding to the origin of SV40 DNA, were removed most rapidly. Three of the six major phosphopeptides of p53 were readily dephosphorylated, while the remaining three were relatively resistant to PP2A. Dephosphorylation of most of the sites in large-T and p53 by the AC form was inhibited by SV40 small-t antigen. The inhibition was most apparent for those sites which were preferentially dephosphorylated. Inhibition was specific for the AC form; no effect was observed on the dephosphorylation of either protein by the free C subunit or the ABC form. The inhibitory effect of small-t on dephosphorylation by PP2A could explain its role in transformation.     

Modulation of p53 protein expression during cellular transformation with simian virus 40.

We analyzed the relation of metabolic stabilization of the p53 protein during cellular transformation by simian virus 40 (SV40) to (i) expression of the transformed phenotype and (ii) expression of the large tumor antigen (large T). Analysis of SV40-tsA28-mutant-transformed rat cells (tsA28.3 cells) showed that both p53 complexed to large T and free p53 (W. Deppert and M. Haug, Mol. Cell. Biol. 6:2233-2240, 1986) were metabolically stable when the cells were cultured at 32 degrees C and expressed large T and the transformed phenotype. At the nonpermissive temperature (39 degrees C), large-T expression is shut off in these cells and they revert to the normal phenotype. In such cells, p53 was metabolically unstable, like p53 in untransformed cells. To determine whether metabolic stabilization of p53 is directly controlled by large T, we next analyzed the metabolic stability of complexed and free p53 in SV40 abortively infected normal BALB/c mouse 3T3 cells. We found that neither p53 in complex with large T nor free p53 was metabolically stable. However, both forms of p53 were stabilized in SV40-transformed cells which had been developed in parallel from SV40 abortively infected cultures. Our results indicate that neither formation of a complex of p53 with large T nor large-T expression as such is sufficient for a significant metabolic stabilization of p53. Therefore, we suggest that metabolic stabilization of p53 during cellular transformation with SV40 is mediated by a cellular process and probably is the consequence of the large-T-induced transformed phenotype.     

Surface T-antigen expression in simian virus 40-transformed mouse cells: correlation with cell growth rate.

Cell growth control appears to be drastically altered as a consequence of transformation. Because the cell surface appears to have a role in modulating cell growth and simian virus 40 (SV40)-transformed cells express large T antigen (T-Ag) in the plasma membrane, we investigated whether surface T-Ag expression varies according to cell growth rate. Different growth states were obtained by various combinations of seeding density, serum concentration, and temperature, and cell cycle distributions were determined by flow microcytofluorometry. Actively dividing SV40-transformed mouse cell cultures were consistently found to express higher levels of surface T-Ag and T-Ag/p53 complex than cultures in which cells were mostly resting. In addition, the T-Ag/p53 complex disappeared from the surface of tsA7-transformed cells cultured under restrictive conditions known to induce complete growth arrest (39.5 degrees C), although the surface complex did not disappear from other tsA transformants able to keep cycling at 39.5 degrees C. These results suggest that surface SV40 T-Ag or surface T-Ag/p53 complex, or both, are involved in determining the growth characteristics of SV40-transformed cells.     

Radioimmunoassay of the cellular protein p53 in mouse and human cell lines

We have developed quantitative radioimmunological solid phase assays for the host protein p53 from mouse cells and from human cells. The first assay, for mouse p53, depends on having two monoclonal antibodies reacting with different determinants on the p53 molecule. With this assay we have shown that SV40-transformed cells have approximately 100-fold more p53 than untransformed mouse cells and that other transformed cells have intermediate levels. Embryonal carcinoma cell lines have approximately 50-fold less p53 than SV40-transformed cells. This is in contrast to the high levels of incorporation of [35S]methionine into p53 in these cells and indicates that metabolic labelling is not a valid approach for measuring p53 levels. The second assay, for human p53, required a different approach and made use of the anti-p53 antibodies detected in the sera of some breast cancer patients. Human tumour cell lines contained amounts of p53 varying from the high level seen in SV40-transformed human fibroblasts down to less than one hundredth of this amount. Normal human cells showed low levels of p53. The data confirm that many, but not all, human tumour cell lines contain more p53 than normal cells.     

Phosphorylation of MDMX mediated by Akt leads to stabilization and induces 14-3-3 binding

The critical tumor suppressor p53 is mutated or functionally inactivated in nearly all cancers. We have shown previously that the MDM2-MDMX complex functions as an integral unit in targeting p53 for degradation. Here we identify the small protein 14-3-3 as a binding partner of MDMX, which binds at the C terminus (Ser367) in a phosphorylation-dependent manner. Importantly, we demonstrate that the serine/threonine kinase Akt mediates phosphorylation of MDMX at Ser367. This phosphorylation leads to stabilization of MDMX and consequent stabilization of MDM2. Previous studies have shown that Akt phosphorylates and stabilizes MDM2. Our data suggest that stabilization of MDMX by Akt may be an alternative mechanism by which Akt up-regulates MDM2 protein levels and exerts its oncogenic effects on p53 in tumor cells.     

