JOURNAL OF VIROLOGY, Mar. 1992, p. 1777-1780
Vol. 66, No. 3
0022-538X/92/031777-04$02.00/0 Copyright © 1992, American Society for Microbiology
Herpesvirus Saimiri Oncogene STP-C488 Encodes
a
Phosphoprotein
JAE U. JUNG AND RONALD C. DESROSIERS* New England Regional Primate Research Center, Harvard Medical School, Southborough, Massachusetts 01772-9102 Received 30 September 1991/Accepted 27 November 1991
The STP-C488 open reading frame of herpesvirus saimiri encodes an oncoprotein that has transforming and tumor-inducing activities independent of the rest of the herpesvirus genome. STP-C488 protein has an unusual, membrane-associated, fibrous structure and is located primarily in perinuclear compartments. We now report that STP-C488 is phosphorylated in vivo. The phosphorylated form, which accounted for about 15% of STP-C488 in transformed cells, migrated slightly more slowly through sodium dodecyl sulfate-polyacrylamide gels than unphosphorylated STP-C488. A serine residue near the amino terminus was shown to be the site of phosphorylation. However, phosphorylation was not required for transformation of Rat-i cells by STP-C488.
Herpesvirus saimiri is a member of the gamma subfamily of herpesviruses. Some members of this group, e.g., EpsteinBarr virus, herpesvirus saimiri, herpesvirus ateles, and herpesvirus sylvilagus, are capable of inducing lymphoproliferative disorders in natural or experimental hosts. Herpesvirus saimiri is apparently not associated with disease in its natural host, the squirrel monkey (Saimin sciureus). However, it does induce rapidly progressing fatal lymphomas, leukemias, and lymphosarcomas in several other species of New World primates (9). Herpesvirus saimiri strains have been divided into three subgroups (A, B, and C) based on the extent of DNA sequence divergence at the left terminus of L-DNA (13). Subgroup A and C viruses are highly oncogenic in vivo and efficiently transform common marmoset peripheral blood T lymphocytes in vitro to interleukin 2-independent growth (7). Mutagenic analyses have demonstrated that the leftmost open reading frame of group A strain 11 is required for the immortalization of common marmoset T lymphocytes in vitro and for lymphoma induction in vivo but not for virus replication (5, 6, 12, 15). This open reading frame, called STP-All, and an analogous open reading frame called STP-C488 from the group C strain 488 genome are capable of transforming Rat-1 cells in culture (11). Transformation by STP-C488 resulted in loss of contact inhibition, formation of foci, growth at reduced concentration of serum, and formation of invasive tumors in nude mice. STP-A11 was less potent in its transforming activity (11). Except for hydrophobic, putative membrane-spanning domains at their carboxyl termini, only limited sequence identities are apparent between STP-A11 and STP-C488 (2). Close inspection of the STP-A11 and STP-C488 sequences, however, reveals a similar organization in terms of the presence and locations of specific structural motifs (11). Both proteins are predicted to have acidic amino termini (pI = 3.5 for the amino-terminal 32 amino acids of STP-A11 and pl = 4.4 for the amino-terminal 17 amino acids of STP-C488). These acidic domains are likely to be the active or ligandbinding sites. STP-C488 is predicted to contain 18 uninterrupted collagenlike repeats, and STP-A11 is predicted to have 9 collagenlike repeats. The collagenlike repeats are Gly-X-Y, where X and/or Y is proline. The collagenlike motifs in STP-A11 are not directly repeated as they are in STP-C488, *
but they seem to be similarly concentrated in the central portion of the protein (11). These collagenlike repeats can be inferred to form a fibrous structure similar to that of cellular collagen and may serve as a hinge to extend the active domain of the protein to its site of action. STP-All and STP-C488 also contain a hydrophobic stretch at their carboxyl termini sufficient for a membrane-spanning domain. Recently, a polyclonal anti-STP-C488 antibody was used to identify the protein encoded by STP-C488 (10). The apparent molecular mass of STP-C488 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (20 to 22 kDa) was considerably higher than that predicted from the DNA sequence (9.9 kDa). STP-C488 was sensitive to treatment with bacterial collagenase, consistent with the 18 uninterrupted collagenlike repeats predicted by the DNA sequence, was associated with cytoplasmic membranes, and was found primarily in perinuclear compartments. These results suggest an unusual, membrane-associated, fibrous structure for the transforming STP-C488 oncoprotein. In addition to the major STP-C488 protein which migrated with an apparent molecular mass of 20 kDa, a less abundant species of slightly slower mobility (approximately 22 kDa) has also been previously observed (10). This 20-kDa-22-kDa doublet of STP-C488 in SDS-PAGE suggested the presence of a modified form. The experiments described here demonstrate that STP-C488 protein is phosphorylated at a serine residue near the amino terminus, that the 20- and 22-kDa species represent unphosphorylated and phosphorylated forms of the protein, and that phosphorylation is not required for transformation of Rat-1 cells by STP-C488. To demonstrate phosphorylation of STP-C488 in vivo, cells were labeled with 32P04. Rat-STP-C488 cells, which stably express STP-C488 protein in Rat-1 cells, and Rat-LXSN cells, which contain only the LXSN retrovirus vector (10), were used for labeling. When cells reached 80 to 90% confluence in a 25-cm2 dish, they were rinsed twice with labeling medium (phosphate-free minimal essential medium plus 5% dialyzed fetal calf serum [GIBCO-Bethesda Research Laboratories, Gaithersburg, Md.]) and preincubated for 30 min with this same medium. 32P04 (New England Nuclear Corp., Boston, Mass.) was added at a concentration of 0.5 mCi/ml, and the incubation was continued for 3 h. Cells were harvested by scraping with a rubber policeman, washed once with phosphate-buffered saline, and lysed with 1 ml of RIPA buffer (0.15 M NaCl, 1% [vol/vol] Nonidet P-40, 0.5% [wtlvol] sodium deoxycholate, 0.1% [wtlvol] SDS, 50 mM Tris [pH
Corresponding author. 1777
1778
J. VIROL.
NOTES
1
2
A.
