Proc. Natl. Acad. Sci. USA
Vol. 76, No. 12, pp. 6396-6400, December 1979 Cell Biology
Enhanced phosphorylation of many endogenous protein substrates in human fibroblasts transformed by simian virus 40 (transformation/protein kinase/cancer)
J. EPSTEIN, J. L. BRESLOW, AND J. H. FONTAINE Metabolism Division, Department of Medicine, Children's Hospital Medical Center and the Department of Pediatrics, Harvard Medical School,
Boston, Massachusetts 02115
Communicated by Arthur B. Pardee, August 16, 1979
ABSTRACT Protein phosphorylation in normal and in simian virus 40-transformed human skin fibroblasts was assessed by two different methods: incubation of whole-cell homogenates with [y-32P]ATP or labeling of intact cells with Na2H32PO4. Phosphorylated proteins were detected by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiography. With both methods, the Coomassie-blue-stained protein patterns of the three transformed cell lines studied were similar to the patterns of the nontransformed normal human cells. However, although the phosphoprotein autoradiograms of the three transformed cell fines were nearly identical, their patterns were strikingly different from those of the nontransformed cells. Each of the three transformed lines tested showed approximately 25-30 phosphoprotein bands that were significantly enhanced when compared to the patterns of the nontransformed cells. Quantitation of 12 of the enhanced phosphoprotein bands in one of the transformed cell lines showed an average of 4.4 times as much phosphorylation as in the normal cells. The enhanced phosphorylation observed in the transformed cell lines was not dependent on the growth rate of the cells or on cyclic AMP. Furthermore, when homogenates of transformed and nontransformed cells were mixed prior to incubation with [y-32PIATP, the resultant phosphoprotein patterns resembed those obtained with transformed cells alone. In addition, an evaluation of the time course of protein phospho ryation revealed that the initial reaction rate was greater in the transformed than in the normal cells, although in both cell types the reaction was complete after 1 min. The results suggest that the simian virus 40-transformed human fibroblasts possess an increased ability to phosphorylate proteins rather than that the normal cells possess a diffusible inhibitor. There appear to be many endogenous cellular substrates for this increased activity. Studies of malignant cells have been facilitated by the use of in vitro cell-culture systems. In these systems, the transformation phenotype of cells has been associated with a decrease in the amount of serum required for growth, an ability to grow in soft agar, and a capacity to form tumors when introduced into an appropriate host. In contrast, untransformed (normal) cells generally require higher amounts of serum or specific growth factors, are anchorage dependent, and will not form tumors when injected into a host (1). Transformed cells exhibit other abnormalities in vitro, including altered ion and nutrient transport, RNA metabolism, and protein synthesis. Some transformed cells also have aberrant plasma membrane and cytoskeleton features (1). It is tempting to speculate that the induction of many of these changes occurs under a coordinated controlling mechanism that is activated as a result of cellular exposure to a transforming event. Consequently, any biochemical mechanism that could have the potential for significantly altering a large number of cellular proteins might be able to exert a major role in cellular transformation. Because it is believed that the biological activity of many different cellular
proteins can be altered through phosphorylation-dephosphorylation mechanisms, we decided to compare the abilities of normal and simian virus 40 (SV40)-transformed human fibroblasts to phosphorylate proteins (2). EXPERIMENTAL PROCEDURES
Skin fibroblasts were obtained by biopsy and grown and maintained as described (3). SV40-1, SV40-2, and LNSV cell lines were obtained by transforming different human fibroblast strains with SV40 and maintaining until stable T-antigen-positive lines were obtained. Confluent monolayers of normal and transformed cell lines were harvested by trypsinization; 0.5-2.0 X 106 cells were inoculated into 150-mm-diameter petri dishes (Falcon) and renewed with medium every 48 hr. When the cells were approximately 90-100% confluent (6-8 days of growth), the cells were prepared for phosphorylation assays by one of two methods: (i) For in vitro protein phosphorylation experiments, the monolayers were rinsed twice with and scraped into normal saline. The cells were then resuspended in homogenizing buffer (0.25 M sucrose/10 mM Tris-HCl, pH 7.0/1 mM EDTA) at 4°C. The cells were kept at 40C and broken up with a Teflon-glass homogenizer (Kontes). The protein concentration was determined by the Lowry method (4), with bovine serum albumin as a standard. The homogenate was then diluted with homogenizing buffer to the desired final concentration for each experiment. The protein phosphorylation reaction was performed according to a minor variation of the methods developed by DeLorenzo et al. (5), which involves adding [y32P]ATP to the homogenate, incubating for 2 min, and using a sodium dodecyl sulfate (NaDodSO4) solution and boiling to stop the reaction. (ii) For intact cell protein phosphorylation experiments, the subconfluent monolayers were rinsed twice with normal saline, renewed with Eagle's minimal essential medium containing 0.1 mM Na2HPO4 (one-tenth the normal amount of phosphate) and 100 ,uCi of Na2H32PO4 per ml (>1000 Ci/mmol, New England Nuclear; 1 Ci = 3.7 X 1010 becquerels), and incubated at 36.50C in a CO2 incubator for 3 hr. (Preliminary experiments were performed with in vivo labeling at 0.5, 1, 1.5, 3, 6, and 20 hr, which showed that steady-state labeling of the phosphoproteins was achieved by 3 hr.) The incubation was terminated by washing the monolayers twice with normal saline at 40C, dissolving them in 3% NaDodSO4/62.5 mM Tris-HCI, pH 6.8/3 mM EDTA/10% glycerol, and heating in a boiling water bath for 2 min; an aliquot was then removed for protein determination by the method of Lowry (4). Dithiothreitol was then added to the dissolved cells to a final concentration of 120 mM and the solution was again placed in a boiling water bath for 2 min. The phosphorylated cellular material prepared by either of the above two methods was subjected to electrophoresis on
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Abbreviations: SV40, simian virus 40; NaDodSO4, sodium dodecyl sulfate; cAMP, cyclic AMP. 6396
Cell Biology: Epstein et al.
Proc. Natl. Acad. Sci. USA 76 (1979) A
NaDodSO4/polyacrylamide gradient slab gels (6-15%) by the method of Laemmli (6). Completed gels were stained. with Coomassie brilliant blue, destained, and dried. Autoradiographs of the dried gels were made with Kodak Royal X-Omat film, and scans were performed with the Schoeffel Spectrodensitometer, model SD 300. In addition to autoradiographic analysis, the degree of phosphorylation of endogenous protein substrates in dell homogenates was also examined by a direct quantitative assay involving liquid scintillation measurement of radioactivity. The cells were homogenized and treated with the [-y32P]ATP as described above for the in vitro protein phosphorylation experiments. However, the reaction was terminated by addition of 4 ml of ice-cold 5% trichloroacetic acid/5 mM sodium phosphate/2 mM sodium pyrophosphate; the mixture was filtered through a GF/C glass microfiber Whatman filter. The filter was washed three times with 4 ml of ice-cold 5% trichloroacetic acid and dried; radioactivity was measured in 10 ml of Instagel. Results are expressed as pmol of trichloroacetic acid-precipitable phosphate per mg of homogenate protein.
