(Received for publication, September 18, 1995; and in revised form, December 9, 1995)
From the
We have discovered that the ability of the tumor suppressor protein p53 to bind to the viral large T antigen (TAg) oncogene product is regulated by divalent cations. Both proteins were purified from an insect cell line infected with the appropriate baculovirus expression vector. In a two-site capture enzyme-linked immunosorbent assay, complex formation between the purified proteins is strictly dependent on the addition of specific concentrations of divalent metal ions, notably zinc, copper, cadmium, cobalt, manganese, and nickel. In the presence of zinc the pattern of proteolytic fragments obtained when TAg was subjected to proteolysis by endoproteinase Glu-C (V8) was strikingly different, supporting the idea that a conformational change in TAg associated with ion binding is required for it to complex with p53. Monoclonal antibody analysis provides supporting evidence for a conformational change. When TAg was captured onto an enzyme-linked immunosorbent assay plate coated with PAb 419 as opposed to many other anti-TAg antibodies, complex formation was completely independent of the presence of additional divalent cations. Our results suggest that the ability of p53 and TAg to form a stable complex in vitro is dependent upon a regulatory domain residing in the N terminus of TAg, zinc ions or the binding of a specific monoclonal antibody (PAb 419) provoking a conformational change in TAg that facilitates and supports complex formation.
The large T antigen (TAg) ()of the DNA papova virus
SV40 is a 94-kDa multifunctional phosphoprotein composed of 708 amino
acids that is essential for viral replication and transformation of
infected cells(1, 2) .The repertoire of biochemical
activities that characterize TAg, and that mediate replication of SV40,
include its ability to bind specifically to viral DNA, coordinate the
assembly of the replication apparatus, and act as a helicase at the
replication fork and the ability to regulate transcription of viral
genes(3, 4) . However, it is the capacity of TAg to
bind to and inactivate endogenous cellular proteins encoded by the p53
and retinoblastoma (Rb) tumor suppressor genes that is considered to be
a critical event in transformation of infected
cells(5, 6, 7) .
Loss of wild type p53 activity is frequently identified in human tumors (8) . This loss of functional p53 is commonly the result of a mutation within the gene, but complex formation with other endogenous or exogenous (virally encoded) proteins has the same effect(9) . In the absence of wild type p53, which has been described as the ``guardian of the genome,'' the cell is predisposed to acquire genetic abnormalities and thus to neoplastic transformation(10) . Therefore, it is important to define the biochemical mechanisms that regulate the interaction of p53 with other proteins that may inactivate it. The large TAg of the SV40 virus is the archetype of p53 binding proteins, and indeed it is the ability of p53 and TAg to form a stable complex that originally enabled p53 to be identified(5, 6) . The identification of mechanisms that determine the ability of p53 and TAg to bind to each other may provide further insight into the regulation of events that permit viral transformation of cells. Furthermore, since putative TAg-like proteins have also been identified in human tumor cell lines the biochemical basis and pathophysiological significance of these interactions may be elucidated(11) .
The analysis of mutant or truncated forms of TAg has demonstrated that the various biochemical functions of TAg can be assigned to specific domains within the molecule. It has therefore been possible to examine the dependence of the various biological functions of TAg on its specific biochemical activities. Thus, it has been shown that the ability to support viral replication and the capacity to transform cells are independent functions of TAg(12) . However, complex formation with p53 is not an essential prerequisite for TAg to support these activities(13, 14) . The introduction of certain point mutations within a hydrophobic region of TAg between amino acids 570 and 590 may inhibit the ability of TAg to bind to p53. However, despite being unable to bind to p53 these mutants are still competent in some cell transformation assays(15) . Zhu et al.(16) have also observed that loss of the ability to bind to either p53 or the retinoblastoma protein need not impair the transactivation activity of TAg to any significant extent(16) . Conversely, replication and transformation may be defective without affecting the ability of TAg to bind to p53(17) . However in the immortalization of primary cell lines the capacity of TAg to bind to and inactivate p53 appears to be critical(18) .
