Constant Expression and Activity of Protein Phosphatase 2A in ... - NCBI

MOLECULAR AND CELLULAR BIOLOGY, Aug. 1991, p. 4282-4285

Vol. 11, No. 8

0270-7306/91/084282-04$02.00/0 Copyright © 1991, American Society for Microbiology

Constant Expression and Activity of Protein Phosphatase 2A in Synchronized Cells RALF RUEDIGER,' JILL E. VAN WART HOOD,2 MARC MUMBY,2 AND GERNOT WALTER'* Department of Pathology, University of California, San Diego, La Jolla, California 92093-0612,1 and Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235 90412 Received 26 February 1991/Accepted 8 May 1991

The levels of the A, B, and C subunits of protein phosphatase 2A in extracts from synchronized embryonic bovine tracheal cells were determined by immunoblotting with subunit-specific antibodies. A constant amount of each subunit was found in resting cells as well as in growing cells from all stages of the cell cycle. The phosphatase activity of protein phosphatase 2A was also constant. A quantitative comparison showed that the A and C subunits were present in similar amounts, whereas the B subunit was present at a significantly lower level. Together, the A, B, and C subunits represented approximately 0.2% of the total cellular protein. Control of the cell cycle in eukaryotes is achieved by the complex interplay of protein kinases and phosphatases (2, 17-19, 25). Protein phosphatase 2A (PP2A), a serine/threonine-specific phosphatase, has been implicated in specific mitotic events in yeast cells (11) and amphibian oocytes (14). It is composed of a 37-kDa catalytic subunit (termed C) and two regulatory subunits of 65 and 55 kDa, termed A and B, respectively (for a review, see reference 3). The catalytic subunit is highly conserved between species and is a member of a gene family which includes the catalytic subunits of type 1 and 2B protein phosphatases (4). The A subunit, which is also highly conserved, has an unusual structure consisting of 15 nonidentical repeats that are 38 to 40 amino acids in length (10, 28). A key substrate of PP2A in oocytes is maturationpromoting factor (MPF) (14), a protein kinase with a catalytic subunit homologous to p34cd"2 of fission yeast (1, 6, 7, 13, 15) and a regulatory subunit called cyclin (5). PP2A removes phosphate from a specific site of the MPF catalytic subunit that correlates with loss of its enzymatic and maturation-promoting activity. PP2A also plays a role in neoplastic transformation by the small DNA tumor viruses polyomavirus and simian virus 40 (SV40). It forms specific complexes with the polyomavirus medium-sized tumor antigen (medium T) (8, 9, 12, 20, 21, 29) and small tumor antigen (small T) (23) and with SV40 small T (21, 22, 27, 31). Genetic experiments suggest that the complex formation is important for transformation (8). We have recently demonstrated that SV40 small T inhibits the dephosphorylation of various substrates, including SV40 large T and the growth suppressor protein p53, by binding to the A subunit of PP2A (24, 30). This activity of small T might explain its growth-promoting effect, as in the case of the tumor-promoting effect of okadaic acid, which also inhibits PP2A (for a review, see reference 4). Since PP2A plays an important role in growth control, the questions of whether and how it is regulated itself arise. In the present communication, we report on the expression and activity of PP2A during the mitotic cell cycle. Embryonic bovine tracheal (EBTr) cells were synchronized by starvation in medium containing 0.2% fetal calf serum (FCS). After release from growth arrest with medium containing 10% FCS, the cells were harvested every 3 h over a total period *

