MOLECULAR AND CELLULAR BIOLOGY, Apr. 1991, p. 1996-2003 0270-7306/91/041996-08$02.00/0 Copyright C 1991, American Society for Microbiology

Vol. 11, No. 4

Dephosphorylation of Simian Virus 40 Large-T Antigen and p53 Protein by Protein Phosphatase 2A: Inhibition by Small-t Antigen KARL HEINZ

SCHEIDTMANN,l MARC C.

MUMBY,2* KATHLEEN RUNDELL,3 AND GERNOT WALTER4 Institut fur Genetik, Universitat Bonn, Romerstrasse 164, D5300 Bonn, Federal Republic of Germany'; Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-90412; Department of Microbiology-Immunology, Northwestern University McGaw Medical Center, Chicago, Illinois 6061J3; and Department of Pathology, University of California, San Diego, La Jolla, California 920934 Received 1 November 1990/Accepted 14 January 1991

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.

Polyomavirus medium-T antigen, as well as the polyomavirus and simian virus 40 (SV40) small-t antigens (small-t), form complexes with protein phosphatase 2A (PP2A), a serine/threonine-specific phosphatase (31, 49) consisting of three subunits, the catalytic subunit and two regulatory subunits, A and B (11). To elucidate the role of these complexes in neoplastic transformation by polyomavirus and SV40, it is important to determine the effect of T-antigen binding on the enzymatic properties of PP2A. In the accompanying article (50), we demonstrate that purified SV40 small-t binds to the free A subunit and the AC form of PP2A but not to the free C subunit or the ABC form. We also show that small-t inhibits dephosphorylation of myelin basic protein and myosin light chains by the AC form but has no effect on the activity of free C or the ABC form. With phosphorylated histone Hi as an exogenous substrate, small-t stimulates the activity of the AC form. The B subunit and small-t appear to bind to the same site on AC and have similar effects on its activity. In this article, we investigated the substrate specificity of PP2A towards SV40 large-T antigen and the cellular protein p53 and how small-t alters this activity. Large-T is phosphorylated at eight or more sites clustered in two regions of the polypeptide (17, 20, 27, 38, 42, 47). Phosphorylation at Ser-120 and Ser-123 inhibits binding to the origin of SV40 DNA replication (19, 30, 40, 48), whereas phosphorylation at Thr-124 is a prerequisite for DNA binding (27). The catalytic subunit of PP2A stimulates SV40 DNA replication in a cell-free system, presumably by dephosphorylating Ser-120 *

and Ser-123 (40a, 48). p53 is a nuclear protein (see reference 23 for review) which is phosphorylated at multiple sites, some of which have been mapped (28, 36). It has growthsuppressing properties and appears to be involved in a large number of human cancers (1, 46). We demonstrate that PP2A dephosphorylates large-T and p53 preferentially at specific sites and that the activity of the AC form is partially inhibited by small-t.

MATERIALS AND METHODS Purification of proteins. The catalytic subunit (C), the two-subunit (AC), and the three-subunit (ABC) forms of PP2A and recombinant SV40 small-t were purified as described in the accompanying article (50). Cell lines and virus. The SV40-transformed rat SV52 cell line (2) and monkey TC7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. SV40 (large-plaque strain 45-50, originally obtained from P. Tegtmeyer) was used for infection of TC7 cells. The multiplicity of infection was approximately 10. Labeling of cells. Metabolic labeling of SV52 cells and TC7 cells infected with SV40 was carried out on 9-cm plates for 3 h in 2 ml of phosphate-free Eagle's medium containing 1 mCi of 32pi. SV40-infected TC7 cells were labeled from 36 to 39 h postinfection. Immunoprecipitation of large-T and p53. After labeling, cells were washed with cold phosphate-buffered saline and lysed on the plates with isotonic lysis buffer (10 mM sodium phosphate [pH 8], 140 mM NaCl, 3 mM MgCl2, 1 mM dithiothreitol [DTT], 5% Nonidet P-40, 50 ,uM leupeptin). Nuclei were removed by centrifugation for 15 min at 7,000

