Characterization of the in Vitro interaction between SV40 T antigen and p53: Mapping the p53 binding site

VIROLOGY

164, 132-l 40 (1988)

Characterization

of the in vitro Interaction between SV40 T Antigen and ~53: Mapping the p53 Binding Site FLORENCE I. SCHMIEG’

School of Life and Health Sciences,

and DANIEL T. SIMMONS’

University

of Delaware,

Newark,

Delaware

19716

Received July 2 1, 1987; accepted January 8, 1988

An efficient in vitro system for generating soluble complexes between simian virus 40 T antigen and the cellular protein ~53 was developed. A p53 cDNA was inserted 3’ to the SP6 promoter in pGEM-1 (Promega-Biotec) and transcribed by SP6 polymerase. In vitro translation of the cRNA generated p53 which was immunoprecipitable with all five monoclonal antibodies tested (PAbl22, PAb421, PAb242, PAb246, and PAb248). The p53 sedimented at about S-10 S in sucrose gradients, possibly corresponding to a tetramer. T-antigen-p53 complexes were produced by the addition of immunoaffinity-purified T antigen to p53-containing translation lysates. Equivalent amounts of ~53 were immunoprecipitated with the anti-T-antigen antibodies PAb416, PAb419, and PAblOl, suggesting that in vitro made ~53 complexed mostly to a population of T-antigen molecules that had matured at least 15 min in the cell. The complexes sedimented at 18-20 S in sucrose gradients. In order to map the ~53 binding site on T antigen, ~53 was complexed in vitro to labeled proteolytic fragments of T antigen. A 46K fragment, spanning residues 131-517, was immunoprecipitated with the anti-p53 monoclonal PAb122 and therefore is likely to contain the ~53 binding site. This region contains T-antigen sequences necessary for the efficient transformation of nonpermissive cells and for the induction of cellular rRNA synthesis. It also contains the binding sites for DNA polymerase CIand ATP. We suggest a possible role for T-p53 complexes in T-antigen-associated functions. @ 1988 Academic PWSS, IIIC.

INTRODUCTION

antigen in complex with DNA polymerase (Y (Smale and Tjian, 1986; Gannon and Lane, 1987) an enzyme implicated in cellular DNA replication (Chang et al., 1973; Wang et al., 1984; Murakami et a/., 1986). T antigen also binds tightly to the cellular protein p53 (Lane and Crawford, 1979; McCormick and Harlow, 1980). Although the function of p53 is not known, the p53 gene can cooperate with activated ras genes to transform primary cells in culture (Eliyahu et al., 1984; Parada et al., 1984) and by itself can immortalize primary mouse cells (Jenkins et al., 1984). p53 has also been shown to act as a tumor progression factor (Wolf et a/., 1984) as a competence factor in the initiation of cellular DNA synthesis (Mercer et al., 1982, 1984; Kaczmarek et a/., 1986) and to possibly affect SV40 viral DNA replication in infected cells (Braithwaite et a/., 1987). The interaction between T antigen and p53 occurs in both SV40-infected (Harlow et al., 1981 b) and transformed cells (Lane and Crawford, 1979; McCormick and Harlow, 1980; Benchimol et al., 1982), where complexing to T antigen stabilizes p53 and increases its half-life dramatically (Oren et al., 1981; Chandrasekaran eta/., 1982). However, the function of the complex in either infected or transformed cells is unknown. In the present study, we have investigated the interaction between SV40 T antigen and p53 by using an in vitro binding assay. We show that our system mimics