In these studies we have purified TAg and wild type human p53 in a biochemically active state. The purified TAg was able to direct viral DNA synthesis in a standard SV40 replication assay, and this activity could be inhibited with the addition of p53. We sought to determine potential mechanisms that may regulate the interaction of TAg and p53 using a two-site capture ELISA. Our results suggest that a conformational change in TAg mediated by specific concentrations of divalent cations or binding of a specific monoclonal antibody (PAb 419) that recognizes an epitope contained within the N-terminal 82 amino acids defines the ability of TAg to recognize and bind to p53 in a stable complex(19, 20) . Thus, these findings suggest that a conformational change in TAg emanating from or nucleating to the N terminus of TAg may reveal another domain that serves to regulate the interaction of TAg with p53.
The cells
expressing TAg were then pelleted and lysed on ice for 15 min in lysis
buffer (0.3 M NaCl, 10% (v/v) glycerol, 0.5% (v/v) Nonidet
P-40, 20 mM Tris-HCl, pH 9.0, 1 mM EDTA, 0.1 mM PMSF, 0.2 µg/ml leupeptin, 0.2 µg/ml pepstatin, 0.2
µg/ml aprotinin, and 1 mM DTT). Following lysis, the cell
debris was pelleted by centrifugation at 4 °C for 30 min at 14,000
rpm, and the supernatant was dispensed into a sterile Falcon tube and
neutralized with the addition of half the volume of TAg lysate
neutralization buffer, pH 6.8 (0.3 M NaCl, 10 mM Tris-HCl, pH 6.8, 10% (v/v) glycerol, 1 mM EDTA, 0.1
mM PMSF, 0.2 µg/ml leupeptin, 0.2 µg/ml pepstatin, 0.2
µg/ml aprotinin, and 1 mM DTT) before being loaded onto a
DEAE-Sepharose column with a bed volume of 20 ml that had initially
been equilibrated with 10 column volumes of 0.3 M NaCl loading
buffer (0.3 M NaCl, 20 mM Tris-HCl, pH 8.0, 10% (v/v)
glycerol, 1% (v/v) Nonidet P-40, 1 mM EDTA, 0.1 mM PMSF, 0.2 µg/ml leupeptin, 0.2 µg/ml pepstatin, 0.2
µg/ml aprotinin, and 1 mM DTT). The column was then washed
with 10 column volumes of 0.3 M NaCl loading buffer or until
no further protein (as determined by Bradford assay(24) ) was
present in the flow-through. The flow-through was then run on a protein
A-Sepharose column with a bed volume of 1 ml that had been equilibrated
as described above. The flow-through from this column was collected and
loaded onto a PAb 419 immunoaffinity column prepared by binding and
cross-linking 1 mg of the purified anti-TAg monoclonal antibody PAb 419
to protein A-Sepharose beads. This column was equilibrated as described
above after all unbound 419 antibody had been removed from the column
by washing the column with 5 column volumes of TAg elution buffer (20
mM Tris-HCl, pH 8.5, 1 M NaCl, 1 mM MgCl, 1 mM EDTA, 10% (v/v) glycerol, 55%
(v/v) ethylene glycol, 0.1 mM PMSF, 0.2 µg/ml leupeptin,
0.2 µg/ml pepstatin, 0.2 µg/ml aprotinin, and 1 mM DTT). Prior to elution of TAg bound to the 419 column the column
was washed with 10 column volumes of 1 M NaCl buffer (50
mM Tris-HCl, pH 8.0, 1 M NaCl, 10% (v/v) glycerol, 1
mM EDTA, 0.1 mM PMSF, 0.2 µg/ml leupeptin, 0.2
µg/ml pepstatin, 0.2 µg/ml aprotinin, and 1 mM DTT)
and 10 column volumes of 10% ethylene glycol with 0.5 M NaCl
wash buffer (50 mM Tris-HCl, pH 8.5, 0.5 M NaCl, 10%
(v/v) ethylene glycol, 10% (v/v) glycerol, 1 mM EDTA, 0.1
mM PMSF, 0.2 µg/ml leupeptin, 0.2 µg/ml pepstatin, 0.2
µg/ml aprotinin, and 1 mM DTT). The TAg bound to the
column was then eluted with TAg elution buffer. Prior to use in any of
the subsequent assays the TAg eluate was dialyzed in 2
1000 ml
of dialysis buffer (10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 1 mM EDTA, 50% (v/v) glycerol, and 1 mM DTT), after which it was stored at -20 °C. The presence
of TAg within the dialyzed eluate was determined by SDS-polyacrylamide
gel electrophoresis and Western blotting, and the concentration of the
TAg was established by the Bradford assay(24, 25) .