Corresponding author. 1996

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rpm in a Beckman JS-13 rotor. The supernatants were used for immunoprecipitation of large-T or large-T/p53 complexes with large-T-specific monoclonal antibody PAb419 (18) or KT3 (25) as indicated in the figure legends. Immune complexes were adsorbed to protein A-Sepharose and washed three times with RIPA buffer (0.15 M NaCl, 10 mM sodium phosphate [pH 7.2], 1% deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate [SDS], 1 mM DTT, 50 ,uM leupeptin). The washed precipitates were either eluted with SDS sample buffer (2% SDS, 5% 2-mercaptoethanol, 5% glycerol, 10 mM DTT, 0.01% bromphenol blue, 50 mM Tris-HCI [pH 6.8]) or washed three times with 20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.0)-i mM DTT-100 ,ug of bovine serum albumin (BSA) per ml-50 ,uM leupeptin and treated with PP2A as described below. SDS-treated samples were analyzed on 12.5% SDS-polyacrylamide gels (22) and visualized by autoradiography. Phosphatase treatment of p53- and large-T-containing immune complexes. Washed immunoprecipitates were incubated at 30°C with PP2A as described in the figure legends. Reactions were stopped with 1 ml of cold TE buffer (10 mM Tris-HCl [pH 7.5], 0.1 mM EDTA). The TE buffer was collected, and the released 32P was counted in a scintillation counter. The precipitate was washed again with TE, and the 32P remaining associated with large-T and p53 was solubilized with SDS sample buffer. The solubilized material was counted, analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), or subjected to two-dimensional peptide mapping. Two-dimensional peptide analysis. Large-T and p53 were separated on 12.5% SDS-polyacrylamide gels. The bands were localized on the unfixed, dried gel by autoradiography, cut out, and processed for peptide mapping as described previously (39, 40). Briefly, the proteins were extracted from the gel, precipitated with 20% trichloroacetic acid, and oxidized with 3% performic acid. Large-T was digested sequentially with trypsin and pronase; p53 was digested sequentially with trypsin and chymotrypsin. The digests were analyzed on cellulose thin-layer plates by electrophoresis and ascending chromatography. The peptides were visualized by autoradiography. RESULTS

Dephosphorylation of large-T and p53. For studies on dephosphorylation of large-T and p53, we used an SV40transformed cell line, SV52, which expresses high levels of these proteins. The bulk of p53 in these cells is complexed to large-T. The cells were labeled with 32P, and large-T/p53 complexes were immunoprecipitated with monoclonal antibodies against large-T. Immunoprecipitates were then used as substrates for PP2A. Initially, the dephosphorylation assays were carried out with the free C subunit in the absence of divalent cations since PP2A does not require divalent metal ions for activity (11). The extent of dephosphorylation of both substrates increased with increasing amounts of enzyme (Fig. 1A). By densitometry scanning of the autoradiogram, it was determined that 21, 48, and 62% of the phosphate label of large-T and 52, 74, and 81% of the phosphate label of p53 was removed by 0.67, 2.7, and 13.5 pmol of C subunit, respectively. The highest concentration of enzyme used amounts to an enzyme-to-large-T ratio of approximately 20:1, assuming that one 9-cm culture dish of confluent SV52 cells contains 0.5 ,ug of large-T (data not