The simian virus 40 (SV40) T antigen is a remarkable multifunctional protein, intricately involved in molecular events necessary for viral replication. T antigen binds to specific sequences of SV40 DNA in order to initiate viral DNA replication (Shortle et a/., 1979; Shor-tle and Nathans, 1979a,b; Myers and Tjian, 1980; Deb et a/., 1987). It possesses an ATPase (Giacherio and Hager, 1979; Clark et a/., 1981) and a helicaseunwindase activity (Stahl et a/., 1986; Dean et al., 1987) which are probably involved in the replication process. It also regulates transcription of the early genes (Alwine et al., 1977; Khoury and May, 1977; Hansen et al., 1983; Rio and Tjian, 1983) and activates the late genes of SV40 (Khoury and May, 1977; Brady et al., 1984; Keller and Alwine, 1984). Genetic studies have also defined a role for T antigen in the initiation of cellular DNA synthesis in infected cells (Chou and Martin, 1975; Tjian and Graessman, 1978; Galanti et al., 1981) in the induction of cellular rRNA synthesis (Soprano et a/., 1981, 1983) and in the transformation of nonpermissive cells to the malignant phenotype (Kimura and Dulbecco, 1973; Martin and Chou, 1975; Brockman, 1978). T-antigen function is undoubtedly modulated by binding to cellular factors. Recent work has identified T ’ Current address: The Wistar Institute. Philadelphia, PA 19104. ’ To whom requests for reprints should be addressed. 0042-6822/88

$3.00

Copyright 0 1998 by Academic Press, Inc. All rights of reproduction an any form reserved.

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in viva complex formation, as determined by immunoprecipitation reactions and sedimentation anaylsis. Using this in vitro system, we have mapped the p53 binding site on T antigen to a 46K fragment in the middle of the T-antigen molecule. This region contains sequences that are important in the induction of cellular rRNA synthesis and in transformation of nonpermissive cells. This portion of the molecule is also responsible for binding to DNA polymerase (Y and ATP. Our results present the possibility that p53 may play a role in T-antigen-associated functions in infected or transformed cells. MATERIALS

AND METHODS

Materials The following T-antigen-specific monoclonal antibodies were used: PAb416 and PAb419, which recognize the NH,-terminal region (Harlow et al., 1981a), and PAblOl which recognizes a COOH-terminal region of T-antigen molecules that becomes modified approximately 15 min postsynthesis (Gurney et al., 1980; Carroll and Gurney, 1982). The p53-specific monoclonal antibodies used were PAb122 and PAb421 which recognize COOH-terminal determinants (Gurney et al., 1980; Harlow et al., 1981 a), PAb242 which recognizes residues 9-75, and PAb246 and PAb248 which recognize residues 88-l 09 and 157-l 92 on mouse ~53, respectively (Wade-Evans and Jenkins, 1985; Yewdell et al., 1986). The control antibody (lA5-64) against rat lipoprotein E was obtained from Dr. David Usher. Plasmid pSVp53 was obtained from Dr. Arnold Levine and contains a mouse F9 cell-specific p53 cDNA. This plasmid was made from pl l-4 (Tan et al., 1986) by insertion of the SV40 early promoter directly upstream of the p53 cDNA sequences. The 2.1-kb BamHl fragment of pSVp53 was ligated into pGEM-1 (Promega-Biotec), which contains both the SP6 and T7 bacteriophage promoters, using standard subcloning procedures (Maniatis et al., 1982). Nuclease-treated rabbit reticulocyte lysates were obtained from Promega-Biotec. AdSVR284 hybrid virus (obtained from Dr. Robert Tjian) contains the adenovirus major late promoter and the SV40 A gene inserted into the third region of the adenovirus late tripartite leader (Thummel et a/., 1983).

