VIROLOGY

99,

413-416 (197%

Simian Virus 40 Large T Antigen Isoelectric Focuses as Multiple Species with Varying Phosphate Content DANIEL Department

of Pathology,

S. GREENSPAN AND ROBERT B. CARROLL’ New York University

School of Medicine,

New York, New York 10016

Accepted August 24, 1979

Simian Virus 40 large T antigen (T-Ag), radiolabeled with [32P]orthophosphate and with/ without r3H]methionine, was found to isoelectric focus as four distinct species distributed in a highly reproducible pattern. These species have been found to differ in their 32P/3Hratios indicating that individual monomers of large T-Ag are heterogeneous in their phosphate content and suggesting, further, that large T-Ag may be differentially phosphorylated in vivo. Also, by increasing the concentration of large T-Ag the more basic species can be displaced into the more acidic species suggesting that the different isoelectric focusing forms represent aggregates of large T-Ag with varying phosphate content.

Simian Virus 40 large T antigen (T-Ag)Z is encoded by the viral A locus (l-3) which governs the initiation of DNA synthesis and the establishment of viral transformation (4, 5). T-Ag is a phosphoprotein which contains at least one acid-stable phosphoserine on a single tryptic peptide (6). Recently Edwards et al. (7) have reported that the phosphate groups of T-Ag turn over in a biphasic manner suggesting that T-Ag may contain two different populations of phosphate groups. They also found the rate of phosphate turnover to be greater than the turnover rate of T-Ag itself which suggests a physiological role for the phosphorylation and dephosphorylation of T-Ag. T-Ag has been compared to the acidic chromatinassociated proteins of eukaryotic cells in that both bind specific sites on DNA, both are thought to be involved in the regulation of gene activity, and both are phosphorylated (S-9). The phosphorylation and dephosphorylation of the acidic chromatin proteins has been suggested as a control mechanism for the regulation of their fimctional properties. Also the functions of ’ To whom reprint requests should be addressed. * Abbreviations used: SV40, Simian virus 40; T-Ag, Simian virus 40 large T antigen; DMEM, Dulbecco’s modified Eagle’s medium; SDS, sodium dodecyl sulfate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; NP-40, Nonidet P-40.

many cellular enzymes are known to be modulated by the addition and removal of phosphate groups (9). It was our hypothesis that differential phosphate turnover in TAg would generate subfractions differing in their contents of phosphoserine and/or other acid labile phosphate moieties and that these subfractions could then be separated by isoelectric focusing. This communication describes the purification of TAg and its separation by isoelectric focusing into four species differing in phosphate content and aggregation state. SV40-transformed Balb/c 3T3 cells, radiolabeled in culture with [32P]orthophosphate and with/without [3H]methionine, were extracted by freeze-thawing and the T-Ag immunoprecipitated from the clarified extracts with hamster anti-tumor serum (10). T-Ag was then eluted from the immunoprecipitate with sample buffer containing SDS and P-mercaptoethanol and further purified by SDS-PAGE on cylindrical 8.5% gels. Bands of purified T-Ag were located on the frozen SDS-gels by Neither SV40-transautoradiography. formed Balb/c 3T3 cells immunoprecipitated with normal hamster serum nor normal Balb/c 3T3 cells immunoprecipitated with anti-tumor serum had radioisotope migrating with a mobility corresponding to that of T-Ag when analyzed by SDS-PAGE. Ex-