1 2 3
B.
2 3
97-
69-
C.
58 -
69 -
0-
47 -
40.:
47-
i:.
29-
29 -
_U'~
20-
~.
P-SERP-THR-
P-TYR-
29 -
14 -
FIG. 1. Phosphorylation of STP-C488. G418-resistant Rat-LXSN and Rat-STP-C488 cells were labeled with 32PO4. Labeled cell extracts were incubated with anti-STP-C488 antibody, and immunoprecipated proteins were analyzed by SDS-PAGE. Lane 1, Rat-LXSN; lane 2, Rat-STP-C488. The 14C molecular mass markers are lysozyme (14 kDa), carbonic anhydrase (29 kDa), ovalbumin (47 kDa), and bovine serum albumin (69 kDa). The arrow indicates STP-C488.
7.5], 10 ,ug of aprotinin [U.S. Biochemicals, Cleveland, Ohio] per ml) for 30 min at 4°C with shaking. Lysates were cleared by centrifugation for 5 min at 13,600 x g, and the supematant was transferred to a clean tube. Rabbit anti-STP-C488 polyclonal antiserum was used as described previously to identify STP-C488 protein by immunoprecipitation (10). Five microliters of anti-STP-C488 antibody and 30 RI of protein A-agarose (Oncogene Science, Manhasset, N.Y.) were added to 500 RI of RIPA buffer containing 100 RI of supematant and incubated for 4 h at 4°C with shaking. The pellet was washed five times by suspending the pellet in 1 ml of RIPA buffer and vortexing, centrifuging, and aspirating the supernatant. Finally, the pellet was rinsed twice with 10 mM Tris (pH 7.5), suspended in SDS-PAGE sample buffer, and boiled for 5 min. Proteins were resolved by SDS-PAGE (12.5% polyacrylamide), and the gel was dried and exposed to Kodak XAR-5 film at -70°C. Anti-STP-C488 antibody specifically precipitated a 32P-labeled 22-kDa protein only from Rat-STP-C488 cells (Fig. 1, lane 2). No such protein was precipitated from control RatLXSN cells lacking the STP-C488 sequence (Fig. 1, lane 1). This result demonstrated that STP-C488 was phosphorylated in vivo. There are three phosphorylhydroxyamino acids in cellular proteins, phosphoserine, phosphothreonine, and phosphotyrosine. To determine the identity of the 32P-labeled amino acid(s) in STP-C488, Rat-STP-C488 cells were first labeled
14-
14 -
FIG. 2. STP-C488 is phosphorylated at a serine residue near the amino terminus. (A) Immunoprecipitation of STP-C488 from 32p_ labeled cell lysates of Rat-STP-C488, Rat-STP-C488/3S--*G, and Rat-STP-C488/97S-+A cells. Radiolabeling of cells and immunoprecipitation were done as described in the legend to Fig. 1. The molecular mass markers are the same as in Fig. 1. Lane 1, Rat-STPC488; lane 2, Rat-STP-C488/3S--*G; lane 3, Rat-STP-C488/97S-*A. (B) Immunoblot analysis of STP-C488, STP-C488/3S--G, and STPC488/97S-*A. Cell extracts were subjected to SDS-PAGE and immunoblotted as described previously (10). The molecular mass markers are lysozyme (14 kDa), trypsin inhibitor (20 kDa), carbonic anhydrase (29 kDa), alcohol dehydrogenase (40 kDa), catalase (58 kDa), and phosphorylase b (97 kDa). Lane 1, Rat-STP-C488; lane 2, Rat-STPC488/3S-*G; lane 3, Rat-STP-C488/97S-*A. (C) STP-C488 contains a phosphoserine residue. Rat-STP-C488 cells were labeled with 32pO4, and proteins were immunoprecipitated with anti-STP-C488 antibody. The proteins were separated by SDS-PAGE, the STP-C488 protein was extracted from the gel, and phosphoamino acid analysis was carried out by cellulose thin-layer chromatography (4). The positions of origin (0), phosphoserine (P-SER), phosphothreonine (P-THR), and phosphotyrosine (P-TYR) are indicated.
