MOLECULAR AND CELLULAR BIOLOGY, Dec. 1991, p. 6067-6074 0270-7306/91/126067-08$02.00/0 Copyright © 1991, American Society for Microbiology

Vol. 11, No. 12

Analysis of p53 Mutants for Transcriptional Activity LORETTA RAYCROFr, JULIANN R. SCHMIDT, KAREN YOAS, MINGMING HAO, AND GUILLERMINA LOZANO* Department of Molecular Genetics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 Received 16 June 1991/Accepted 11 September 1991

The wild-type p53 protein functions to suppress transformation, but numerous mutant p53 proteins are transformation competent. To examine the role of p53 as a transcription factor, we made fusion proteins containing human or mouse p53 sequences fused to the DNA binding domain of a known transcription factor, GALA. Human and mouse wild-type p53/GALA specifically transactivated expression of a chloramphenicol acetyltransferase reporter in HeLa, CHO, and NIH 3T3 cells. Several mutant p53 proteins, including a mouse p53 mutant which is temperature sensitive for suppression, were also analyzed. A p53/GALA fusion protein with this mutation was also transcriptionally active only at the permissive temperature. Another mutant p53/GALA fusion protein analyzed mimics the mutation inherited in Li-Fraumeni patients. This fusion protein was as active as wild-type p53/GALA in our assay. Two human p53 mutants that arose from alterations of the p53 gene in colorectal carcinomas were 30- to 40-fold less effective at activating transcription than wild-type p53/GALA fusion proteins. Thus, functional wild-type p53/GAL4 fusion proteins activate transcription, while several transformation competent mutants do so poorly or not at all. Only one mutant p53/GALA fusion protein remained transcriptionally active.

The protein encoded by the normal p53 gene functions to transformation. In tissue culture experiments, the addition of wild-type p53 suppresses the transformation of rat embryo fibroblasts by two cooperating oncogenes (8, 11). Also indicative of a suppressor function for p53 is the presence of rearrangements, deletions, and point mutations in p53 in many human tumors, including colorectal tumors, lung cancer, breast cancer, osteosarcomas, and leukemias (1, 2, 4, 7, 25, 27, 36). Detailed molecular analysis of a colorectal tumor showed that no wild-type p53 gene product was made. The reintroduction of a plasmid expressing the wild-type human p53 gene into a colorectal cell line developed from this tumor reversed the transformed phenotype (3). In addition, mutations in p53 are inherited in patients exhibiting the Li-Fraumeni syndrome (LFS) and correlate with early-onset tumor development (23, 34). In contrast, mutations in p53 result in a protein that is transformation competent. Mutant p53 and activated ras cooperate to transform rat embryo fibroblasts under conditions in which neither gene product alone transforms (9, 18, 20, 29). Thus, mutations in p53 result not only in loss of suppression function but also in the gain of function, i.e., the ability to actively transform. How does p53 suppress transformation? Wild-type p53 may function as a transcription factor to suppress transformation (28, 30). To assay for p53 function as a transcription factor, p53/GAL4 fusion proteins were made by using mouse p53 sequences (amino acids 1 to 343 of 390) fused to the GAL4 DNA binding domain (amino acids 4 to 147) which by itself has no transactivation ability (30). The reporter plasmid, CAT (chloramphenicol acetyltransferase), contained the DNA binding sites for the GAL4 domain. After transfection of p53/GAL4 (activator) and CAT plasmids (with or without binding sites for GAL4) into HeLa cells, wild-type p53/GAL4 fusion proteins specifically activated transcrip-