Sf9 cells expressing p53 were lysed on ice for 20 min with a lysis buffer containing 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1% (v/v) Nonidet P-40, 5 mM DTT, 1 mM PMSF, and 0.15 M NaCl. The lysate was then centrifuged at 14,000 rpm at 4 °C for 20 min, and the supernatant was diluted 3-fold in Buffer B (15% (v/v) glycerol, 25 mM HEPES, pH 7.6, 0.1% (v/v) Triton X-100, 5 mM DTT, and 1 mM benzamidine) before filtration through a 0.45-µm filter. Thereafter, the sample was loaded onto a 5-ml heparin-Sepharose Hi-Trap column (Pharmacia Biotech Inc.) and eluted with a linear KCl gradient from 0.05 to 1 M in Buffer B. This purification procedure resolves three forms of p53 that are produced in Sf9 cells; two forms are in an activated state (forms I and II), and one is in a latent state (form III) for sequence-specific DNA binding. The p53 used in the experiments described in this report is termed activated form II.
The ability of p53 to form
stable complexes with TAg was further investigated using a standard
ELISA modified as follows(29) . The anti-p53 antibody DO-1 was
used to bind p53 (1 µg/ml in a buffer solution containing 100
mM NaCl, 10 mM HEPES, pH 7.5, 5 mM KCl, and
0.01% (v/v) Nonidet P-40 in mille-Q water) to the ELISA plate overnight
at 4 °C. After the plate had been washed, TAg diluted to 1
µg/ml in the same buffer as the p53 was added to each well, and the
incubation was continued for 3 h at 4 °C. After further washes,
bound TAg was detected by probing with the rabbit polyclonal anti-TAg
antibody 115, horseradish peroxidase-conjugated swine anti-rabbit
immunoglobulins, and 3,3`,5,5`-tetramethylbenzidine developing solution
as described previously. The development reaction was arrested with the
addition of an equivalent volume of 1 M HSO
, and the absorbance of the reaction
products at 450 nm was recorded by a Dynatech 5000 plate reader.
The
influence of zinc ions on the ability of p53 and TAg to form stable
complexes in the ELISA was investigated by titrating Zn (from the chloride salt) into the reaction. The dependence of
complex formation on the presence of Zn
was further
established by titrating EDTA into the reaction. Since it has been
shown that other divalent cations may bind to p53, the influence of
other such ions (Ca
, Cd
,
Co
, Cu
, Mg
,
Mn
, and Ni
) on the formation of
TAg-p53 complexes in this ELISA was investigated in the same manner as
for Zn
; all ion solutions were prepared from the
chloride salt(30) .
A further series of assays was
undertaken in which the ELISA plate was coated with PAbs 101, 204, 210,
211, 219, 221, 251, 268, 414, and 419, which recognize epitopes along
the length of the N-terminal and central domains of
TAg(19, 20, 31) , with PAbs 421 and 1620,
which bind to epitopes along the length of the p53 molecule different
from that recognized by DO-1, and with DO-7, an independent antibody
that binds to the same region as DO-1(28, 32) . The
dependence of TAg-p53 complex formation on Zn in
these systems was investigated in the same manner as described above.
In this instance detection of complex formation was as described above
with the exception of those instances where the plate had been coated
with antibodies to TAg, in which case the primary antibody used in the
detection reaction was the anti-p53 polyclonal serum CM-1(33) .
TAg is also able to form a stable complex with the p110 protein product of the Rb gene(7) . The binding site for the Rb protein on TAg is distinct from that which recognizes p53(34, 35, 36) , and to determine whether the effect of either zinc ions or of the antibody used to bind TAg to the ELISA specifically regulated p53-TAg complex formation, their influence on the interaction between TAg and purified underphosphorylated p110 Rb (Canji Inc.), which is the form of Rb competent to bind to TAg, was also investigated(37) . Rb protein bound to TAg captured on the ELISA plate was detected with the rabbit polyclonal antibody C-15 (Santa Cruz Biotechnology Inc.).
Figure 1: The biochemical activity of purified TAg and p53 in the SV40 replication assay. The ability of TAg to direct viral DNA synthesis was determined in the presence and absence of p53. In the absence of p53 the purified TAg enabled viral DNA replication to occur (A). The purified p53 was analyzed by a standard ELISA procedure to ensure that it was in the wild type (1620+) and not a mutant (240+) conformation (B). Titration of this wild type p53 into the SV40 replication reaction effectively abrogated TAg-dependent DNA replication (C).