with 32p04. 32P-labeled STP-C488 was immunoprecipitated, localized in polyacrylamnide gels by direct autoradiography, and cut out from multiple lanes of the gel. The labeled STP-C488 was extracted from the gel and hydrolyzed for 1 h at 110°C in 6 N HCl as described by Cooper et al. (4). Phosphoamino acids were resolved by one-dimensional cellulose thin-layer chromatography (EM Science, Gibbstown, N.J.) in isobutyric acid-0.5 M ammonium hydroxide (5:3) with unlabeled phosphoamino acids phosphoserine, phosphothreonine, and phosphotyrosine (Sigma Chemical Co., St. Louis, Mo.) as standards for the chromatography. The positions of unlabeled phosphoamino acid standards were determined by staining with ninhydrin. The phosphoamino acid analysis revealed only the presence of phosphoserine among the three potential phosphorylated amino acids (Fig. 2C). The DNA sequence of STP-C488 predicts two serines, at residue 3 near the amino terminus (3S) and at residue 97 near the carboxyl terminus (97S). To determine which serine residue was phosphorylated, amino acid substitutions at these serine residues of STP-C488 were generated by oligonucleotide-directed site-specific mutagenesis by using polymerase chain reaction. Oligonucleotide primers from
VOL. 66, 1992
complementary strands representing the 5' and 3' ends of STP-C488 were synthesized with EcoRI and BamHI sites at the 5' ends to facilitate cloning into retrovirus vector LXSN (14). For mutagenesis, oligonucleotide primers for 3S or 97S mutation contained a nucleotide change from AGC (Ser) to GGC (Gly) or from TCT (Ser) to GCT (Ala), respectively. Polymerase chain reaction cycling was accomplished with a DNA thermal cycler (Perkin-Elmer Cetus Instruments, Norwalk, Conn.) with the following conditions: 30 cycles of 2 min at 53°C for annealing, 2 min at 72°C for polymerization, and 1 min at 94°C for denaturation. The amplified DNA fragments containing STP-C488/3S--G or STP-C488/97S--3A were purified and cloned into the EcoRI and BamHI cloning site of the retrovirus vector LXSN. STP-C488/3S--G and STP-C488/97S--A DNAs were completely sequenced to verify the presence of the mutations and the absence of any other changes. Ten micrograms of recombinant retrovirus vector DNA LXSN-STP-C488/3S-+G or LXSN-STP-C488/ 97S- A, was introduced into Rat-1 cells by electroporation (Bio-Rad, Richmond, Calif.) at 250 V and 960 ,uF in serumfree Dulbecco's modified Eagle's medium (GIBCO-Bethesda Research Laboratories). Cells were incubated with Dulbecco's modified Eagle's medium supplemented with 10% donor bovine calf serum (Hazleton, Lenexa, Kans.). After 48 h of incubation, resistant cells were selected with 500 ,ug of G418 (GIBCO-Bethesda Research Laboratories) per ml and called Rat-STP-C488/3S-*G or Rat-STP-C488/97S--+A. Immunoblot analysis revealed a major STP-C488 protein migrating at 20 kDa (approximately 85%) and a less abundant species migrating at 22 kDa (approximately 15%) in Rat-STP-C488 and Rat-STP-C488/97S--+A cells (Fig. 2B, lanes 1 and 3). This is the same pattern of reactivity we have described previously for authentic STP-C488 protein (10). However, only the 20-kDa STP-C488 protein was detected on immunoblots from Rat-STP-C488/3S--)G cells (Fig. 2B, lane 2). In a separate experiment, cells were labeled with 32P04 and proteins were immunoprecipitated with anti-STP-C488 antibody. 32P-labeled STP-C488 protein migrating at 22 kDa was detected from Rat-STP-C488 and Rat-STP-C488/97S--A cells (Fig. 2A, lanes 1 and 3), but labeled STP-C488 was not detected from Rat-STP-C488/3S-'G cells (Fig. 2A, lane 2). These results indicate that the serine residue at position 3 in the STP-C488 reading frame is the site of phosphorylation in vivo. Previous studies (10) and the results described above show that STP-C488 migrates as a 20-kDa-22-kDa doublet in SDSPAGE. Furthermore, our current results indicate that the doublet at 20 to 22 kDa represents phosphorylated and nonphosphorylated forms of STP-C488. To provide further evidence for this, we attempted to convert the more slowly migrating species to the predominant 20-kDa form by phosphatase treatment. Cell extracts of Rat-STP-C488, Rat-STPC488/3S-)G, and Rat-STP-C488/97S--*A were incubated with or without calf intestinal alkaline phosphatase (Boehringer Mannheim, Indianapolis, Ind.) for 30 min, resolved by SDSPAGE, and immunoblotted. Without alkaline phosphatase, wild-type Rat-STP-C488 and mutant Rat-STP-C488/97S--.A cells yielded the 20-kDa-22-kDa doublet in SDS-PAGE (Fig. 3, lanes 1 and 3). However, mutant Rat-STP-C488/3S--+G cells yielded only the 20-kDa species (Fig. 3, lane 2). After treatment with alkaline phosphatase, the slowly migrating species from STP-C488 and STP-C488/97S--A cells were indeed converted to the faster-migrating species (Fig. 3, lanes 4 and 6). The amounts of wild-type and mutant STP-C488 proteins were approximately the same after all reactions with or without alkaline phosphatase, suggesting that the removal
NOTES
1779
12 3 4 5 6
FIG. 3. Identification of the phosphorylated species of STPC488. The same amounts of cell extracts from Rat-STP-C488 (lanes 1 and 4), Rat-STP-C488/3S--G (lanes 2 and 5), and Rat-STP-C488/ 97S--A (lanes 3 and 6) were incubated with (lanes 4, 5, and 6) or without (lanes 1, 2, and 3) 5 U of calf intestinal alkaline phosphatase for 30 min. Reaction mixtures were loaded onto SDS-PAGE and immunoblotted with anti-STP-C488 antibody. The arrow indicates the more slowly migrating species of STP-C488.