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CTP, and UTP, 0.72 mM MgAc, 64 mM KAc, 1 mM dithiothreitol, and 37.5 units of SP6 polymerase (Promega-Biotec) for 1 hr at 37”. When synthesizing labeled transcripts, lo-20 PCi [a3*P]ATP (ICN, 650 Ci/ mmol) was included in the reaction mix. Expected runoff transcript was approximately 1.96 kb. Following in vitro transcription, 10 pg tRNA carrier and 1 unit of DNase (Promega-Biotec) per microgram of DNA template were added at 37” for 15 min. cRNA was brought to 2% SDS and the sample was extracted once with an equal volume of phenol, once with an equal volume of phenol-chloroform, and once with an equal volume of chloroform. cRNA was precipitated twice with 0.4 vol of 5 mM NH,Ac and 2.5 vol of 95% ethanol, and once with 0.2 vol of 2 R/I KAc and 2.5 vol of 95% ethanol at -20”. When the RNA was used for translation, ethanol pellets were washed once with 70% ethanol and resuspended in 25 ~1 of sterile, deionized water. In vi&o translation One microgram of p53 cRNA (0.1 mg/ml) was added to 35 yl nuclease-treated rabbit reticulocyte lysate (Promega-Biotec), 1 mM amino acid mixture minus methionine, and 50 &i of L-[35S]methionine (New England Nuclear, 1086 Ci/mmol). When making unlabeled ~53, 1 mM L-methionine was substituted for the labeled methionine. Reactions were carried out for 1 hr at 30”. Following in vitro translation, RNase A (60 pug/ml) was added and incubated at 30” for 15 min. If necessary, lysates were stored at -80”. lmmunoaffinity

purification

of T antigen

T antigen was obtained from HeLa cells infected with Ad-SVR284 virus (Thummel et al., 1983) and purified on protein A-Sepharose columns containing covalently bound monoclonal antibody PAb419 as described (Simanis and Lane, 1985). In vitro complex formation RNase-treated translation lysates (50 ~1) containing p53 were thawed on ice and incubated with 1 ~1 immunoaffinity-purified T antigen (640 pg/ml) or with 10 ~1 of labeled T-antigen fragments at 30” for 30 min. Samples were either diluted immediately for immunoprecipitation or stored at -80”.

In vitro transcription Five micrograms of template DNA was linearized with F/pal restriction endonuclease (ICN) and incubated with 40 m/v! Tris-HCI, pH 7.5, 6 mM MgCI,, 2 mM spermidine, 10 mM NaCI, 500 PM ATP, GTP,

lmmunoprecipitation Lysates containing p53 or T-antigen-p53 complexes were diluted with 0.6 ml of NET buffer (50 mM Tris-HCI, pH 7.4, 150 mM NaCI, 5 mM EDTA, 0.2%

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albumin, 0.02% NaN,), 0.1 ml Eagle’s minimum essential medium (MEM) + 10% globulin-free FCS, and 60 ~1 of lysis buffer (20 mM Tris-HCI, pH 8.0, 1% Nonidet-P40). Diluted lysates were precleared with 100 ~1 Staphylococcus aufeus (S. aufeus) for 30 min on ice. S. aureus was pelleted and supernatants were subjected to immunoprecipitation with monoclonal or polyclonal antibodies as previously described (Levitsky et al., 1983). Gel electrophoresis Acrylamide slab gel electrophoresis of sodium dodecyl sulfate-denatured proteins was performed as previously described (Simmons, 1980). Dried gels were exposed to Kodak XAR-5 film for 1 to 42 days at -80”. cRNA was denatured in 90% formamide at 65” for 5 min in buffer containing 36 mM Tris-HCI, pH 7.5, 30 m/l/l NaH,P04, and 1 mM EDTA. cRNA transcripts were applied to a 3.5% acrylamide-7 IL1 urea gel and subjected to electrophoresis at 115 V for 2.5 hr. To visualize the in vitro transcripts, the gels were soaked in 0.5 pg/ml ethidium bromide for 30 min. To detect labeled transcripts, gels were dried and exposed to film as described above for 1 day at -80”.

Sucrose gradient centrifugation p53 or p53-T-containing lysates were subjected to centrifugation in 5-200~ sucrose gradients containing 0.01 M Tris, pH 7.4, 0.14 M NaCI, and 1 mM dithiothreitol. Gradients containing free p53 were spun for 41 hr at 20,600 rpm and 4” in a Spinco SW41 rotor. Sedimentation coefficients were estimated from the sedimentation of fscherichia co/i ,&galactosidase (Sigma Chemical Co.). Gradients containing T-antigen-p53 complexes were spun for 17 hr at 20,400 rpm and 4”. Sedimentation coefficients were estimated from sedimentation of HeLa cell 28 and 18 S rRNAs.

