VIROLOGY
66,291-294
(1978)
The Fidelity of Phosphorylation of the Adenovirus DNA-Binding Protein by an in Vitro Nuclear Protein Kinase from Virus-infected Cells E. POSTEL, Department
of Biochemical
H. KLEIN,
Sciences,
AND
Princeton
Accepted
A. J. LEVINE’
University,
December
Princeton,
New Jersey
08540
24, 1977
The type 5 adenovirus single-strand-specific DNA-binding protein is phosphorylated and can be labeled by the addition of 32P04 to the medium of infected cells. An infected cell nuclear protein kinase activity is capable of phosphorylating the single-strand-specific DNA-binding protein in vitro (A. D. Levinson, E. H. Postel, and A. J. Levine, Virology 79, 144-159, 1977). The fidelity of phosphorylation by this in vitro protein kinase activity was tested by comparing the ““P-labeled partial peptides generated by Staphylococcus aureus protease digestion of the 32P-labeled DNA-binding protein which had been phosphorylated in vivo or in vitro. Identical 32P-labeled partial peptides were obtained from the DNAbinding protein whether it was phosphorylated in uivo or in vitro. A second nuclear protein of 36,000 MW was also faithfully phosphorylated by the in vitro nuclear protein kinase activity.
lar weights of 100,000, 72,000, and 36,000. Proteins of identical molecular weight are also labeled by 32P04 added to adenovirusinfected cells in vivo (5). Although these experiments demonstrated that the same adenovirus-induced proteins (based upon their molecular weight) were phosphorylated in vivo and in vitro, the fidelity of the in vitro phosphorylation reaction was not proven. Whether the same amino acids and peptides of these proteins were phosphorylated in vivo and in vitro was not clear. In order to test this, a partial peptide analysis (9,10) was performed on the 72,000 and 36,000 MW proteins labeled with 32P04 in vivo or in vitro. To do this, KB cells were infected with type 5 adenovirus (5) and at 15 or 20 hr after infection one-half of the cultures were labeled with 50 $X/ml of 3”P04 in phosphate-free Dulbecco’s modified Eagle’s medium (5). After 2 hr of labeling the cells were harvested and nuclei were prepared as described elsewhere (5). At the same time as these labeled cells were harvested, unlabeled infected cells were also obtained and nuclei were prepared which were employed to label, with [y32P04JATP, the endogenous nuclear proteins in adenovirus-infected cells (5). The
At early times after adenovirus infection least six virus-specific proteins are synthesized (I). One of these proteins is a 72,006 MW single-strand-specific DNA-binding protein (2). The 72,000 MW protein is required for viral DNA synthesis (3, 4) although its role in this process is not yet defined. A temperature-sensitive mutant, H5tsl25, in the structural gene of this protein, produces an altered protein which is thermolabile for binding to single-stranded DNA (4). Recently, this 72,000 MW protein has been shown to undergo several posttranslational modifications (5-8). One of these modifications is the phosphorylation of the DNA binding protein. In vivo, the phosphorylation has been demonstrated by labeling, via a covalent linkage, the purified 72,000 MW protein with 32P04 added to the culture medium of virus-infected cells ( 5-8). This same 72,000 MW protein can also be 32P-labeled in vitro by a nuclear protein kinase activity using [v-~‘PO~]ATP (5). This nuclear protein kinase activity(ies) specifically phosphorylates three adenovirus-induced nuclear proteins with molecu-
at
’ Author addressed.
to whom
requests
for reprints
should
be 291
0042-6822/78/0861-0291%02.09/O Copyright All rights
0 1978 by Academic Press, of reproduction in any form
Inc. reserved.
292
SHORT
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nuclei containing the 32P-labeled phosphoproteins produced in vivo or in vitro were analyzed by electrophoresis on SDSpolyacrylamide gels (5). Figure 1 presents the results of this experiment. The 72,000 and 36,000 MW phosphoproteins are the major proteins labeled with 32P04 under both in vivo and in vitro conditions. The appropriate gel slices containing the in vivo and in vitro 72,000 and 36,000 MW 32P-labeled proteins were cut out of the gel and treated with the Staphylococcus aureus protease under conditions that pro-
A
B
duced a set of overlapping partial peptides (9, IO). These peptides were then analyzed on 15% polyacrylamide-SDS gels. The autoradiograms showing the 32P-labeled partial peptides generated from the 72,000 and 36,000 MW proteins labeled in vivo or in vitro are presented in Fig. 2. The 72,000 MW protein generated six discrete 32P-labeled peptides after S. aureus protease digestion of either the in vivo or the in vitro prepared phosphoprotein. All of the 32Plabeled peptides produced by the digestion of the in vitro phosphorylated 72,000 MW
C
D
72,000
72,000
36,000
36,000
IN VITRO FIG. 1. SDS-acrylamide gel electrophoresis of phosphoproteins labeled in vivo or in vitro. KB cells were mock-infected or infected with adenovirus type 5. At 15 to 20 hr postinfection half the cells were labeled with 32P04. Nuclei were prepared from these cells and the 32P04-labeled proteins were analyzed by SDS polyacrylamide gel electrophoresis. Nuclei were also prepared from the unlabeled cells and labeled in vitro with [Y-~~PO~] ATP. The products of this reaction were then analyzed by SDS-polyacrylamide gel electrophoresis. An autoradiogram of this gel is shown. 32P0Jabeled phosphoproteins labeled in vivo fkom (A) mock-infected cells and (B) adenovirus-infected cells. “*Pod-labeled phosphoproteins labeled in vitro from (C) mock-infected cells and (D) adenovirus-infected cells.