Excess wild-type p53 blocks initiation and maintenance of simian virus 40 transformation.

Wild-type (wt) murine p53 has been tested for its ability to block and reverse the transforming effects of simian virus 40 (SV40) large T antigen. Established and precrisis mouse cells overexpressing exogenously introduced wt p53 became resistant to SV40 transformation. The introduction of excess wt p53 into SV40-transformed precrisis cells reverted their transformed phenotype. However, the phenotype of SV40-transformed established cells was not reverted by excess wt p53. We conclude that an antioncogenic action of wt p53 is exerted during SV40 transformation and that in precrisis cells, the antitransforming action of wt p53 can be exerted both at initiation and during the maintenance of transformation.     

Effects of the cellular p53 protein on Simian-virus-40-T-antigen-catalyzed DNA unwinding in vitro

It is known that large T antigen, the regulatory protein encoded by Simian virus 40 (SV40), forms tight complexes with the cellular p53 protein in SV40-transformed rodent cells. Using immunoaffinity procedures we have purified large T antigen and, in separate experiments, the cellular p53 protein. The two proteins formed complexes in vitro which bound well to double-stranded DNA fragments although in a sequence-unspecific manner. Free, uncomplexed T antigen readily converted double-stranded DNA into a single-stranded form whereas in-vitro-formed p53-T-antigen complexes were inactive in this reaction. We conclude that one function of p53 in SV40-transformed mouse cells could be the inhibition of the replication initiating activity of T antigen.     

Simian virus 40 large T antigen disrupts genome integrity and activates a DNA damage response via Bub1 binding

Simian virus 40 (SV40) large T antigen (LT) is a multifunctional protein that is important for viral replication and oncogenic transformation. Previously, infection of monkey or human cells with SV40 was shown to lead to the induction of DNA damage response signaling, which is required for efficient viral replication. However, it was not clear if LT is sufficient to induce the damage response and, if so, what the genetic requirements and functional consequences might be. Here, we show that the expression of LT alone, without a replication origin, can induce key DNA damage response markers including the accumulation of γ-H2AX and 53BP1 in nuclear foci. Other DNA damage-signaling components downstream of ATM/ATR kinases were induced, including chk1 and chk2. LT also bound the Claspin mediator protein, which normally facilitates the ATR activation of chk1 and monitors cellular replication origins. Stimulation of the damage response by LT depends mainly on binding to Bub1 rather than to the retinoblastoma protein. LT has long been known to stabilize p53 despite functionally inactivating it. We show that the activation of a DNA damage response by LT via Bub1 appears to play a major role in p53 stabilization by promoting the phosphorylation of p53 at Ser15. Accompanying the DNA damage response, LT induces tetraploidy, which is also dependent on Bub1 binding. Taken together, our data suggest that LT, via Bub1 binding, breaches genome integrity mechanisms, leading to DNA damage responses, p53 stabilization, and tetraploidy.     

Simian virus 40 can overcome the antiproliferative effect of wild-type p53 in the absence of stable large T antigen-p53 binding.

In simian virus 40 (SV40)-transformed cells, a tight complex is formed between the viral large T antigen (large T) and p53. It has been proposed that this complex interferes with the antiproliferative activity of p53. This notion was tested in primary rat fibroblasts by assessing the ability of SV40-mediated transformation to be spared from the inhibitory effect of wild-type (wt) p53. The data indicate that relative to transformation induced by myc plus ras, SV40-plus-ras-mediated focus formation was indeed much less suppressed by p53 plasmids. A majority of the resultant cell lines made a p53 protein with properties characteristic of a wt conformation. Furthermore, cell lines expressing stably both SV40 large T and a temperature-sensitive p53 mutant continued to proliferate at a temperature at which this p53 assumes wt-like properties and normally causes a growth arrest. Surprisingly, at least partial resistance to the growth-inhibitory effect of wt p53 was also evident when transformation was mediated by an SV40 deletion mutant, encoding a large T which does not bind p53 detectably. In addition to supporting the idea that SV40 can overcome the growth-restrictive activity of wt p53, these findings strongly suggest that at least part of this effect does not require a stable association between p53 and large T.     

Altered phosphorylation of free and bound forms of monkey p53 and simian virus 40 large T antigen during lytic infection.