tion of CAT only when the CAT plasmid contained the GAL4 DNA recognition sequence. Because tissue culture experiments and tumor analysis suggested the existence of mutations in p53 that would make it transformation competent, we also analyzed the effects of two mutations in the mouse p53 gene on transcriptional activation (30). Both of the mutations studied resulted in a p53 protein that cannot suppress transformation (11). Both mutations instead resulted in a p53 that cooperated with ras to transform primary rat embryo fibroblasts (12). In cotransfection experiments with the CAT reporter plasmid into HeLa cells, neither could function to transactivate expression of CAT, thus showing a direct correlation between transcriptional activation by p53 and tumor suppressor function (30). The wild-type p53 protein, a tumor suppressor, activates transcription. Mutations within the p53 protein that result in both loss of the suppression phenotype and gain of transformation ability show no transactivation function. Since our initial observations, numerous questions remained about the ability of p53 to transactivate gene expression. The experiments presented here show that the human wild-type p53/GAL4 fusion protein also functions as a specific transactivator in HeLa cells. In addition, both mouse and human wild-type p53/GAL4 fusion proteins activate transcription in other cell lines studied. The mouse p53/ GAL4 fusion protein containing an Ala-to-Val substitution is temperature sensitive for transformation and for transcriptional activation. One p53/GAL4 fusion protein containing an Arg-to-Trp substitution at p53 amino acid 245, a mutation inherited in LFS patients, was transcriptionally active. Two p53 mutations that arose from alterations in human colorectal carcinomas were also cloned into the p53/GAL4 plasmid and analyzed in our assay. Neither of these mutations resulted in a functional p53/GAL4 fusion protein. Mutations analyzed thus far which fall in the conformation domain of p53 are transformation competent and transcriptionally inactive. One mutation analyzed outside of this domain retained its transactivation function.

suppress

*

Corresponding author. 6067

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MATERIALS AND METHODS Plasmid constructions. Plasmid p53-cWT (a gift from A. J. Levine) containing the human wild-type p53 sequences under control of the cytomegalovirus (CMV) enhancer/ promoter was used to generate the human p53/GAL4 fusion (19). p53-cWT was cut at a unique StuI restriction endonuclease site near the 3' end of the p53 coding sequences and fused in frame to a GAL4 sequence encoding amino acids 4 to 147. The neo gene was also removed from this construct by using XbaI and Hindlll. Sequence analysis showed the fusion protein to be in frame. This human p53/GAL4 fusion construct was used to generate plasmids carrying two independent mutations. Plasmid p53C143A, which contains a human cDNA with a Val-to-Ala mutation at amino acid 143, was cut with BamHI, and the 0.9-kb fragment replaced the wild-type p53 sequences to generate p53-CX3/GAL4. Plasmid p53-175H was used to generate a new p53/GAL4 plasmid containing a mutation at amino acid 175. Since p53-175H contains introns, a partial NcoI digest and complete XhoI restriction fragment were used to generate a p53 cDNA/ GAL4 fusion identical to the others except for the single base substitution. The mouse p53/GAL4 fusion plasmid has been described previously (30). An Arg (CGC)-to-Trp (TGG) mutation at p53 amino acid 245 was created by using site-directed polymerase chain reaction (17, 31). Sequence analysis confirmed the presence of the mutations. For Cos cell transfections, the CMV promoter and P-globin intron of the human p53/GAL4 constructs were replaced with the simian virus 40 (SV40) enhancer/promoter (KpnI-StuI). All other plasmids used in these experiments have been described elsewhere (30). Transfection experiments. HeLa, CHO, or NIH 3T3 cells were plated at a density of 0.5 x 106 cells per 100-mm petri dish 1 day before transfection. The activator and reporter plasmids (10 F.g of each) were cotransfected with a plasmid expressing P-galactosidase (5 ,ug), using calcium phosphate precipitation (6). For temperature sensitivity experiments, 24 h after transfection, duplicate plates were incubated at 32 or 37°C for an additional 24 h. All plates were harvested 48 h after transfection. ,-Galactosidase activity was measured to monitor and normalize for transfection efficiency (35). The activity of CAT was measured by the conversion of [14C]chloramphenicol to acetyl and diacetyl chloramphenicol (15). The percent conversion was analyzed by direct measurement of thinlayer chromatography plates, using a Betagen Betascanner. Protein analysis. Approximately 0.5 x 106 Cos cells were plated per 100-mm petri dish and transfected 24 h later, using calcium phosphate precipitation (6). Transfected cells were labelled with a mixture of [35S]Cys and [35S]Met. Immunoprecipitations were performed as described with p53 monoclonal antibody p53 Ab-2 (Oncogene Science) or PAb240 and 5 x 106 trichloroacetic acid-precipitable counts (12). Samples were run on a 7.5% sodium dodecyl sulfate-polyacrylamide denaturing gel and fluorographed as described previously (12). Immunofluorescence. Cos cells were plated on coverslips in 100-mm petri dishes at a density of 0.5 x 106 cells and transfected 24 h later, using calcium phosphate precipitation (6). Approximately 36 h after transfection, cells were fixed in 3% formalin in phosphate-buffered saline (PBS) for 30 min and then fixed in cold 50% acetone-50% methanol for 15 min. Nonspecific binding was blocked by using goat serum. p53 expression was detected by using undiluted monoclonal antibody cell culture supernatants from PAb240 (a gift from