of 36 h. The amounts of the A, B, and C subunits were determined by immunoblots with subunit-specific antibodies. EBTr cells were seeded at 5 x 105 per 10-cm dish and grown for 12 h in Dulbecco's modified Eagle's medium supplemented with 10% FCS. The medium was then replaced with medium containing 0.2% FCS. After 48 h, the cells were arrested as determined by flow cytofluorometry. Medium with 10% FCS was added to the cells at time zero. Cells were harvested every 3 h by washing with 50 mM Tris, pH 7.5, containing 150 mM NaCl and by lysis in 500 ,ul of 50 mM Tris, pH 7.5, containing 0.5% Triton X-100 for 10 min on ice. Lysates were centrifuged at 12,000 x g for 10 min at 4°C. One aliquot of the supernatant was used for protein determination with the bicinchoninic acid assay (Pierce). Another part was adjusted to 0.5 mM EDTA-0.05 mM leupeptin-1 mM dithiothreitol-20% glycerol and frozen in aliquots at -70°C for later phosphatase assays. Another aliquot was prepared for electrophoresis by the addition of one volume of 2 x concentrated sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (100 mM Tris, pH 6.8-4% SDS-10% 2-mercaptoethanol-40 mM dithiothreitol-10% glycerol-0.01% bromophenol blue). Protein (8 p.g) from each sample and 0.05 ,ug of purified PP2A1 (ABC form) were loaded on an SDS-10% polyacrylamide gel. After transfer of the proteins to nitrocellulose paper (Hybond ECL; Amersham), the subunits were visualized with a mouse anti-bovine C subunit monoclonal antibody at 0.02 ,ug/ml and with a rabbit anti-human A subunit antiserum (immunoglobulin G fraction) at a 1:500 dilution in Trisbuffered saline containing 0.2% Tween 20. The ECL developing system was applied. Figure 1 demonstrates that the amounts of the A and C subunits were constant throughout the entire period. This includes the GJGl, S, G2, and M phases as determined by flow cytofluorometry (see Fig. 3A). The amounts of the B subunit in resting and growing EBTr cells were also constant in all phases of the cell cycle as shown in Fig. 2. Cells from duplicate plates of the experiment described above were lysed at room temperature with 100 [l of 50 mM Tris, pH 6.8-2% SDS-5% glycerol. The lysates were sheared and centrifuged at 12,000 x g for 10 min. Aliquots of the supernatants were used for protein assays. A total of 100 [lI was adjusted to 5% mercaptoethanol-1 mM dithiothreitol-3 ,ul of saturated bromophenol blue, boiled for 5 min, and frozen at -70°C. Protein (24 ,ug) from

Corresponding author. 4282

VOL. 11, 1991

NOTES

4283

EBTr 0

1

0

3

6

9

12

15

18

21

24

27

30

33

36

48

51

-"mm"-

PP2A

-

FIG. 1. Expression of the A and C subunits of PP2A in synchronized EBTr cells. Cells were cultured and lysates were subjected to SDS-PAGE as described in the text. The ECL developing system was used, and the exposure time was 5 min. Numbers at top indicate hours after release from growth arrest.

each sample and 0.08 ,ug of PP2A1 holoenzyme as a control were loaded. After transfer of the proteins to nitrocellulose, the B subunit was visualized as described above, with a rabbit anti-bovine B subunit antiserum (immunoglobulin G fraction) at a 1:50 dilution in Tris-buffered saline containing 0.2% Tween 20, 3% nonfat dry milk, and 3% bovine serum albumin Cohn fraction V. Since the antiserum against the B subunit was weaker than those against the A and C subunits and since it contained antibodies against proteins unrelated to the B subunit, the background of nonspecific proteins in the immunoblots was high. As controls, immunoblots were carried out with preimmune serum and immune serum in the presence of purified PP2A holoenzyme consisting of all three subunits (Fig. 2, lanes 1 through 6). In all immunoblots, the B subunit appears as a doublet that is not recognized by the preimmune serum and that is specifically blocked by the addition of purified PP2A. Synchronization experiments were also carried out with mouse fibroblasts (10T1/2) and polyomavirus medium T- and small T-transformed 10T1/2 cells. In all cases, the amounts of the A and C subunits were constant throughout the cell cycle (data not shown). Since the antiserum against the B subunit, which was raised against purified bovine B subunit, did not recognize the