of the phosphate group, rather than nonspecific proteolytic cleavage, caused the shift in migration. These results provide further evidence that the species with a slower mobility (22 kDa) is the phosphorylated form of STP-C488. Previous studies demonstrated that expression of the STP-C488 gene phenotypically transformed Rat-1 cells resulting in formation of foci, growth at reduced concentration of serum, and growth to higher cell densities (11). Since protein phosphorylation is a major mechanism by which activity and specificity of proteins can be altered, we investigated whether phosphorylation was important for transformation by STP-C488. Morphologic change was used to assess the transforming activity of STP-C488 in Rat-1 cells. G418-resistant Rat-LXSN, Rat-STP-C488, and Rat-STPC488/3S- G cells (105) were plated in 25-cm2 flasks and maintained with Dulbecco's modified Eagle's medium supplemented with 10% serum changed every 4 days. By day 14, Rat-STP-C488 and Rat-STP-C488/3S-+G cells formed many foci of heaped cells with loss of contact inhibition (Fig. 4B and C). However, foci of cells transformed by STP-C488/ 3S-*G did not appear as dense or as deeply piled as cells transformed by the parental STP-C488. In contrast, RatLXSN cells containing only the retrovirus vector grew into a flat monolayer, like normal Rat-1 cells, and did not pile (Fig. 4A). Rat-1 cells transformed by STP-C488/3S-+G formed tumors in nude mice, similar to cells transformed by wildtype STP-C488 (data not shown). This report demonstrates that the herpesvirus saimiri oncoprotein STP-C488 is phosphorylated in vivo at a serine residue near the amino terminus. Labeling with 32P04 and treatment with phosphatase demonstrated that the more slowly migrating species is phosphorylated and that phosphorylation is responsible for the slower migration. However, phosphorylation does not appear to be essential for STP-C488 transformation of Rat-1 cells. Phosphorylation and dephosphorylation of proteins catalyzed by protein kinases and protein phosphatases are recognized as major processes for regulating cellular functions. Particularly, protein phosphorylation is prominent for the role that it plays in signal transduction. Signals impinging on cells have their effects amplified and disseminated by a network of protein phosphorylation-dephosphorylation reactions. Many serine and threonine protein kinases contain consensus sequences for phosphorylation sites (8). The amino acid sequence around the phosphorylation site in STP-C488 is not highly homologous to these previously identified consensus sequences. Nonetheless, there is some similarity to the site of phosphorylation by casein kinase II. Genetic variants of casein that are phosphorylated by casein kinase II are characterized by a cluster of acidic residues on
1780
J. VIROL.
NOTES
C.
B.
A
Rat- LX SN
Rat- STP-C488
Rat-STP-C488 /3S-oG
FIG. 4. Transformation ability of STP-C488 and STP-C488/3S-*G. Rat-1 cells were electroporated with plasmid LXSN, LXSN-STP-C488, and LXSN-STP-C488/3S--+G and incubated with 500 ,ug of G418 per ml for selection. G418-resistant Rat-LXSN, Rat-STP-C488, and Rat-STP-C488/3S-+G cells (105) were plated in 25-cm2 flasks, incubated with medium changes every 4 days, and photographed at 14 days at a magnification of x80 to show morphologic transformation.
the carboxyl side of the phosphorylated serine (8). STP-C488 does contain a cluster of acidic amino acids just downstream of the phosphorylated serine residue. Casein kinase II has been implicated in the regulation of RNA and protein synthesis as well as DNA metabolism by phosphorylating the enzymes and proteins mediating those processes (8). Human papillomavirus oncoprotein E7 has also been shown to be a substrate for casein kinase II (1). Our results suggest that phosphorylation is not essential for STP-C488 transformation of Rat-1 cells. Similar results have been reported previously in studies with simian virus 40 large T antigen (16) and in some (3, 17) but not all (1) studies with human papillomavirus oncoprotein E7. Phosphorylation-negative mutants of simian virus 40 large T antigen retained focus-forming activity in Rat-2 cells (16). Nonphosphorylated E7 protein was reported to still transform primary baby rat kidney and rat embryo fibroblasts with the cooperation of an activated ras gene (3, 17). However, the potential importance of phosphorylation should not be discounted, at least in the case of STP-C488. First, a retrovirus vector was used for STP-C488 gene transfer and expression. Strong transcription from the murine leukemia virus promoter may result in overexpression which could possibly overcome the loss of phosphorylation. Secondly, the microscopic appearance of cells transformed by STP-C488/3S--+G was slightly different from that of cells transformed by the parental STP-C488 (Fig. 4). Finally, the biological role of STP-C488 phosphorylation may be cell specific. Since herpesvirus saimiri is T lymphotropic, phosphorylation of STPC488 may be important for the action of this oncoprotein in T lymphocytes. Further studies are thus needed to clarify the importance of phosphorylation for STP-C488 activity. We thank Beverly Blake for critical reading of the manuscript. This work was supported by Public Health Service grant 31363 from the National Cancer Institute and RR00168 from the Division of Research Resources.
REFERENCES 1. Barbosa, M. S., C. Edmonds, C. Fisher, J. T. Schiller, D. R. Lowy, and K. H. Vousden. 1990. The region of the HPV E7 oncoprotein homologous to adenovirus Ela and SV40 large T antigen contains separate domains for Rb binding and casein kinase II phosphorylation. EMBO J. 9:153-160. 2. Biesinger, B., J. J. Trimble, R. C. Desrosiers, and B. Fleckenstein. 1990. The divergence between two oncogenic Herpesvirus
3.
4. 5.
6. 7.
8. 9.
10. 11.
12. 13.
14. 15. 16.
17.