RESULTS Protein Phosphorylation in Cell Homogenates. In the experiment presented in Fig. 1, the transformed and untransformed cells were compared for their ability to phosphorylate endogenous cellular proteins by the in vitro method. The stained protein patterns of the normal human fibroblast strain S-238 and the SV40-transformed human fibroblast line SV40-1 are similar (Fig. 1A). In Fig. 1B, however, the autoradiograms of the same gel patterns presented in Fig. 1A show a greatly enhanced phosphorylation of approximately 25-30 protein bands in the transformed cells. A possible role of cyclic AMP (cAMP) in explaining the differences observed was also studied by examining cAMP-dependent protein phosphorylation in the presence and absence of the heat-stable inhibitor of the catalytic subunit of the cAMP-dependent protein kinase (Sigma). The inhibitor diminished the phosphorylation of only a few protein bands in the transformed and untransformed cells but did not appreciably diminish the overall difference in protein phosphorylation between the two cell types (data not shown), thus showing that cAMP had little or no effect on the difference in phosphorylation. To obtain a quantitative assessment of enhanced protein phosphorylation in the transformed cells, we analyzed the results-in Fig. 1B (lanes a and c) by scanning densitometry (Table 1). For a given peak, SV40-1 showed 2.3-8.0 times the amount of phosphorylation when compared to the normal cells. The bands observed were sensitive to Pronase but not to DNase or RNase (data not shown). Time Course of Protein Phosphorylation. In the experiments thus far presented, the time of the reaction of the cell homogenates with ['y-32P]ATP was 2 min. Because it was possible that the kinetics of the reactions in untransformed and transformed cells were different, the in vitro reaction time was varied and aliquots of the same homogenates were incubated with ['y-32P]ATP for 10, 20, 30, 45, 60, 90, and 120 sec. The results showed that the initial reaction rate was greater for transformed cells than for untransformed cells although the reaction for both types was completed after approximately 1 min (data not shown). These reaction kinetics are compatible with an enhanced protein phosphorylating ability in the transformed cells. Mixing Experiments. What appears to be enhanced phosphorylation in SV40-transformed cells in the experiments reported here could be the result of a suppression of phosphorylation in the normal cells. To determine if normal cells contain an inhibitor of phosphorylation, we mixed homogenates of normal and transformed cells, incubated them with [ky-
6397 B
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FIG. 1. In vitro phosphorylation of endogenous cellular proteins. Monolayers of normal and SV40-transformed human fibroblasts were homogenized. In vitro protein phosphorylation was assessed with [vy-32P]ATP in the presence of 10 mM Mg2+ at pH 6.5. The reaction was terminated by addition of NaDodSO4-containing buffer; 30 Ag of cell homogenate protein was then subjected to electrophoresis on NaDodSO4/polyacrylamide gradient slab gels (6-15%). The completed gels were stained with Coomassie brilliant blue, destained, and dried. Autoradiograms were made of the dried gels. (A) Coomassie brilliant blue-stained pattern of normal human fibroblast strain S-238 (lanes a and b) and the SV40-transformed human fibroblast line SV40-1 (lanes c and d). Arrow, 68,000 Mr standard, bovine serum albumin. (B) Autoradiogram of the same gel patterns presented in A. Lanes a and c, phosphorylation patterns without cAMP in reaction mixtures. Lanes b and d, patterns after addition of 10 1uM cAMP. The differences in protein phosphorylation between S-238 and SV40-1 were also observed between other normal human fibroblast strains and SV40-2 and LNSV (data not shown). (For arrows 1-12, see Table 1.)
32P]ATP, and subjected the mixture to NaDodSO4/polyacrylamide gel electrophoresis and autoradiography (Fig. 2). The pattern of the mixed homogenates (lane c) resembles the pattern of the transformed cells alone (lane b) and not the normal cells (lane a). These experiments suggest that the phosphoprotein pattern in normal cells is not due to an active diffusible inhibitor. This question was also examined by studying protein phosphorylation in the presence of 2 mM NaF, a phosphatase inhibitor, or of 5 mM ZnCl2, which inhibited protein kinase activity in this system. Inhibition of phosphatase with NaF showed a minor increase in the overall pattern of protein phosphorylation in both normal and transformed cells. However, the substantial difference between the two types remained. Inhibition of protein kinase activity with ZnCI2 re-
Cell Biology: Epstein et al.
PU-0098
Table 1. In vitro protein phosphorylation in normal and transformed cells* Protein Untransformed Transformed band cells cells Ratiot 1 10 80 8.0 2and3 89 422 4.7 4 and 5 204 1107 5.4 6 49 230 4.7 7 53 180 3.4 8 and 9 56 127 2.3 10 and 11 57 184 3.2 12 44 117 2.7
1-12t 2447 562 4.4 * To quantitatively assay protein phosphorylation, we analyzed the results in Fig. 1B, lanes a and c, by scanning denslitometry. The peaks from the densitometer tracings for the 12 enhanced phosphoprotein bands (arrows, Fig. 1B) were then cut out and weighed. The weight of each peak was taken to be proportional to the amount of protein phosphorylation and is shown in the table. t Ratio of the weights of the peaks of the transformed cells to those of the untransformed cells. Total weights of the peaks for all 12 proteins.
duced protein phosphorylation in the transformed cells to the level seen in normal cells (data not shown). These studies further suggest that enhanced phosphorylation in the transformed cells is due to either increased protein kinase activity or to a new ZnCl2-sensitive protein kinase and not to an active diffusible inhibitor in the normal cells.
Proc. Natl. Acad. Sci. USA 76 (1979)
Protein Phosphorylation at Different Stages of Confluency. In the experiments thus far described, the normal and transformed cells were compared after they had grown to 90-100% confluency. At this density, the contact-inhibited normal cells have a diminished growth rate whereas the noncontact-inhibited transformed cells maintain a rapid growth rate. To verify that the enhanced protein phosphorylation observed in the tumor cells was an intrinsic property of the transformed cell and not merely related to growth rate, we performed experiments with normal and transformed cells at different stages of confluency. Protein phosphorylation in normal and transformed cells was assessed by autoradiography (Fig. 3) and by a direct quantitative assay involving liquid scintillation measurement of radioactivity (Table 2). At 25, 50, and 75% confluency, with both types of phosphorylation methods, tumor cells showed enhanced protein phosphorylation when compared to normal cells. This was at a time when the normal cells were actually growing faster than the tumor cells. At confluence, the contact-inhibited normal cells had a diminished growth rate whereas the noncontact-inhibited transformed cells maintained a rapid growth rate. Thus, the dramatic enhancement of protein phosphorylation seen in transformed cells when compared to normal cells at all stages of confluency does not appear to be merely a function of growth rate. However, growth rate or cell density could have important effects on protein phosphorylation; at confluence, the normal cells may show a slight decrease in phosphorylation (apparent
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FIG. 2. Mixing experiments. Autoradiogram from an in vitro protein phosphorylation assay in which both untransformed and transformed cell homogenates were mixed. Arrow, 68,000 Mr standard, bovine serum albumin. Lanes a and b, phosphorylation patterns of S-238 and SV40-1, normal and transformed cells, respectively; lane c, results of mixing the S-238 and SV40-1 homogenates prior to the in vitro phosphorylation assay.