SHORT
293
COMMUNICATIONS
IN VITRO
IN VIVO
IN VITRO 72, 36,
B
A FIG. 2. Polyacrylamide gel electrophoresis of the partial peptide fragments vitro 32P-labeled 72,003 and 36,000 MW proteins. The 72,600 and 36,000 MW SDS-polyacrylamide gel of in uiuo and in vitro 32P-labeied nuclear extracts
derived from the in uiuo and in proteins were cut out from the shown in Fig. 1. Proteins were extracted from the gel slices and divided into four samples each. Samples were treated with (from left to right) 10, 1, 0.1, and 0.01 pg of S. aureas protease. (A) 72,000 MW protein; (B) 36,000 MW protein.
protein migrated with an identical mobility as those 32P-labeled peptides from the in viuo phosphorylated 72,000 MW protein (Fig. 2A). Similarly, the 32P-labeled partial peptides of the 36,000 MW protein phosphorylated in uivo or in vitro migrated in an identical fashion (Fig. 2B). This analysis demonstrates that the in vitro nuclear protein kinase activity(ies) faithfully and specifically phosphorylate endogenous adenovirus-specific proteins in the same peptides as the in uiuo protein. While this nuclear protein kinase activity phosphorylates the endogenous 72,000 MW protein, from the level of phosphorylation in vitro, it seems unlikely that this enzyme only phosphorylates newly synthesized proteins that do not yet contain phosphate groups. Indeed, it is clear that the phosphate group(s) on the 72,000 MW protein turns over and phosphorylation can occur in the absence of the synthesis of this protein ( 7; Postel, unpublished observations). Thus a protein kinase-phosphatase system is most likely active in these nuclear preparations and works faithfully to remove and replace phosphate residues. The native conformation of the 72,006 MW protein appears to be essential for the phosphoryla-
tion (recognition) of this protein by the kinase. In H5ts125 infected cells at the nonpermissive temperature, the 72,060 MW protein is synthesized (and in the nucleus) but it is not phosphorylated (5). This then seems to be an excellent system to study the recognition of specific serine residues (6) in a protein by a protein kinasephosphatase activity. As both the phosphorylated and dephosphorylated forms of the 72,000 MW protein bind specifically to single-stranded DNA (.5), the functional significance of the phosphate modification of this protein remains unclear. ACKNOWLEDGMENTS The authors thank A. K. Teresky, N. Tick, and S. Wyckoff for their assistance. H.L.K. was a postdoctoral of the Damon Runyon-Walter Winchell Cancer Fund and E.H.P. was a postdoctoral of the NC1 (CAO9167-03). This work was supported by a grant from the American Cancer Society (VC57E). REFERENCES 1. LEVINE, A. J., B&him. Biophys. Acta 466, 213-241 (1976). 2. VAN DER VLIET, P. C., and LEVINE, A. J., Nature New Bid. 246, 170-174 (1973). 3. ENSINGER, M. J., and GINSBERG, H. S., J. Vi&.
294
SHORT
COMMUNICATIONS
10,328-339 (1972). 4. VAN DER VLIET, P. C., LEVINE, A. J., ENSINGER, M. J., and GINSBERG, H. S., J. Viral. 15.348-354 (1975). 5. LEVINSON, A. D., POSTEL, E. H., and LEVINE, A. J., Virology 79, 144-159 (1977). 6. JENG, V. H., WOLD, W. S. M., SUGAWARA, K., GILEAD, Z., and GREEN, M., J. Viral. 22,
402-411 (1977). 7. RUSSEL, W. C., and BLAIR, G. E., J. Gen. Vi&. 34,19-35 (1977). 8. AXELROD, N., Virology, in press. 9. CLEVELAND, D., FISHER, S., KIRSCHNER, M., and LAEMMLI, U., J. Biol. Chem. 252,1102-1106. 10. LEVINSON, A. D., and LEVINE, A. J., Cell 11, 8714379 (1977).
