Cell, Vol. 60, 167-176,

January

12, 1990, Copyright

0 1990 by Cell Press

Polyoma Small and Middle T Antigens and SV40 Small t Antigen Form Stable Complexes with Protein Phosphatase 2A David C. Pallas,’ Lilian K. Shahrik,’ Bruce L. Martint Stephen Jaspers,* Thomas B. Miller,t David L. Brautigan,t and Thomas M. Roberts’ Division of Cellular and Molecular Biology Dana-Farber Cancer Institute and Department of Pathology Harvard Medical School Boston, Massachusetts 02115 tsection of Biochemistry Division of Biology and Medicine Brown University Providence, Rhode Island 02912 $ Department of Biochemistry University of Massachusetts Medical Center Worcester, Massachusetts 01655 l

Summary We have purified the 36 and 63 kd cellular proteins known to associate with polyomavirus middle and small tumor (T) antigens and SV40 small t antigen. Microsequenclng of the 36 kd protein indicated that it was probably identical to the catalytic subunit of protein phosphatase 2A (PPSA). Identity was confirmed by comigration on two-dimensional (2D) gels and by 20 analysis of complete chymotryptic digests. In addition, PPSA-like phosphatase activity was detected in immunoprecipitates of wild-type middle T. Immunoblotting experiments, comigration on 2D gels, and 20 analysis of limit chymotryptic digests demonstrated that the 63 kd protein, present in the middle T complex in approximately equimolar ratio to the 36 kd protein, is a known regulatory subunit of the PPSA holoenzyme. Finally, the 36 kd PPSA catalytic subunit can be immunoprecipitated by anti-pp60c-src antisera only from cells expressing wild-type middle T. These results suggest that complex formation between PPSA and T antigens may be important for T antigen-mediated transformation. Introduction In recent years the identities of several of the host cell proteins that form stable complexes with papova virus tumor (T) antigens have been determined. In each case, the newly identified species has proven to be a protein thought to have an important cellular function. The interactions between T antigens and the host protein are divided into two general classes: One class is typified by the interaction between SV40 large T antigen and the retinoblastoma susceptibility gene product, where large T antigen is thought to interfere in a negative manner with the normal functioning of the Rb protein as an antioncogene product (DeCaprio et al., 1968; Ludlow et al., 1989). A second class is typified by the interaction between middle T antigen (MT) of polyoma virus and ppGoc=

(Courtneidge and Smith, 1983), where the T antigen is thought to enhance the normal activity of pp60c-sz as a tyrosine kinase (Bolen et al., 1984; Courtneidge, 1985). Other examples of the latter class are the interactions of MT with a second tyrosine kinase, p62c-yes (Kornbluth et al., 1986), and with a phosphatidylinositide kinase tentatively assigned to an 85 kd species (Kaplan et al., 1987; Courtneidge and Heber, 1987; Pallas et al., 1988). In each case knowledge of the normal function of the host protein has helped in the formulation of hypotheses concerning the mode of action of the T antigen. Two cellular proteins that bind to polyoma small t and middle T antigens and to SV40 small t antigen have been studied, but their identities are not yet determined. These are polypeptides of 36 kd and 63 kd, which are found complexed to small t antigen of SV40 (Yang et al., 1979; Murphy et al., 1988; Pallas et al., 1988; Walter et al., 1988) and to both small and middle T antigens of polyomavirus (Schaffhausen et al., 1978; Schaffhausen and Benjamin, 1981; Flundell et al., 1981; Grussenmeyer et al., 1985; Noda et al., 1986; Schaffhausen et al., 1987; Pallas et al., 1988). The 36 and 63 kd proteins associated with these three different T antigens have been shown to be the same by the criteria of comigration on two-dimensional (2D) gels and one-dimensional (1D) peptide mapping (Pallas et al., 1988; Walter et al., 1988). The association of these two protein species with T antigens appears to be interdependent: mutations in either the SV40 or polyomavirus system that abolish binding of one of the proteins concomitantly destroy the binding of the other (Yang et al., 1979; Schaffhausen et al., 1978; Pallas et al., 1988). Consistent with this finding, the two proteins also associate with small t and middle T at a constant molar ratio relative to one another (Rundell, 1987; Pallas et al., 1988). Analysis of mutations in polyomavirus small t and MT suggests that the interaction with 36 and 63 kd proteins is necessary for the functioning of the T antigen (Schaffhausen et al., 1978; Grussenmeyer et al., 1985; Pallas et al., 1988). For MT which is the transforming gene of polyoma, the binding of these two proteins is clearly not sufficient for MT’s transformation function, because transformation-defective mutant MTs exist that apparently bind the two proteins normally (Magnusson and Berg, 1979; Smolar and Griffin, 1981; Morgan et al., 1988; Pallas et al., 1988). However, the binding of 36 and 63 kd proteins may indeed be sufficient for small t function. The 36 and 63 kd proteins are the only proteins found associated with small t antigens in cell lysates. In addition, no mutant small t has been found that has lost function and retained binding of the 36 and 63 kd proteins. The small t antigens cannot by themselves transform cells but are thought to play a role in some cell types in viral replication (Shenk et al., 1976; Topp, 1980; Garcea et al., 1989; Berger and Wintersberger, 1986; Templeton et al., 1986; Martens et al., 1989) and transformation (Bouck et al., 1978; Sleigh et al., 1978; Feunteun et al., 1978; Fluck and Benjamin, 1979; Martin et al., 1979; Noda et al., 1987; Martens et al., 1989).

