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in proliferation (8) and oncogenic transformation (reviewed in (9)) and is itself a target for the cell cycle kinase cdc2 (10). The kinase is found in the nucleus or the cytoplasm depending on the growth state of the cell (11,12). MATERIALS AND METHODS
Cells and tissue culture. The cell line M-ABA has been described elsewhere (13). The cells were maintained in RPMI 1640 medium as described (7). M-ABA cells contain a complete EBV genome (subtype A). Radioactive labeling, cell extraction and immunoprecipitation. H32po4 (32pi) and [q,_32p] ATP were purchased from New England Nuclear or from Amersham. Radioactive labeling of proteins with 32pi in vivo, cell extraction, immunoprecipitation of EBNA-2 from cell extract and dephosphorylation with potato acid phosphatase was caried out as described (7).
In vitro phosphorylation. The purification of CK-1 from yeast and of recombinant CK-2 has been described (14-16). Kinase reactions using untreated or dephosphorylated EBNA-2 were carried out for 30-45 rain at 37oC in kinase buffer composed of 20 mM 2-[N-morpholino] ethanesulfonic acid (MES), pH 6.9, 130 mM KC1, 10 mM MgCI 2, 3 mM dithioerythritol (DTE), and 50/~M ATP. In a typical asssay, EBNA-2 from 3 x 107 cells bound to 25/zl of protein A Sepharose was suspended in 25 /~1 reaction mix that contained 1 /~1 CK-1 or 1 /zl of recombinant CK-2 (25 or 37 ~M) and 0.1-100/zCi ['7-32p] ATP. The activity of the enzymes was confirmed by phosphorylation of casein (1% w/v). CK-1 from yeast was a generous gift of Dr. N. Grankowski, Lublin. In some cases, the immunoprecipitated EBNA-2 was dephosphorylated with potato acid phosphatase (Boehringer, Mannheim) at pH 5.5 (7). The dephosphorylated EBNA-2 was then treated with CK,1 or CK-2 as described above. The final products were analyzed by SDS-PAGE, immunoblotting and autoradiography. The phosphoamino acid content of EBNA-2 phosphorylated with CK-2 was determined as described (7). Partial digestion with trypsin or V8 protease. For partial tryptic digestion, in vivo or in vitro
labeled EBNA-2 was purified further by a second round of immunoprecipitation as described previously (7). The digest with TPCK-treated trypsin (Worthington) was carried out in low salt lysis buffer for 30 rain at 0oC at a trypsin concentration of 30/~g/ml in a total volume of 50 >1 (17). V8-digestion (Boehringer, Mannheim) was carried out as described (18). Expression of the N-terminal 56 amino acids of EBNA-2 as a bacterial trpE-fusion protein. Recombinant DNA work was carried out according to published procedures (19). The 58 EBNA-2 N-terminal amino acids of EBNA-2 were expressed as a trpE fusion protein using the tryptophan-inducible vector pATH (20). The DNA encoding these 58 EBNA-2-specific amino acids was amplified by PCR from a cosmid clone containing the M-ABA EBNA-2 gene (21). The N-terminal fusion protein was induced in parallel with the C-terminal expression construct g88-1 (22) encoding the C-terminal 140 amino acids of EBNA-2 and the pATH 11 vector as a control in the E.coli strain BL21/DE3 (23). Cell extract was prepared by lysing the bacteria in buffer containing 50 mM Tris-HC1, pH 7.8, 300 mM NaCI, 2 mM EDTA, 2 mM dithiothreitol (DTT), 10% glycerol, 0.01 Fzg/ml aprotinin (Boehringer, Mannheim), 1 mM phenyl-methylsulfonyl-fluoride (PMSF) and 1 mg/ml lysozyme. After centrifugation, the supernatant containing the trpE proteins was dialyzed extensively against kinase buffer (above) without ATP. Phosphorylation of a synthetic peptide with CK-2. The synthetic peptide FETTESPSSDEDY-
(amide), "FY", MPS, San Diego, Ca, USA) corresponds to amino acids 462-474 of EBNA-2. The peptide was dissolved to a final concentration of 0.25 mg/ml in a final volume of 10 ~1 kinase buffer and the reaction was started by the addition of 1-2/~Ci [../_32p] ATP and 1/11 of CK-2 (25 ~M). 1 /~1 of the reaction mixture was spotted onto a cellulose thin-layer chromatography plate (Merck) and electrophoresed for 20 rain at 1500 V in pH 1.9 buffer and visualized by autoradiography (17). The molar ratio of phosphate incorporation into peptide FY was determined essentially as described (24). 1695
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RESULTS AND DISCUSSION EBNA-2 is phosphorylated in vivo at the C-terminus. To localize the in vivo phosphorylation sites, E B N A - 2 was immunoprecipitated with an antiserum directed against its C-terminus and subjected to a partial tryptic digestion as described (17).