corresponding mouse protein, the cell cycle expression of the B subunit in 1OT1/2 cells could not be determined. Constant expression of the PP2A subunits was found whether cytoplasmic extracts or whole-cell lysates were used for the immunoblots. We estimated that the bulk of the enzyme (approximately 80%) was contained in the cytoplasmic fraction. To determine the activity of PP2A in synchronized EBTr cells, aliquots from the extracts which were analyzed by immunoblotting (Fig. 1) were used for measuring phosphatase activity. As shown in Fig. 3, the activity was constant during the cell cycle. Phosphatase assays were carried out in the absence (Fig. 3B, upper curve) and presence (lower curve) of 5 nM okadaic acid with myosin light chain as the substrate. This concentration of okadaic acid inhibited over 95% of the PP2A activity and had little effect on protein phosphatase 1. This result demonstrates that under the conditions used, approximately 70% of the total cytoplasmic phosphatase activity is derived from PP2A. Different forms of PP2A have been isolated, the most common one being composed of equimolar amounts of the A, B, and C subunits. A two-subunit form consisting of A and C has also been identified (for a review, see reference 3).

m

< CIA

EBTr

m_

m

CMC4

0. a.0 * CL so a~~~~~~~~~~~~~~~~~~. a.

CL

0

0

1

3

6

9

12

15

182124

27303333644851

EL

aurn.

-

4i

-

"Mtt,'

"~

-

rn

-

.-

-

4

--A

*

,

=

h.

6

Qc

cm 0. a.

m_~r Ct'CM E fUL

0.. ILE

4m

_

=

-

1 2 3 4 5 6 FIG. 2. Expression of the B subunit of PP2A in synchronized EBTr cells. Cells were cultured and lysed as described in the text. Protein (24 ,ug) from each sample and PP2A1 holoenzyme (0.08 ,ug) as a control were loaded onto the gel (left panel). After transfer of the proteins to nitrocellulose, the B subunit was visualized as described in the text. The exposure time was 20 min. Lanes 1 through 3 represent 24 ,ug of protein of a nonsynchronized EBTr cell lysate immunoblotted with either preimmune serum at a 1:10 dilution (pre), anti-B subunit antiserum (1:50) (a-PP2AB), or anti-B subunit antiserum (1:50) in the presence of 20 p.g of SDS-dissociated PP2A1 holoenzyme (a-PP2AB + PP2A1). The exposure time was 2 h. Lanes 4 through 6 represent 0.08 ,ug of PP2A1 immunoblotted as described for lanes 1 through 3. The exposure time was 3 min.

P-

MOL. CELL. BIOL.

NOTES

4284

ioo-I A

0-

C') cv

co CO

50

C.)

0

I

30

~0

20

amco

15

a.

10

CD

coJ cm

.12 .08 .05 .03

8

16

I

PP2A c

E0. 25

EBTr

_

_

B

c

1-

PP2A1

o~~~~~~~~-a\-

I

I

PP2AB 1

5 0 0

.12 .08 .05 .03 20 40

6

12

18

24

30

Time after Release from

36

42

48

Go (h)

FIG. 3. Protein phosphatase activity of PP2A in synchronized EBTr cells. (B) Aliquots of the lysates described in the text were subjected to phosphatase activity assays with (V) or without (v) 5 nM okadaic acid. Phosphatase assays were carried out as described previously (16) with 2 ,ug of total cellular protein and 2 ,uM 32P-labeled myosin light chain as the substrate in a final volume of 50 pI. Incubations were for 10 min at 30°C. The samples were analyzed in triplicates. The values for each time point differed by less than 10%. (A) The percentages of cells in S phase (V) and M phase (O) were determined by flow cytofluorometry. For each time point, one plate was washed with 50 mM Tris (pH 7.5) containing 150 mM NaCl. The cells were trypsinized and adjusted to 50%o ethanol in phosphate-buffered saline.