saimiri strains in a genomic region related to the transforming phenotype. Virology 176:505-514. Chesters, P. M., K. H. Vousden, C. Edmonds, and D. J. McCance. 1990. Analysis of human papillomavirus type 16 open reading frame E7 immortalizing function in rat embryo fibroblast cells. J. Gen. Virol. 71:449-453. Cooper, J. A., B. M. Sefton, and T. Hunter. 1983. Detection and quantification of phosphotyrosine in proteins. Methods Enzymol. 99:387-402. Desrosiers, R. C., A. Bakker, J. Kamine, L. A. Falk, R. D. Hunt, and N. W. King. 1985. A region of the Herpesvirus saimini genome required for oncogenicity. Science 228:184-187. Desrosiers, R. C., R. L. Burghoff, A. Bakker, and J. Kamine. 1984. Construction of replication-competent Herpesvirus saimin deletion mutants. J. Virol. 49:343-348. Desrosiers, R. C., D. P. Silva, L. M. Waldron, and N. L. Letvin. 1986. Nononcogenic deletion mutants of herpesvirus saimiri are defective for in vitro immortalization. J. Virol. 57:701-705. Edelman, A. M., D. K. Blumenthal, and E. G. Krebs. 1987. Protein serine/threonine kinases. Annu. Rev. Biochem. 56:567-613. Fleckenstein, B., and R. C. Desrosiers. 1982. Herpesvirus saimiri and herpesvirus ateles, p. 253-332. In B. Roizman (ed.), The herpesviruses, vol. 1. Plenum Press, New York. Jung, J. U., and R. C. Desrosiers. 1991. Identification and characterization of the herpesvirus saimiri oncoprotein STPC488. J. Virol. 65:6953-6960. Jung, J. U., J. J. Trimble, N. W. King, B. Biesinger, B. W. Fleckenstein, and R. C. Desrosiers. 1991. Identification of transforming genes of subgroup A and C strains of Herpesvirus saimin. Proc. Natl. Acad. Sci. USA 88:7051-7055. Koomey, J. M., C. Mulder, R. L. Burghoff, B. Fleckenstein, and R. C. Desrosiers. 1984. Deletion of DNA sequences in a nononcogenic variant of Herpesvirus saimiri. J. Virol. 50:662-665. Medveczky, P., E. Szomolanyi, R. C. Desrosiers, and C. Mulder. 1984. Classification of herpesvirus saimiri into three groups based on extreme variation in a DNA region required for oncogenicity. J. Virol. 52:938-944. Miller, A. D., and G. J. Rosman. 1989. Improved retroviral vectors for gene transfer and expression. BioTechniques 7:980-990. Murthy, S. C. S., J. J. Trimble, and R. C. Desrosiers. 1989. Deletion mutants of herpesvirus saimiri define an open reading frame necessary for transformation. J. Virol. 63:3307-3314. Schneider, J., and E. Fanning. 1988. Mutations in the phosphorylation sites of simian virus 40 (SV40) T antigen alter its origin DNA-binding specificity for sites I or II and affect SV40 DNA replication activity. J. Virol. 62:1598-1605. Storey, A., N. Almond, K. Osborn, and L. Crawford. 1990. Mutations of the human papillomavirus type 16 E7 gene that affect transformation, transactivation and phosphorylation by the E7 protein. J. Gen. Virol. 71:965-970.
Vol. 66, No. 3
0022-538X/92/031777-04$02.00/0 Copyright © 1992, American Society for Microbiology
Herpesvirus Saimiri Oncogene STP-C488 Encodes
a
Phosphoprotein
JAE U. JUNG AND RONALD C. DESROSIERS* New England Regional Primate Research Center, Harvard Medical School, Southborough, Massachusetts 01772-9102 Received 30 September 1991/Accepted 27 November 1991
The STP-C488 open reading frame of herpesvirus saimiri encodes an oncoprotein that has transforming and tumor-inducing activities independent of the rest of the herpesvirus genome. STP-C488 protein has an unusual, membrane-associated, fibrous structure and is located primarily in perinuclear compartments. We now report that STP-C488 is phosphorylated in vivo. The phosphorylated form, which accounted for about 15% of STP-C488 in transformed cells, migrated slightly more slowly through sodium dodecyl sulfate-polyacrylamide gels than unphosphorylated STP-C488. A serine residue near the amino terminus was shown to be the site of phosphorylation. However, phosphorylation was not required for transformation of Rat-i cells by STP-C488.
Herpesvirus saimiri is a member of the gamma subfamily of herpesviruses. Some members of this group, e.g., EpsteinBarr virus, herpesvirus saimiri, herpesvirus ateles, and herpesvirus sylvilagus, are capable of inducing lymphoproliferative disorders in natural or experimental hosts. Herpesvirus saimiri is apparently not associated with disease in its natural host, the squirrel monkey (Saimin sciureus). However, it does induce rapidly progressing fatal lymphomas, leukemias, and lymphosarcomas in several other species of New World primates (9). Herpesvirus saimiri strains have been divided into three subgroups (A, B, and C) based on the extent of DNA sequence divergence at the left terminus of L-DNA (13). Subgroup A and C viruses are highly oncogenic in vivo and efficiently transform common marmoset peripheral blood T lymphocytes in vitro to interleukin 2-independent growth (7). Mutagenic analyses have demonstrated that the leftmost open reading frame of group A strain 11 is required for the immortalization of common marmoset T lymphocytes in vitro and for lymphoma induction in vivo but not for virus replication (5, 6, 12, 15). This open reading frame, called STP-All, and an analogous open reading frame called STP-C488 from the group C strain 488 genome are capable of transforming Rat-1 cells in culture (11). Transformation by STP-C488 resulted in loss of contact inhibition, formation of foci, growth at reduced concentration of serum, and formation of invasive tumors in nude mice. STP-A11 was less potent in its transforming activity (11). Except for hydrophobic, putative membrane-spanning domains at their carboxyl termini, only limited sequence identities are apparent between STP-A11 and STP-C488 (2). Close inspection of the STP-A11 and STP-C488 sequences, however, reveals a similar organization in terms of the presence and locations of specific structural motifs (11). Both proteins are predicted to have acidic amino termini (pI = 3.5 for the amino-terminal 32 amino acids of STP-A11 and pl = 4.4 for the amino-terminal 17 amino acids of STP-C488). These acidic domains are likely to be the active or ligandbinding sites. STP-C488 is predicted to contain 18 uninterrupted collagenlike repeats, and STP-A11 is predicted to have 9 collagenlike repeats. The collagenlike repeats are Gly-X-Y, where X and/or Y is proline. The collagenlike motifs in STP-A11 are not directly repeated as they are in STP-C488, *