Cell 168

In this paper we demonstrate that the 36 and 63 kd proteins associated with the polyoma and S/40 T antigens are, respectively, catalytic and regulatory subunits of protein phosphatase 2A (PP2A). The possible nature of the interactions between T antigens and this major cellular phosphatase is discussed. Results Purification of the 36 and 63 kd Small t- and Middle T-Associated Cellular Proteins The 36 and 63 kd proteins were purified by separating the components of large scale MT immunoprecipitates on preparative 2D gels as described in the Experimental Procedures. Briefly, human 293 cells infected with a recombinant adenovirus expressing MT (Berkner et al., 1987) at high levels were lysed, and MT was immunoprecipitated with rabbit antiserum directed against polyomavirus T antigens. The immune complexes were then incubated with saturated urea and reducing agent to break up protein interactions, and the proteins were separated by preparative 2D gel electrophoresis. The proteins in the 2D gels were electrophoretically transferred to nitrocellulose and visualized by staining with Ponceau S prior to excision. Forty 15 cm dishes of infected cells yielded mO.3 ttg and 1 ug, respectively, of the 36 and 63 kd proteins. Isolation and Sequence Analysis of Peptides An attempt to obtain amino-terminal sequences of both the purified proteins by Edman degradation was unsuccessful, indicating that their amino-termini are blocked. Therefore, digests of the two proteins were made with various proteolytic enzymes, and the peptides were purified by reverse-phase HPLC. For the 36 kd protein, five tryptic

Table 1. Amino Acid Sequences from the 36 kd MTAP

of Tryptic

Peptides

Isolated

Peptide

Sequencea,b

Position in the Sequence of PP2AC

36NT23a 36NT19 38NT30 36NT15a 36NT15b

YSFLQFDPAPR SPDTNYLFMGDYVDR GAGYTFGQDISETFNHANGL (-GNQA)AIMELDDTLK (RTPDY)FL

284-294 75-89 215-234 269-283 303-309

a Peptides 36NT15a and b coeluted on HPLC and were therefore sequenced simultaneously. Assignments after cycle 5 were made to a or b based on relative abundance of the hvo residues detected at each cycle. At cycles 2-5 relative assignment of the two residues at each cycle could not be made, because they were present in equimolar yield. However, upon comparison with the published sequence of PP2A catalytic subunit, the pairs of residues obtained at cycles 2-5 were found to be consistent with the assignments shown in parentheses. In addition, cysteine at position 269 of PPZA catalytic subunit was not detected (‘-” in first position of peptide 36NT15a), since we did not reduce and alkylate prior to sequencing. b Approximately 12-33 pmol of peptide or peptide mixture was sequenced in each case. All peptide sequences from the 36 kd MTAP correspond precisely to sequences in PPPA at the positions indicated. c Da Cruz e Silva et al., 1987.

peptides were isolated and sequenced. In each case the sequence corresponded precisely to sequences found in the 36 kd catalytic subunit of PP2A at the positions indicated in Table 1. These same peptides were only 42% homologous to the related protein phosphatase 1 (PPl) catalytic subunit, which is of similar size. For the 63 kd protein, none of the peptide sequences obtained corresponded to sequences present in the GenBank data base (data not shown).

Figure 1. The 36 kd MTAP Comigrates on 2D Gels with PPPA Catalytic Subunit but Not with PPl Catalytic Subunit Purified PPBA catalytic subunit (1.7 pg) and 2 ng of purified PPl catalytic subunit each were mixed with a small amount of 35S-labeled wildtype MT immunoprecipitate, and the mixtures were analyzed on separate 2D gels. The gels were silver stained and dried, and radioactive proteins were visualized by autoradiography. The amount of 35S-labeled MT immunoprecipitate added in the mixtures was predetermined to contain amounts of the 38 kd MTAP undetectable by silver staining. The autoradiograms were superimposed on the silver stained gels, and the positions of migration of the purified catalytic subunits were traced on the autoradiograms with dots. (a, b, d, e): Autoradiograms of the gels containing PP2A (a and b) or PPl (d and e). Arrowheads indicate the positions of the radiolabeled 36 kd MTAP species. The minor species of this protein are best seen in (a) and (d), which are identical,. respectively, to (b) and (e) except for the traced dots in the latter. (c) and (f): Silver-stained gels containing purified PPSA (c) or PPl (f) catalytic subunits. Brackets indicate the position of the purified catalytic subunits of PPZA and PPl.