66,291-294
(1978)
The Fidelity of Phosphorylation of the Adenovirus DNA-Binding Protein by an in Vitro Nuclear Protein Kinase from Virus-infected Cells E. POSTEL, Department
of Biochemical
H. KLEIN,
Sciences,
AND
Princeton
Accepted
A. J. LEVINE’
University,
December
Princeton,
New Jersey
08540
24, 1977
The type 5 adenovirus single-strand-specific DNA-binding protein is phosphorylated and can be labeled by the addition of 32P04 to the medium of infected cells. An infected cell nuclear protein kinase activity is capable of phosphorylating the single-strand-specific DNA-binding protein in vitro (A. D. Levinson, E. H. Postel, and A. J. Levine, Virology 79, 144-159, 1977). The fidelity of phosphorylation by this in vitro protein kinase activity was tested by comparing the ““P-labeled partial peptides generated by Staphylococcus aureus protease digestion of the 32P-labeled DNA-binding protein which had been phosphorylated in vivo or in vitro. Identical 32P-labeled partial peptides were obtained from the DNAbinding protein whether it was phosphorylated in uivo or in vitro. A second nuclear protein of 36,000 MW was also faithfully phosphorylated by the in vitro nuclear protein kinase activity.
lar weights of 100,000, 72,000, and 36,000. Proteins of identical molecular weight are also labeled by 32P04 added to adenovirusinfected cells in vivo (5). Although these experiments demonstrated that the same adenovirus-induced proteins (based upon their molecular weight) were phosphorylated in vivo and in vitro, the fidelity of the in vitro phosphorylation reaction was not proven. Whether the same amino acids and peptides of these proteins were phosphorylated in vivo and in vitro was not clear. In order to test this, a partial peptide analysis (9,10) was performed on the 72,000 and 36,000 MW proteins labeled with 32P04 in vivo or in vitro. To do this, KB cells were infected with type 5 adenovirus (5) and at 15 or 20 hr after infection one-half of the cultures were labeled with 50 $X/ml of 3”P04 in phosphate-free Dulbecco’s modified Eagle’s medium (5). After 2 hr of labeling the cells were harvested and nuclei were prepared as described elsewhere (5). At the same time as these labeled cells were harvested, unlabeled infected cells were also obtained and nuclei were prepared which were employed to label, with [y32P04JATP, the endogenous nuclear proteins in adenovirus-infected cells (5). The
At early times after adenovirus infection least six virus-specific proteins are synthesized (I). One of these proteins is a 72,006 MW single-strand-specific DNA-binding protein (2). The 72,000 MW protein is required for viral DNA synthesis (3, 4) although its role in this process is not yet defined. A temperature-sensitive mutant, H5tsl25, in the structural gene of this protein, produces an altered protein which is thermolabile for binding to single-stranded DNA (4). Recently, this 72,000 MW protein has been shown to undergo several posttranslational modifications (5-8). One of these modifications is the phosphorylation of the DNA binding protein. In vivo, the phosphorylation has been demonstrated by labeling, via a covalent linkage, the purified 72,000 MW protein with 32P04 added to the culture medium of virus-infected cells ( 5-8). This same 72,000 MW protein can also be 32P-labeled in vitro by a nuclear protein kinase activity using [v-~‘PO~]ATP (5). This nuclear protein kinase activity(ies) specifically phosphorylates three adenovirus-induced nuclear proteins with molecu-
at
’ Author addressed.
to whom
requests
for reprints
should
be 291
0042-6822/78/0861-0291%02.09/O Copyright All rights
0 1978 by Academic Press, of reproduction in any form
Inc. reserved.
292
SHORT
COMMUNICATIONS
nuclei containing the 32P-labeled phosphoproteins produced in vivo or in vitro were analyzed by electrophoresis on SDSpolyacrylamide gels (5). Figure 1 presents the results of this experiment. The 72,000 and 36,000 MW phosphoproteins are the major proteins labeled with 32P04 under both in vivo and in vitro conditions. The appropriate gel slices containing the in vivo and in vitro 72,000 and 36,000 MW 32P-labeled proteins were cut out of the gel and treated with the Staphylococcus aureus protease under conditions that pro-
A
B
duced a set of overlapping partial peptides (9, IO). These peptides were then analyzed on 15% polyacrylamide-SDS gels. The autoradiograms showing the 32P-labeled partial peptides generated from the 72,000 and 36,000 MW proteins labeled in vivo or in vitro are presented in Fig. 2. The 72,000 MW protein generated six discrete 32P-labeled peptides after S. aureus protease digestion of either the in vivo or the in vitro prepared phosphoprotein. All of the 32Plabeled peptides produced by the digestion of the in vitro phosphorylated 72,000 MW
C
D
72,000
72,000
36,000
36,000
IN VITRO FIG. 1. SDS-acrylamide gel electrophoresis of phosphoproteins labeled in vivo or in vitro. KB cells were mock-infected or infected with adenovirus type 5. At 15 to 20 hr postinfection half the cells were labeled with 32P04. Nuclei were prepared from these cells and the 32P04-labeled proteins were analyzed by SDS polyacrylamide gel electrophoresis. Nuclei were also prepared from the unlabeled cells and labeled in vitro with [Y-~~PO~] ATP. The products of this reaction were then analyzed by SDS-polyacrylamide gel electrophoresis. An autoradiogram of this gel is shown. 32P0Jabeled phosphoproteins labeled in vivo fkom (A) mock-infected cells and (B) adenovirus-infected cells. “*Pod-labeled phosphoproteins labeled in vitro from (C) mock-infected cells and (D) adenovirus-infected cells.