but they seem to be similarly concentrated in the central portion of the protein (11). These collagenlike repeats can be inferred to form a fibrous structure similar to that of cellular collagen and may serve as a hinge to extend the active domain of the protein to its site of action. STP-All and STP-C488 also contain a hydrophobic stretch at their carboxyl termini sufficient for a membrane-spanning domain. Recently, a polyclonal anti-STP-C488 antibody was used to identify the protein encoded by STP-C488 (10). The apparent molecular mass of STP-C488 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (20 to 22 kDa) was considerably higher than that predicted from the DNA sequence (9.9 kDa). STP-C488 was sensitive to treatment with bacterial collagenase, consistent with the 18 uninterrupted collagenlike repeats predicted by the DNA sequence, was associated with cytoplasmic membranes, and was found primarily in perinuclear compartments. These results suggest an unusual, membrane-associated, fibrous structure for the transforming STP-C488 oncoprotein. In addition to the major STP-C488 protein which migrated with an apparent molecular mass of 20 kDa, a less abundant species of slightly slower mobility (approximately 22 kDa) has also been previously observed (10). This 20-kDa-22-kDa doublet of STP-C488 in SDS-PAGE suggested the presence of a modified form. The experiments described here demonstrate that STP-C488 protein is phosphorylated at a serine residue near the amino terminus, that the 20- and 22-kDa species represent unphosphorylated and phosphorylated forms of the protein, and that phosphorylation is not required for transformation of Rat-1 cells by STP-C488. To demonstrate phosphorylation of STP-C488 in vivo, cells were labeled with 32P04. Rat-STP-C488 cells, which stably express STP-C488 protein in Rat-1 cells, and Rat-LXSN cells, which contain only the LXSN retrovirus vector (10), were used for labeling. When cells reached 80 to 90% confluence in a 25-cm2 dish, they were rinsed twice with labeling medium (phosphate-free minimal essential medium plus 5% dialyzed fetal calf serum [GIBCO-Bethesda Research Laboratories, Gaithersburg, Md.]) and preincubated for 30 min with this same medium. 32P04 (New England Nuclear Corp., Boston, Mass.) was added at a concentration of 0.5 mCi/ml, and the incubation was continued for 3 h. Cells were harvested by scraping with a rubber policeman, washed once with phosphate-buffered saline, and lysed with 1 ml of RIPA buffer (0.15 M NaCl, 1% [vol/vol] Nonidet P-40, 0.5% [wtlvol] sodium deoxycholate, 0.1% [wtlvol] SDS, 50 mM Tris [pH
Corresponding author. 1777
1778
J. VIROL.
NOTES
1
2
A.
1 2 3
B.
2 3
97-
69-
C.
58 -
69 -
0-
47 -
40.:
47-
i:.
29-
29 -
_U'~
20-
~.
P-SERP-THR-
P-TYR-
29 -
14 -
FIG. 1. Phosphorylation of STP-C488. G418-resistant Rat-LXSN and Rat-STP-C488 cells were labeled with 32PO4. Labeled cell extracts were incubated with anti-STP-C488 antibody, and immunoprecipated proteins were analyzed by SDS-PAGE. Lane 1, Rat-LXSN; lane 2, Rat-STP-C488. The 14C molecular mass markers are lysozyme (14 kDa), carbonic anhydrase (29 kDa), ovalbumin (47 kDa), and bovine serum albumin (69 kDa). The arrow indicates STP-C488.
7.5], 10 ,ug of aprotinin [U.S. Biochemicals, Cleveland, Ohio] per ml) for 30 min at 4°C with shaking. Lysates were cleared by centrifugation for 5 min at 13,600 x g, and the supematant was transferred to a clean tube. Rabbit anti-STP-C488 polyclonal antiserum was used as described previously to identify STP-C488 protein by immunoprecipitation (10). Five microliters of anti-STP-C488 antibody and 30 RI of protein A-agarose (Oncogene Science, Manhasset, N.Y.) were added to 500 RI of RIPA buffer containing 100 RI of supematant and incubated for 4 h at 4°C with shaking. The pellet was washed five times by suspending the pellet in 1 ml of RIPA buffer and vortexing, centrifuging, and aspirating the supernatant. Finally, the pellet was rinsed twice with 10 mM Tris (pH 7.5), suspended in SDS-PAGE sample buffer, and boiled for 5 min. Proteins were resolved by SDS-PAGE (12.5% polyacrylamide), and the gel was dried and exposed to Kodak XAR-5 film at -70°C. Anti-STP-C488 antibody specifically precipitated a 32P-labeled 22-kDa protein only from Rat-STP-C488 cells (Fig. 1, lane 2). No such protein was precipitated from control RatLXSN cells lacking the STP-C488 sequence (Fig. 1, lane 1). This result demonstrated that STP-C488 was phosphorylated in vivo. There are three phosphorylhydroxyamino acids in cellular proteins, phosphoserine, phosphothreonine, and phosphotyrosine. To determine the identity of the 32P-labeled amino acid(s) in STP-C488, Rat-STP-C488 cells were first labeled
14-
14 -
FIG. 2. STP-C488 is phosphorylated at a serine residue near the amino terminus. (A) Immunoprecipitation of STP-C488 from 32p_ labeled cell lysates of Rat-STP-C488, Rat-STP-C488/3S--*G, and Rat-STP-C488/97S-+A cells. Radiolabeling of cells and immunoprecipitation were done as described in the legend to Fig. 1. The molecular mass markers are the same as in Fig. 1. Lane 1, Rat-STPC488; lane 2, Rat-STP-C488/3S--*G; lane 3, Rat-STP-C488/97S-*A. (B) Immunoblot analysis of STP-C488, STP-C488/3S--G, and STPC488/97S-*A. Cell extracts were subjected to SDS-PAGE and immunoblotted as described previously (10). The molecular mass markers are lysozyme (14 kDa), trypsin inhibitor (20 kDa), carbonic anhydrase (29 kDa), alcohol dehydrogenase (40 kDa), catalase (58 kDa), and phosphorylase b (97 kDa). Lane 1, Rat-STP-C488; lane 2, Rat-STPC488/3S-*G; lane 3, Rat-STP-C488/97S-*A. (C) STP-C488 contains a phosphoserine residue. Rat-STP-C488 cells were labeled with 32pO4, and proteins were immunoprecipitated with anti-STP-C488 antibody. The proteins were separated by SDS-PAGE, the STP-C488 protein was extracted from the gel, and phosphoamino acid analysis was carried out by cellulose thin-layer chromatography (4). The positions of origin (0), phosphoserine (P-SER), phosphothreonine (P-THR), and phosphotyrosine (P-TYR) are indicated.