The release of phosphorylated
fragments during the digestion indicates that phosphorylation site(s) were located towards the N-terminus, while fragments that remained bound to the antibody-resin should be derived from C-terminal phosphorylation site(s).
Figures la and l b display the result of such an
experiment. The material shown in Figure la was separated on a 15% SDS-gel, the material displayed in Figure l b was separated on a 7.5-15% linear SDS-gradient gel. Undigested E B N A - 2 is shown in lane 1; lanes 2 and 3 represent the fragments that were retained and released during the digestion, respectively.
In addition to some undigested E B N A - 2 and a
minor band with a Mr of 62,000, two major fragments remained b o u n d to the antibody resin, indicating that a major phosphorylation site(s) is located at the C-terminus of the protein. In the digest shown in Figure la, lane 2, we observed fragments with a Mr of approximately
in vivo
CK-2
in vivo
1.PAP
b 1
2
3 1| 4
5
6
2.CK-2 1
2
3[
4
5
200 92.5 68 46
30
14.3
Fig. 1.(a). Partial tryptic digestion of EBNA-2 labeled metabolically and in vitro with casein kinase 2 (CK-2). EBNA-2 was immunopurified from M-ABA cells labeled in vivo with 32pi and subjected to partial tryptic digestion. The digestion products were separated on a 15% SDS-potyacrylamidc gel (SDS-PAGE) and visualized by autoradiography. Untreated material is shown in lane 1, material that remained bound is shown in lane 2, fragments released are shown in lane 3. The same experiment was carried out with EBNA-2 labeled in vitro with CK-2. The undigested EBNA-2 is shown in lane 4, bound and released fragments were separated in lanes 5 and 6, respectively. (b). Partial tryptic digestion of EBNA-2 labeled in vivo (lanes 1-3) and of EBNA-2 dephosphorylated with potato acid phosphatase prior to phosphorylation with CK-2 (lanes 4-6). The samples were separated by 7.5-15% SDS-PAGE. A partial tryptic digest of in vivo labeled EBNA-2 carried out in parallel is shown in lanes 1-3, in vitro .phosphorylated material is displayed in lanes 4-6. Untreated material is shown m lanes 1 and 4, material that remained bound is shown in lanes 2 and 5, fragments released are shown in lane 3 and 6. a4C-methylated molecular mass markers were from Amersham (x10-3). 1696
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15,000 and 31,000. In the digest shown in Figure lb, lane 2, we detected fragments with a Mr of approximately 13,000 and 26,000. The difference in mobilities are either the result of the acrylamide concentration of the gels or may be due to a further digestion of the material in Figure lb. The signal observed at 26,000 (lanes 2) appeared to consist of two or possibly three bands. The material released during the reaction contained several fragments. The material in Figure la, lane 3, shows some undigested EBNA-2 and a band migrating at approximately 29,000 indicated by an arrow. The released material shown in Figure lb, lane 3, contained two fragments that migrated with the same mobilities as the ones retained during the digest and two additional bands at a Mr of approximately 16,000 and 20,000.
The largest fragment
released during the reaction is indicated with an arrow. The fragments with identical mobilities might have been released to the supernatant due to digestion of the antibody. The origin of the two additional fragments could not be determined. We conclude from the intensity of the various bands in the bound and the released fractions that the C-terminus contains one of the major phosphorylation site(s) of EBNA-2, with additional site(s) not yet identified. Due to the low amount of radioactive label present we did not determine the phosphoamino acid composition of the various fragments. In vitro phosphorylation of EBNA-2 with recombinant CK-2.
Ser457 in the sequence
AspGluSer457TrpAsp and Ser469 and Ser470 in the sequence Ser469SerAspGluAsp of EBNA-2 represent possible phosphorylation sites for the CK-1 and CK-2, respectively (25,26) EBNA-2 was purified by immunoprecipitation
from M-ABA
cells, not treated
or
dephosphorylated with potato acid phosphatase (7) and then incubated with recombinant CK2 (14,15). The CK-2 transfered phosphate efficiently to either untreated or dephosphorylated EBNA-2 as demonstrated in Figure la, lane 4 and Figure lb, lane 4, respectively. We found that only serine residues were phosphorylated by this enzyme (data not shown). Partial tryptic digest of EBNA-2 phosphorylated by CK-2 in vitro.