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these. glarshw. Aro,6,00M tandrd boieseu l a b c d e f f a b c d 000 e Noma hua firbatsrinS29Rgt bui. 3.(Left FIG. Protein at different of phosphorylation stages confluency. Monolayers of normal and SV40-transformed human fibroblasts at different stages of confluency were homogenized and subjected to in vitro protein phosphorylation. The reaction was terminated with NaDodSO4 containing buffer; 25 ,sg of cell homogenate protein was then subjected to electrophoresis on NaDodSO4/polyacrylamide gradient slab gels (6-15%). The completed gels were stained with Coomassie brilliant blue, destained, and dried. Autoradiograms of
transformed line SV40-1. Lanes a, b, c, and d, dishes that were 25, 50, 75, and 100% confluent, respectively. Lanes e and f, dishes that were maintained in culture for 3 and 12 days, respectively, after confluence.
Cell Biology: Epstein et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
Table 2. In vitro protein phosphorylation in normal (S-239) and transformed (SV40-1) cells at different stages of conflency* v pmol P04/mg Cells/disht Days X 10-6 protein in culture Confluency,t % S239 SV40-1 S239 SV40-1 S239 SV40-1 649 2 518 2 0.64 1.2 2.8 919 6.2 530 5 9 3.1 572 1063 7.4 6 13 50 560 1815 7 6.6 23 * Monolayers of normal and SV40-transformed human fibroblasts at different stages of confluency were homogenized and subject to in vitro protein phosphorylation. The reaction was terminated with trichloroacetic acid; the precipitate was collected on a filter which was washed and dried; and radioactivity was measured. The results are expressed as pmol of acid-precipitable phosphate per mg of cell homogenate protein. t Confluency was assessed visually by scanning the culture dishes with a phase-contrast microscope. Cells per dish was determined by treating a replicate petri dish with trypsin and performing a cell count on a Coulter Counter.
25 50 75 100
by autoradiography but not by quantitative assay) whereas the transformed cells show an increase in phosphorylation. Protein Phosphorylation in Intact Cells. Because it was possible that a unique feature of the in vitro phosphorylation assay could have artificially enhanced protein phosphorylat: on in the transformed cells, we decided to repeat the experiments by using a more physiological, intact-cell assay method. As with the first method, the stained protein patterns of the normal human diploid fibroblast strains were similar to those of the
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shown. Arrows indicate the 200,000, 68,000, and 44,000 Mr standards, myosin, bovine serum albumin, and actin, respectively. Lanes a and b, intact cell phosphorylation pattern for the untransformed strain S-238. Lanes c and d, pattern for the transformed cell line SV40-1. Results shown in lanes b and d were obtained with cultures incubated with 1 ,uM isoproterenol and 1 mM theophylline
during the last 15 min of exposure Na2H32PO4.
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6399
SV40-transformed human fibroblast lines. However, the autoradiograms showed greatly enhanced protein phosphorylation in SV40-1 cells as compared to S-238 (normal) cells (Fig. 4). Thus, the use of intact cells for measurement of phosphorylation confirms the results obtained with cell homogenates. The actual protein bands phosphorylated in intact cells differ somewhat from those phosphorylated in experiments using cell homogenates. This may be due to the fact that the protein kinase(s) involved appears to be membrane bound and that in intact cells it may have access only to certain proteins and not to others. DISCUSSION The results presented demonstrate enhanced phosphorylation of at least 25-30 endogenous cellular proteins in three SV40transformed human fibroblast cell lines when compared with several different untransformed human fibroblast strains. These results were obtained by two different methods, and it was apparent that the enhanced protein phosphorylation was not merely a function of the growth rate of the transformed cell lines, nor was it dependent on cAMP. Mixing of homogenates of transformed and normal cells prior to incubation with [y32P]ATP resulted in a phosphoprotein pattern similar to that of the transformed cells alone. These data suggest that enhanced protein phosphorylation ability observed in the transformed cell lines could perhaps be due to increased protein kinase activity or to a new protein kinase activity. SV40 is a small DNA virus that codes for about 200,000 daltons of protein. Three distinct gene products have been identified, only one of which, that of gene A, is correlated with an early function that appears to be required for viral DNA synthesis. Gene A has been shown to code for the SV40 T antigen, a 100,000-dalton protein that is believed to initiate viral DNA synthesis, and is also required for the transformation phenotype (7), although the mechanism by which the T antigen performs these functions is not clear. It is possible that the T antigen may be responsible, directly or indirectly, for the enhanced protein phosphorylation seen in our experiments with SV4O-transformed human fibroblasts. The T antigen may either possess protein kinase activity itself or stimulate an endogenous cellular protein kinase activity (or both). Recently, Tjian and Robbins (8) showed that protein kinase and ATPase activities copurify with a protein that is antigenically related to the T antigen. Furthermore, protein kinase activity has also been associated with the transforming proteins found in other systems. For example, the src gene of the avian sarcoma virus, an RNA tumor virus, has been shown by workers in two different laboratories to code for a protein of Mr 60,000 that exhibits protein kinase activity (9, 10). Protein kinase activity has also been associated with the transforming proteins of the DNA virus, adenovirus type 5 (11). These recent reports, as well as the data presented in the current paper, suggest that protein phosphorylation may be an important mechanism intimately involved in the events through which several different tumor viruses induce cellular transformation. It is important to emphasize that the overall pattern of enhanced protein phosphorylation observed with SV40-transformed cells in the experiments reported here was not cAMP dependent. cAMP-dependent processes have been implicated in the arrest of cell growth and reversion of some transformed cells to a more normal phenotype (12, 13). Cell cAMP-dependent effects are thought to be mediated by a protein kinase (14), and variants with mutations in a cAMP-dependent protein kinase have been isolated that resist the growth inhibitory effect of cAMP (15). In addition, changes detected in the two principal types of soluble cAMP-dependent protein kinases (16) have also been related to growth regulation and cellular transformation. Activation of type II cAMP-dependent protein kinase has been
6400
Cell Biology: Epstein et al.