Small and Middle T Antigen-Associated 169

Phosphatase

:;c

0

Figure 2. Comparison Chymotryptic Peptide

of the 36 kd MTAP and Mapping

PP2A by Complete

MT immunoprecipitates were prepared from human 293 cells infected with a recombinant adenovirus expressing MT (Berkner et al., 1967) as described in Experimental Procedures. These immunoprecipitates and purified PPPA catalytic subunit were analyzed on parallel 2D gels. The proteins were visualized by Coomassie blue staining, and the 36 kd MTAP and the PP2A catalytic subunit were excised and radiolabeled with [lssl]Bolton-Hunter labeling reagent as described in Experimental Procedures. The two proteins were then digested exhaustively with chymotrypsin. The peptides were prepared as described in Experimental Procedures and analyzed on thin-layer plates by electrophoresis (vertical axis) followed by chromatography (horizontal axis). During electrophoresis, the peptides migrate toward the negative electrode (top of figure). (a) Peptides of the 36 kd protein from MT immunoprecipitates; (b) peptides of the PPZA 36 kd catalytic subunit. The circles denote the origins. The arrowheads indicate the position of unbound Bolton-Hunter labeling reagent.

Confirmation of the identity of the 36 kd Protein as the Catalytic Subunit of PPZA Two experimental protocols were used to confirm the initial sequence analysis results. First, a%-labeled MT immunoprecipitates containing the 36 kd associated protein were mixed with unlabeled purified catalytic subunit of either PP2A or PPl and analyzed on 2D gels. The 36 kd associated protein comigrated on these gels with PP2A catalytic subunit but not with PPl catalytic subunit, which migrated at a more basic position in the isoelectric focusing dimension (Figure 1). Second, limit chymotryptic digests of the 36 kd associated protein and the PP2A catalytic subunit were analyzed in parallel by 2D thin-layer electrophoresis and chromatography (Figure 2). The resultant peptide maps were indistinguishable, indicating that the proteins probably had identical primary structures. Identification of the 63 kd Protein as a Regulatory Subunit of PPSA One possible explanation for the apparent interdependent binding of the 36 and 63 kd T antigen-associated proteins and their constant ratio relative to one another in the T antigen complexes (see above) is that these two proteins are complexed with one another (Rundell, 1967). Because the

catalytic subunit of PP2A is normally found associated with a 60 kd regulatory subunit (for review, see Cohen, 1989) we wanted to determine whether the 63 kd T antigen-associated protein is the regulatory subunit of PP2A. Indeed, this is the case. The results shown in Figure 3 demonstrate that antibodies directed against the 60 kd PP2A regulatory subunit specifically recognize the 63 kd associated protein in 2D immunoblots. In addition, when s5.Slabeled MT immunoprecipitates containing the 63 kd associated protein were mixed with unlabeled purified PP2A 60 kd regulatory subunit and analyzed on 2D gels, the 63 kd associated protein comigrated on these gels with the PP2A 60 kd regulatory subunit (Figure 4). Moreover, when limit chymotryptic digests of the 63 kd associated protein and the PP2A 60 kd regulatory subunit were analyzed in parallel by 2D thin-layer electrophoresis and chromatography (Figure 5), the resultant peptide maps were indistinguishable, indicating that the proteins probably had identical primary structures. Subsequently, we were able to compare seven of our peptide sequences obtained for the 63 kd T antigen-associated protein with the sequence of a human PP2A 60 kd regulatory subunit a form cDNA clone (B. Hemmings, unpublished data). Fifty-three of 54 amino acids determined with high confidence and 11 of 14 amino acids determined with medium confidence corresponded to sequences in this cDNA. Detection of Phosphatase Activity in MT Immunoprecipitates lmmunoprecipitates of MT contain PP2A activity. Of the four major cytosolic protein serine/threonine phosphatases, only PPl and PP2A have appreciable activity with phosphorylase as substrate (Ingebritsen and Cohen, 1983). Dephosphorylation of phosphorylase a converts phosphorylase to its inactive form, phosphorylase b. Phosphatase activity can be monitored by loss of phosphorylase activity in a two-stage coupled enzyme assay. lmmunoprecipitates prepared with anti-MT serum, but not those prepared with preimmune serum, caused the conversion of phosphorylase a to phosphorylase b. Addition of AMP to phosphorylase b activates the enzyme by an allosteric mechanism (Madsen, 1986). This reactivation was observed in all assays with MT immunoprecipitates, demonstrating that phosphorylase a inactivation was caused by dephosphorylation, not by proteolysis or some irreversible process. The phosphorylase phosphatase activity in MT immunoprecipitates was not affected by phosphatase inhibitor-2 (60 ng) but was sensitive to 25 nM okadaic acid. In control experiments, purified PPl was completely neutralized by 60 ng inhibitor-2 but was unaffected by 25 nM okadaic acid. Purified PPSA, on the other hand, was sensitive to 25 nM okadaic acid but not affected by 60 ng inhibitor-2. Initial evaluation of specific activity of the PP2A in MT immunoprecipitates with phosphorylase a as substrate yields a preliminary estimate of 300 U/mg. Coimmunoprecipitation of PPSA Catalytic Subunit with pp60c-s’c from Cells Expressing Wild-Type MT PPPA may potentially associate with MT with or without