Little is known about the molar ratios of the A, B, and C subunits within cells. By immunoblotting, we quantitated the different subunits in whole-cell lysates from bovine and mouse cells by using known quantities of holoenzyme for calibration. In order to obtain accurate values by this procedure, we loaded a range of holoenzyme amounts that covered the amount of PP2A in the unknown test sample. We found that in bovine and mouse cells, the A and C subunits were always present in similar concentrations. On the other hand, the B subunit was found at a significantly lower concentration than the A and C subunits in EBTr cells. As shown in Fig. 4, four concentrations of PP2A holoenzyme, ranging from 0.03 to 0.12 ,ug, were compared with two amounts of EBTr whole-cell lysate. By scanning the fluorograms, it was determined that 16 ,ug of EBTr protein contains 0.04 to 0.05 ,ug of the A subunit and 0.03 ,ug of the C subunit, whereas 40 jig of EBTr protein contains only 0.03 ,ug of the B subunit. This amounts to a molar ratio of approximately 1.5:1:0.3 for the A, C, and B subunits, respectively. Our findings might indicate that the AC form exists in cells in addition to the three-subunit enzyme. It could also suggest that the AC form interacts with different forms of the B subunit, only one of which is recognized by the antibodies used in our study, or that the AC form interacts with other unidentified subunits unrelated to the B subunit. It is unlikely that the lower concentration of the B subunit is the result of selective degradation, since the cells used for immunoblotting were lysed in PAGE sample buffer and boiled immediately. The combined amounts of the A, B, and C subunits constituted approximately 0.2% of the total

2

3

4

5

6

FIG. 4. Quantification of PP2A subunits in lysates of EBTr cells. Immunoblots with different amounts of purified PP2A1 (0.03 to 0.12 ,ug) were carried out with antibodies against the A, B, and C subunits and compared with immunoblots with different amounts of EBTr cell lysates prepared in SDS-PAGE sample buffer. Either 8 or 16 ,ug of lysate protein was used for the A and C subunits; 20 and 40 ,ug of lysate protein were used for the B subunit. The fluorograms were quantitated by densitometry. The same relative amounts of the A, B, and C subunits were found in lysates prepared with either SDS-PAGE sample buffer or Triton X-100 buffer. No degradation of PP2A in Triton X-100 extracts was observed.

cell protein. We also determined the amounts of the A, B, and C subunits in various bovine organs. In most organs, the ratios of the A and C subunits were close to 1, with the exception of brain white matter, which contained a large excess of the C subunit. Kidney and liver, on the other hand, contained somewhat more A than C subunit. The B subunit was generally found in smaller quantities than the A and C subunits, except in heart tissue, where it was present in a similar amount. We have demonstrated that the amounts of the three major subunits and the activity of PP2A remain constant during the cell cycle. These data are consistent both with the findings of Virshup et al. (26), who demonstrated that in HeLa cell extracts, the level of the PP2A C subunit is constant throughout G1, S, and G2, and with those of Kinoshita et al. (11), who showed that the ppal+ and ppa2+ gene products from fission yeast, which are homologous to the C subunit of mammalian PP2A, do not fluctuate during the cell cycle. How can these results be reconciled with the notion that PP2A plays a key role in regulating specific events at defined points of the cell cycle? To answer this question, one has to consider that the phosphatase assays were carried out with myosin light chain as a substrate. It is conceivable that the activity of PP2A could vary during the cell cycle if a more physiological substrate, such as MPF, were used. In fact, recent evidence indicates that the activity which dephosphorylates MPF is a subfraction of the total PP2A in Xenopus extracts (14). Our data do not exclude the possibility that the activity of a small subfraction of PP2A is regulated by modification, e.g., phosphorylation, and that this subfraction is involved in specific events of the cell cycle. However, at present there exists no evidence that any of the subunits of PP2A are modified. On the other hand, in order to control specific cell cycle events, it is not necessary that PP2A activity be regulated. If PP2A is equally active at all times, as suggested by our data, it would be sufficient for specific