demonstrated as CHO cells traverse G1, and de novo synthesis of this enzyme occurs at the G1/S border (17, 18). In addition, alterations in type I cAMP-dependent protein kinase have been associated with mitogenesis, neoplastic cell lines, or tumors, whereas hormone-dependent mammary tumor regression has been associated with an increase in type II cAMP-dependent protein kinase (19-22). Moreover, SV40 transformation of 3T3 cells is associated with altered growth properties of the cells and the appearance of a type I cAMP-dependent protein kinase (23). The regulation of cell growth has also been associated with alterations in cAMP-dependent protein phosphorylation. Induction of the quiescent state in BHK cells resulted in the phosphorylation of five cytosolic proteins, four of them only in the presence of cAMP (24). In the present experiments, the effects of cAMP on protein phosphorylation were minor regardless of whether the cell homogenates were treated in vitro with cAMP or the intact cells were treated in vivo to stimulate endogenous cAMP levels. These results further suggest that the phenomenon reported here of enhanced phosphorylation of many endogenous cellular proteins observed in SV40-transformed human fibroblasts is independent of cAMP. However, the results reported here do not, of course, preclude a role for cAMP in important processes related to growth control or expression of tumorgenicity in SV40-transformed human fibroblasts. Studies of protein substrates for enhanced phosphorylation in SV40-transformed cells have, to our knowledge, been reported twice previously. Segawa et al. (25) have observed a highly phosphorylated protein of approximately 90,000 daltons detected in both chromatin and ribosomes of SV40-transformed rodent cells; this protein was either not present or was present only in very small amounts in the organelles of untransformed cells. They suggested that the expression of this protein was regulated by the SV40 T antigen because the amount of phosphorylation of this protein in cells transformed with a temperature-sensitive mutant of SV40 was greatly decreased when the cells were cultured at the restrictive temperature (25). Segawa et al. (26) have also observed this highly phosphorylated, 90,000-dalton protein in cells transformed by adenovirus, murine sarcoma virus, or methylcholanthrene. They therefore proposed that the phosphorylation of this protein is closely related to the appearance of the transformed state in these cells. In our studies, we have detected a highly phosphorylated protein band of Mr approximately 90,000 in the SV40-transformed human fibroblasts. However, we also see phosphorylation of the same protein band in rapidly growing untransformed fibroblasts studied at subconfluence (Fig. 3). When these cells were confluent, the degree of phosphorylation of this protein species decreased (Fig. 3), suggesting that this phenomenon is related to growth rate rather than transformation. However, a closer examination of the autoradiographic patterns presented by Segawa et al. (25, 26) reveals that there is also an enhanced phosphorylation of many other endogenous cellular proteins in the transformed cells they have studied when the patterns were compared to those obtained with untransformed cells. Our data agree with this observation, but more importantly, also demomstrate that the degree of enhanced phosphorylation observed for many of these other endogenous protein substrates in the transformed cells is not merely related to the growth rate of the cells. A second study showing enhanced phosphorylation of proteins in SV40-transformed cells was done by Pumo et al. (27). These investigators showed a 10-fold enhancement of phosphorylation of nonhistone chromosomal proteins in SV40transformed when compared with normal human diploid fibroblasts. These studies showed that the increased phosphorylation in the transformed cells could not be accounted for
Proc. Natl. Acad. Sci. USA 76 (1979)
by either an increased rate of phosphate transport or the synthesis of new species of nonhistone chromosomal proteins which subsequently become phosphorylated. The exact identity and physiological functions of the proteins that exhibited enhanced phosphorylation in the transformed cells used in our experiments remain to be determined. However, because the biological role of many different proteins can be changed by phosphorylation, alterations in the degree of phosphorylation that occur after viral transformation may be part of the cell's pleiotropic response coordinating the many functional changes observed in the transformation from a normal to a malignant phenotype (1, 2). Note Added in Proof. After this manuscript was submitted for review, Griffin et al. (28) reported that the large T antigen of SV40 exhibits protein kinase acitivity. This work was supported by U. S. Public Health Service Grants HL15895 and AM20010 and by grants from The National Foundation-March of Dimes, and the Medical Foundation, Inc., Boston, MA. 1. Tooze, J., ed. (1973) The Molecular Biology of Tumor Viruses (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). 2. Greengard, P. (1978) Science 199, 146-151. 3. Epstein, J. & Breslow, J. L. (1977) Proc. Natl. Acad. Sci. USA 74, 1676-1679. 4. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 5. DeLorenzo, R. J., Walton, K. G., Curran, P. F. & Greengard, P. (1973) Proc. Natl. Acad. Sci. USA 70,880-884. 6. Laemmli, U. K. (1970) Nature (London) 227,680-685. 7. Fried, M. & Griffin, B. E. (1977) Adv. Cancer Res. 24, 67113. 8. Tjian, R. & Robbins, A. (1979) Proc. Natl. Acad. Sci. USA 76, 610-614. 9. Collett, M. S. & Erikson, R. L. (1978) Proc. Natl. Acad. Sci. USA 75,2021-2024. 10. Levinson, A. D., Oppermann, H., Levintow, L., Varmus, H. E. & Bishop, J. M. (1978) Cell 15,561-572. 11. Lassan, N. J., Bayley, S. T., Graham, F. L. & Branton, P. E. (1979) Nature (London) 277,241-243. 12. Pastan, I. H., Johnson, G. S. & Anderson, W. B. (1975) Annu. Rev. Biochem. 44, 491-511. 13. Pastan, I. H. & Willingham, M. (1978) Nature (London) 274, 645-650. 14. Kuo, J. F. & Greengard, P. (1969) Proc. Natl. Acad. Sci. USA 64, 1349-1353. 15. Insel, P. A., Bourne, H. R., Coffino, P. & Tomkins, G. M. (1975) Science 190, 896-898. 16. Rubin, C. S. & Rosen, 0. M. (1975) Annu. Rev. Biochem. 44, 831-887. 17. Costa, M., Gerner, E. W. & Russell, D. H. (1976) Biochim. Biophys. Acta 425, 246-255. 18. Costa, M., Gerner, E. W. & Russell, D. H. (1976) J. Biol. Chem. 251,3313-3319. 19. Byus, C. V., Klimpel, G. R., Lucas, D. 0. & Russell, D. H. (1977) Nature (London) 268,63-64. 20. Gutomann, N. S., Rae, P. A. & Schimmer, B. P. (1978) J. Cell. Physiol. 97, 451-460. 21. Fossberg, T. M., Doskeland, S. D. & Ueland, P. M. (1978) Arch. Biochem. Biophys. 189,372-381. 22. Cho-Chung, Y. S., Clair, J. & Zubialde, J. P. (1978) Biochem. Biophys. Res. Commun. 85, 1150-1155. 23. Glarrett, A. J., Malkinson, A. M. & Sheppard, J. R. (1976) Nature (London) 264, 673-675. 24. Kletzien, R. F., Milles, M. R. & Pardee, A. B. (1977) Nature (London) 270,57-59. 25. Segawa, K., Yamaguchi, N. & Oda, K. (1977) J. Virol. 22, 679-693. 26. Segawa, K., Oda, K., Yuasa, Y., Shiroki, K. & Shimojo, H. (1978) J. Virol. 27,800-808. 27. Pumo, D. E., Stein, G. S. & Kleinsmith, L. J. (1975) Biochim. Biophys. Acta 402,125-130. 28. Griffin, J. D., Spangler, G. & Livingston, D. M. (1979) Proc. Natl. Acad. Sci. USA 76,2610-2614.