Cell 170

Figure 3. The 63 kd MTAP Specifically Immunoblots with Antisera Directed against the PP2A 60 kd Regulatory Subunit

a

Cell lysatesfrom NIH 3T3 cellsexpressing wildtype MT labeled with [ssS]methionine for 5 hr were immunoprecipitated with rabbit anti-T antigen serum (a and c) or preimmune antiserum (b and d). lmmunoprecipitates were analyzed in parallel on 2D gels, and the proteins were transferred electrophoretically to nitrocellulose membranes and probed with antibody directed against the 60 kd regulatory subunit of PPPA. lmmunoblots were developed with color development substrates as described in Experimental Procedures. The radiolabeled proteins on the membranes were visualized by autoradiography (a and b). lmmunoblots are shown in (c) and (d). The positions of the 27, 29, 36, 61 (which includes pp6WsE), and 63 kd MTAPs are indicated by arrowheads. The positions of actin (A) and tubulin (T) are marked for reference.

b

63, .

d

C

r I u

63,) u

.: A

*

.

Figure 4. The 63 kd MTAP Comigrates Regulatory Subunit

on 2D Gels with the 60 kd PPPA

Approximately 4 ug of purified 60 kd PPPA regulatory subunit was mixed with a small amount 35S-labeled wild-type MT immunoprecipitate, and the mixture was analyzed on a 2D gel. The gel was stained with Coomassie blue and dried, and radioactive proteins were visualized by autoradicgraphy. The amount of %-labeled MT immunoprecipitate added in the mixture was predetermined to contain a quantity of the 36 kd MTAP undetectable by staining. The autoradiogram shown was superimposed on the stained gel, and the position of migration of the purified 60 kd PPPA regulatory subunit was traced on it with dashes. The 63 kd MTAP is indicated by the numbered arrowhead. The positions of actin (A) and tubulin (T) are marked for reference.

other associated proteins in the same complex or complexes. If PPPA forms a complex with MT in association with another protein, then PP2A should coprecipitate with antisera directed against the other associated protein, provided an appropriate epitope is available in the complex in MT-expressing cells. To determine whether PP2A coimmunoprecipitates with pp60C-s’c from cells expressing wild-type MT, pp60c-s’c and control immunoprecipitates were prepared from MT-expressing NIH 3T3 cells. The immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis, and the proteins were transferred to nitrocellulose and probed with antibody directed against the catalytic subunit of PP2A. As shown in Figure 6, although a small amount of catalytic subunit was present in immunoprecipitates prepared from wildtype MT-expressing cells with nonimmune serum (lane l), a significant amount of the 36 kd PP2A catalytic subunit clearly coimmunoprecipitated specifically with pp60c-s’c from these cells (lane 2). If this coimmunoprecipitation was due to MT’s facilitation of the formation of a complex containing both PPPA and pp6W-s’c, it should require the presence of a MT capable of complexing with PP2A and pp60c-sK. To test this prediction, pp60C-s’c immunoprecipitates were prepared from NIH 3T3 cells expressing NG59 MT,‘ a mutant MT that does not associate with the PPPA catalytic subunit. No PP2A catalytic subunit was detected in the pp60c-sC immunoprecipitates from these cells (lane 3).

~~11

and Middle

T Antigen-Associated

Phosphatase

a

123

0 b

0 Figure 5. Comparison of the 63 kd MTAP and the 60 kd PP2A Regulatory Subunit by Complete Chymotryptic Peptide Mapping MT immunoprecipitates were prepared from human 293 cells infected with a recombinant adenovirus expressing MT (Berkner et al., 1967) as described in Experimental Procedures. These immunoprecipitates and purified 60 kd PPPA regulatory subunit were analyzed on parallel 2D gels. The proteins were visualized by Coomassie blue staining and the 63 kd MTAP and the PP2A 60 kd subunit were excised and radiolabeled with [1251]Bolton-Hunter labeling reagent as described in Experimental Procedures. The two proteins were then digested exhaustively with chymotrypsin. The peptides were prepared as described in Experimental Procedures and analyzed on thin-layer plates by electrophoresis (vertical axis) followed by chromatography (horizontal axis). During electrophoresis the peptides migrate towards the negative electrode (top of figure). (a) Peptides of the 63 kd protein from MT immunoprecipitates; (b) peptides of the PPZA 60 kd regulatory subunit. The circles denote the origins. The arrowheads indicate the position of unbound Bolton-Hunter labeling reagent.