with 32p04. 32P-labeled STP-C488 was immunoprecipitated, localized in polyacrylamnide gels by direct autoradiography, and cut out from multiple lanes of the gel. The labeled STP-C488 was extracted from the gel and hydrolyzed for 1 h at 110°C in 6 N HCl as described by Cooper et al. (4). Phosphoamino acids were resolved by one-dimensional cellulose thin-layer chromatography (EM Science, Gibbstown, N.J.) in isobutyric acid-0.5 M ammonium hydroxide (5:3) with unlabeled phosphoamino acids phosphoserine, phosphothreonine, and phosphotyrosine (Sigma Chemical Co., St. Louis, Mo.) as standards for the chromatography. The positions of unlabeled phosphoamino acid standards were determined by staining with ninhydrin. The phosphoamino acid analysis revealed only the presence of phosphoserine among the three potential phosphorylated amino acids (Fig. 2C). The DNA sequence of STP-C488 predicts two serines, at residue 3 near the amino terminus (3S) and at residue 97 near the carboxyl terminus (97S). To determine which serine residue was phosphorylated, amino acid substitutions at these serine residues of STP-C488 were generated by oligonucleotide-directed site-specific mutagenesis by using polymerase chain reaction. Oligonucleotide primers from
VOL. 66, 1992
complementary strands representing the 5' and 3' ends of STP-C488 were synthesized with EcoRI and BamHI sites at the 5' ends to facilitate cloning into retrovirus vector LXSN (14). For mutagenesis, oligonucleotide primers for 3S or 97S mutation contained a nucleotide change from AGC (Ser) to GGC (Gly) or from TCT (Ser) to GCT (Ala), respectively. Polymerase chain reaction cycling was accomplished with a DNA thermal cycler (Perkin-Elmer Cetus Instruments, Norwalk, Conn.) with the following conditions: 30 cycles of 2 min at 53°C for annealing, 2 min at 72°C for polymerization, and 1 min at 94°C for denaturation. The amplified DNA fragments containing STP-C488/3S--G or STP-C488/97S--3A were purified and cloned into the EcoRI and BamHI cloning site of the retrovirus vector LXSN. STP-C488/3S--G and STP-C488/97S--A DNAs were completely sequenced to verify the presence of the mutations and the absence of any other changes. Ten micrograms of recombinant retrovirus vector DNA LXSN-STP-C488/3S-+G or LXSN-STP-C488/ 97S- A, was introduced into Rat-1 cells by electroporation (Bio-Rad, Richmond, Calif.) at 250 V and 960 ,uF in serumfree Dulbecco's modified Eagle's medium (GIBCO-Bethesda Research Laboratories). Cells were incubated with Dulbecco's modified Eagle's medium supplemented with 10% donor bovine calf serum (Hazleton, Lenexa, Kans.). After 48 h of incubation, resistant cells were selected with 500 ,ug of G418 (GIBCO-Bethesda Research Laboratories) per ml and called Rat-STP-C488/3S-*G or Rat-STP-C488/97S--+A. Immunoblot analysis revealed a major STP-C488 protein migrating at 20 kDa (approximately 85%) and a less abundant species migrating at 22 kDa (approximately 15%) in Rat-STP-C488 and Rat-STP-C488/97S--+A cells (Fig. 2B, lanes 1 and 3). This is the same pattern of reactivity we have described previously for authentic STP-C488 protein (10). However, only the 20-kDa STP-C488 protein was detected on immunoblots from Rat-STP-C488/3S--)G cells (Fig. 2B, lane 2). In a separate experiment, cells were labeled with 32P04 and proteins were immunoprecipitated with anti-STP-C488 antibody. 32P-labeled STP-C488 protein migrating at 22 kDa was detected from Rat-STP-C488 and Rat-STP-C488/97S--A cells (Fig. 2A, lanes 1 and 3), but labeled STP-C488 was not detected from Rat-STP-C488/3S-'G cells (Fig. 2A, lane 2). These results indicate that the serine residue at position 3 in the STP-C488 reading frame is the site of phosphorylation in vivo. Previous studies (10) and the results described above show that STP-C488 migrates as a 20-kDa-22-kDa doublet in SDSPAGE. Furthermore, our current results indicate that the doublet at 20 to 22 kDa represents phosphorylated and nonphosphorylated forms of STP-C488. To provide further evidence for this, we attempted to convert the more slowly migrating species to the predominant 20-kDa form by phosphatase treatment. Cell extracts of Rat-STP-C488, Rat-STPC488/3S-)G, and Rat-STP-C488/97S--*A were incubated with or without calf intestinal alkaline phosphatase (Boehringer Mannheim, Indianapolis, Ind.) for 30 min, resolved by SDSPAGE, and immunoblotted. Without alkaline phosphatase, wild-type Rat-STP-C488 and mutant Rat-STP-C488/97S--.A cells yielded the 20-kDa-22-kDa doublet in SDS-PAGE (Fig. 3, lanes 1 and 3). However, mutant Rat-STP-C488/3S--+G cells yielded only the 20-kDa species (Fig. 3, lane 2). After treatment with alkaline phosphatase, the slowly migrating species from STP-C488 and STP-C488/97S--A cells were indeed converted to the faster-migrating species (Fig. 3, lanes 4 and 6). The amounts of wild-type and mutant STP-C488 proteins were approximately the same after all reactions with or without alkaline phosphatase, suggesting that the removal
NOTES
1779
12 3 4 5 6
FIG. 3. Identification of the phosphorylated species of STPC488. The same amounts of cell extracts from Rat-STP-C488 (lanes 1 and 4), Rat-STP-C488/3S--G (lanes 2 and 5), and Rat-STP-C488/ 97S--A (lanes 3 and 6) were incubated with (lanes 4, 5, and 6) or without (lanes 1, 2, and 3) 5 U of calf intestinal alkaline phosphatase for 30 min. Reaction mixtures were loaded onto SDS-PAGE and immunoblotted with anti-STP-C488 antibody. The arrow indicates the more slowly migrating species of STP-C488.