Vol. 76, No. 12, pp. 6396-6400, December 1979 Cell Biology
Enhanced phosphorylation of many endogenous protein substrates in human fibroblasts transformed by simian virus 40 (transformation/protein kinase/cancer)
J. EPSTEIN, J. L. BRESLOW, AND J. H. FONTAINE Metabolism Division, Department of Medicine, Children's Hospital Medical Center and the Department of Pediatrics, Harvard Medical School,
Boston, Massachusetts 02115
Communicated by Arthur B. Pardee, August 16, 1979
ABSTRACT Protein phosphorylation in normal and in simian virus 40-transformed human skin fibroblasts was assessed by two different methods: incubation of whole-cell homogenates with [y-32P]ATP or labeling of intact cells with Na2H32PO4. Phosphorylated proteins were detected by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiography. With both methods, the Coomassie-blue-stained protein patterns of the three transformed cell lines studied were similar to the patterns of the nontransformed normal human cells. However, although the phosphoprotein autoradiograms of the three transformed cell fines were nearly identical, their patterns were strikingly different from those of the nontransformed cells. Each of the three transformed lines tested showed approximately 25-30 phosphoprotein bands that were significantly enhanced when compared to the patterns of the nontransformed cells. Quantitation of 12 of the enhanced phosphoprotein bands in one of the transformed cell lines showed an average of 4.4 times as much phosphorylation as in the normal cells. The enhanced phosphorylation observed in the transformed cell lines was not dependent on the growth rate of the cells or on cyclic AMP. Furthermore, when homogenates of transformed and nontransformed cells were mixed prior to incubation with [y-32PIATP, the resultant phosphoprotein patterns resembed those obtained with transformed cells alone. In addition, an evaluation of the time course of protein phospho ryation revealed that the initial reaction rate was greater in the transformed than in the normal cells, although in both cell types the reaction was complete after 1 min. The results suggest that the simian virus 40-transformed human fibroblasts possess an increased ability to phosphorylate proteins rather than that the normal cells possess a diffusible inhibitor. There appear to be many endogenous cellular substrates for this increased activity. Studies of malignant cells have been facilitated by the use of in vitro cell-culture systems. In these systems, the transformation phenotype of cells has been associated with a decrease in the amount of serum required for growth, an ability to grow in soft agar, and a capacity to form tumors when introduced into an appropriate host. In contrast, untransformed (normal) cells generally require higher amounts of serum or specific growth factors, are anchorage dependent, and will not form tumors when injected into a host (1). Transformed cells exhibit other abnormalities in vitro, including altered ion and nutrient transport, RNA metabolism, and protein synthesis. Some transformed cells also have aberrant plasma membrane and cytoskeleton features (1). It is tempting to speculate that the induction of many of these changes occurs under a coordinated controlling mechanism that is activated as a result of cellular exposure to a transforming event. Consequently, any biochemical mechanism that could have the potential for significantly altering a large number of cellular proteins might be able to exert a major role in cellular transformation. Because it is believed that the biological activity of many different cellular
proteins can be altered through phosphorylation-dephosphorylation mechanisms, we decided to compare the abilities of normal and simian virus 40 (SV40)-transformed human fibroblasts to phosphorylate proteins (2). EXPERIMENTAL PROCEDURES
Skin fibroblasts were obtained by biopsy and grown and maintained as described (3). SV40-1, SV40-2, and LNSV cell lines were obtained by transforming different human fibroblast strains with SV40 and maintaining until stable T-antigen-positive lines were obtained. Confluent monolayers of normal and transformed cell lines were harvested by trypsinization; 0.5-2.0 X 106 cells were inoculated into 150-mm-diameter petri dishes (Falcon) and renewed with medium every 48 hr. When the cells were approximately 90-100% confluent (6-8 days of growth), the cells were prepared for phosphorylation assays by one of two methods: (i) For in vitro protein phosphorylation experiments, the monolayers were rinsed twice with and scraped into normal saline. The cells were then resuspended in homogenizing buffer (0.25 M sucrose/10 mM Tris-HCl, pH 7.0/1 mM EDTA) at 4°C. The cells were kept at 40C and broken up with a Teflon-glass homogenizer (Kontes). The protein concentration was determined by the Lowry method (4), with bovine serum albumin as a standard. The homogenate was then diluted with homogenizing buffer to the desired final concentration for each experiment. The protein phosphorylation reaction was performed according to a minor variation of the methods developed by DeLorenzo et al. (5), which involves adding [y32P]ATP to the homogenate, incubating for 2 min, and using a sodium dodecyl sulfate (NaDodSO4) solution and boiling to stop the reaction. (ii) For intact cell protein phosphorylation experiments, the subconfluent monolayers were rinsed twice with normal saline, renewed with Eagle's minimal essential medium containing 0.1 mM Na2HPO4 (one-tenth the normal amount of phosphate) and 100 ,uCi of Na2H32PO4 per ml (>1000 Ci/mmol, New England Nuclear; 1 Ci = 3.7 X 1010 becquerels), and incubated at 36.50C in a CO2 incubator for 3 hr. (Preliminary experiments were performed with in vivo labeling at 0.5, 1, 1.5, 3, 6, and 20 hr, which showed that steady-state labeling of the phosphoproteins was achieved by 3 hr.) The incubation was terminated by washing the monolayers twice with normal saline at 40C, dissolving them in 3% NaDodSO4/62.5 mM Tris-HCI, pH 6.8/3 mM EDTA/10% glycerol, and heating in a boiling water bath for 2 min; an aliquot was then removed for protein determination by the method of Lowry (4). Dithiothreitol was then added to the dissolved cells to a final concentration of 120 mM and the solution was again placed in a boiling water bath for 2 min. The phosphorylated cellular material prepared by either of the above two methods was subjected to electrophoresis on
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Abbreviations: SV40, simian virus 40; NaDodSO4, sodium dodecyl sulfate; cAMP, cyclic AMP. 6396
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Proc. Natl. Acad. Sci. USA 76 (1979) A
NaDodSO4/polyacrylamide gradient slab gels (6-15%) by the method of Laemmli (6). Completed gels were stained. with Coomassie brilliant blue, destained, and dried. Autoradiographs of the dried gels were made with Kodak Royal X-Omat film, and scans were performed with the Schoeffel Spectrodensitometer, model SD 300. In addition to autoradiographic analysis, the degree of phosphorylation of endogenous protein substrates in dell homogenates was also examined by a direct quantitative assay involving liquid scintillation measurement of radioactivity. The cells were homogenized and treated with the [-y32P]ATP as described above for the in vitro protein phosphorylation experiments. However, the reaction was terminated by addition of 4 ml of ice-cold 5% trichloroacetic acid/5 mM sodium phosphate/2 mM sodium pyrophosphate; the mixture was filtered through a GF/C glass microfiber Whatman filter. The filter was washed three times with 4 ml of ice-cold 5% trichloroacetic acid and dried; radioactivity was measured in 10 ml of Instagel. Results are expressed as pmol of trichloroacetic acid-precipitable phosphate per mg of homogenate protein.