Discussion In this paper we identified two of the cellular proteins associated with the T antigens of polyoma and SV40 viruses as two subunits of the PP2A holoenzyme: the 36 kd catalytic subunit and one of its regulatory subunits. For the 36 kd protein there are five independent lines of evidence to support this conclusion: First, sequences of the five internal tryptic peptides obtained from the 36 kd MT-associated protein (MTAP) are identical to sequence of PPPA catalytic subunit (Table 1). Second, the two proteins have identical sizes and isoelectric points (Figure 1). The 36 kd protein purified with MT can be separated into three species on 2D gels (Figure 1; unpublished data). Each of these species is also seen when PP2A purified from rabbit skeletal muscle is analyzed on these same gels (Figure 1). These polypeptides probably correspond to different isotypes of PP2A (Stone et al., 1987; Da Cruz e Silva and Cohen, 1987; Da Cruz e Silva et al., 1987; Arino et al., 1988). The peptide sequences we obtained are from regions of the protein conserved between the different isotypes. Third, the 2D limit chymotryptic map of the 36 kd protein associated with MT is identical to that of purified PPBA catalytic subunit (Figure 2). Fourth, PP2A-like phosphatase activity is found specifically associated with MT immunoprecipitates containing these proteins. Finally, anti-

Figure 6. Coimmunoprecipitation of PPPA pp60c-” Serum from Cells Expressing MT

Catalytic

Subunit

with

Cell lysates from NIH 3T3 cells expressing wild-type MT were immunoprecipitated with normal mouse serum (lane 1) or GDll, a mouse antipp60c-s’c monoclonal antibody (lane 2). Cell lysates from NIH 3T3 cells expressing NG59 mutant MT were immunoprecipitated with GDll (lane 3). The immunoprecipitates were analyzed on a 10% SDS-polyacrylamide gel, and the proteins were electrophoretically transferred to a nitrocellulose membrane and probed with antibody directed against the PPPA 36 kd catalytic subunit. The arrowhead indicates the position of the PP2A catalytic subunit. The molecular size markers in both outside lanes are included for reference; their approximate molecular sizes are 200, 97, 66, 43, 29, and 16 kd.

bodies directed against purified PP2A catalytic subunit immunoblot the 36 kd protein in MT immunoprecipitates (data not shown). In contrast, PPl could not be detected in MT immunoprecipitates analyzed on 2D gels, and no PPl-like activity was present. For the 63 kd protein associated with MT, there are four lines of evidence that indicate it is the 60 kd PP2A regulatory subunit: First, antibodies directed specifically against the purified 60 kd PP2A regulatory subunit specifically immunoblot the 63 kd MTAP (Figure 3). Second, 94% of the peptide sequence obtained with medium to high confidence from the 63 kd MTAP is identical to sequence of the a form of the PPPA 60 kd regulatory subunit (B. Hemmings, unpublished data). Next, the two proteins have identical sizes and isoelectric points (Figure 4). The difference between the originally reported size of 60 kd for the regulatory subunit and our estimate of 63 kd is apparently due to variation in the estimation procedures used. In addition, a second, much less abundant 60 kd species is seen in MT immunoprecipitates (Figures 1 and 4) that migrates just slightly more acidic than the major species and may correspond to the 6 form of the protein (B. Hemmings, unpublished data). And finally, the 2D limit chymotryptic maps of the 63 kd protein associated with MT are identical to that of purified PP2A 60 kd regulatory subunit (Figure 5). Phosphatase 2A is active in vitro against a number of

Cdl 172

enzymes involved in metabolic pathways and is thought to have an important role in the regulation of various metabolic processes (for review, see Cohen, 1989). The precise physiological role for this enzyme, however, remains unclear. Recently, PP2A was shown to dephosphorylate and inactivate S6 kinase (Ballou et al., 1988a; Jeno et al., 1988), which is activated early in the cell cycle at the GO/G1 transition (Ballou et al., 1988b). Virshup and Kelly (1989) have purified acellular factor, RP-C, that potentiates SV40 DNA replication in vitro. This factor has been partially sequenced and seems to be identical to the catalytic subunit of PP2A. Dephosphorylation of serine groups on SV40 large T has been shown to activate its specific DNA binding to site II within the origin of SV40 DNA replication and to increase its ability to support DNA replication in vitro (Mohr et al., 1987; Klausing et al., 1988). These results are consistent with a hypothesis that small t may activate the ability of PP2A to dephosphorylate large T, thereby activating large T. As pointed out by Virshup and Kelly (1989), it would be interesting to determine whether RP-CIPPPA is identical to an unidentified activity in extracts that potentiates SV40 DNA replication and appears to vary in amount or activity during the cell cycle (deficient in Gl in relation to S phase) (Roberts and D’Urso, 1988). Another possible role for the interaction of SV40 small t with PPPA is in the ability of SV40 small t to trans-activate certain promoters requiring RNA polymerase II and III (Loeken et al., 1988). Loeken and co-workers proposed that because small t lacks detectable DNA binding activity (Prives and Beck, 1977; Spangler et al., 1980), it probably trans-activates by modifying the activity of certain transcription factors. As the only known cellular enzyme with which small t interacts, PP2A is an obvious candidate for an intermediate in this process. The interaction with small t antigens and/or MTs could have several possible effects on the activity of PPPA, including inactivation, activation, or redirection of PP2A to new targets. These possibilities are not mutually exclusive. Our preliminary estimate of specific activity of the PP2A in MT immunoprecipitates with phosphorylase a as substrate falls in the expected range for the 36 kd-60 kd PP2A heterodimer. This result argues against simple inactivation of PP2A by MT, but it does not preclude the possibility that MT may either increase or decrease the activity of PPPA toward any given substrate. The data of Virshup and Kelly are consistent with activation or redirection of PP2A by SV40 small t. On the other hand, it is interesting to note that okadaic acid, a powerful tumor promoter (Fujiki et al., 1987; Suganuma et al., 1988), is a potent inhibitor of PPPA and PPl in vitro (Bialojan and Takai, 1988; Hescheler et al., 1988; lshihara et al., 1989). However, an adequate determination of the effect of T antigens on PPPA would require mixing the purified proteins and assaying with each individual substrate of interest, because modulation of the activity of the catalytic subunit could be influenced by the substrate. This substrate-specific type of modulation is the case both for the binding of the 60 kd protein to the 36 kd catalytic subunit (Chen et al., 1989; Usui et al., 1988) and for interactions of the 36 kd-60 kd