VOL . 1 l, 1991

substrates like MPF or p53 to become available only during restricted time intervals of the cell cycle. This work was supported by Public Health Service grants CA21327 (to G.W.) and HL-31107 and HL-17669 (to M.M.). J.V.W.H. was supported by Public Health Service Training grant HL-0736012. R.R. was supported by a postdoctoral fellowship (300-402-529-9) from the Dr. Mildred Scheel Foundation for Cancer Research, Bonn, Germany. REFERENCES 1. Arion, D., L. Mejer, L. Brizuela, and D. Beach. 1988. cdc2 is a component of the M phase-specific histone Hi kinase: evidence for identity with MPF. Cell 55:371-378. 2. Booher, R., and D. Beach. 1989. Involvement of a type 1 protein phosphatase encoded by bws1+ in fission yeast mitotic control. Cell 57:1009-1016. 3. Cohen, P. 1989. The structure and regulation of protein phosphatases. Annu. Rev. Biochem. 58:453-508. 4. Cohen, P., and P. T. W. Cohen. 1989. Protein phosphatases come of age. J. Biol. Chem. 264:21435-21438. 5. Draetta, G., F. Luca, J. Westendorf, L. Brizuela, J. Ruderman, and D. Beach. 1989. cdc2 protein kinase is complexed with both cyclin A and B: evidence for proteolytic inactivation of MPF. Cell 56:829-838. 6. Dunphy, W. G., and J. W. Newport. 1988. Fission yeast p13 blocks mitotic activation and tyrosine dephosphorylation of the Xenopus cdc2 protein kinase. Cell 54:423-431. 7. Gautier, J., C. Norbury, N. Lohka, P. Nurse, and J. Maller. 1988. Purified maturation promoting factor contains the product of a Xenopus homolog of the fission yeast cell cycle control gene cdc2+. Cell 54:433-439. 8. Grussemmeyer, T., A. Carbone-Wiley, K. H. Scheidtmann, and G. Walter. 1987. Interactions between polyomavirus medium T antigen and three cellular proteins of 88, 61, and 37 kilodaltons. J. Virol. 61:3902-3909. 9. Grussenmeyer, T., K. H. Scheidtmann, W. Hutchinson, and G. Walter. 1985. Complexes of polyoma virus medium T antigen and cellular proteins. Proc. Natl. Acad. Sci. USA 82:7952-7954. 10. Hemmings, B. A., C. Adams-Pearson, F. Maurer, P. Muller, L. Goris, W. Merlevede, J. Hofsteenge, and S. R. Stone. 1990. Alpha and beta-forms of the 65-kDa subunit of protein phosphatase 2A have a similar 39 amino acid repeating structure.

Biochemistry 29:3166-3173. 11. Kinoshita, N., H. Ohkura, and M. Yanagida. 1990. Distinct, essential roles of type 1 and 2A protein phosphatases in the control of the fission yeast cell division cycle. Cell 63:405-415. 12. Koch, W., A. Carbone, and G. Walter. 1986. Purified polyoma virus medium T antigen has tyrosine-specific protein kinase activity but no significant phosphatidylinositol kinase activity. Mol. Cell. Biol. 6:1866-1874. 13. Labbe, J. C., M. Lee, P. Nurse, A. Picard, and M. Doree. 1988. Activation at M-phase of a protein kinase encoded by a starfish homolog of the cell cycle control gene cdc2+. Nature (London) 355:251-254. 14. Lee, T. H., M. J. Solomon, M. C. Mumby, and M. W. Kirschner. 1991. INH, a negative regulator of MPF, is a form of protein phosphatase 2A. Cell 64:415-423.