RESULTS Protein Phosphorylation in Cell Homogenates. In the experiment presented in Fig. 1, the transformed and untransformed cells were compared for their ability to phosphorylate endogenous cellular proteins by the in vitro method. The stained protein patterns of the normal human fibroblast strain S-238 and the SV40-transformed human fibroblast line SV40-1 are similar (Fig. 1A). In Fig. 1B, however, the autoradiograms of the same gel patterns presented in Fig. 1A show a greatly enhanced phosphorylation of approximately 25-30 protein bands in the transformed cells. A possible role of cyclic AMP (cAMP) in explaining the differences observed was also studied by examining cAMP-dependent protein phosphorylation in the presence and absence of the heat-stable inhibitor of the catalytic subunit of the cAMP-dependent protein kinase (Sigma). The inhibitor diminished the phosphorylation of only a few protein bands in the transformed and untransformed cells but did not appreciably diminish the overall difference in protein phosphorylation between the two cell types (data not shown), thus showing that cAMP had little or no effect on the difference in phosphorylation. To obtain a quantitative assessment of enhanced protein phosphorylation in the transformed cells, we analyzed the results-in Fig. 1B (lanes a and c) by scanning densitometry (Table 1). For a given peak, SV40-1 showed 2.3-8.0 times the amount of phosphorylation when compared to the normal cells. The bands observed were sensitive to Pronase but not to DNase or RNase (data not shown). Time Course of Protein Phosphorylation. In the experiments thus far presented, the time of the reaction of the cell homogenates with ['y-32P]ATP was 2 min. Because it was possible that the kinetics of the reactions in untransformed and transformed cells were different, the in vitro reaction time was varied and aliquots of the same homogenates were incubated with ['y-32P]ATP for 10, 20, 30, 45, 60, 90, and 120 sec. The results showed that the initial reaction rate was greater for transformed cells than for untransformed cells although the reaction for both types was completed after approximately 1 min (data not shown). These reaction kinetics are compatible with an enhanced protein phosphorylating ability in the transformed cells. Mixing Experiments. What appears to be enhanced phosphorylation in SV40-transformed cells in the experiments reported here could be the result of a suppression of phosphorylation in the normal cells. To determine if normal cells contain an inhibitor of phosphorylation, we mixed homogenates of normal and transformed cells, incubated them with [ky-
6397 B
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FIG. 1. In vitro phosphorylation of endogenous cellular proteins. Monolayers of normal and SV40-transformed human fibroblasts were homogenized. In vitro protein phosphorylation was assessed with [vy-32P]ATP in the presence of 10 mM Mg2+ at pH 6.5. The reaction was terminated by addition of NaDodSO4-containing buffer; 30 Ag of cell homogenate protein was then subjected to electrophoresis on NaDodSO4/polyacrylamide gradient slab gels (6-15%). The completed gels were stained with Coomassie brilliant blue, destained, and dried. Autoradiograms were made of the dried gels. (A) Coomassie brilliant blue-stained pattern of normal human fibroblast strain S-238 (lanes a and b) and the SV40-transformed human fibroblast line SV40-1 (lanes c and d). Arrow, 68,000 Mr standard, bovine serum albumin. (B) Autoradiogram of the same gel patterns presented in A. Lanes a and c, phosphorylation patterns without cAMP in reaction mixtures. Lanes b and d, patterns after addition of 10 1uM cAMP. The differences in protein phosphorylation between S-238 and SV40-1 were also observed between other normal human fibroblast strains and SV40-2 and LNSV (data not shown). (For arrows 1-12, see Table 1.)
32P]ATP, and subjected the mixture to NaDodSO4/polyacrylamide gel electrophoresis and autoradiography (Fig. 2). The pattern of the mixed homogenates (lane c) resembles the pattern of the transformed cells alone (lane b) and not the normal cells (lane a). These experiments suggest that the phosphoprotein pattern in normal cells is not due to an active diffusible inhibitor. This question was also examined by studying protein phosphorylation in the presence of 2 mM NaF, a phosphatase inhibitor, or of 5 mM ZnCl2, which inhibited protein kinase activity in this system. Inhibition of phosphatase with NaF showed a minor increase in the overall pattern of protein phosphorylation in both normal and transformed cells. However, the substantial difference between the two types remained. Inhibition of protein kinase activity with ZnCI2 re-
Cell Biology: Epstein et al.
PU-0098
Table 1. In vitro protein phosphorylation in normal and transformed cells* Protein Untransformed Transformed band cells cells Ratiot 1 10 80 8.0 2and3 89 422 4.7 4 and 5 204 1107 5.4 6 49 230 4.7 7 53 180 3.4 8 and 9 56 127 2.3 10 and 11 57 184 3.2 12 44 117 2.7
1-12t 2447 562 4.4 * To quantitatively assay protein phosphorylation, we analyzed the results in Fig. 1B, lanes a and c, by scanning denslitometry. The peaks from the densitometer tracings for the 12 enhanced phosphoprotein bands (arrows, Fig. 1B) were then cut out and weighed. The weight of each peak was taken to be proportional to the amount of protein phosphorylation and is shown in the table. t Ratio of the weights of the peaks of the transformed cells to those of the untransformed cells. Total weights of the peaks for all 12 proteins.
duced protein phosphorylation in the transformed cells to the level seen in normal cells (data not shown). These studies further suggest that enhanced phosphorylation in the transformed cells is due to either increased protein kinase activity or to a new ZnCl2-sensitive protein kinase and not to an active diffusible inhibitor in the normal cells.
Proc. Natl. Acad. Sci. USA 76 (1979)
Protein Phosphorylation at Different Stages of Confluency. In the experiments thus far described, the normal and transformed cells were compared after they had grown to 90-100% confluency. At this density, the contact-inhibited normal cells have a diminished growth rate whereas the noncontact-inhibited transformed cells maintain a rapid growth rate. To verify that the enhanced protein phosphorylation observed in the tumor cells was an intrinsic property of the transformed cell and not merely related to growth rate, we performed experiments with normal and transformed cells at different stages of confluency. Protein phosphorylation in normal and transformed cells was assessed by autoradiography (Fig. 3) and by a direct quantitative assay involving liquid scintillation measurement of radioactivity (Table 2). At 25, 50, and 75% confluency, with both types of phosphorylation methods, tumor cells showed enhanced protein phosphorylation when compared to normal cells. This was at a time when the normal cells were actually growing faster than the tumor cells. At confluence, the contact-inhibited normal cells had a diminished growth rate whereas the noncontact-inhibited transformed cells maintained a rapid growth rate. Thus, the dramatic enhancement of protein phosphorylation seen in transformed cells when compared to normal cells at all stages of confluency does not appear to be merely a function of growth rate. However, growth rate or cell density could have important effects on protein phosphorylation; at confluence, the normal cells may show a slight decrease in phosphorylation (apparent
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FIG. 2. Mixing experiments. Autoradiogram from an in vitro protein phosphorylation assay in which both untransformed and transformed cell homogenates were mixed. Arrow, 68,000 Mr standard, bovine serum albumin. Lanes a and b, phosphorylation patterns of S-238 and SV40-1, normal and transformed cells, respectively; lane c, results of mixing the S-238 and SV40-1 homogenates prior to the in vitro phosphorylation assay.