MOL. CELL. BIOL.

A. J. Levine [13]). After washing in PBS, goat-anti-mouse fluorescein isothiocyanate conjugate (Sigma) was added at a 1:200 dilution for 30 min. Samples were mounted on glass slides in 50% glycerol in PBS and photographed on a Zeiss Axiophot microscope. All samples were exposed for 30 s at x 63 magnification.

RESULTS Wild-type p53/GAL4 fusion proteins are transcriptionally active independent of cell line. We had previously shown that mouse wild-type p53 when fused to a GAL4 DNA binding domain specifically activated CAT expression in HeLa cells (30). Numerous biological and immunological differences between mouse and human p53 proteins prompted us to examine the ability of human wild-type p53 to activate transcription. Mouse and human p53 are 80% conserved at the amino acid level. However, the first 73 amino acids which are required for transactivation are less than 50% conserved (10, 33). In addition, we wished to examine p53 transactivation function in various cell lines since HeLa cells contain human papillomavirus (HPV)-derived sequences and may represent an artificial system to analyze p53 function. The HPV E6 protein binds p53 and may affect p53 function (37). Therefore, to determine whether p53/GAL4 fusions could function in other cell lines, we also assayed for mouse and human wild-type p53/GAL4 transcriptional activity in NIH 3T3 and CHO cells. First, we made a human wild-type p53/GAL4 fusion plasmid containing human p53 amino acids 1 to 346 followed by linker sequences and GAL4 amino acids 4 to 147 (Fig. 1A). Sequence analysis across the fusion site showed that the GAL4 sequences were in frame (data not shown). The CMV enhancer/promoter was used to express this fusion protein. In addition, a P-globin intron was used to increase expression of p53/GAL4. An expression plasmid containing the entire yeast GAL4 protein which activates transcription in mammalian cells was used as a positive control (30). Expression of GAL4 amino acids 4 to 147, which does not activate transcription or plasmid DNA alone, was used as a negative control. Wild-type p53/GAL4 plasmids and control plasmids were individually transfected into HeLa, CHO, or NIH 3T3 cells with the reporter CAT plasmid containing either zero or four GAL4 binding sites upstream of the start of transcription. All transfection experiments were performed by using a ,-galactosidase-expressing plasmid to monitor and correct for transfection efficiencies. Forty-eight hours after transfection, cells were harvested and protein extracts were made. The CAT activity in these extracts was assayed by using ['4C]chloramphenicol. The data for a CAT reporter plasmid with four GAL4 binding sites are presented in Fig. 2. In HeLa cells, both human and mouse wild-type p53/GAL4 fusion proteins activated CAT expression greater than 25fold above the basal level of expression (Fig. 2A). In CHO cells, both human and mouse wild-type p53/GAL4 fusion proteins could specifically activate CAT expression 17- and 30-fold, respectively, above basal level of CAT activity (Fig. 2B). In NIH 3T3 fibroblasts, both human and mouse wildtype p53/GAL4 fusion proteins were also active (Fig. 2C). The extent to which wild-type p53/GAL4 fusion proteins are activated in different cell lines varies and may be due to the strength of the promoter driving p53/GAL4 in each cell type and/or species differences between other transcription factors interacting with p53/GAL4. The addition of neither carrier plasmid DNA nor DNA encoding GAL4 amino acids