Preparation of T-antigen fragments BSC-1 monkey cells were infected with SV40 at a multiplicity of 10 PFU per cell. At 39 hr postinfection, cells were labeled for 1 hr with L-[35S]methionine (100 &i/ml) and lysed with NP-40 lysis buffer (20 mlLl TrisHCI, pH 8.0, 500 mM NaCI, 1% Nonidet-P40) contain6 ing 800 pg of leupeptin per milliliter. Lysates were diluted with 3 vol of NP-40 lysis buffer lacking NaCl and subjected to immunoprecipitation with PAb416. Partial proteolysis was performed on the immunoprecipitated T antigen as previously described (Simmons, 1986), using 20 pg/ml N-tosyl-L-phenylalanine chloromethyl ketone-trypsin (Worthington Diagnostics). The S.

FIG. 1. lmmunoreactivityof p53 synthesizedin vitro. cRNA for p53 was translated in rabbit reticulocyte lysates in the presence of 50 &i L-[35Sl]methionine. Translation products were analyzed either directly (lane 1) or after immunoprecipitation with nonimmune control MEM t 10% globulin-free FCS (lane 2), monoclonal antibodies PAb122, PAb421, Pab242, PAb246, PAb248 (lanes 3-7), normal hamster serum (lane 8), or anti-T tumor serum (lane 9). Proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 13% gels, and labeled bands were detected by fluorography.

aureus was pelleted, and the labeled, solubilized fragments were used for p53 binding reactions.

RESULTS Subcloning of ~53 cDNA and in vitro transcription The aim of this present work was to develop an efficient SV40 T-antigen-p53 binding assay to probe the structure of this complex and to deduce its possible functions in SV40-infected and transformed cells. Our approach was to prepare highly labeled p53 by in vitro translation of in vitro made cRNA and bind it to purified T antigen. To achieve this end, we first subcloned a 2.1-kb BarnHI digestion fragment of plasmid pSVp53 (a gift from Dr. Arnold Levine) into a vector (Gemini 1, Promega-Biotec) containing a promoter recognized by SP6 polymerase. This fragment contained the entire p53 cDNA insert, plus approximately 550 bp of DNA which included the SV40 small t splice site, as described by Tan et al. (1986). The fragment was oriented in the vector in such a way that the sense +cRNA was read off the SP6 promoter.

ln vitro translation and characterization

of the p53

The p53 cRNA made by SP6 polymerase was translated in rabbit reticulocyte lysates and the p53 product was characterized to determine its authenticity. As a first test, L-[35S]methionine-labeled translation products were analyzed by immunoprecipitation with monoclonal antibodies directed against various epitopes of ~53. Figure 1 shows an SDS-acrylamide gel of the p53 translation products analyzed directly (lane 1) or after immunoprecipitation with monoclonal anti-

SV40 T Ag-p53

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FIG. 2. Sedimentatron profile of p53 synthesized in v&o. A rabbit retrculocyte lysate containing p53 was layered on a 5-20% sucrose gradient and centrifuged at 20,600 rpm for 41 hr at 4’. Twenty (0.6 ml each) fractions were collected. Odd numbered fractions (l-l 9) were subjected to immunoprecipitation by monoclonal antibody PAb122 and precipitated proteins were analyzed as described in Ftg. 1. The bottom of the gradient IS to the left. fscherichia co/i p-galactosidase (16 S) was run in a parallel gradient and assayed for enzymatic activrty, as determined by ODbZOabsorption following incubation with o-nttrophenyl-P-o-galactosidase at 30” for 1 hr (Pardee et a/., 1959)