Figure 2: The ability of TAg to bind to and form a stable complex with p53 in a two-site capture ELISA. Purified p53 was captured onto a 96-well plate that had been coated with the anti-p53 monoclonal antibody DO-1. In the absence of zinc ions at a concentration of 0.1 mM, there was negligible complex formation. The addition of 0.1 mM zinc ions greatly enhanced the ability of TAg to bind to p53 in a stable complex.
We therefore examined whether a series of metal ions, which may have been depleted by the presence of EDTA in the purification buffers, could restore TAg-p53 complex formation. Although TAg could not form a stable complex with p53 captured on a plate coated with the anti-p53 monoclonal antibody DO-1, complex formation could be restored by specific concentrations of zinc or copper ions (Fig. 3A). Other divalent cations such as cadmium, cobalt, nickel, and manganese could substitute at higher concentrations for zinc or copper ions, although neither calcium nor magnesium had any demonstrable effect on the formation of the p53-TAg complex (Fig. 3, A and B). Complex formation promoted by 0.1 mM zinc could be inhibited by titrating EDTA into the reaction (Fig. 4).
Figure 3: The ability of divalent metal cations to restore the ability of p53 and TAg to form a stable complex in a two-site capture ELISA. The effect of zinc and copper was greatest at a concentration of 0.1 mM. Apart from calcium and magnesium, which had no effect, the other ions promoted complex formation at higher concentrations. For ease of analysis the titration curves for the ions studied have been displayed on two graphs (A and B).
Figure 4: Titration of EDTA into the reaction inhibits the effect of zinc ions on restoring the ability of TAg and p53 to form a stable complex. Furthermore, titration of EDTA into the reaction once complex formation has occurred also impairs the ability of the two proteins to form a stable complex (data not shown).
Figure 5: Partial proteolysis of TAg and p53 by endoproteinase Glu-C (V8). In the presence of 0.1 mM zinc (+), the pattern of proteolytic fragments of TAg generated by the enzyme is different from that obtained in the absence of zinc(-). In contrast, the size of fragments of p53 generated in the same way is not affected by zinc ions. However, in the presence of zinc it does appear that the efficiency of the enzyme is altered, as the intensity of the signal of particular paired bands in the samples derived from digestion of p53 is not the same. In each case the results illustrated were obtained when 100 ng of purified protein was incubated with 625 ng of enzyme at 30 °C for 1 h prior to arresting the reaction as described under ``Materials and Methods.''
Regardless of which p53-specific
antibodies were used to capture p53 onto the ELISA plate, binding to
TAg was strictly dependent upon the added presence of zinc ions, (Fig. 6A). When TAg was first captured onto the ELISA plate
with any one of nine different TAg-specific monoclonal antibodies (PAbs
101, 204, 210, 211, 219, 221, 251, 268, and 414) complex formation with
p53 remained strictly dependent upon the addition of zinc ions (Fig. 6B). Furthermore, when bound to the ELISA plate with
these same monoclonal antibodies, TAg was able to form a stable complex
with p110 Rb (Fig. 6C), suggesting that the effect of
Zn is apparently on a domain of TAg specifically
involved in the interaction with p53 and not other TAg-binding
proteins.
Figure 6:
The effect of using different monoclonal
antibodies to capture either p53 or TAg onto the ELISA plate on the
ability of the purified proteins to form a complex. A, in
addition to using DO-1 to capture p53 on to the 96-well plate, the
experiments were repeated using DO-7, 421, and 1620 as the capture
antibodies. In each case the ability of p53 and TAg to form a complex
remained strictly dependent upon the availability of additional zinc
ions. B, the reciprocal experiments in which TAg was first
captured onto the 96-well plate using a range of monoclonal antibodies
were also carried out. When the monoclonal antibodies 101, 204, 210,
211, 219, 221, 251, 268, and 414 were used, complex formation remained
dependent on the addition of zinc ions; the results for PAbs 204, 211,
219, and 251 are illustrated and are representative. The use of PAb 419
as the capture antibody renders complex formation completely
independent of zinc ions. C, even in the absence of
Zn, TAg can form a stable complex with p110 Rb,
although the signal is slightly greater in the presence of 0.1 mM Zn
.