To localize the site
phosphorylated by CK-2, the in vitro labeled EBNA-2 was subjected to partial tryptic digest as outlined above. As can be seen in Figure la and lb, lanes 5, the in vitro labeled material remained bound to the resin during the digestion indicating that only C-terminal serines were modified.
The peptides that remained bound during the digest of EBNA-2 that was not
dephosphorylated prior to the kinase reaction migrated with the same mobility as the corresponding peptides from the in vivo labeled material. 1697
In contrast, the peptides
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Fig. 2. V8 mapping of EBNA-2 labeled with 32Pi in vivo and in vitro using CK-2. EBNA-2 was immunopurified from M-ABA cells labeled in vivo with 32pi. In parallel, EBNA-2 immunoprecipitated from unlabeled cells was phosphorylated with CK-2. The reaction products were separated on a 7.5% SDS-gel, EBNA-2 was visualized by autoradiography excised and reapplied to a 9.5-15% linear gradient gel for VS-digestion. Lanes 1-5, EBNA-2 labeled in vitro by CK-2. Lanes 6-10:EBNA-2 labeled in vivo. Lanes 1 and 6, no enzyme, lane 2 and 7, 0.05 /~g protease, lanes 3 and 8, 0.1 /~g protease, lanes 4 and 9, 1/zg protease, lanes 5 and 10, 10/~g of protease. Part (b) shows a shorter exposure (ld) of the same autoradiograph. Molecular mass markers were as in Figure 1.
phosphorylated following dephosphorylation of the substrate migrated differently than the in vivo labelled material. This difference might reflect the observation that dephosphorylation of EBNA-2 results in an increase of the electrophoretic mobility of the protein (8). Partial proteolytic (V8) mapping of in vivo and in vitro labeled EBNA-2. EBNA-2 labeled metabolically with 32pi was compared to EBNA-2 phosphorylated in vitro with the CK-2 by one-dimensional mapping as described by Cleveland et al. (18). As can be seen in Figure 2, the majority of the fragments obtained from both the in vivo and the in vitro labeled material migrated to identical positions in the gel. The in vitro labeled material is shown in lanes 1-5, the in vivo labeled EBNA-2 is displayed in lanes 6-10. Panel b represents a shorter exposure of the same autoradiograph.
We note that most of the
radioactive label of the kinase treated material was found in the smaller fragments indicated by vertical bars. Most importantly, we observed no incorporation into fragments not found in the in vivo labeled material. The in vivo labeled EBNA-2 yielded a fragment migrating at approximately 32,000 (denoted with an arrow) that was not observed in the in vitro labeled EBNA-2. Phosphorylation of a C-terminal trpE-EBNA-2 fusion protein by CK-2.
To confirm the
observation that C-terminal residue(s) were phosphorylated by CK-2, a bacterial trpE-Cterminal EBNA-2 fusion protein described earlier (26) served as a substrate for CK-2. This 1698
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1 trpE -
NH 2 +
+
2 @
COOH -
÷
-Pj
iii!iiiii~
-ATP 1
......
O
-Ori
Q
FY
e
Fig. 3. Phnsphnrylatinn of recombinant trpE-fusinn proteins with easein ldnase 2 (CK.2). Bacteria transformed with the parental vector pATHll (20), lanes designated trpE, the pATH vector encoding the N-terminal trpE-fusion protein, lanes designated NH2 and the pATH vector encoding the C-terminal fusion protein, lanes designated COOH, were incubated with [,./_32p] ATP without (-) or with (+) added kinase. The position of the labeled C-terminal trpE-fusion protein is indicated by an arrow. Molecular mass markers were as in Figure 1. Fig. 4. Phosphorylation of the synthetic peptide FY by CK-2. The synthetic peptide "FY" corresponding to amino acids 462-474 of EBNA-2 was incubated without (lane 1) or with CK-2 (lane 2) in the presence of ['7-32P] ATP and analyzed by electrophoresis on a cellulose thin-layer chromatography plate in buffer at pH 1.9, The dried plate was subjected to autoradiography. The position of labeled ATP, the phosphorylated peptide and the origin are indicated.
fusion protein contained the 140 C-terminal amino acids of EBNA-2. A trpE- fusion protein that contained amino acids 1-58 of EBNA-2 (see materials and methods) as well as the wildtype trpE protein were used as controls. As shown in Figure 3, the CK-2 did phosphorylate the C-terminal fusion protein, which migrated to the expected position of an approximate Mr of 57,000 indicated by an arrow. In contrast, only a marginal phosphorylation of the trpE protein and essentially no incorporation into the N-terminal fusion protein was detectable.