bodies which recognize determinants located at the COOH-terminal end of p53 (PAb122 and PAb421, lanes 3 and 4, respectively), or those which recognize determinants in the middle of the p53 protein (PAb242, PAb246, and PAb248, lanes 5, 6, and 7, respectively). A control reaction using nonimmune medium was also included (lane 2). All antibodies used immunoprecipitated only full-length p53 protein. Our anti-T tumor serum contains antibodies to p53 (Simmons, 1980) and it immunoprecipitated a small amount of p53 (lane 9). All immunoprecipitation reactions were done at antibody excess as determined by titration analysis. Despite that, only a small percentage (l-39/0) of the total p53 synthesized in the reticulocyte lysate was immunoprecipitated by any monoclonal antibody used. The remaining p53 was not precipitated with additional antibody. The explanation for this is not known, but appears to be due to a block in the precipitation of the majority of p53 (and probably other proteins) from reticulocyte lysates. Other investigators have made similar observations with in vitro translated human p53 (Harlow et al., 1985). We then wished to test the ability of the in vitro synthesized p53 to self-associate. In viva made p53 from various cell types exists in dimeric and tetrameric states (McCormick et a/., 1981). To determine the size of the in vitro made ~53, 35S-labeled translation lysates were subjected to sucrose gradient centrifugation.

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The p53 in the odd-numbered fractions (Fig. 2, lanes l-l 9) was immunoprecipitated with monoclonal antibody PAb122. Figure 2 shows an SDS-acrylamide gel analysis of these immunoprecipitation products. The p53 sedimented with a peak between 8 and 10 S, which most likely represents a tetramer (McCormick et a/., 1981). There were also appreciable amounts of larger aggregates (216 S), and a significant amount of smaller molecules, probably dimers and monomers. No p53 was immunoprecipitated from the even-numbered fractions using a control antibody (not shown). These results paralleled those obtained by direct analysis of the gradient fractions without immunoprecipitation (data not shown), and therefore are representative of all the p53 synthesized in the lysate. The p53 obtained in these experiments corresponded very closely in size to the p53 extracted from F9 cells by McCormick et al. (1981) who reported a size of lo-12 S. [The original p53 cDNA in pSVp53 was also specific to F9 cells (Tan et al., 1986).] Characterization made in vitro

of p53-T-antigen

complexes

SV40 T antigen was extracted from Ad-SVR284 (Thummel et al., 1983) -infected HeLa cells and purified by immunoaffinity chromatography on PAb419 columns (Simanis and Lane, 1985). The purified T antigen was complexed to the in vitro made p53 protein by direct addition to the translation reaction. After 30 min of incubation at 30”, the resulting complexes were analyzed by immunoprecipitation assays (Fig. 3, +T antigen). Parallel reactions lacking T antigen were also

-T antigen 123456

+T antigen 1234

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FIG. 3. lmmunoreactivity of p53--T-antrgen complexes made in virro. p53-containrng rabbit retrculocyte lysates were incubated with or wrthout immunoaffinity-purified T anttgen Lysates were subjected to immunoprecipitation with control MEM + 10% globulinfree FCS (lanes l), unrelated antrbody lA5-64 against rat lipoprotein E (lanes 2) or monoclonal antibodies PAb416, PAb419, PAbl 01, or PAb122 (lanes 3-6). Gel analysis was as described in Fig. 1.