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cised bands of T-Ag were then minced, polymerized onto the basic ends of polyacrylamide isoelectric focusing gels, and isoelectric focused in the presence of urea and NP-40 as in O’Farrell(11). SDS dissociates from protein samples upon isoelectric focusing in this system and forms mixed micelles with the NP-40. The detergent micelles then migrate to the anodal end of the gel where they do not interfere with the isoelectric focusing (21). To avoid carbamylation ultrapure urea was made up fresh for each experiment and T-Ag was never boiled in the presence of urea. Typically the cylindrical isoelectric focusing gels contained 1.6% ampholines, pH 5 to 8, and 0.4% ampholines, pH 3.5 to 10. When T-Ag was eluted from immunoprecipitates with either O’Farrell’s lysis buffer A (11) or SDS-p-mercaptoethanol buffer and directly subjected to isoelectric focusing without prior purification by SDSPAGE only a small percentage of T-Ag entered the isoelectric focusing gel and was distributed as a long streak rather than as discrete species. Similar results have been reported by others (12). Treatment of cell extracts with 50 pglml pancreatic deoxyribonuclease and 50 pglml pancreatic ribonuclease prior to immunoprecipitation had no effect on these results suggesting that the streaking was not due to binding of nucleic acids. Another possibility, also suggested by Crawford and O’Farrell(12) was that the streaking was due to the selfassociation of T-Ag into aggregates. Alternatively, the aggregation may have involved the aggregation of T-Ag with other proteins present as contaminants in the immunoprecipitate. Our method of using SDS-PAGE prior to isoelectric focusing, however, serves the function of dissociating protein aggregates while further isolating T-Ag from bound contaminants. After about 9000 V-hr T-Ag previously excised from SDS-gels focuses as four species (Pl, P2, P3, and P4) distributed in a characteristic and reproducible pattern with apparent isoelectric points of 7.9, 7.0, 6.7, and 6.2, respectively. When these four isoelectric focusing species are reelectrophoresed from the isoelectric focusing gel into a second dimension containina“~ SDS (11 ~I j

each migrates with a mobility corresponding to the monomer molecular weight of large T-Ag. This suggests that the appearance of T-Ag as four isoelectric focusing forms is not due to proteolysis. To ascertain whether the various species differ in phosphate content, T-Ag, doubly labeled with [32P]orthophosphate and 13H]methionine was purified and isoelectric focused as above. Each isoelectric focusing gel was then sliced and the content of both 3H and 32Pdetermined for each slice by liquid scintillation counting. Over the course of six such experiments the most basic species, Pl, consistently had the lowest 32P/3Hratio with increasing 32P/3Hratios in P2 and P3 (Fig. 1). The 32P/3Hratios of species Pl, P2, and P3 illustrated in Fig. 1 were 0.37, 0.70, and 0.97, respectively. This suggests a correlation between acidity and 32P/3H ratios for these three species. Gels containing high numbers of counts in Pl contain few counts in P4 and visa versa due to concentration dependent displacement as outlined below (see Fig. 2). Consequently, the gel in Fig. 1 contained insufficient counts in P4 for an accurate estimate of its 32P/“H ratio. In gels in which P4 did contain high numbers of counts, its 32P/3H ratio was roughly equal to that of P2. The acidity of P4, therefore, is not simply a function of its phosphate content. In a reequilibration experiment the four species were cut from an isoelectric focusing gel and separately reisoelectric focused (13). Each species was found to generate the other species indicating that the different isoelectric focusing forms are in equilibrium with one another. To determine if this equilibrium involved the aggregation of TAg, equal aliquots of dilute 32P-labeledT-Ag were isoelectric focused in the presence of increasing amounts of concentrated, unlabeled T-Ag (Fig. 2). Increasing the concentration of an aggregating protein will shift the association equilibrium towards formation of higher aggregates (14). As observed by either autoradiography (Fig. 2A) or liquid scintillation counting (Fig. 2B), at very low concentrations T-Ag isoelectric focuses predominantly as species Pl and P3 (gel a). However, as the concentration is increased by the addition of increasing