heterodimer with another regulatory subunit of 55 kd (Tung et al., 1985; Chen et al., 1989). The PPPA 36 kd-60 kd heterodimer is often found associated with a third subunit in its native state (for review, see Cohen, 1989). Several species of additional regulatory subunits, of molecular masses of 54, 55, and 72 kd, have been identified. We did not detect any of these species in T antigen immunoprecipitates from NIH 3T3 cells expressing small t or MT (data not shown). However, it is possible that a third regulatory subunit of PP2A is associated with the T antigen complexes in vivo but is lost during the immunoprecipitation procedure. Alternatively, interaction of the heterodimer with small t or MT in vivo may substitute for binding of a normal regulatory subunit of PP2A. It appears that the T antigens may interact directly with the 60 kd subunit of the heterodimer, since the 36 kd subunit can be dissociated from SV40 small t by treatment with N-ethylmaleimide, leaving the 60 kd subunit complexed with small t (K. Rundell, personal communication). lmaoka et al. (1983) have shown that the 55 kd regulatory subunit of PP2A does not bind directly to the 36 kd catalytic subunit in vitro nor alter its activity when added, but it does bind to the 36 kd-60 kd PP2A heterodimer and alters its activity. These results suggest that the 55 kd PPPA regulatory subunit may also interact with the 60 kd subunit rather than directly with the 36 kd catalytic subunit. It is interesting to speculate that the T antigens may alter PPPA function by replacing a normal cellular regulatory subunit of the PP2A holoenzyme. If this were the case, SV40 small t may alter the function of the entire population of PP2A, since it is known for SV40 that all of the 36 kd cellular protein is in complex with small t (Rundell, 1987). The targets, if any, for the membrane-bound complex of phosphatase and MT antigen remain to be determined. However, it is interesting to note that only pp60C-s’c molecules that carry no phosphate on a regulatory tyrosine, tyrosine 527, are found in association with MT (Cartwright et al., 1986). Phosphorylation of pp60c-s’c on serine and threonine appears to be unaffected by complexation with MT. It is known that mutations in pp60c-sm that prevent phosphorylation of tyrosine 527 activate it as a kinase and render the molecule transforming (Cartwright et al., 1987; Kmiecik and Shalloway, 1987; Piwnica-Worms et al., 1987). Our results suggest that PPPA may be in the same MT complex as p~60~-~” (Figure 4). Because purified PPPA is known to have low but measurable tyrosine phosphatase activity, it is tempting to speculate that the PP2A complexed with MT may dephosphorylate pp60c-sn: at tyrosine 527 and activate it as a tyrosine kinase. Experimental

Procedures

Cell Culture Murine cells that express wild-type polyomavirus MT and G418 resistance (Cheririgton et al., 1986), polyomavirus NG59 (Carmichael and Benjamin, 1960) transformation-defective mutant MT and G418 resistance (Morgan et al., 1988), or only G418 resistance (Cherington et al., 1986) were described previously. All rodent cell lines were maintained

~~3all and Middle T Antigen-Associated

Phosphatase

in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented 10% calf serum. Human 293 cells (Graham et al., 1977) were tained in DMEM supplemented with 10% fetal calf serum.