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subjected to immunoprecipitation (Fig. 3, -T antigen). lmmunoprecipitations were performed with either control medium (lanes l), pAblA5-64, an unrelated antibody recognizing an epitope on rat lipoprotein E (lanes 2) PAb416 and PA641 9, which recognize the NH,-terminal region of T antigen (lanes 3 and 4), PAbl 01, which recognizes a COOH-terminal determinant present only on T antigens that have matured at least 15 min in the cell (lanes 5) or the anti-p53 antibody PAb122 (lanes 6). It is apparent that in the presence of T antigen, p53 was immunoprecipitated with all monoclonal antibodies specific for T antigen, indicating the presence of complexes between T and ~53. No p53 is evident in the corresponding lanes of the control reaction without added T antigen. Additionally, in the presence of T antigen, equivalent amounts of p53 were immunoprecipitated with PAb122 and with the monoclonal antibodies specific for T antigen (lanes 3-6 of the +T-antigen sample). Similar amounts of p53 were also immunoprecipitated with PAblOl (lane 5) suggesting that the p53 bound to the older T-antigen molecules that are recognized by this monoclonal antibody. This mimics the behavior of in vivo made p53 as noted by us (Schmieg and Simmons, 1984) and by others (Carroll and Gurney, 1982). We feel that the minor amount of p53 visible in lane 1 of the +T sample is due to nonspecific binding of the protein to the S. aureus when T antigen is added to the lysates. The reason for this is unclear, but the addition of T antigen somehow interferes with efficient washing of the immunoprecipitates. Sequential immunoprecipitation reactions of T-antigen-containing p53 lysates (not shown) demonstrated that the same class of p53 which was precipitable with PAb122 could be precipitated with anti-T monoclonal antibodies and was therefore bound to T antigen. The remaining p53 also appeared to be bound to T antigen (see below) but could not be precipitated with either antibody. The in vitro association between p53 and T antigen is a very rapid one. Kinetic experiments indicated that the binding reaction was essentially complete 10 min after addition of T antigen (data not shown). This is similar to the behavior of newly made p53 in transformed mouse and rat cells (Schmieg and Simmons, 1984). The in vitro binding reaction also occurred readily at 0”. We then wanted to determine the size of the complex made in vitro. It has been reported that the mature in viva complex of T antigen and mouse ~53 sediments at 22-24 S (Greenspan and Carroll, 1981) and consists of four molecules of T antigen and four to five molecules of p53 (Freed et a/., 1983). This complex apparently matures with time through a 16 S interme-

diate (Greenspan and Carroll, 1981). We subjected p53-T-containing lysates to sucrose gradient centrifugation and incubated the even-numbered fractions with PAb419 and the odd-numbered fractions with PAb122. Figure 4A shows the p53 immunoprecipitated with PAb419, representing, therefore, molecules in complex with T antigen. [PAb419 does not react with p53 (see Fig. 2, lane 4).] It is apparent that the complex sedimented with a peak of approximately 18 S. A very small amount of 22-24 S oligomers as well as smaller complexes was also present. The p53 in the gradient fractions precipitated with PA1 22 (Fig. 4B) displayed a similar sedimentation profile. The p53 sedimented with a peak at fraction number 13 (18 S). A nearly identical profile was obtained without precipitation (data not shown). This indicated that nearly all of the p53 was associated with T antigen, not merely the fraction that was immunoprecipitable.

Mapping of the p53 binding site on T antigen Since SV40 T antigen is a multifunctional protein that participates in many molecular events in permissively and nonpermissively infected cells, and since it has a specific binding affinity for ~53, it is not unreasonable to propose a possible role for p53 in T-antigen-associated functions. For this reason, it is important to know the functional domain of T antigen which contains the binding site for ~53. In order to map this site, we reacted p53 with radioactively labeled trypsinized fragments of T antigen. These fragments have previously been characterized by tryptic peptide fingerprinting in our laboratory and carefully mapped on the T-antigen molecule (Simmons, 1986). We incubated the fragment mixture with either unlabeled p53-containing lysates or unlabeled control lysates to which no cRNA had been added, and immunoprecipitated the complexed T-antigen fragments with the anti-p53 antibody PAb122. Figure 5 shows that a 46K T-antigen fragment was immunoprecipitated with PAb122 from the p53-containing lysate but not from the control lysate (lanes 2 and 3, respectively). A small amount of a 76K fragment was also specifically immunoprecipitated with PAb122 (lane 2). Our interpretation is that both of these fragments are able to bind to p53 in the in vitro reaction. We do not feel that the 17K fragment present in lane 2 represents true binding to ~53, since ttiis band is also present in the control lane (lane 3). The 76K and 46K T-antigen fragments originate from approximately residues 13 l-708 and 13 l-5 17, respectively (Simmons, 1986). Under the trypsinization conditions used, a family of 30K fragments (lane 1) was generated consisting of COOH-terminal fragments (residues 5 18-708) as well