The
bacterial extracts contained an unknown protein of an approximate Mr of 14,500 that was strongly phosphorylated by CK-2. The phosphoamino acid analysis of the labeled C-terminal fusion protein revealed the presence of phosphoserine (data not shown). Phosphorylation of a synthetic peptide corresponding to amino acids 462-474 of EBNA-2 by CK-2. A synthetic peptide encompassing residues 462-474 of EBNA-2 (FETTESPSSDEDY, "FY") was used as a substrate for CK-2. As shown in Figure 4, a single spot corresponding to phosphorylated peptide was observed (lane 2). The control reaction without added kinase is shown in lane 1. 1699
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Control peptides unreleated to EBNA-2 lacking obvious kinase recognition sites were not modified, while a synthetic peptide (RRRDDDSDDD) containing a standard recognition site for the kinase (17) was efficiently phosphorylated (data not shown). Phosphoamino acid analysis of the phosphorylated peptide revealed the presence of phosphoserine (data not shown). The incorporation into the FY peptide was 0.69 mol of phosphate/mol peptide indicating that only one residue was modified. This is important inasmuch as Ser469 and Ser470 are both potential substrates for CK-2. Phosphorylation of EBNA-2 with CK-1.
We also carried out experiments to determine
whether EBNA-2 is a substrate for CK-I. Neither the complete protein isolated from EBVtransformed B-cells nor the C-terminal trpE-fusion protein expressed in bacteria which contained the potential recognition site Ser457 were phosphorylated by the kinase while casein was efficiently modified (data not shown).
ACKNOWLEDGMENTS
This work was supported by the Deutsche Forschungsgemeinschaft (Mu 452/2-2) and Sonderforschungsbereich 241/133 (B.B. and O.G.I.). We thank N. Grankowski, Lublin, for the generous gift of purified CK-1.
REFERENCES
1. Sugden, B. (1989) Cell 57, 5-7. 2. Miller, G. (1990) In Virology (B.N. Fields and D.M. Knipe, Eds.), pp. 1921-1958. Raven Press, New York. 3. Herbst, H., Dallenbach, F., Hummel, M., Niedobitek, G., Pileri, S., Mueller-Lantzsch, N., and Stein, H. (1991) Proc. Natl.Acad.Sci. U.S.A.88, 4766-4770. 4. Dambaugh, T., Hennessy, K., Fennewald, S., and Kieft, E. (1986) In The Epstein-Barr virus:recent advances (A. Epstein and B.G. Achong, Eds.), pp. 13-45. William Heinemann Medical Books Ltd., London. 5. Hammerschmidt, W. and Sugden, B. (1989) Nature 340, 393-397. 6. Petti, L., Sample, C., and Kieff, E. (1990) Virology 176, 563-574. 7. Grfisser, F.A., Haiss, P., G6ttel, S., and Mueller-Lantzsch, N. (1991) J. Virol. 65, 3779-3788. 8. Schneider, H.R., and Issinger, O.G. (1989) BioTechForum 6,82-88. 9. Meisner, H., and Czech, M.P. (1991) Curr.Opin.Cell.Biol. 3,474-483. 10. Mulner-Lorillon, O., Cormier, P., Labbe, J.C., Doree, M., Poulhe, R., Osborne, H., and Belle, R. (1990) Eur.J.Biochem. 193, 529-534. 11. Yu, I.J,, Spector, D.L., Bae, Y.-S., and Marshak, D.R. (1991) J.Cell Biol. 114, 1217-1232. 12. Krek, W., Maridor. G., and Nigg, E.A. (1992) J.Cell.Biol. 116,43-55. 13. Crawford, D.H., Epstein, M.A., Bornkamm, G.W., Achong, B.G., Finerty, S., and Thompson, J. (1979) Int.J. Cancer 24, 294-302. 14. Grankowski, N., Boldyreff, B., and Issinger, O.G. (1991) Eur.J.Biochem. 198, 25-30. 15. Boldyreff, B., Piontek, K., Schmidt-Spaniol, I., and Issinger, O.G. (1991) Biochim. Biophys. Acta 1088, 439-441. 16. Kudlicki, W., Szyska, R., Palen, E., and Gasior, E. (1980) Biochim.Biophys.Acta 633, 376385. 1700
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17. Grfisser, F.A., Scheidtmann, K.H., Tuazon, P.T., Traugh, J.A., and Walter, G. (1988) Virology 165, 13-22. 18. Cleveland, D.W., Fischer, S.G., Kirschner, M.W., and Laemmli, U.K. (1977) J.Biol.Chem. 252, 1102-1106. 19. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular cloning:a laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 20. Koerner, T.J., Hill, J.E., Myers, A.M., and Tzagoloff, A. (1991) Methods Enzymol. 194, 477-490. 21. Polack, A., Hartl, G., Zimber, U., Freese, K.-U., Laux, G., Takadi, K., Hohn, B., Gissmann, I.., and Bornkamm, G.W. (1984) Gene 27, 279-288. 22. Billaud, M., Busson, P., Huang, D., Mueller-Lantzsch, N., Rousselet, G., Pavlish, O., Wakasugi, H., Seigneurin, J.M., Tursz, T., and Lenoir, G.M. (1989) J.Virol. 63, 41214128. 23. Studier, F.W., and Moffatt, B.A. (1986) J.Mol.Biol. 189, 113-130. 24. Cola, C., Brunati, A.M., Borin, G., Ruzza, P., Calderan, A., De-Castiglione, R., and Pinna, L.A. (1989) Biochim.Biophys.Acta 1012, 191-195. 25. Kemp, B.E. and Pearson, R.B. (1990) Trends Biochem.Sci. 15,342-346. 26. Meggio, F., Marchiori, F., Borin, G., Chessa, G., and Pinna, L.A. (1984) J.Biol.Chem.259, 14576-14579.