with main-

Radiolabeling and Extraction of Cells For metabolic labeling of rodent cells with methionine, subconfluent dishes of cells were labeled for 5 hr with [35S]methionine (300 uCi/ml) in DMEM minus methionine supplemented with 0.5% dialyzed fetal bovine serum. For metabolic labeling of 293 cells, cells were labeled with [?9]methionine (100 t&i/ml) for 24 hr postinfection in DMEM minus methionine supplemented with 2.5% fetal calf serum and 5% of the normal level of methionine. Cell lysates were prepared as described (Whitman et al., 1985). Purification of MTAPs Forty 15 cm dishes of 60% confluent 293 cells were infected with 1.5 ml of Ad5(pymT) virus stock (Berkner et al., 1967) and cells were harvested at 24 hr. Cells were lysed in 1% NP40, 10% glycerol, 150 mM NaCI, 50 mM Tris containing 1 mM PMSF and 0.02 Trypsin inhibitory units/ml aprotinin (pH 6.0) for 20 min, and the cell lysate was centrifuged at 13,000 x g for 10 min. The supernatant was immunoprecipitated with 0.6 ml of rabbit polyclonal antiT antigen serum (Pallas et al., 1966) and 6.4 ml of protein A-Sepharose beads (Sigma). Immune complexes were collected by centrifugation at 2000 x g for 30 s and washed once with phosphate-buffered saline, twice with 0.5 M LiCI, and twice again with phosphate-buffered saline. The immune complexes were divided into three aliquots, and 1 ml of 2D gel loading buffer (6 M urea, 2% ampholines [LKB], 7.5% P-mercaptoethanol, 16% NP40) was added to one aliquot and saturated with urea by the addition of solid urea beads (Malincrott). The supernatant was then transferred sequentially to the other aliquots and the same procedure was repeated. The three aliquots were reextracted in the same manner with another 0.75 ml of 2D gel loading buffer. The supernatants containing antibody, MT, and MTAPs were analyzed on preparative 2D gels as described below. 1D and 2D Gel Electrophoresis and Fluorography SDS-polyacrylamide gel electrophoresis (10% acrylamide) was performed according to Laemmli (1970). 2D gel analysis was performed as described (Pallas and Solomon, 1962). Gradients of pH 4.6-7.4 were obtained by mixing 400 Kl of pH 3.5-10 ampholine (LKB) with 150 ttl of pH 6-6 ampholine (LKB) per 10 ml gel solution. Preparative 2D gels were similar except that the first-dimension gels were 9 cm or 14 cm long tube gels, 5.5 mm in diameter, with loading capacities of 2 or 4 ml, respectively. of note, these gels allow the loading of large sample volumes and large quantities of polyclonal antibody (>lO mg) on a single gel. The second-dimension gel, however, is a standard 1.5 mm thick slab gel. A J/4 in thick notched plate is used in the second dimension to support the tube gel when it is loaded on the second dimension. The loading limit of protein for this technique appears to depend mainly upon the capacity of the second-dimension gel. Polyclonal antibodies, in particular, do not pose an overloading problem, because they do not focus in discrete spots and therefore do not overload the second-dimension gel. Gels of methionine-labeled proteins were preincubated with En3Hance before exposure; gels of phosphatelabeled proteins were exposed using intensifying screens. All exposures were on XAR-5 film (Kodak) at -70°C. Gels were silver stained by the procedure of Wray et al. (1961) except that after electrophoresis the gels were sequentially incubated 10 min in distilled water (200 ml), 10 min in 95% ethanol (200 ml), 1 hr in 50% methanol (100 ml), and 30 min in distilled water (100 ml) prior to staining. Protein Sequence Analysis Initial attempts to obtain amino-terminal sequences of either the 36 or 63 kd MTAPs failed, indicating blockage of the amino-termini of these proteins. Except for HPLC, internal amino acid sequence analysis was obtained by the method of Aebersold et al. (1987). Resultant tryptic, chymotryptic, or staphylococcal V-6 protease digestion mixtures were run essentially as described (Stone et al., 1969) on a Hewlett Packard 1090 HPLC equipped with a 1040 diode array detector and a Vydac Cl6 column (2.1 mm inside diameter by 150 mm length). Peptides were eluted essentially using the gradient described elsewhere (Stone et al.,

1969). Peptides were applied to an Applied Biosystems Model pulsed liquid protein sequencer equipped with an online Model HPLC using the manufacturer’s fast cycle program.