Sv40 T Ag-p53

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FIG. 4. Sedrmentation profile of in v/tro made p53-T-antigen complexes. Reticulocyte lysates contarning ~533T complexes were layered on a 5-20% sucrose gradient and centrifuged for 17 hr at 20,400 rpm at 4’. Twenty (0.6 ml each) fractions were collected and Incubated with either monoclonal antibody PAb419 (even-numbered fractions, Z-20) or PAb122 (odd-numbered fractions, l-l 9). Gels were run as described in Frg. 1. A parallel gradient was run with rRNA markers (28 and 18 S). Gel A: preciprtation wrth PAb419. Gel B: precipitation with PAb122.

as a subfragment of the 46K fragment (residues 131-371). Because the subfragment was generated in minor amounts, it is not possible to determine if it bound to ~53. However, the COOH-terminal 30K fragments did not appear to bind (Fig. 5, lane 2), consistent with our interpretation that the 46K fragment binds to ~53. The binding site for p53 therefore maps between residues 131 and 517.

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FIG. 5. Mapping of the ~53 binding site on T antigen. Labeled T-antigen fragments were incubated with rabbit reticulocyte lysates containing unlabeled ~53 or with a control lysate without p53 for 30 min at 30”. p53-containing lysates were subjected to immunoprecipitation with either anti-T serum (lane 1) or PAb122 (lane 2); the control lysate was subjected to immunoprecipitation with PAb122 (lane 3). Precipitated peptides were analyzed on acrylamide gels as described in Fig. 1.

DISCUSSION In vitro complex

formation

In this study, we have developed an in vitro system for generating T-antigen-p53 complexes. The system that we have employed is efficient and because of the large amounts of labeled soluble complexes that can be made it is useful in the biochemical dissection of the T-antigen-p53 binding reaction and possibly in the purification of these complexes. The protein used in the formation of our in vitro complexes was produced in rabbit reticulocyte lysates by translation of in vitro made cRNA for ~53. We feel that the p53 made in this manner is representative of that produced in cells, since it is recognized by a panel of monoclonal antibodies directed against epitopes in several different regions of the p53 molecule. In fact, the epitopes recognized by many anti-p53 monoclonal antibodies have been mapped by reactions with in vitro made p53 (Wade-Evans and Jenkins, 1985). Also, the p53 synthesized in our in vitro system associates into oligomers similar to those in F9 cells (McCormick et a/., 1981). [F9 cells were used in the original p53 cDNA cloning work (Tan et a/., 1986).] The binding of T antigen and p53 in vitro is similar in several respects to the reaction in transformed mouse and rat cells. We and others have previously shown that, in these cells, p53 complexes to a population of T-antigen molecules recognized by PAbl 01 (Carroll and Gurney, 1982; Schmieg and Simmons, 1984). This antibody recognizes a class of T antigen that has undergone some structural change or modification about 15 min postsynthesis. In the present experi-