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conformation-dependent

A. HUMAN globin intron _

CMV wt p53/GAL4 X

S

monoclonal antibody PAb246. At

however, the ability of p53Val to transform is lost. 32°C, Instead, p53Val now suppresses growth, no longer binds

GAL4

p53

B

p53-CX3/GAL4

hsc70, and is recognized by PAb246. These data suggest that the Val mutation disrupts a critical conformational domain within the p53 protein. We therefore tested the ability of the p53Val/GAL4 fusion protein to activate transcription at permissive temperatures. and plasmids (Fig. 1B) Wild-type were independently transfected into HeLa cells with a CAT vector containing four GAL4 DNA binding sites and a P-galactosidase reporter under standard conditions. Twentyfour hours after transfection, duplicate plates were shifted to 32 or and harvested 24 h later. After normalization for transfection efficiency, CAT assays were performed. As was active and shown in Fig. 3, at wild-type mutant was inactive. However, at 32°C the 15% of the P53Val/GAL4 mutant regained approximately The second protein. activity of the wild-type mutant (KH215/GAL4) tested contains an in-frame insertion at amino acid 215 and is not known to be temperature sensitive; it had no transactivation capability at either temperature (Fig. 3). Thus, the mutant p53Val/GAL4 fusion protein also exhibits a temperature-sensitive phenotype for its transactivation function. A p53 mutation identified in LFS patients was transcriptionally active as a p53/GAL4 fusion protein. LFS defines a familial cancer syndrome in which families are prone to various types of cancers at an unusually early age (22). Affected individuals in these families inherit a mutant p53 allele, while nonaffected individuals inherit two wild-type p53 alleles (23, 34). The most common mutation inherited appears to be a C-to-T transition at the first nucleotide of codon 248 resulting in an Arg-to-Trp substitution. This mutation falls in a highly conserved domain of the p53 molecule and corresponds to amino acid 245 in the mouse p53 protein. Using site-directed polymerase chain reaction, we replaced the CGC codon (Arg) with TGG (Trp). After sequence analysis confirmed that this was the only mutation introduced into our clone, the mutation was introduced into the mouse p53/GAL4 fusion protein. Either wild-type p53/ GAL4 or (Fig. 1B) was transfected into cultured cells with CAT and P-galactosidase plasmids. As fusion proshown in Fig. 4, wild-type and teins were equally effective at transactivation of a CAT plasmid with four GAL4 binding sites. This activity was also seen in CHO and NIH 3T3 cells (data not shown). Thus, the p53 mutation inherited in most LFS patients can transactivate CAT expression as a p53/GAL4 fusion protein. p53 mutations from colorectal carcinomas when introduced into p53/GAL4 fusion proteins cannot activate transcription. Studies of human colorectal carcinoma cells have been critical to our understanding of p53 function as a tumor suppressor (2, 3). Progressive development of colorectal carcinomas results in or is a result of progressive loss or mutation of both p53 alleles. Two mutations, a Val-to-Ala substitution at p53 amino acid 143 and an Arg-to-His alteration at amino acid 175, have recently been identified in colorectal carcinomas. Both mutations result in a p53 protein that no longer suppresses growth and now cooperates with ras to transform rat embryo fibroblasts (19). These mutations within the context of the p53 protein also result in an increased half-life for p53. Since the analogy had been made that the progressive development of cancer results in a nonfunctional p53 protein, we tested the ability of these mutants to function as transcriptional activators.