We have identified the phosphorylation sites in monkey p53 as well as specific changes in the phosphorylation state of free and complexed forms of simian virus 40 (SV40) large T antigen (T) and monkey p53 isolate from SV40 lytically infected CV1 cells. Phosphopeptide analyses of free T and p53 (To and p53o) and complexed T and p53 (T+ and p53+) fractions indicated several quantitative increases in the specific phosphorylation of complexed forms of both proteins. The N terminus of monkey p53+ is phosphorylated at Ser-9, Ser-15, Ser-20, either Ser-33 or Ser-37, and at least one of Ser-90 to Ser-99. The C-terminal sites are Ser-315 and Ser-392. On comparing p53+ with p53o, we found that labeling of the two N-terminal phosphotryptic peptides encompassing residues 1 to 20 and 33 to 101 was increased fivefold and that Ser-315 was sevenfold more labeled than was Ser-392. When T+ was compared with To, the N-terminal peptide containing phosphorylation sites Ser-106 through Thr-124 was twofold more labeled, the peptide containing Ser-657 through Ser-679 was sixfold more labeled and contained up to four phosphorylated serine residues, and Ser-639 and Thr-701 appeared unchanged. Overall, T+ molecules appeared to contain 3.5 mol more of labeled phosphate than did To, with the N-terminal peptide appearing fully phosphorylated. The phosphopeptide patterns obtained for lytic T+ and To fractions were nearly identical to those found for wild-type SV40 T (stably complexed with mouse p53) and mutant 5080 T (defective for p53 binding) expressed in transformed C3H10T1/2 cells (L. Tack, C. Cartwright, J. Wright, A. Srinivasan, W. Eckhart, K. Peden, and J. Pipas, J. Virol. 63:3362-3367, 1989). These results indicate that increases in specific phosphorylation sites in both T+ and p53+ correlate with the association of T with p53. The enhanced phosphorylation state may be a consequence of complex formation between T and p53 or reflect an increased affinity of p53 for highly phosphorylated forms of T.     

SV40 large T-antigen and transformation related protein p53 are associated in situ with nuclear RNP structures containing hnRNA of transformed cells

The localization of SV40 large T-antigen (T-Ag) and the cellular protein p53 in the nuclei of mouse and human SV40-transformed cells and of a methylcholanthrene-transformed mouse cell line, was studied. Their detection by ultrastructural immunocytochemistry with specific monoclonal antibodies employed two complementary methods used in parallel. These consisted of indirect immunoperoxidase labelling carried out before embedment on Triton-permeabilized cells, or indirect immunogold labelling applied to thin sections of cells embedded in Lowicryl K4M. The results indicate that in SV40-transformed cells both proteins are chiefly localized on peri- and interchromatin RNP fibrils. This shows that they occur in structures involved in the synthesis and processing of hnRNA. The nucleoli and chromatin did not appear to be labelled. In methylcholanthrene-transformed cells the protein p53 (in the absence of large T-Ag) was also detected on peri- and interchomatin fibrils. Taken together with recent results which demonstrated that, during lytic infection, T-Ag was associated chiefly with cellular chromatin (Harper, F, Florentin, Y & Puvion, E, Exp cell res 161 (1985) 434) [33], our experiments provide evidence that the transforming function of SV40 large T-Ag is dissociable from its function in SV40 lytic infection in terms of its subnuclear distribution.     

Phosphorylation of human p53 at serine 46 determines promoter selection and whether apoptosis is attenuated or amplified

The capacity of DNA damaging agents to induce apoptosis is regulated by target gene induction by p53. We found that p53 targeted MDM2 in cells in which DNA repair was occurring, but persistent DNA damage induced by chemotherapy led p53 to selectively target PTEN. High dose chemotherapy induced the phosphorylation of p53 on serine 46, whereas low dose chemotherapy did not. A nonphosphorylatable serine 46 to alanine p53 mutant (S46A) targeted the MDM2 promoter in preference to that for PTEN. A serine 46 to aspartate mutant (S46D, a phosphorylation mimic) targeted PTEN in preference to MDM2. These observations show that phosphorylation of serine 46 in p53 is sufficient for it to induce the PTEN (phosphatase and tensin homolog deleted on chromosome ten) tumor suppressor protein in preference to MDM2. S46A induced significantly less cell death than the S46D in cells. The phosphorylation-induced change of p53 promoter targeting suppresses the induction of MDM2 and the formation of the autoregulatory feedback loop. Induction of PTEN by p53 followed by expression of PTEN inhibits AKT-induced translocation of MDM2 into the nucleus and sustains p53 function. The protection of p53 from MDM2 by PTEN and the damage-induced activation of PTEN by phosphorylated p53 leads to the formation of an apoptotic amplification cycle in which p53 and PTEN coordinately increase cellular apoptosis.