In striking contrast to all other monoclonal antibodies studied, the formation of TAg-p53 complexes was independent of zinc ions when PAb 419, a monoclonal antibody directed against an epitope at the N-terminal of TAg, was used as the capture antibody (Fig. 6B). These results indicate that the the effect of zinc ions on restoring the ability of purified p53 and TAg to form a stable complex in this assay system can be recapitulated by specific antibody binding to TAg. Furthermore, given that the epitope of PAb 419 lies within the N terminus of TAg, the conformational change induced by zinc ions may emanate from or nucleate to the N-terminal domain of TAg.
Figure 7: Western blots of the same samples as illustrated in Fig. 5were also probed with monoclonal antibodies specific for TAg. In the presence of zinc (+), the 419 epitope is lost, and no signal is seen in the Western blot; the sample subjected to proteolysis in the absence of zinc(-) retains the 419 epitope.
TAg is a transforming oncogene that is believed to specifically bind and inactivate the biochemical function of two key tumor suppressor proteins, p53 and Rb. Although the domains of TAg that are required for interaction with Rb and p53 have been shown to be different, the mechanisms regulating the assembly of TAg with these tumor suppressor proteins have not been clarified(3, 7, 34, 35, 36, 42) . In particular, it has been demonstrated that the p53 binding site on TAg lies out with the N-terminus between amino acids 217 and 517(3, 34) , while the region of TAg required for Rb binding is the LXCXE motif between amino acids 104 and 111(43) . Interest in the factors regulating the interaction of TAg and p53 may have important relevance for elucidating how mutations alter the conformation of p53 and TAg and determine whether these proteins can form a stable complex. Furthermore, since putative endogenous TAg-like proteins have been identified in human tumor cell lines it is possible that detailed knowledge of the biochemical parameters that govern the interaction between TAg and p53 may be applicable to these other proteins and offer the potential to devise novel strategies to modulate the activity of p53 within cells(11) .
We have used highly purified proteins to study the factors affecting the stability of the TAg-p53 protein complex. The novel findings reported in this paper indicate that the amino terminus of TAg contains a motif that specifically regulates the association of TAg with p53 and that factors influencing the conformation of this domain can regulate the binding of these proteins. We have identified two distinct factors that restore TAg-p53 complex formation; one involves the zinc-dependent conformational changes in TAg that activate its binding to p53, and the second is a protein-protein interaction involving monoclonal antibody PAb 419 that is effective in the absence of zinc.
Depletion of zinc ions from p53 during purification has previously been shown to alter the conformation of wild type (PAb 1620+/240-), generating a mutant conformation (PAb 1620-/240+), which was inactive in a sequence-specific DNA binding assay and would be unable to bind to TAg(44, 45) . The consequences of similar depletion of zinc from TAg during purification have not been studied. TAg contains zinc finger motifs, and the presence of EDTA in the buffer solutions used throughout the purification of TAg would provide a basis by which zinc bound to TAg could be depleted, thus providing an explanation of why zinc alters the conformation of TAg and restores p53 binding activity. However, point mutations disrupting the proposed zinc finger structures in TAg do not affect p53 binding but do inactivate the replicative function of TAg(46) . It is possible, therefore, that the zinc requirement seen here for TAg binding to p53 may involve sites in TAg other than the postulated zinc finger.
The hypothesis that an important aspect of the mechanism by which zinc and PAb 419 restore the p53 binding activity of TAg is a conformational change within the N terminus of TAg is strongly supported by the observation that proteolytic cleavage of TAg in the presence of zinc actually occurs within the PAb 419 epitope. Since binding to p110 Rb is independent of zinc ions, these results further suggest that the conformational changes induced in TAg are occurring in a specific domain that serves to facilitate and support complex formation with p53.
The N terminus of TAg has previously been implicated in the regulation of the replicative and transforming activity(16, 47) . The novel findings presented in this paper further indicate that the N-terminal contains a motif, probably within the epitope of the Pab 419 (which has been mapped within the amino-terminal 82 amino acids(19, 20) , which can negatively regulate the ability of TAg to bind to p53 in a stable complex. The basis of this regulatory function is dependent upon conformational changes within the TAg molecule that can be provoked by metal ions such as zinc and by interactions with other proteins. It is indeed possible that similar mechanisms may control the interaction of p53 with other proteins.