pS3/GAL4

aa 143 val --mala

p53-CX22VGAL4

37°C

pS3Val/GAL4

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p53/GAL4

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37°C

aa 175 arg --his

LTR

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FIG. 1. Wild-type (wt) and mutant p53/GAL4 plasmids. (A) Plasmids encoding human wild-type or mutant p53 (amino acids 1 to 346) fused to the GAL4 DNA binding domain (amino acids [aa] 4 to 147). These plasmids are regulated by the CMV promoter/enhancer and a P-globin intron. p53-CX3/GAL4 contains a single T-to-C substitution resulting in a Val-to-Ala alteration at p53 amino acid 143. p53-CX22/GAL4 contains a G-to-A substitution resulting in an Arg-to-His change at p53 amino acid 175. (B) Plasmids encoding mouse wild-type or mutant p53 (amino acids 1 to 343) fused to the GAL4 DNA binding domain (amino acids 4 to 147). These plasmids are expressed by the Rous sarcoma virus long terminal repeat (LTR). p53vaI/GAL4 contains a C-to-T substitution resulting in an Ala-to-Val alteration at amino acid 135. p53LFS/GAL4 contains an Arg (CGC)-to-Trp (TGG) mutation at p53 amino acid 245. Restriction sites: X, XbaI; S, SalI; B, BamHI; E/Sm, EcoRI/SmaI. Vertical dashed lines denote the SV40 polyadenylation signal.

4 to 147 to transfection experiments activated CAT expression. Transactivation did not occur when a CAT reporter plasmid without GAL4 binding sites was used (data not shown). Thus, these experiments demonstrate that specific

transcriptional activation of both human and mouse wildtype p53/GAL4 fusion proteins is independent of the cell line used for the analyses. Temperature-sensitive transcriptional activation of p53va/ GAL4. The mouse wild-type p53 fused to a GAL4 DNA binding domain specifically transactivates expression of CAT (30) (Fig. 2). Transformation-competent mutations within the p53 amino acid sequence resulted in p53/GAL4 fusion proteins that did not activate transcription (30). One of the mutants analyzed in this assay contains an Ala-to-Val substitution at p53 amino acid 135. This p53 Val mutant is temperature sensitive (26): at 37°C, it (i) is more stable than wild-type p53, (ii) readily transforms rat embryo fibroblasts in cooperation with activated ras, (iii) binds the heat shock protein hsc70, and (iv) is not well recognized by the p53

p53LFS/GAL4

p53LFS/GAL4

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RAYCROFT ET AL.

6070

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FIG. 2. Evidence that human and mouse wild-type p53/GAL4 fusion proteins function in several cell lines. The wild-type p53/GAL4 plasmids were cotransfected with a CAT reporter plasmid containing four GAL4 binding sites and a 3-galactosidase plasmid into HeLa (A), CHO (B), or NIH 3T3 (C) cells. Cells were harvested 48 h after transfection and assayed for CAT activity. Numbers above the lanes denote percent conversion of [14C]chloramphenicol (lower spot) to acetyl and diacetyl chloramphenicol (upper two spots) relative to the value for the plasmid-transfected control (lane 1 in each panel). Radioactivity was measured with a Betagen Betascanner.

We made two individual p53/GAL4 fusion constructs that carried these mutations and were identical to the wild-type p53/GAL4 fusion except for a single nucleotide change which altered one amino acid residue (either 143 or 175) (Fig. 1A). Sequence analysis verified the presence of the single base pair substitution. These plasmids were cotransfected with a CAT reporter and P-galactosidase plasmids into HeLa cells. CAT assays were performed on cell extracts harvested '25

2

82

48 h after transfection. Neither mutant was efficient at activating transcription of a CAT reporter plasmid containing four GAL4 binding sites (Fig. 5). Compared with wildtype p53, both mutants showed a 30- to 40-fold decrease in transcriptional activation. Inactive p53/GAL4 fusion proteins are made at levels comparable to wild-type levels. The lack of transactivation function by mutant p53/GAL4 fusion proteins may be due to production of the mutant proteins at lower than wild-type levels. It is unlikely that this is true, since wild-type and

8.

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FIG. 3. Temperature-sensitive activation of pS3Val/GAL4. A CAT plasmid containing four GAL4 binding sites was cotransfected

with plasmids expressing wild-type p53/GAL4 or mutant p53/GAL4 into HeLa cells. One mouse mutant (KH215/GAL4) contained a linker insertion at p53 amino acid 215 which alters a Val-Pro dipeptide to Pro-Ser-Leu-Ala. The second mutant contains an Ala-to-Val substitution at p53 amino acid 135 (p53Val/GAL4). A ,B-galactosidase-expressing plasmid was also used in transfection experiments to monitor and normalize for transfection efficiency. Twenty-four hours after transfection, plates were shifted to the appropriate temperature. Cells were harvested 48 h after transfection and assayed for CAT activity. Numbers above the lanes represent the fold induction of CAT activity with respect to the negative controls (lanes 1 and 2).