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7 2 3 4 5 6 FIG. 1. Dephosphorylation of large-T and p53 by PP2A. (A) Variation of enzyme concentrations. For the immunoprecipitation of each sample, 100 Il of 32P-labeled SV52 cell extract, 20 p.l of PAb419 hybridoma supematant, and 20 RI of protein A-Sepharose were used. Dephosphorylation was carried out at 30°C for 30 min in 30 ,ul of 20 mM HEPES (pH 7.0)-i mM DTT-100 ,ug of BSA per ml-50 p.M leupeptin with 0, 0.05, 0.2, and 1 pLg of a 50% pure preparation of the catalytic subunit, corresponding to 0, 0.67, 2.7, and 13.5 pmol, respectively. The molar concentrations of enzyme were 22.5, 90, and 450 nM, respectively. Dried gels were exposed to film for 3 h. (B) Effect of Mg2" and Mn2+ on phosphatase activity. Immunoprecipitations were carried out with 100 ,u1 of extract, 20 p.l of PAb419, and 20 ,ul of protein A-Sepharose per assay. Dephosphorylations were carried out with 13.5 pmol of catalytic subunit (C) per sample at 30°C for 30 min in HEPES buffer as described for panel A. The MnCl2 concentration was varied from 0.01 to 1 mM, and the MgCl2 concentration was held constant at 7 mM, as indicated. Exposure time was 36 h. (C) Comparison of three forms of PP2A. Large-scale immunoprecipitations were carried out with 1 ml of 32P-labeled SV52 extract, 200 .l of PAb419, and 100 p.l of protein A-Sepharose for each sample. The C, AC, and ABC forms were used at approximately equimolar amounts (135 pmol per assay). The Mg2' and the Mn2+ concentrations were 7 and 1 mM, respectively. HEPES buffer was used as described for panel A. A small aliquot (5%) from each sample was analyzed on the analytical gel shown here; the bulk of the material was loaded on a preparative gel and used for the peptide mapping experiment shown in Fig. 2. Exposure time was 2 days with an enhancer screen. LT, Large-T. 1

shown). The dephosphorylation of p53 was considerably faster than that of large-T. As was observed with myosin light chains as the substrate (50), significantly more dephosphorylation of large-T and p53 was obtained in the presence of magnesium chloride and/or manganese chloride. As determined by densitometry of the autoradiogram shown in Fig. 1B, with 0.1 mM Mn2 , 66% of large-T and 94% of p53 were dephosphorylated, while 49 and 84%, respectively, were dephosphorylated in the presence of 7 mM Mg2+. Addition of 0.01 mM Mn2+ to 7 mM Mg2+ increased the dephosphorylation of large-T from 49 to 94% and of p53 from 84 to 99%. These data demonstrate that the combined effects of the two cations were more than additive.

1998

SCHEIDTMANN ET AL.

Increasing the Mn2" concentration from 0.1 to 1 mM, in the absence of Mg2", increased dephosphorylation of large-T from 66 to 96% and of p53 from 94 to 99%. Figure 1C shows a comparison of the activity of the three forms of PP2A. In the presence of 7 mM Mg2+, dephosphorylation of large-T with the C, AC, and ABC forms was 35, 18, and 9%, respectively, and that of p53 was 79, 86, and 63%, respectively. These results demonstrate that with large-T and p53 as substrates, all forms of PP2A are stimulated to a greater extent by Mn2+ than by Mg2+ and that the free catalytic subunit is more active than the two-subunit form, which in turn is more active than the three-subunit form. SV40 large-T is well characterized with regard to the location and function of individual phosphorylation sites. The following sites which are phosphorylated in vivo have been identified: Ser-106, Ser-112, Ser-120, Ser-123, Thr-124, Ser-639, Ser-677 or Ser-679, and Thr-701 (38, 40, 42). In order to determine which sites were dephosphorylated by the different forms of PP2A, large-T was dephosphorylated and individual sites were examined by peptide mapping. Large-T was digested with trypsin and pronase, and the resulting phosphopeptides were analyzed by two-dimensional peptide mapping (Fig. 2). The map of SV40 large-T dephosphorylated by free C in the presence of 7 mM Mg2+ (Fig. 2B) showed an increase in phosphopeptide 12 and decreases in peptides 7 and 11 compared with the control. When the dephosphorylation was carried out in the presence of 1 mM Mn2 , the amounts of most of the phosphopeptides decreased. Mapping experiments were also carried out with AC and ABC with results similar to those obtained with C (data not shown), although dephosphorylation with the ABC form appeared to be less complete, as judged from a higher proportion of peptides 7 and 11 relative to 12. We have shown previously that peptides 7, 11, and 12 are identical in sequence and differ only in the number of phosphate residues (37, 40). Peptide 7 is labeled on Ser-120, Ser-123 and Thr-124, peptide 11 is labeled on Ser-120 or Ser-123 and on Thr-124, whereas peptide 12 contains phosphate only on Thr-124. Thus, the decrease in peptides 7 and 11 after PP2A treatment and the increase in the labeling of peptide 12 reflect dephosphorylation of Ser-120 and Ser-123 and a corresponding accumulation of the peptide labeled only on Thr-124. We know from previous work that the two threonine phosphates in large-T (Thr-124 and Thr-701, the latter being contained in peptide 13) are considerably more resistant to phosphatases than the serine phosphates. Although the dephosphorylation of Ser-120 and Ser-123 is most easily detected, all of the sites in large-T were dephosphorylated by PP2A. Even under mild conditions (7 mM Mg2+), some dephosphorylation of threonine residues took place, although at a slower rate than that of Ser-120 and Ser-123. The rates of dephosphorylation of the remaining serine phosphates (Ser-106, -112, -639, and -677 or -679) were also rather slow. When large-T from SV40-infected TC7 cells was dephosphorylated with PP2A, we obtained peptide maps identical to those obtained with large-T from SV52 cells (data not shown). This demonstrates that the dephosphorylation of large-T is not affected by bound p53, since large-T from infected TC7 cells is not complexed to