1701
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in proliferation (8) and oncogenic transformation (reviewed in (9)) and is itself a target for the cell cycle kinase cdc2 (10). The kinase is found in the nucleus or the cytoplasm depending on the growth state of the cell (11,12). MATERIALS AND METHODS
Cells and tissue culture. The cell line M-ABA has been described elsewhere (13). The cells were maintained in RPMI 1640 medium as described (7). M-ABA cells contain a complete EBV genome (subtype A). Radioactive labeling, cell extraction and immunoprecipitation. H32po4 (32pi) and [q,_32p] ATP were purchased from New England Nuclear or from Amersham. Radioactive labeling of proteins with 32pi in vivo, cell extraction, immunoprecipitation of EBNA-2 from cell extract and dephosphorylation with potato acid phosphatase was caried out as described (7).
In vitro phosphorylation. The purification of CK-1 from yeast and of recombinant CK-2 has been described (14-16). Kinase reactions using untreated or dephosphorylated EBNA-2 were carried out for 30-45 rain at 37oC in kinase buffer composed of 20 mM 2-[N-morpholino] ethanesulfonic acid (MES), pH 6.9, 130 mM KC1, 10 mM MgCI 2, 3 mM dithioerythritol (DTE), and 50/~M ATP. In a typical asssay, EBNA-2 from 3 x 107 cells bound to 25/zl of protein A Sepharose was suspended in 25 /~1 reaction mix that contained 1 /~1 CK-1 or 1 /zl of recombinant CK-2 (25 or 37 ~M) and 0.1-100/zCi ['7-32p] ATP. The activity of the enzymes was confirmed by phosphorylation of casein (1% w/v). CK-1 from yeast was a generous gift of Dr. N. Grankowski, Lublin. In some cases, the immunoprecipitated EBNA-2 was dephosphorylated with potato acid phosphatase (Boehringer, Mannheim) at pH 5.5 (7). The dephosphorylated EBNA-2 was then treated with CK,1 or CK-2 as described above. The final products were analyzed by SDS-PAGE, immunoblotting and autoradiography. The phosphoamino acid content of EBNA-2 phosphorylated with CK-2 was determined as described (7). Partial digestion with trypsin or V8 protease. For partial tryptic digestion, in vivo or in vitro
labeled EBNA-2 was purified further by a second round of immunoprecipitation as described previously (7). The digest with TPCK-treated trypsin (Worthington) was carried out in low salt lysis buffer for 30 rain at 0oC at a trypsin concentration of 30/~g/ml in a total volume of 50 >1 (17). V8-digestion (Boehringer, Mannheim) was carried out as described (18). Expression of the N-terminal 56 amino acids of EBNA-2 as a bacterial trpE-fusion protein. Recombinant DNA work was carried out according to published procedures (19). The 58 EBNA-2 N-terminal amino acids of EBNA-2 were expressed as a trpE fusion protein using the tryptophan-inducible vector pATH (20). The DNA encoding these 58 EBNA-2-specific amino acids was amplified by PCR from a cosmid clone containing the M-ABA EBNA-2 gene (21). The N-terminal fusion protein was induced in parallel with the C-terminal expression construct g88-1 (22) encoding the C-terminal 140 amino acids of EBNA-2 and the pATH 11 vector as a control in the E.coli strain BL21/DE3 (23). Cell extract was prepared by lysing the bacteria in buffer containing 50 mM Tris-HC1, pH 7.8, 300 mM NaCI, 2 mM EDTA, 2 mM dithiothreitol (DTT), 10% glycerol, 0.01 Fzg/ml aprotinin (Boehringer, Mannheim), 1 mM phenyl-methylsulfonyl-fluoride (PMSF) and 1 mg/ml lysozyme. After centrifugation, the supernatant containing the trpE proteins was dialyzed extensively against kinase buffer (above) without ATP. Phosphorylation of a synthetic peptide with CK-2. The synthetic peptide FETTESPSSDEDY-