p53

wt

LFS/ p53/ GAL4 GAL4 GAL4

-

FIG. 4. Evidence that the mutant p53LFS/GAL4 fusion protein retains transactivation function. An Arg-to-Trp mutation was introduced into p53 amino acid 245. This mutant p53LFS/GAL4 fusion plasmid was transfected into HeLa cells with a CAT reporter plasmid containing four GAL4 DNA binding sites and a 0-galactosidase plasmid to monitor transfection efficiency. Numbers above the lanes denote percent conversion of [14C]chloramphenicol to acetyl and diacetyl chloramphenicol relative to the value for the control transfection (lane 4). The pS3LFS/GAL4 fusion protein was on average 1.5 times more active than wild-type (wt) p53/GAL4 with a standard deviation of 0.4.

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1

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d fusionproteins thath humana mutant n FIG. 5. 5 Evidence p53/GAL4 fusion proteins are po or transcriptional activators. Human wild-type (wt) and mutant p53/GAL4 plasmids were cotransfected with CAT reporter and 1-igalactosidase plasmids into HeLa cells. CAT activity was assayedI in a 2-h reaction (twice normal). Numbers above the lanes denote percent conversion as measured with a Betagen Betascanner. mutanit p53/GAL4 proteins are expressed by the same promoter and differ in a point mutation in the coding region. Howe)ver, we analyzed for the presence of the fusion proteins s,ince a very unstable mutant p53/GAL4 fusion would also be transcriptionally inactive. Plasmids containing wildtype, IVal-to-Ala, or Arg-to-His p53/GAL4 fusion proteins and th e SV40 promoter/enhancer were transfected into Cos and HieLa cells. After transfection, cells were labelled with [35S]Mlet and [5S]Cys. Proteins were immunoprecipitated with p'53-specific monoclonal antibody p53 Ab-2 or PAb240. In Cos;cells, both mutants (Fig. 6A, lanes 1 and 2) are made at levcels comparable to wild-type p53 levels (lane 3). In

A 1

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FIG. 6. Immunoprecipitation of p53/GAL4 fusion proteins. (A) Cos cells were transfected with p53/GAL4-expressing plasmids, labeled with [35S]Met and [35S]Cys, and immunoprecipitated with human p53 antibody p53 Ab-2 (lanes 1 to 3), T-antigen antibody PAb412 (lane 4), or mutant p53 antibody PAb240 (lanes 5 to 7). were performed with p53-CX3/GAL4 (lanes 1 and 5), p53-CX22/GAL4 (lanes 2 and 6), human wild-type p53/GAL4 (lanes

Transfections

3 and 7), or no DNA (lane 4). (B) HeLa cells were transfected with wild-type p53/GAL4 (lane 1), p53-CX22/GAL4 (lanes 2), or p53-