p53.

Dephosphorylation of p53 by PP2A was also monitored by peptide mapping. As demonstrated in Fig. 3, dephosphorylation of p53 by PP2A shows a remarkable specificity in that phosphate from peptides 1, 5, and 6 was almost completely removed, whereas peptides 2, 3, and 4 were resistant.

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FIG. 2. Phosphopeptide analysis of large-T dephosphorylated with PP2A. Large-scale immunoprecipitations and dephosphorylations of large-T and p53 in the presence of 7 mM Mg2" or 1 mM Mn2+ with the C, AC, and ABC forms of PP2A were carried out as described in the legend to Fig. 1C. Peptide mapping was carried out as described in the text. Panels B and C show maps of large-T dephosphorylated by the catalytic subunit with 7 mM Mg2' and 1 mM Mn2+, respectively. A control map of undephosphorylated large-T was done in parallel (panel A). Exposure times with an enhancer screen were 2 days (A), 3 days (B), and 9 days (C). Numbers refer to specific peptides (see text).

DEPHOSPHORYLATION INHIBITION BY SMALL-T ANTIGEN

VOL. 1 l, 1991

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FIG. 3. Phosphopeptide analysis of p53 dephosphorylated with PP2A. Immunoprecipitations and dephosphorylations of p53 with the C, AC, and ABC forms of PP2A were carried out as described in the legends to Fig. 1C and 2. (A) Control map of undephosphorylated p53; (B) map of p53 dephosphorylated with the C subunit in the presence of 7 mM Mg2+. Exposure times with an enhancer screen were 16 h (A) and 2 days (B).

Inhibition of dephosphorylation of large-T and p53 by SV40 small-t. Initial experiments with small-t were carried out at a high ratio of PP2A to substrate as in the experiments described above; under these conditions, no effect of small-t on PP2A was observed. When the ratio of enzyme to large-T was reduced from an estimated ratio of 20:1 to 1:4, inhibition of dephosphorylation of large-T and p53 became apparent. Further studies showed that the ratio had to be decreased to 1:3 or less. Figure 4 shows the effect of small-t on the dephosphorylation of large-T and p53 immunoprecipitated from 32P-labeled SV52 extracts by the different forms of PP2A. The data demonstrate that neither C nor the ABC form was significantly affected by small-t, whereas the AC

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FIG. 4. Inhibition of dephosphorylation of large-T and p53 by SV40 small-t. Individual large-T/p53 samples were precipitated from 400 ,ul of 32P-labeled SV52 extract with 8 ,ud of monoclonal antibody KT3 (ascites fluid) and 40 ,ul of protein A-Sepharose. Dephosphorylations were carried out with 18 ng (0.5 pmol) of C subunit or an equivalent amount of the AC and ABC forms, in the presence (+) and absence (-) of 0.7 ,ug (33 pmol) of small-t in a final volume of 11 >1± of 20 mM HEPES (pH 7.0)-i mM DTT-100 ,ug of BSA per ml-50 ,uM leupeptin-1 mM MnCl2-0.05% Nonidet P-40. The small-t/PP2A mixtures were incubated at 30°C for 10 min before being added to the precipitates. Incubations were for 45 min at 30°C. The reactions were stopped, and the 32P released was determined by scintillation counting as described in the text.