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ments, we immunoprecipitated equivalent amounts of p53 with PAb416 (which reacts with all forms of T antigen), PAb419, and PAblOl (Fig. 3) indicating that the precipitated p53 bound only to the T-antigen population with the PAbl 01 epitope. Second, virtually all of the p53 that was immunoprecipitated with PAb122 was associated with T antigen in vitro, as determined before and after sucrose gradient sedimentation and by sequential immunoprecipitation reactions. These complexes sedimented as 16-18 S oligomers. All of these properties also apply to T-antigen-p53 complexes isolated from transformed mouse cells (Greenspan and Carroll, 1981; Schmieg and Simmons, 1984). Although the p53 and p53-T-antigen complexes generated in vitro are not readily immunoprecipitated under our conditions, it appears that they are nevertheless in a native form because they form oligomeric structures which are very similar to those found in transformed cells, and because all of the p53 binds to added T antigen. The complexes produced in these reactions should therefore be useful in obtaining relevant biochemical information about T-antigen-p53 interactions. Mapping of the ~53 binding site on T antigen To determine the p53 binding site on T antigen, we reacted a T-antigen fragment mixture with in vitro made ~53. A T-antigen 46K fragment was readily precipitated with PAb122, indicating that this fragment contains the p53 binding region. The 46K fragment maps to residues 13 l-5 17 on the T-antigen molecule (Simmons, 1986), corresponding to map units 0.51-0.28. A 76K fragment which contains all the sequences present in the 46K fragment (Simmons, 1986) also showed a low level of binding to ~53. The lower level of binding to the 76K fragment could be due to the presence of additional peptide regions outside the binding site which reduce the efficiency of interaction between the fragment and ~53. These results indicate that the NH*- and COOH-terminal regions of T antigen are dispensable for p53 binding. Our mapping data are supported by results obtained in other laboratories. The D2 protein produced by the AD2+ 02 hybrid virus’binds to p53 (Montenarh, et al., 1986) and is missing the NH,-terminal 115 amino acids of T antigen. Additionally, the deletion mutant CTSVl , which codes for a T antigen missing residues 1 1O-l 52 can complex to p53 (Montenarh eta/., 1986). Recently, Mole et al. (1988) have shown that a T antigen missing residues l-272 can complex to p53 in another in vitro system. These data suggest that residues l-272 are not necessary for p53 binding. Simi-

larly, genetic studies indicate that the COOH-terminal region of T antigen is dispensable for p53 binding. Deletion mutants dl 2194, dl 2198, dl 1263, and dl 1265 all produce T antigens that complex to p53 (Kress et al., 1982). These deletions generate truncated T antigens that are missing 20-30 COOH-terminal residues. Recently, it has been demonstrated that T antigen coded by dl 1066 (missing residues 670-708) (Montenarh et al., 1986) and tsA 1499 (missing residues 635-661) (Montenarh et al., 1985) can complex to ~53. Taken together, these results eliminate the COOH-terminal region of T antigen from residue 635 onward as the site of p53 binding. By combining these data with our results, we can localize the p53 binding site to residues 272-517. This corresponds to map units 0.41-0.28 on the SV40 genome. This significantly narrows down the region of T antigen which binds to ~53. The p53 binding site on T antigen maps outside of the origin-specific DNA-binding domain (Simmons, 1986) and the region necessary for induction of cellular DNA synthesis (Soprano et a/., 1983). However, the binding site contains, or overlaps with, the sequences necessary for T antigen to bind to DNA polymerase a in vitro (approximate residues 335-626) (Smale and Tjian, 1986; Gannon and Lane, 1987) to bind to ATP (residues 418-528) (Bradley et a/., 1987; Smale and Tjian, 1986) to induce rRNA synthesis (317-426) (Soprano et a/., 1983) and to fully transform certain nonpermissive cells (Pipas et a/., 1983; Montenarh et al., 1985). Although the NH>-terminal region of T antigen alone can immortalize hamster and rat cells, and certain T-antigen mutants that cannot bind p53 appear able to transform in certain cases, additional sequences in the p53 binding region are necessary for many nonpermissive cells to acquire transformation phenotypes such as focus formation, growth in low serum, or growth in soft agar (Hirschorn et al., 1984; Tevethia, 1984; Sompayrac and Danna, 1985). The possibility that ~53, or the T-p53 complex, plays a role in one of more of these activities should be considered. It is worth noting that p53 acts as a competence factor for the initiation of DNA synthesis in quiescent mouse cells (Kaczmarek et a/., 1986). Its ability to immortalize primary cell cultures (Jenkins et al., 1984) and to cooperate with the ras gene to transform primary cells (Eliyahu et a/., 1984; Parada et al., 1984) also suggests a role in’growth regulatory events. Our in vitro complexing system can be used to generate T-p53 complexes for use in both biochemical and enzymatic studies to clarify the role of p53 in Tantigen-related functions.

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COMPLEXING

ACKNOWLEDGMENTS This work was supported by a Public Health Service grant (CA361 18) from the National Cancer Institute. We thank A. Levine for the pSVp53 plasmid.

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