CX3/GAL4 (lane 3), labeled, and immunoprecipitated with p53 Ab-2. Lane 4 is

an

addition, p53 Ab-2 immunoprecipitates p53 and pS3/GAL4 but not T antigen, suggestive of a lack of interaction between p53/GAL4 and T antigen in Cos cells. However, in nontransfected Cos cells, p53 Ab-2 immunoprecipitates only p53 and not p53 complexed to T antigen (data not shown). Immunoprecipitations with PAb240, a mutant p53-specific antibody (13), identify the mutant p53/GAL4 fusion proteins but not the wild-type fusion protein or endogenous p53 (lanes 5 to 7). In addition, PAb240 recognizes mutant p53-CX22/GAL4 better than mutant p53-CX3/GAL4. In transfected HeLa cells, both mutant p53/GAL4 fusion proteins (Fig. 6B, lanes 2 and 3) are more readily detected than the wild-type pS3/GAL4 (lane 1). Thus, the p53 mutants identified in colorectal carcinomas when fused to GAL4 are made at or Cos cells and comparable levels in transfected HeLa cellsreporter are inefficient at transactivation of a CAT AreAllinfin ct ivAtion a repore lasmid nonfunctional pS3/GALA fusion proteins are located in the nucleus. Another possible explanation for lack of function of mutant p53/GALA fusion proteins is that they are not translocated to the nucleus. Recent data in fact suggest that p53V , the temperature-sensitive p53 protein with an Ala-toVal substitution at amino acid 135, does not make it to the nucleus at restrictive temperatures (14, 24). Even though the GAL4 DNA binding domain contains a nuclear localization signal, it was important to determine the localization of mutant p53/GAL4 fusion proteins (32). Cos cells were plated on coverslips, transfected with mutant p53/GAL4 plasmids, and stained with the mutant-specific p53 monoclonal antibody PAb240. Cos cells containing endogenous wild-type p53 did not stain with PAb240 (Fig. 7A). Cos cells transfected with the p53Val/GAL4 temperature-sensitive mutant or the two mutant p53/GAL4 fusions from colorectal carcinomas had nuclear staining (Fig. 7B to D). Since mutant p53/GAL4 fusion proteins cannot complex to SV40 T antigen (Fig. 6), it is unlikely that T antigen is pulling p53/GAL4 into the nucleus. Thus, the inability of these mutants to function as transactivators is also not due to inappropriate localization.

plasmid.

7 _-

0

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DISCUSSION Mutations in p53 are numerous and are found in tumors as diverse as breast cancer and osteosarcoma. Since the normal protein functions as a growth suppressor, cells must inactivate p53 and overcome a G1 block to become tumorigenic (24). How does p53 prevent cells from growing? Recent evidence suggests that the wild-type p53 protein can function as a transcriptional activator (28, 30). Two p53 mutations analyzed cannot suppress growth and cannot activate transcription (30). These observations led to the hypothesis that p53 functions as a growth suppressor by activating transcription of some other gene(s). The study presented in this report extends these observations by analysis of wild-type human and mouse p53 transactivation function in various cell lines. This analysis is important since the original study examined the transactivation function of wild-type p53 only in HeLa cells. HeLa cells contain the protein HPV E6, which binds p53 (37). The question remained as to whether wild-type p53 without HPV E6 could activate transcription. As shown in Fig. 2, wild-type human and mouse p53/GAL4 fusion proteins can transactivate CAT expression in various cell lines. The extent to which activation occurs varies in the different cell lines and may be due to the strength of the promoters in each cell type or species differences between p53/GAL4 fusion proteins and the cell lines. In addition, this study analyzes the transactivation func-

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FIG. 7. Immunostaining of mutant p53/GAL4. Cos cells were grown on coverslips and transfected with mutant p53/GAL4 fusion proteins. Mutant p53/GAL4 was detected by using a mutant p53-specific monoclonal antibody (PAb240) and goat anti-mouse fluorescein isothiocyanate conjugate. (A) Nontransfected Cos cells; (B) Cos cells transfected with p53Val/GAL4; (C) Cos cells transfected with p53-CX3/GAL4; (D) Cos cells transfected with p53-CX22/GAL4.

tion of other p53 mutants. One mutation, an Ala-to-Val substitution at mouse p53 amino acid 135, resulted in a temperature-sensitive p53 protein (26). At 37°C, p53Va' behaved as a mutant protein. p53Val cooperated with activated ras to transform primary rat embryo fibroblasts. p53Val cannot suppress transformed cell growth. It binds hsc70, a member of the heat shock proteins, p53Val is not well recognized by the p53 conformation-dependent monoclonal antibody PAb246. At 370C, pS3Val/GAL4 is transcriptionally inactive (30) (Fig. 3) even though the protein is stable (30) and is located in the nucleus (Fig. 7). At 32°C, however, p53 Val has the properties of wild-type p53: it no longer binds hsc70, most is now recognized by PAb246, and it functions to suppress transformed cell growth. At 320C, p53Val/GAL4 also had transcriptional activity. This temperature-sensitive mutant provides the best example of the correlation between transactivation and growth suppression. Another important observation is that 15% of wild-type transactivation function is sufficient for p53 Val to suppress transformation. To extend our observations to p53 mutations that actually arise in human tumors, we analyzed naturally occurring mutations from human colorectal carcinomas and from patients inheriting LFS. Two point mutations in colorectal carcinomas occurring at either p53 amino acid 143 or 175 allow these mutant p53 proteins to cooperate with ras for transformation. In our transcription activation assay, these two point mutants were inefficient transcriptional activators. Inappropriate localization of the mutants or protein instability is not the cause of the lack of activity, since we show that the mutant proteins are made at levels comparable to wildtype levels (Fig. 6) and are translocated to the nucleus (Fig. 7). These mutations fall in the conformation domain and alter the conformation of p53 as monitored by antibody binding. Both human p53 mutants from colorectal carcinomas are recognized by the mutant-specific monoclonal antibody