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these. glarshw. Aro,6,00M tandrd boieseu l a b c d e f f a b c d 000 e Noma hua firbatsrinS29Rgt bui. 3.(Left FIG. Protein at different of phosphorylation stages confluency. Monolayers of normal and SV40-transformed human fibroblasts at different stages of confluency were homogenized and subjected to in vitro protein phosphorylation. The reaction was terminated with NaDodSO4 containing buffer; 25 ,sg of cell homogenate protein was then subjected to electrophoresis on NaDodSO4/polyacrylamide gradient slab gels (6-15%). The completed gels were stained with Coomassie brilliant blue, destained, and dried. Autoradiograms of
transformed line SV40-1. Lanes a, b, c, and d, dishes that were 25, 50, 75, and 100% confluent, respectively. Lanes e and f, dishes that were maintained in culture for 3 and 12 days, respectively, after confluence.
Cell Biology: Epstein et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
Table 2. In vitro protein phosphorylation in normal (S-239) and transformed (SV40-1) cells at different stages of conflency* v pmol P04/mg Cells/disht Days X 10-6 protein in culture Confluency,t % S239 SV40-1 S239 SV40-1 S239 SV40-1 649 2 518 2 0.64 1.2 2.8 919 6.2 530 5 9 3.1 572 1063 7.4 6 13 50 560 1815 7 6.6 23 * Monolayers of normal and SV40-transformed human fibroblasts at different stages of confluency were homogenized and subject to in vitro protein phosphorylation. The reaction was terminated with trichloroacetic acid; the precipitate was collected on a filter which was washed and dried; and radioactivity was measured. The results are expressed as pmol of acid-precipitable phosphate per mg of cell homogenate protein. t Confluency was assessed visually by scanning the culture dishes with a phase-contrast microscope. Cells per dish was determined by treating a replicate petri dish with trypsin and performing a cell count on a Coulter Counter.
25 50 75 100
by autoradiography but not by quantitative assay) whereas the transformed cells show an increase in phosphorylation. Protein Phosphorylation in Intact Cells. Because it was possible that a unique feature of the in vitro phosphorylation assay could have artificially enhanced protein phosphorylat: on in the transformed cells, we decided to repeat the experiments by using a more physiological, intact-cell assay method. As with the first method, the stained protein patterns of the normal human diploid fibroblast strains were similar to those of the
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shown. Arrows indicate the 200,000, 68,000, and 44,000 Mr standards, myosin, bovine serum albumin, and actin, respectively. Lanes a and b, intact cell phosphorylation pattern for the untransformed strain S-238. Lanes c and d, pattern for the transformed cell line SV40-1. Results shown in lanes b and d were obtained with cultures incubated with 1 ,uM isoproterenol and 1 mM theophylline
during the last 15 min of exposure Na2H32PO4.
to
6399
SV40-transformed human fibroblast lines. However, the autoradiograms showed greatly enhanced protein phosphorylation in SV40-1 cells as compared to S-238 (normal) cells (Fig. 4). Thus, the use of intact cells for measurement of phosphorylation confirms the results obtained with cell homogenates. The actual protein bands phosphorylated in intact cells differ somewhat from those phosphorylated in experiments using cell homogenates. This may be due to the fact that the protein kinase(s) involved appears to be membrane bound and that in intact cells it may have access only to certain proteins and not to others. DISCUSSION The results presented demonstrate enhanced phosphorylation of at least 25-30 endogenous cellular proteins in three SV40transformed human fibroblast cell lines when compared with several different untransformed human fibroblast strains. These results were obtained by two different methods, and it was apparent that the enhanced protein phosphorylation was not merely a function of the growth rate of the transformed cell lines, nor was it dependent on cAMP. Mixing of homogenates of transformed and normal cells prior to incubation with [y32P]ATP resulted in a phosphoprotein pattern similar to that of the transformed cells alone. These data suggest that enhanced protein phosphorylation ability observed in the transformed cell lines could perhaps be due to increased protein kinase activity or to a new protein kinase activity. SV40 is a small DNA virus that codes for about 200,000 daltons of protein. Three distinct gene products have been identified, only one of which, that of gene A, is correlated with an early function that appears to be required for viral DNA synthesis. Gene A has been shown to code for the SV40 T antigen, a 100,000-dalton protein that is believed to initiate viral DNA synthesis, and is also required for the transformation phenotype (7), although the mechanism by which the T antigen performs these functions is not clear. It is possible that the T antigen may be responsible, directly or indirectly, for the enhanced protein phosphorylation seen in our experiments with SV4O-transformed human fibroblasts. The T antigen may either possess protein kinase activity itself or stimulate an endogenous cellular protein kinase activity (or both). Recently, Tjian and Robbins (8) showed that protein kinase and ATPase activities copurify with a protein that is antigenically related to the T antigen. Furthermore, protein kinase activity has also been associated with the transforming proteins found in other systems. For example, the src gene of the avian sarcoma virus, an RNA tumor virus, has been shown by workers in two different laboratories to code for a protein of Mr 60,000 that exhibits protein kinase activity (9, 10). Protein kinase activity has also been associated with the transforming proteins of the DNA virus, adenovirus type 5 (11). These recent reports, as well as the data presented in the current paper, suggest that protein phosphorylation may be an important mechanism intimately involved in the events through which several different tumor viruses induce cellular transformation. It is important to emphasize that the overall pattern of enhanced protein phosphorylation observed with SV40-transformed cells in the experiments reported here was not cAMP dependent. cAMP-dependent processes have been implicated in the arrest of cell growth and reversion of some transformed cells to a more normal phenotype (12, 13). Cell cAMP-dependent effects are thought to be mediated by a protein kinase (14), and variants with mutations in a cAMP-dependent protein kinase have been isolated that resist the growth inhibitory effect of cAMP (15). In addition, changes detected in the two principal types of soluble cAMP-dependent protein kinases (16) have also been related to growth regulation and cellular transformation. Activation of type II cAMP-dependent protein kinase has been
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Cell Biology: Epstein et al.