(amide), "FY", MPS, San Diego, Ca, USA) corresponds to amino acids 462-474 of EBNA-2. The peptide was dissolved to a final concentration of 0.25 mg/ml in a final volume of 10 ~1 kinase buffer and the reaction was started by the addition of 1-2/~Ci [../_32p] ATP and 1/11 of CK-2 (25 ~M). 1 /~1 of the reaction mixture was spotted onto a cellulose thin-layer chromatography plate (Merck) and electrophoresed for 20 rain at 1500 V in pH 1.9 buffer and visualized by autoradiography (17). The molar ratio of phosphate incorporation into peptide FY was determined essentially as described (24). 1695
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RESULTS AND DISCUSSION EBNA-2 is phosphorylated in vivo at the C-terminus. To localize the in vivo phosphorylation sites, E B N A - 2 was immunoprecipitated with an antiserum directed against its C-terminus and subjected to a partial tryptic digestion as described (17).
The release of phosphorylated
fragments during the digestion indicates that phosphorylation site(s) were located towards the N-terminus, while fragments that remained bound to the antibody-resin should be derived from C-terminal phosphorylation site(s).
Figures la and l b display the result of such an
experiment. The material shown in Figure la was separated on a 15% SDS-gel, the material displayed in Figure l b was separated on a 7.5-15% linear SDS-gradient gel. Undigested E B N A - 2 is shown in lane 1; lanes 2 and 3 represent the fragments that were retained and released during the digestion, respectively.
In addition to some undigested E B N A - 2 and a
minor band with a Mr of 62,000, two major fragments remained b o u n d to the antibody resin, indicating that a major phosphorylation site(s) is located at the C-terminus of the protein. In the digest shown in Figure la, lane 2, we observed fragments with a Mr of approximately
in vivo
CK-2
in vivo
1.PAP
b 1
2
3 1| 4
5
6
2.CK-2 1
2
3[
4
5
200 92.5 68 46
30
14.3
Fig. 1.(a). Partial tryptic digestion of EBNA-2 labeled metabolically and in vitro with casein kinase 2 (CK-2). EBNA-2 was immunopurified from M-ABA cells labeled in vivo with 32pi and subjected to partial tryptic digestion. The digestion products were separated on a 15% SDS-potyacrylamidc gel (SDS-PAGE) and visualized by autoradiography. Untreated material is shown in lane 1, material that remained bound is shown in lane 2, fragments released are shown in lane 3. The same experiment was carried out with EBNA-2 labeled in vitro with CK-2. The undigested EBNA-2 is shown in lane 4, bound and released fragments were separated in lanes 5 and 6, respectively. (b). Partial tryptic digestion of EBNA-2 labeled in vivo (lanes 1-3) and of EBNA-2 dephosphorylated with potato acid phosphatase prior to phosphorylation with CK-2 (lanes 4-6). The samples were separated by 7.5-15% SDS-PAGE. A partial tryptic digest of in vivo labeled EBNA-2 carried out in parallel is shown in lanes 1-3, in vitro .phosphorylated material is displayed in lanes 4-6. Untreated material is shown m lanes 1 and 4, material that remained bound is shown in lanes 2 and 5, fragments released are shown in lane 3 and 6. a4C-methylated molecular mass markers were from Amersham (x10-3). 1696
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15,000 and 31,000. In the digest shown in Figure lb, lane 2, we detected fragments with a Mr of approximately 13,000 and 26,000. The difference in mobilities are either the result of the acrylamide concentration of the gels or may be due to a further digestion of the material in Figure lb. The signal observed at 26,000 (lanes 2) appeared to consist of two or possibly three bands. The material released during the reaction contained several fragments. The material in Figure la, lane 3, shows some undigested EBNA-2 and a band migrating at approximately 29,000 indicated by an arrow. The released material shown in Figure lb, lane 3, contained two fragments that migrated with the same mobilities as the ones retained during the digest and two additional bands at a Mr of approximately 16,000 and 20,000.