PAb240 (Fig. 6), bind hsc70, have increased half-lives, and cooperate with activated ras to transform primary rat embryo fibroblasts (19). These transformation-competent p53 mutants are proposed to inactivate wild-type p53 in a dominant negative fashion (11, 19). This hypothesis suggests that oligomerization of wild-type and mutant p53 results in an inactive oligomer. On the other hand, little is known about the LFS mutation. It is a p53 mutation inherited in families with LFS, and its presence correlates with early-onset tumor development (23, 34). The LFS mutation that we analyzed resides at pS3 amino acid 245, outside the conformation domain but within another highly conserved p53 domain (33). It is unknown whether the p53LFS mutation cooperates with ras to transform cells. We predict that this mutation is not a dominant negative mutation. By definition, a dominant negative mutation suggests that most p53 oligomers would be inactive. If this is the case, the LFS mutation would not be inherited but would result in a lethal phenotype. In our assay, the p53LFS/ GAL4 fusion protein is transcriptionally active. But is pS3LFS transcriptionally active? The GAL4 amino acids 4 to 147 fused to pS3LFS provide three functions: DNA binding, dimerization, and nuclear localization (5, 21, 32). Thus, if the LFS mutation causes a loss of DNA binding or a lack of oligomerization, it would remain transcriptionally active in a GAL4 fusion. Identification of p53 targets will allow us to answer this question. The LFS mutation analyzed here is not the only p53 mutation that results in a transcriptionally active p53/GAL4 fusion protein. An Arg-to-His mutation at human p53 amino acid 273 also results in a p53/GAL4 fusion protein that is transcriptionally active (10). This amino acid also falls in a highly conserved region of p53 near the LFS mutation and, like the LFS mutation, may disrupt other domains of p53 important for function. This Arg-to-His mutation produces a

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mutant p53 that is weakly transforming (19). Thus, different p53 mutants have distinct biological activities (16). From these results, we conclude that the conformation domain is critical for p53 transactivation function. A possible role for this domain is to position the acidic domain in proper context with the rest of the transcriptional machinery. The amino-terminal 73 amino acids of p53 have been shown to contain several negatively charged residues. This part of p53 is able to transactivate gene expression when fused directly to GAL4 (10). Interestingly, this region of p53 is poorly conserved among human, mouse, and Xenopus genomes except for the overall negative charge (33). This phenomenon is typical of other transcription factors: negative transactivation domains are not highly conserved. Since transactivation domains are not conserved, the negatively charged amino terminus of p53 must be analyzed in the context of the

whole molecule. The p53 acidic domain by itself is not the only requirement for transcriptional activation. Three mutations analyzed in this study fall within p53 amino acids 135 to 215, the conformation domain, and result in a p53 protein that cannot activate transcription. These mutations do not alter any of the first 73 amino acids. The LFS mutation and the Arg-to-His mutation at p53 amino acid 273 occur outside

of the conformation domain and remain transcriptionally active. ACKNOWLEDGMENTS

We thank C. A. Finlay and A. J. Levine for the human wild-type and mutant p53-expressing plasmids and G. S. May for help with immunofluorescence. We also thank J. Heath for use of his microscope and P. McCauley for preparation of the manuscript. This work was supported by Public Health Service grant CA47296 from the National Cancer Institute.

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