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amounts of cold T-Ag the labeled T-Ag is displaced first into species P2 (gel b) and finally into species P4 (gels c-f). We conclude, therefore, that P2 and P4 represent higher aggregation states of T-Ag than do Pl and P3. The presence of four different isoelectric focusing species of T-Ag might have been due to (i) the existence of subfractions of monomeric T-Ag differing in their phosphate contents and, therefore, separable on isoelectric focusing gels; (ii) the association of T-Ag into various aggregation states (changes in association states are assumed to change the pKs of ionizable groups on macromolecules thus leading to a change in p1 (15); or (iii) a combination of the two. The differing 32P/3H ratios of peaks Pl, P2, and P3 strongly suggest that individual monomers of large T-Ag are heterogeneous in their phosphate contents. This difference may be due to differences in the number of phosphoserines or other acid-labile phosphate groups carried by different molecules. 32P/3Hratios increased coordinately with acidity for species Pl, P2, and P3 over the course of six double-label experiments. Further these experiments suggested that 32P/3H ratios increased as whole number multiples for Pl, P2, and P3 in the ratio 1:2:3. Differing phosphate content, however, is not the only chemical property contributing to the change difference by which the different species are separated during isoelectric focusing. This is evident in P4 which, although the most acidic species, has a 32P/3Hratio intermediate between those of Pl and P3. Our displacement experiment suggests that some of the different isoelectric focusing forms of T-Ag are in differing states of self-association. We, therefore, conclude that the four major isoelectric focusing forms of T-Ag have differing charges due to a combination of differing phosphate contents and differing states of association as in (iii) above. Possibly the different association states of T-Ag present in the isoelectric focusing gels differ in relative amounts of highly phosphorylated and less phosphorylated subunits of T-Ag. It must be emphasized, however, that the differential phosphorylation of T-Ag is the physiologically significant finding, while the

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FIG. 1. Isoelectric focusing of double-labeled T-Ag. SV40-Transformed Balbic 3T3 cells were preincubated in labeling medium (DMEM with 2% the normal concentration of both phosphate and methionine and containing 5% dialyzed fetal calf serum). After 2 hr the cells were changed to fresh labeling medium containing178 &i/ml carrier-freeorthophosphate and 89 &i/ml [3H]methionine. The cells were labeled for 6 hr and harvested in 0.02% Versene in Ca2+, Mg2+deficient PBS after which T-Ag was extracted, pmfied, and isoelectric focused on 2.5-mm-diameter gels as described in the text. After 9200 V-hr the gels were frozen and sliced into 2-mm sections each of which was dissolved in 0.5 ml 30% H,O1 overnight at 60”. After cooling the counts in each dissolved gel slice were determined by liquid scintillation counting in the presence of 10 ml Aquasol. 50h glacial acetic acid was added to each vial in some experiments to eliminate the possibility of differential quenching due to the differing pHs of the gel slices. Use of variable discrimators made spillage negligable with 3.0% of total 32Pcounts per minute appearing in the 3H window and only 0.3% of total 3H counts per minute appearing in the 3zP window. The basic end of the isoelectric focusing gel is located at the left of the graph. ( - - - ) “H; ( ) =P.

apparent aggregation of T-Ag under the conditions present in isoelectric focusing gels may bear no relationship to the aggregation of T-Ag under physiological conditions. T-Ag shows heterogeneity both in its affinity for double-stranded calf thymus DNA and in its sedimentation behavior (16). Differential phosphorylation states may affect the binding affinities of subunits for each other as in the phosphofructokinase system (17’). Both differential phosphorylation and