SHORT
293
COMMUNICATIONS
IN VITRO
IN VIVO
IN VITRO 72, 36,
B
A FIG. 2. Polyacrylamide gel electrophoresis of the partial peptide fragments vitro 32P-labeled 72,003 and 36,000 MW proteins. The 72,600 and 36,000 MW SDS-polyacrylamide gel of in uiuo and in vitro 32P-labeied nuclear extracts
derived from the in uiuo and in proteins were cut out from the shown in Fig. 1. Proteins were extracted from the gel slices and divided into four samples each. Samples were treated with (from left to right) 10, 1, 0.1, and 0.01 pg of S. aureas protease. (A) 72,000 MW protein; (B) 36,000 MW protein.
protein migrated with an identical mobility as those 32P-labeled peptides from the in viuo phosphorylated 72,000 MW protein (Fig. 2A). Similarly, the 32P-labeled partial peptides of the 36,000 MW protein phosphorylated in uivo or in vitro migrated in an identical fashion (Fig. 2B). This analysis demonstrates that the in vitro nuclear protein kinase activity(ies) faithfully and specifically phosphorylate endogenous adenovirus-specific proteins in the same peptides as the in uiuo protein. While this nuclear protein kinase activity phosphorylates the endogenous 72,000 MW protein, from the level of phosphorylation in vitro, it seems unlikely that this enzyme only phosphorylates newly synthesized proteins that do not yet contain phosphate groups. Indeed, it is clear that the phosphate group(s) on the 72,000 MW protein turns over and phosphorylation can occur in the absence of the synthesis of this protein ( 7; Postel, unpublished observations). Thus a protein kinase-phosphatase system is most likely active in these nuclear preparations and works faithfully to remove and replace phosphate residues. The native conformation of the 72,006 MW protein appears to be essential for the phosphoryla-
tion (recognition) of this protein by the kinase. In H5ts125 infected cells at the nonpermissive temperature, the 72,060 MW protein is synthesized (and in the nucleus) but it is not phosphorylated (5). This then seems to be an excellent system to study the recognition of specific serine residues (6) in a protein by a protein kinasephosphatase activity. As both the phosphorylated and dephosphorylated forms of the 72,000 MW protein bind specifically to single-stranded DNA (.5), the functional significance of the phosphate modification of this protein remains unclear. ACKNOWLEDGMENTS The authors thank A. K. Teresky, N. Tick, and S. Wyckoff for their assistance. H.L.K. was a postdoctoral of the Damon Runyon-Walter Winchell Cancer Fund and E.H.P. was a postdoctoral of the NC1 (CAO9167-03). This work was supported by a grant from the American Cancer Society (VC57E). REFERENCES 1. LEVINE, A. J., B&him. Biophys. Acta 466, 213-241 (1976). 2. VAN DER VLIET, P. C., and LEVINE, A. J., Nature New Bid. 246, 170-174 (1973). 3. ENSINGER, M. J., and GINSBERG, H. S., J. Vi&.
294
SHORT
COMMUNICATIONS
10,328-339 (1972). 4. VAN DER VLIET, P. C., LEVINE, A. J., ENSINGER, M. J., and GINSBERG, H. S., J. Viral. 15.348-354 (1975). 5. LEVINSON, A. D., POSTEL, E. H., and LEVINE, A. J., Virology 79, 144-159 (1977). 6. JENG, V. H., WOLD, W. S. M., SUGAWARA, K., GILEAD, Z., and GREEN, M., J. Viral. 22,
402-411 (1977). 7. RUSSEL, W. C., and BLAIR, G. E., J. Gen. Vi&. 34,19-35 (1977). 8. AXELROD, N., Virology, in press. 9. CLEVELAND, D., FISHER, S., KIRSCHNER, M., and LAEMMLI, U., J. Biol. Chem. 252,1102-1106. 10. LEVINSON, A. D., and LEVINE, A. J., Cell 11, 8714379 (1977).