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FIG. 5. Gel analysis of the inhibition of dephosphorylation of large-T and p53 by SV40 small-t. Individual large-T/p53 samples were immunoprecipitated and treated with PP2A as described in the legend to Fig. 4. Dephosphorylation reaction mixes contained the free catalytic subunit (C), the AC form of PP2A (AC), the ABC form (ABC), or no phosphatase (CONT.) in the presence (+) or absence (-) of small-t (ST). The mobilities of SV40 large-T antigen (LT) and p53 (p53) are indicated. Small-t was also present in lane 7.

form was inhibited by 50%. Interestingly, the extent of dephosphorylation by the AC form in the presence of small-t was similar to that obtained with ABC. Thus, the B subunit of PP2A and small-t seem to have a similar influence on enzyme activity. This result is consistent with the effects of small-t on dephosphorylation of myosin light chains and myelin basic protein. That 32p label was being removed from large-T and p53 during phosphatase treatment was verified by SDS-PAGE (Fig. 5). Further studies showed that a 50-fold molar excess of small-t over enzyme was required for maximum inhibition of the phosphatase activity (data not shown), also consistent with the effects of small-t on exogenous substrates. Effect of small-t on the dephosphorylation of specific sites. To determine whether small-t inhibits dephosphorylation in general or only of particular sites on large-T and p53, the dephosphorylated proteins were analyzed by two-dimensional peptide mapping. The dephosphorylation reactions were carried out with all three forms of PP2A in the presence and absence of small-t and with 1 mM Mn2+ or 7 mM Mg2+ plus 0.1 mM Mn2+. With Mn2' alone, release of total phosphate from the large-T/p53 complex by the AC form was inhibited 40% by small-t. With Mg2+ plus Mn2+, release was inhibited by 25%. No effect of small-t on the C subunit or the ABC form was observed under any conditions. As shown in Fig. 6, small-t decreased the intensity of peptide 12, with a corresponding increase in peptides 7 and 11, during dephosphorylation by AC. As described above, this reflects decreased dephosphorylation of Ser-120 and Ser-123 and decreased conversion to the monophosphorylated peptide (peptide 12). The map obtained in the presence of small-t is very similar to that with untreated large-T. These results suggest that small-t preferentially inhibited the dephosphorylation of Ser-120 and Ser-123. Two other peptides which were dephosphorylated by AC and reappeared in the presence of small-t are located just below and to the right of peptide 11 and above and to the left of peptide 11. They probably represent peptides 9 and 10, respectively, containing Ser-677 and presumably Ser-679. The effect of small-t on the dephosphorylation of p53 by PP2A is shown in Fig. 7. Without small-t, the amounts of

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FIG. 6. Phosphopeptide analysis of large-T dephosphorylated by the two-subunit form of PP2A in the presence and absence of small-t. Large-scale immunoprecipitations with 1.4 ml of 32P-labeled SV52 extract per reaction were carried out with monoclonal antibody KT3 under the conditions described in the legend to Fig. 4. Dephosphorylations of the precipitates with equimolar amounts of C, AC, and ABC were done with the same ratio of enzyme to precipitate described in the legend to Fig. 4. The reactions were carried out in the presence of 1 mM Mn2' and of 7 mM Mg2+ plus 0.1 mM Mn2+. The reactions were stopped and processed for loading onto preparative gels as described in the text. A control precipitation of large-T not treated with phosphatase was processed in parallel. (A) Map of untreated control large-T; (B) large-T dephosphorylated by AC in the absence of small-t (-ST) and in the presence of 7 mM Mg2+ plus 0.1 mM Mn2+; (C) large-T dephosphorylation by AC in the presence of small-t (+ST) and 7 mM Mg2+ plus 0.1 mM Mn2+. Exposure times: 3 to 5 days with enhancer screens.

peptides 1, 5, and 6 were diminished relative to the control, indicating dephosphorylation of those sites by AC (Fig. 7B) and ABC (Fig. 7D). The AC form was much more active toward these sites than the ABC form. In the presence of