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FIG. 2. Concentration-dependent displacement of the multiple isoelectric focusing forms of SV40 large T-Ag. An equal amount of dilute 3ZP-labeledimmunoprecipitate of SV3T3 cells was applied in SDS-sample buffer to each of six SDS-polyacrylamide gels, In addition, increasing amounts of highly concentrated nonradioactive T-Ag, estimated to contain 0.1 mg/ml were then added to five of the six gels. After electrophoresis the bands of mixed “hot” and “cold” T-Ag were excised and subjected to isoelectric focusing on six separate gels for a total of 9150 V-hr. On gel a the labeled T-Ag was isoelectric focused alone. In gels b through f labeled large T-Ag was isoelectric focused with increasing amounts of concentrated cold T-Ag, (b) 0.5, (c) 1, (d) 2, (e) 4, and (f) 8 pg, respectively. Species were detected (A) by exposing the frozen isoelectric focusing gels to Kodak X-OMAT R film at -70” or (B) by slicing the frozen gel and determining 32Pcounts per section as in Fig. 1. The counts observed above Pl at the very tops of gels a-f and at the top of the gel in Fig. 1 were present in almost all our isoelectric focusing gels. They correspond to T-Ag as they migrate with a mobility corresponding to that of large T-Ag when reelectrophoresed into SDS-slab gels. However, they have not entered the isoelectric focusing gel and, consequently, they are not considered to represent an isoelectric focusing species.

various association states may affect the binding to nucleic acids by T-Ag. We, therefore, hope to determine if the heterogeneity of T-Ag in phosphate content contributes to the formation of the two DNA-binding forms and multiple sedimenting forms of native T-Ag. ACKNOWLEDGMENTS We are thankful to Sandy Gruss for helpful discussions and to Drs. M. Lamm, A. Ferreira, and J. Melero for critical reading of the manuscript. This investigation was supported by Grants 5 T32 CA09161 and CA20802 awarded by the National Cancer Institute, DHEW. R.B.C. is an Irma T. Hirsch1 Career Scientist Awardee. REFERENCES 1. LAI, C. J., and NATHANS, D., Virology 60, 466-475 (1974). 2. RUNDELL, K., COLLINS, J. K., TEGTMEYER, P., OZER, H. L., LAI, C. J., and NATHANS, D., J. Viral. 21, 636-646 (1977). 3. PAUCHA, E., HARVEY, R., and SMITH, A. E., J. viroz.

28, 154-170 (1978).

4. TEGTMEYER, P., J. Virol. 10, 591-598 (1972).

5. CHOU, J. Y. and MARTIN, R. G., J. Viral. 15, 145-150 (1975). 6. TEGTMEYER, P., RUNDELL, K., and COLLINS, J. K., J. Viral. 21, 647-657 (1977). 7. EDWARDS,C. A. F., KHOURY, G., and MARTIN, R. G., J. Viral. 29, 753-762 (1979). 8. TENG, C. S., TENG, C. T., and ALLFREY, V. G., J. Biol. Chem. 246, 3597-3609 (1971). 9. RUBIN, C. S., and ROSEN,0. M., Ann. Rev. Biothem. 44, 831-887 (1975). 10. CARROLL,R. B., GOLDFINE,S. M., and MELERO, J. A., Virology 87, 194-198 (1978). 11. O’FARRELL, P. H., J. Biol. Chem. 250, 40074021 (1975). 12. CRAWFORD,L. V. and O’FARRELL, P. Z., J. Viral.

29, 587-596 (1979). 13. WRIGLEY, C. W., In “Isoelectric Focusing” (N. Catsimpoolas, ed.), Academic Press, New York, 1976. 14. KLOTZ, I. M., LANGERMAN,N. R., and DARNALL, D. W., Annu. Rev. B&hem. 39,25-62 (1970). 15. CANN, J. R., STIMPSON,D. I., and Cox, D. J., Anal.

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16. CARROLL,R. B., HAGER, L., and DULBECCO,R., Proe. Nat. Acad. Sci. USA 71, 3754-3757 (1974). 17. UYEDA, K., MIYATAKE, A., LUBY, L. J., and RICHARDS, E. G., J. Biol. Chem. 250, 83198327 (1978).

Simian virus 40 large T antigen isoelectric focuses as multiple species with varying phosphate content.

VIROLOGY 99, 413-416 (197% Simian Virus 40 Large T Antigen Isoelectric Focuses as Multiple Species with Varying Phosphate Content DANIEL Department...
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