NOTES

4285

15. Lohka, M. J., M. K. Hayes, and J. L. Maller. 1988. Purification of maturation-promoting factor, an intracellular regulator of early mitotic events. Proc. Natl. Acad. Sci. USA 85:3009-3013. 16. Mumby, M. C., D. D. Green, and K. K. Russel. 1985. Structural characterization of cardiac protein phosphatase with a monoclonal antibody. J. Biol. Chem. 260:13763-13770. 17. Nurse, P. 1990. Universal control mechanism regulating onset of M-phase. Nature (London) 344:503-508. 18. Ohkura, H., N. Kinoshita, S. Miyatani, T. Toda, and M. Yanagida. 1989. The fission yeast dis2+ gene required for chromosome disjoining encodes one of two putative type 1 protein phosphatases. Cell 57:997-1007. 19. Ohkura, H., and M. Yanagida. 1991. S. pombe gene sds22+ essential for midmitotic transition encodes a leucine-rich repeat protein that positively modulates protein phosphatase-1. Cell 64:149-157. 20. Pallas, D. C., V. Cherington, W. Morgan, J. DeAnda, D. Kaplan, B. Schaffhausen, and T. M. Roberts. 1988. Cellular proteins that associate with the middle and small T antigens of polyomavirus. J. Virol. 62:3934-3940. 21. Pallas, D. C., L. K. Shahrik, B. L. Martin, S. Jaspers, T. B. Miller, D. L. Brautigan, and T. M. Roberts. 1990. Polyoma small and middle T antigens and SV40 small t antigen form stable complexes with protein phosphatase 2A. Cell 60:167-176. 22. Rundell, K. 1987. Complete interaction of cellular 56,000- and 32,000-Mr proteins with simian virus 40 small-t antigen in productively infected cells. J. Virol. 61:1240-1243. 23. Rundell, K., E. 0. Major, and M. Lampert. 1981. Association of cellular 56,000- and 32,000-molecular-weight proteins with BK virus and polyoma virus t-antigens. J. Virol. 37:1090-1093. 24. Scheidtmann, K. H., M. C. Mumby, K. Rundell, and G. Walter. 1991. Dephosphorylation of simian virus 40 large-T antigen and p53 protein by protein phosphatase 2A: inhibition by small-t antigen. Mol. Cell. Biol. 11:1996-2003. 25. Solomon, M. J., M. Glotzer, T. H. Lee, M. Philippe, and M. W. Kirschner. 1990. Cyclin activation of p34cdc2. Cell 63:1013-1024. 26. Virshup, D. M., M. G. Kauffman, and T. J. Kelly. 1989. Activation of SV40 DNA replication in vitro by cellular protein phosphatase 2A. EMBO J. 8:3891-3898. 27. Walter, G., A. Carbone-Wiley, B. Joshi, and K. Rundell. 1988. Homologous cellular proteins associated with simian virus 40 small T antigen and polyomavirus medium T antigen. J. Virol. 62:4760-4762. 28. Walter, G., F. Ferre, 0. Espiritu, and A. Carbone-Wiley. 1989. Molecular cloning and sequence of cDNA encoding polyoma medium tumor antigen-associated 61-kDa protein. Proc. Natl. Acad. Sci. USA 86:8669-8672. 29. Walter, G., R. Ruediger, C. Slaughter, and M. Mumby. 1990. Association of protein phosphatase 2A with polyoma virus medium tumor antigen. Proc. Natl. Acad. Sci. USA 87:25212525. 30. Yang, S.-I., R. L. Lickteig, R. Estes, K. Rundell, G. Walter, and M. C. Mumby. 1991. Control of protein phosphatase 2A by simian virus 40 small-t antigen. Mol. Cell. Biol. 11:1988-1995. 31. Yang, Y.-C., P. Hearing, and K. Rundell. 1979. Cellular proteins associated with simian virus 40 early gene products in newly infected cells. J. Virol. 32:147-154.