The largest fragment
released during the reaction is indicated with an arrow. The fragments with identical mobilities might have been released to the supernatant due to digestion of the antibody. The origin of the two additional fragments could not be determined. We conclude from the intensity of the various bands in the bound and the released fractions that the C-terminus contains one of the major phosphorylation site(s) of EBNA-2, with additional site(s) not yet identified. Due to the low amount of radioactive label present we did not determine the phosphoamino acid composition of the various fragments. In vitro phosphorylation of EBNA-2 with recombinant CK-2.
Ser457 in the sequence
AspGluSer457TrpAsp and Ser469 and Ser470 in the sequence Ser469SerAspGluAsp of EBNA-2 represent possible phosphorylation sites for the CK-1 and CK-2, respectively (25,26) EBNA-2 was purified by immunoprecipitation
from M-ABA
cells, not treated
or
dephosphorylated with potato acid phosphatase (7) and then incubated with recombinant CK2 (14,15). The CK-2 transfered phosphate efficiently to either untreated or dephosphorylated EBNA-2 as demonstrated in Figure la, lane 4 and Figure lb, lane 4, respectively. We found that only serine residues were phosphorylated by this enzyme (data not shown). Partial tryptic digest of EBNA-2 phosphorylated by CK-2 in vitro.
To localize the site
phosphorylated by CK-2, the in vitro labeled EBNA-2 was subjected to partial tryptic digest as outlined above. As can be seen in Figure la and lb, lanes 5, the in vitro labeled material remained bound to the resin during the digestion indicating that only C-terminal serines were modified.
The peptides that remained bound during the digest of EBNA-2 that was not
dephosphorylated prior to the kinase reaction migrated with the same mobility as the corresponding peptides from the in vivo labeled material. 1697
In contrast, the peptides
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b 1 2 3 45 i
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I
I
I
I
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92694630-
14.3-
Fig. 2. V8 mapping of EBNA-2 labeled with 32Pi in vivo and in vitro using CK-2. EBNA-2 was immunopurified from M-ABA cells labeled in vivo with 32pi. In parallel, EBNA-2 immunoprecipitated from unlabeled cells was phosphorylated with CK-2. The reaction products were separated on a 7.5% SDS-gel, EBNA-2 was visualized by autoradiography excised and reapplied to a 9.5-15% linear gradient gel for VS-digestion. Lanes 1-5, EBNA-2 labeled in vitro by CK-2. Lanes 6-10:EBNA-2 labeled in vivo. Lanes 1 and 6, no enzyme, lane 2 and 7, 0.05 /~g protease, lanes 3 and 8, 0.1 /~g protease, lanes 4 and 9, 1/zg protease, lanes 5 and 10, 10/~g of protease. Part (b) shows a shorter exposure (ld) of the same autoradiograph. Molecular mass markers were as in Figure 1.
phosphorylated following dephosphorylation of the substrate migrated differently than the in vivo labelled material. This difference might reflect the observation that dephosphorylation of EBNA-2 results in an increase of the electrophoretic mobility of the protein (8). Partial proteolytic (V8) mapping of in vivo and in vitro labeled EBNA-2. EBNA-2 labeled metabolically with 32pi was compared to EBNA-2 phosphorylated in vitro with the CK-2 by one-dimensional mapping as described by Cleveland et al. (18). As can be seen in Figure 2, the majority of the fragments obtained from both the in vivo and the in vitro labeled material migrated to identical positions in the gel. The in vitro labeled material is shown in lanes 1-5, the in vivo labeled EBNA-2 is displayed in lanes 6-10. Panel b represents a shorter exposure of the same autoradiograph.