477A 120A

lmmunoprecipitations lmmunoprecipitates were prepared as described with rabbit antipolyoma tumor antigen sera raised against purified small t antigen (Pallas et al., 1986). For phosphatase assays, MT immunoprecipitates were prepared from Ad5(pymT)-infected 293 cells as described for purification of MTAPs (see above), except that for some experiments the immune complexes were washed only in phosphatase assay buffer (3 times). Preparation of Phosphatase Catalytic Subunits The catalytic subunits of PPls and PP2As were isolated according to the protocol of Brautigan et al. (1985) with the following modifications: EDTA and EGTA were excluded from all buffers; the tryptic digestion step was omitted; after loading, the poly(tysine)-agarose was washed with a column vol of buffer plus 50 mM NaCl and eluted with a 10 volume linear gradient from 50-400 mM NaCI; Ultrogel-AcA44 was substituted for G-75 Sephadex; and the final step was chromatography on a Mono-Q FPLC column (Pharmacia) with elution by a linear gradient (80 ml) from 50-400 mM NaCI. The two enzymes were separated during chromatography on poly(lysine)-agarose with PPl eluting later. The subsequent chromatography steps were performed on the separated samples. The PPls and PP2As obtained were distinguished by activity with phosphorylase a as the substrate, sensitivity to the phosphatase inhibitor-2 (Brautigan et al., 1966) and reactivity with anti(type-1) antibodies. Assay of Phosphorylase Phosphatase Activity Phosphorylase phosphatase activity was assayed as previously described (Gruppuso et al., 1965). Samples were mixed with an equal volume of 6 mg/ml phosphorylase a (30 f~tlfor immunoprecipitates and 20 ul for solution samples) and incubated at 3oOC. Reaction time was 60 min with immunoprecipitates or immunoprecipitatederived samples and 10 min with purified catalytic subunits. The immunoprecipitate samples were periodically dispersed by gentle agitation during the reaction period. After the appropriate reaction time, the reaction mixture was diluted to 15 ug/ml phosphorylase for assay of the remaining phosphorylase a activity. To measure total phosphorylase a and b activity, 1 mM AMP was included at this point to reactivate phosphorylase b. One unit of activity is required to convert 1 nmol of phosphorylase a to phosphorylase b in 1 min. Peptide Mapping For maps of limit digests of iodine-labeled proteins, a modification of several published procedures was used (Pallas and Solomon, 1962; Boxberg, 1968). Coomassie blue stained spots were excised from 2D gels, incubated twice in 5 ml of 30% methanol for at least 8 hr at 3pC, and vacuum dried. Five microliter (25 NCi) aliquots of [1251]BoltonHunter labeling reagent (Amersham) were pipetted into microfuge tubes and dried gently with nitrogen gas. A dried gel spot was added to each of these tubes followed sequentially by 10 ul of ACS grade DMSO and 20 al of labeling buffer (140 mM NaCI, 5 mM KCI, 6 mM NaHCOs, 1.5 mM CaCIs [pH 7.51) per tube. After 1 hr incubation at room temperature, 100 ul of labeling buffer was added. Five minutes later, the spots were washed twice for 5 min with 125 nl of distilled deionized water (ddHs0) and four times for 15 min with 50 mM ammonium bicarbonate. The spots were transferred to new tubes and incubated 18 hr at 37oC with 300 pglml chymotrypsin (Worthington) in 50 mM ammonium bicarbonate. The supernatant was vacuum dried, resuspended in ddHs0, and then vacuum dried four times to remove the ammonium bicarbonate. The peptides were analyzed by published procedures (Elder et al., 1977; Gibson, 1974). The final samples (2 ~1) were spotted on plastic-backed cellulose-coated thin-layer chromatography plates (10 x 10 cm; EM Laboratories). Electrophoresis and chromatography were carried out as described (Pallas and Solomon, 1982).

Immunoblotting lmmunoblotting

was performed

by standard

procedures

vowbin

et al.,

Cell 174

1979). Briefly, [35S]methionine-labeled proteins in 2D gels or unlabeled protein in 1D gels were electrophoretically transferred to nitrocellulose membranes at 0.5 A for 3.5 hr in an electroblotting apparatus (Hoefer). Prestained molecular size markers (Bethesda Research Laboratories) were included in the 1D gels for reference. The membranes were blocked with 3% bovine serum albumin in phosphatebuffered saline, washed, and probed 3 hr at 22% with affinity-purified sheep antibody directed primarily against the 36,55, or 60 kd subunits, respectively, of rat skeletal muscle PPPA (S. Jaspers and T Miller, unpublished data). They were washed again and then probed with alkaline phosphatase-conjugated second antibody (Jackson ImmunoResearch). The blots were incubated with color development substrates and air dried, and the radiolabeled proteins were visualized by autoradiography. All antibody solutions incubated with the blots contained 0.5% bovine serum albumin to reduce nonspecific binding.

Boxberg, Y. V. (1986). Protein analysis on two-dimensional polyacrylamide gels in the femtogram range: use of a new sulfur-labeling reagent. Anal. Biochem. 169, 372-375.

We thank William S. Lane and David Andrews of the Harvard Microchemistry Facility, Harvard Biological Laboratories, for providing their expertise in performing in situ protease digestions, HPLC, and microsequencing. We are indebted to Brian Hemmings for communicating sequence data in advance of publication. We are also grateful to Brian Schaffhausen for his generous gift of monoclonal antibody GDll, to David Hartshorne for providing okadaic acid, and to David Livingston, Mark Ewen, and Greg Duff for their critical reviews of this manuscript. This work was supported by grants CA30002 Cr. M. R.) and CA45265 (D. C. t?) awarded by the National Cancer Institute, Department of Health and Human Services, and grants DK 31374 (D. L. B.) and DK 16269 (T B. M.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 16 U.S.C. Section 1734 solely to indicate this fact.

Cartwright, C. A., Eckhart, transformation by p~6oC-~ domain. Cell 49, 83-91.

Received

September

26, 1989; revised

November

20, 1969.

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