FIG. 7. Phosphopeptide analysis of p53 dephosphorylated by AC in the presence and absence of small-t. The same preparative immunoprecipitates used for the dephosphorylation of large-T described in the legend to Fig. 6 were used for the peptide analysis of p53. Shown are the maps of p53 dephosphorylated by AC enzyme in the presence of 7 mM Mg2" plus 0.1 mM Mn2". (A) Untreated p53 (CONT.); (B) p53 dephosphorylated by AC in the absence of small-t (-ST); (C) p53 dephosphorylated by AC in the presence of small-t (+ST); (D) p53 dephosphorylated by ABC in the absence of small-t; (E) p53 dephosphorylated by ABC in the presence of small-t.

small-t, dephosphorylation of peptides 1, 5, and 6 was markedly inhibited (Fig. 7C), resulting in a map similar to that of untreated p53. Thus, as in the case of large-T, small-t partially inhibits the dephosphorylation of p53 by the AC form of PP2A. No effect of small-t on dephosphorylation of p53 by the ABC form was observed (Fig. 7E). When large-T and p53 were dephosphorylated with the catalytic subunit (C) and subsequently analyzed by peptide mapping, no effect of small-t was detected (data not shown). Similar maps were obtained when the dephosphorylation was carried out in the presence of 1 mM Mn2+ or 7 mM Mg2+ plus 0.1 mM Mn2+. DISCUSSION Phosphorylation probably plays a role in the function of all oncogenic as well as growth suppressor proteins. The state of phosphorylation is determined by the interplay between protein kinases and phosphatases. In order to study how PP2A might control phosphorylation of these proteins and how this control might be altered by interaction with tumor antigens, we chose SV40 large-T and p53 as model substrates. Large-T is one of the best-characterized transforming proteins with regard to the location and function of phosphorylation sites. The specific removal of phosphates from Ser-120 and Ser-123 in SV40 large-T by the catalytic subunit of PP2A and its effect on large-T-dependent DNA replication in vitro have been demonstrated previously (40a, 48). p53 may function as a suppressor of neoplastic growth, possibly by regulating transcription (15, 32). The role of phosphorylation in its function is presently unknown. We have demonstrated here that all forms of PP2A preferentially dephosphorylate a subset of sites in both proteins. As was

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DEPHOSPHORYLATION INHIBITION BY SMALL-T ANTIGEN

the case with the exogenous substrates myosin light chain and myelin basic protein (50), small-t specifically inhibited dephosphorylation by the AC form but not the C or ABC form. The inhibitory effect was qualitatively and quantitatively similar to that of the B subunit of PP2A. This raises the question of whether the B subunit may also act as a stimulator of cell growth, as does small-t. It will be interesting to elucidate the sequence of the B subunit and to search for structural homology with small-t. The dephosphorylation of large-T and p53 was strongly activated by magnesium and/or manganese. At high enzyme concentration and in the presence of magnesium and manganese, all phosphates were removed from large-T and p53, including those bound to Thr-124 and Thr-701 of large-T, which are rather resistant to most phosphatases. The finding that a small amount (0.01 mM) of manganese strongly enhances the effect of 7 mM magnesium might be physiologically important, since in mammals magnesium is approximately 1,000- to 10,000-fold more abundant than manganese. Many investigators have studied the role of small-t in transformation. Whereas large-T alone can be transforming under certain conditions (6, 21), it is clear that small-t cannot transform cells by itself. However, there is convincing evidence that it plays a supplementary role in both the establishment and the maintenance of transformation (3, 5, 8, 10, 14, 26, 33, 41, 43, 44). Whether or not this role becomes apparent depends on the particular transformation assay, the cell type used, and the growth conditions. Thus, the establishment function of small-t is required for transformation of resting but not of growing cells (26), suggesting that a serum growth factor can substitute for small-t. Small-t can also be partially replaced by the tumor promoter 12-0tetradecanoylphorbol-13-acetate (26). The crucial question is whether the growth-promoting function of small-t can be explained by its inhibitory effect on PP2A. The fact that okadaic acid acts as a cocarcinogen by binding to and inhibiting PP2A and/or PP1 (12) strongly suggests that the growth-promoting or cocarcinogenic activity of small-t is the result of its complex formation with PP2A and the subsequent inhibition of its activity. It has been suggested that PP2A might counteract protein kinase C by dephosphorylating substrates phosphorylated by this kinase (12). Tumor promotion by okadaic acid would then result from inhibition of PP2A and increased phosphorylation of specific protein kinase C substrates. By analogy, the growthstimulatory effects of SV40 small-t might be explained in a similar fashion (i.e., an effect on protein kinase C substrates). This model (Fig. 8) may explain why small-t is not required for the transformation of growing cells (26), in which protein kinase C is maximally activated by growth factors or tumor-promoting phorbol esters. High protein kinase C activity would overwhelm any effect of small-t on PP2A. In resting cells, protein kinase C activity would be low and inhibition of PP2A by small-t would indirectly increase the phosphorylation state and activity of growthstimulatory protein kinase C substrates. The model predicts that small-t would have less growth-promoting activity in cells with a low level of PP2A than in cells with a high level. It might also explain why, in certain cell lines, the amount of small-t required for transformation is lower than in others (8, 43), if these lines differ in their PP2A content. The other known properties of small-t, such as its ability to induce dissolution of actin cables (16), to decrease the sensitivity of cellular DNA synthesis to theophylline inhibition (35), and to transactivate transcription (24), could involve an inhibition of PP2A. If the major effect of small-t is inhibition of PP2A,