We note that most of the
radioactive label of the kinase treated material was found in the smaller fragments indicated by vertical bars. Most importantly, we observed no incorporation into fragments not found in the in vivo labeled material. The in vivo labeled EBNA-2 yielded a fragment migrating at approximately 32,000 (denoted with an arrow) that was not observed in the in vitro labeled EBNA-2. Phosphorylation of a C-terminal trpE-EBNA-2 fusion protein by CK-2.
To confirm the
observation that C-terminal residue(s) were phosphorylated by CK-2, a bacterial trpE-Cterminal EBNA-2 fusion protein described earlier (26) served as a substrate for CK-2. This 1698
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1 trpE -
NH 2 +
+
2 @
COOH -
÷
-Pj
iii!iiiii~
-ATP 1
......
O
-Ori
Q
FY
e
Fig. 3. Phnsphnrylatinn of recombinant trpE-fusinn proteins with easein ldnase 2 (CK.2). Bacteria transformed with the parental vector pATHll (20), lanes designated trpE, the pATH vector encoding the N-terminal trpE-fusion protein, lanes designated NH2 and the pATH vector encoding the C-terminal fusion protein, lanes designated COOH, were incubated with [,./_32p] ATP without (-) or with (+) added kinase. The position of the labeled C-terminal trpE-fusion protein is indicated by an arrow. Molecular mass markers were as in Figure 1. Fig. 4. Phosphorylation of the synthetic peptide FY by CK-2. The synthetic peptide "FY" corresponding to amino acids 462-474 of EBNA-2 was incubated without (lane 1) or with CK-2 (lane 2) in the presence of ['7-32P] ATP and analyzed by electrophoresis on a cellulose thin-layer chromatography plate in buffer at pH 1.9, The dried plate was subjected to autoradiography. The position of labeled ATP, the phosphorylated peptide and the origin are indicated.
fusion protein contained the 140 C-terminal amino acids of EBNA-2. A trpE- fusion protein that contained amino acids 1-58 of EBNA-2 (see materials and methods) as well as the wildtype trpE protein were used as controls. As shown in Figure 3, the CK-2 did phosphorylate the C-terminal fusion protein, which migrated to the expected position of an approximate Mr of 57,000 indicated by an arrow. In contrast, only a marginal phosphorylation of the trpE protein and essentially no incorporation into the N-terminal fusion protein was detectable.
The
bacterial extracts contained an unknown protein of an approximate Mr of 14,500 that was strongly phosphorylated by CK-2. The phosphoamino acid analysis of the labeled C-terminal fusion protein revealed the presence of phosphoserine (data not shown). Phosphorylation of a synthetic peptide corresponding to amino acids 462-474 of EBNA-2 by CK-2. A synthetic peptide encompassing residues 462-474 of EBNA-2 (FETTESPSSDEDY, "FY") was used as a substrate for CK-2. As shown in Figure 4, a single spot corresponding to phosphorylated peptide was observed (lane 2). The control reaction without added kinase is shown in lane 1. 1699
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Control peptides unreleated to EBNA-2 lacking obvious kinase recognition sites were not modified, while a synthetic peptide (RRRDDDSDDD) containing a standard recognition site for the kinase (17) was efficiently phosphorylated (data not shown). Phosphoamino acid analysis of the phosphorylated peptide revealed the presence of phosphoserine (data not shown). The incorporation into the FY peptide was 0.69 mol of phosphate/mol peptide indicating that only one residue was modified. This is important inasmuch as Ser469 and Ser470 are both potential substrates for CK-2. Phosphorylation of EBNA-2 with CK-1.
We also carried out experiments to determine
whether EBNA-2 is a substrate for CK-I. Neither the complete protein isolated from EBVtransformed B-cells nor the C-terminal trpE-fusion protein expressed in bacteria which contained the potential recognition site Ser457 were phosphorylated by the kinase while casein was efficiently modified (data not shown).
ACKNOWLEDGMENTS
This work was supported by the Deutsche Forschungsgemeinschaft (Mu 452/2-2) and Sonderforschungsbereich 241/133 (B.B. and O.G.I.). We thank N. Grankowski, Lublin, for the generous gift of purified CK-1.
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