demonstrated as CHO cells traverse G1, and de novo synthesis of this enzyme occurs at the G1/S border (17, 18). In addition, alterations in type I cAMP-dependent protein kinase have been associated with mitogenesis, neoplastic cell lines, or tumors, whereas hormone-dependent mammary tumor regression has been associated with an increase in type II cAMP-dependent protein kinase (19-22). Moreover, SV40 transformation of 3T3 cells is associated with altered growth properties of the cells and the appearance of a type I cAMP-dependent protein kinase (23). The regulation of cell growth has also been associated with alterations in cAMP-dependent protein phosphorylation. Induction of the quiescent state in BHK cells resulted in the phosphorylation of five cytosolic proteins, four of them only in the presence of cAMP (24). In the present experiments, the effects of cAMP on protein phosphorylation were minor regardless of whether the cell homogenates were treated in vitro with cAMP or the intact cells were treated in vivo to stimulate endogenous cAMP levels. These results further suggest that the phenomenon reported here of enhanced phosphorylation of many endogenous cellular proteins observed in SV40-transformed human fibroblasts is independent of cAMP. However, the results reported here do not, of course, preclude a role for cAMP in important processes related to growth control or expression of tumorgenicity in SV40-transformed human fibroblasts. Studies of protein substrates for enhanced phosphorylation in SV40-transformed cells have, to our knowledge, been reported twice previously. Segawa et al. (25) have observed a highly phosphorylated protein of approximately 90,000 daltons detected in both chromatin and ribosomes of SV40-transformed rodent cells; this protein was either not present or was present only in very small amounts in the organelles of untransformed cells. They suggested that the expression of this protein was regulated by the SV40 T antigen because the amount of phosphorylation of this protein in cells transformed with a temperature-sensitive mutant of SV40 was greatly decreased when the cells were cultured at the restrictive temperature (25). Segawa et al. (26) have also observed this highly phosphorylated, 90,000-dalton protein in cells transformed by adenovirus, murine sarcoma virus, or methylcholanthrene. They therefore proposed that the phosphorylation of this protein is closely related to the appearance of the transformed state in these cells. In our studies, we have detected a highly phosphorylated protein band of Mr approximately 90,000 in the SV40-transformed human fibroblasts. However, we also see phosphorylation of the same protein band in rapidly growing untransformed fibroblasts studied at subconfluence (Fig. 3). When these cells were confluent, the degree of phosphorylation of this protein species decreased (Fig. 3), suggesting that this phenomenon is related to growth rate rather than transformation. However, a closer examination of the autoradiographic patterns presented by Segawa et al. (25, 26) reveals that there is also an enhanced phosphorylation of many other endogenous cellular proteins in the transformed cells they have studied when the patterns were compared to those obtained with untransformed cells. Our data agree with this observation, but more importantly, also demomstrate that the degree of enhanced phosphorylation observed for many of these other endogenous protein substrates in the transformed cells is not merely related to the growth rate of the cells. A second study showing enhanced phosphorylation of proteins in SV40-transformed cells was done by Pumo et al. (27). These investigators showed a 10-fold enhancement of phosphorylation of nonhistone chromosomal proteins in SV40transformed when compared with normal human diploid fibroblasts. These studies showed that the increased phosphorylation in the transformed cells could not be accounted for
Proc. Natl. Acad. Sci. USA 76 (1979)
by either an increased rate of phosphate transport or the synthesis of new species of nonhistone chromosomal proteins which subsequently become phosphorylated. The exact identity and physiological functions of the proteins that exhibited enhanced phosphorylation in the transformed cells used in our experiments remain to be determined. However, because the biological role of many different proteins can be changed by phosphorylation, alterations in the degree of phosphorylation that occur after viral transformation may be part of the cell's pleiotropic response coordinating the many functional changes observed in the transformation from a normal to a malignant phenotype (1, 2). Note Added in Proof. After this manuscript was submitted for review, Griffin et al. (28) reported that the large T antigen of SV40 exhibits protein kinase acitivity. This work was supported by U. S. Public Health Service Grants HL15895 and AM20010 and by grants from The National Foundation-March of Dimes, and the Medical Foundation, Inc., Boston, MA. 1. Tooze, J., ed. (1973) The Molecular Biology of Tumor Viruses (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). 2. Greengard, P. (1978) Science 199, 146-151. 3. Epstein, J. & Breslow, J. L. (1977) Proc. Natl. Acad. Sci. USA 74, 1676-1679. 4. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 5. DeLorenzo, R. J., Walton, K. G., Curran, P. F. & Greengard, P. (1973) Proc. Natl. Acad. Sci. USA 70,880-884. 6. Laemmli, U. K. (1970) Nature (London) 227,680-685. 7. Fried, M. & Griffin, B. E. (1977) Adv. Cancer Res. 24, 67113. 8. Tjian, R. & Robbins, A. (1979) Proc. Natl. Acad. Sci. USA 76, 610-614. 9. Collett, M. S. & Erikson, R. L. (1978) Proc. Natl. Acad. Sci. USA 75,2021-2024. 10. Levinson, A. D., Oppermann, H., Levintow, L., Varmus, H. E. & Bishop, J. M. (1978) Cell 15,561-572. 11. Lassan, N. J., Bayley, S. T., Graham, F. L. & Branton, P. E. (1979) Nature (London) 277,241-243. 12. Pastan, I. H., Johnson, G. S. & Anderson, W. B. (1975) Annu. Rev. Biochem. 44, 491-511. 13. Pastan, I. H. & Willingham, M. (1978) Nature (London) 274, 645-650. 14. Kuo, J. F. & Greengard, P. (1969) Proc. Natl. Acad. Sci. USA 64, 1349-1353. 15. Insel, P. A., Bourne, H. R., Coffino, P. & Tomkins, G. M. (1975) Science 190, 896-898. 16. Rubin, C. S. & Rosen, 0. M. (1975) Annu. Rev. Biochem. 44, 831-887. 17. Costa, M., Gerner, E. W. & Russell, D. H. (1976) Biochim. Biophys. Acta 425, 246-255. 18. Costa, M., Gerner, E. W. & Russell, D. H. (1976) J. Biol. Chem. 251,3313-3319. 19. Byus, C. V., Klimpel, G. R., Lucas, D. 0. & Russell, D. H. (1977) Nature (London) 268,63-64. 20. Gutomann, N. S., Rae, P. A. & Schimmer, B. P. (1978) J. Cell. Physiol. 97, 451-460. 21. Fossberg, T. M., Doskeland, S. D. & Ueland, P. M. (1978) Arch. Biochem. Biophys. 189,372-381. 22. Cho-Chung, Y. S., Clair, J. & Zubialde, J. P. (1978) Biochem. Biophys. Res. Commun. 85, 1150-1155. 23. Glarrett, A. J., Malkinson, A. M. & Sheppard, J. R. (1976) Nature (London) 264, 673-675. 24. Kletzien, R. F., Milles, M. R. & Pardee, A. B. (1977) Nature (London) 270,57-59. 25. Segawa, K., Yamaguchi, N. & Oda, K. (1977) J. Virol. 22, 679-693. 26. Segawa, K., Oda, K., Yuasa, Y., Shiroki, K. & Shimojo, H. (1978) J. Virol. 27,800-808. 27. Pumo, D. E., Stein, G. S. & Kleinsmith, L. J. (1975) Biochim. Biophys. Acta 402,125-130. 28. Griffin, J. D., Spangler, G. & Livingston, D. M. (1979) Proc. Natl. Acad. Sci. USA 76,2610-2614.