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PROLIFERATION

FIG. 8. Proposed model depicting the negative effects of okadaic acid (OA), the B subunit of PP2A (B), and small-t (ST) on the dephosphorylation of substrates of protein kinase C (PKC) by PP2A. The B subunit and small-t are shown interacting with the A subunit of PP2A (shaded area labeled A), while okadaic acid is shown interacting with the C subunit (black area labeled C). TPA, 12-0-Tetradecanoylphorbol-13-acetate; DG, diacylglycerol; (®, positive effector; E, negative effector. The figure labeled S in the center represents a substrate whose function in gene expression or proliferation is altered by phosphorylation.

it would be important that sufficient levels of small-t be present to bind PP2A in cells. This suggestion has been made for infected monkey cells, in which small-t appears to be present in excess over PP2A (34). PP2A might function in growth control not only by inactivating growth-stimulatory proteins but also by activating growth-suppressing proteins. One example of a potential substrate is the retinoblastoma (RB) protein, whose growthsuppressing function is controlled by phosphorylation (7, 9, 13, 29). The RB protein is unphosphorylated and active in resting cells. Mitogenic stimulation induces phosphorylation of the RB protein, which might in turn inactivate its suppressor function. PP2A could play a role in maintaining the RB protein in an unphosphorylated active (growth suppressing) state. Inhibition of PP2A by small-t might lead to enhanced phosphorylation of the RB protein. We have shown that PP2A has a striking effect on the phosphorylation state of p53, another growth-suppressing protein. It remains to be seen whether phosphorylation plays a role in the tumorsuppressing function of p53. The finding that p53 is in a higher state of phosphorylation in transformed cells than in normals cells is consistent with the idea that its function is controlled by phosphorylation (28, 39). Recent work suggests that p53 activity might be regulated by p34cdc2, the cell cycle control protein kinase (4, 45). The sites in rat p53 which are most susceptible to PP2A have not been mapped yet. By comparison with the similar phosphopeptide map of mouse p53 (28, 36), it seems likely that they are located close to the amino terminus. In this regard, it is interesting that the N-terminal 73 amino acids of p53 contain a strong transcription activator domain (15). It is also interesting that PP2A has been identified as an inhibitor of the formation of active p34cdc2 (23a). It is an open question how polyomavirus small-t and medium-T antigens influence PP2A activity. Because of its structural similarity to SV40 small-t, one would expect that polyomavirus small-t also inhibits PP2A. Since medium-T is a membrane protein, the potentially available substrates for PP2A that might be affected by medium-T may be quite different from those for the small-t antigens. The presence in membranes of many enzymes involved in the regulation of cell growth makes this possibility an interesting one. ACKNOWLEDGMENTS We thank Juanita Coley for preparation of the manuscript. This work was supported by Public Health Service grants HL31107 and HL-17669 (to M.C.M.), CA-21327 (to K.R.), and CA-

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