of the phosphate group, rather than nonspecific proteolytic cleavage, caused the shift in migration. These results provide further evidence that the species with a slower mobility (22 kDa) is the phosphorylated form of STP-C488. Previous studies demonstrated that expression of the STP-C488 gene phenotypically transformed Rat-1 cells resulting in formation of foci, growth at reduced concentration of serum, and growth to higher cell densities (11). Since protein phosphorylation is a major mechanism by which activity and specificity of proteins can be altered, we investigated whether phosphorylation was important for transformation by STP-C488. Morphologic change was used to assess the transforming activity of STP-C488 in Rat-1 cells. G418-resistant Rat-LXSN, Rat-STP-C488, and Rat-STPC488/3S- G cells (105) were plated in 25-cm2 flasks and maintained with Dulbecco's modified Eagle's medium supplemented with 10% serum changed every 4 days. By day 14, Rat-STP-C488 and Rat-STP-C488/3S-+G cells formed many foci of heaped cells with loss of contact inhibition (Fig. 4B and C). However, foci of cells transformed by STP-C488/ 3S-*G did not appear as dense or as deeply piled as cells transformed by the parental STP-C488. In contrast, RatLXSN cells containing only the retrovirus vector grew into a flat monolayer, like normal Rat-1 cells, and did not pile (Fig. 4A). Rat-1 cells transformed by STP-C488/3S-+G formed tumors in nude mice, similar to cells transformed by wildtype STP-C488 (data not shown). This report demonstrates that the herpesvirus saimiri oncoprotein STP-C488 is phosphorylated in vivo at a serine residue near the amino terminus. Labeling with 32P04 and treatment with phosphatase demonstrated that the more slowly migrating species is phosphorylated and that phosphorylation is responsible for the slower migration. However, phosphorylation does not appear to be essential for STP-C488 transformation of Rat-1 cells. Phosphorylation and dephosphorylation of proteins catalyzed by protein kinases and protein phosphatases are recognized as major processes for regulating cellular functions. Particularly, protein phosphorylation is prominent for the role that it plays in signal transduction. Signals impinging on cells have their effects amplified and disseminated by a network of protein phosphorylation-dephosphorylation reactions. Many serine and threonine protein kinases contain consensus sequences for phosphorylation sites (8). The amino acid sequence around the phosphorylation site in STP-C488 is not highly homologous to these previously identified consensus sequences. Nonetheless, there is some similarity to the site of phosphorylation by casein kinase II. Genetic variants of casein that are phosphorylated by casein kinase II are characterized by a cluster of acidic residues on
1780
J. VIROL.
NOTES
C.
B.
A
Rat- LX SN
Rat- STP-C488
Rat-STP-C488 /3S-oG
FIG. 4. Transformation ability of STP-C488 and STP-C488/3S-*G. Rat-1 cells were electroporated with plasmid LXSN, LXSN-STP-C488, and LXSN-STP-C488/3S--+G and incubated with 500 ,ug of G418 per ml for selection. G418-resistant Rat-LXSN, Rat-STP-C488, and Rat-STP-C488/3S-+G cells (105) were plated in 25-cm2 flasks, incubated with medium changes every 4 days, and photographed at 14 days at a magnification of x80 to show morphologic transformation.
the carboxyl side of the phosphorylated serine (8). STP-C488 does contain a cluster of acidic amino acids just downstream of the phosphorylated serine residue. Casein kinase II has been implicated in the regulation of RNA and protein synthesis as well as DNA metabolism by phosphorylating the enzymes and proteins mediating those processes (8). Human papillomavirus oncoprotein E7 has also been shown to be a substrate for casein kinase II (1). Our results suggest that phosphorylation is not essential for STP-C488 transformation of Rat-1 cells. Similar results have been reported previously in studies with simian virus 40 large T antigen (16) and in some (3, 17) but not all (1) studies with human papillomavirus oncoprotein E7. Phosphorylation-negative mutants of simian virus 40 large T antigen retained focus-forming activity in Rat-2 cells (16). Nonphosphorylated E7 protein was reported to still transform primary baby rat kidney and rat embryo fibroblasts with the cooperation of an activated ras gene (3, 17). However, the potential importance of phosphorylation should not be discounted, at least in the case of STP-C488. First, a retrovirus vector was used for STP-C488 gene transfer and expression. Strong transcription from the murine leukemia virus promoter may result in overexpression which could possibly overcome the loss of phosphorylation. Secondly, the microscopic appearance of cells transformed by STP-C488/3S--+G was slightly different from that of cells transformed by the parental STP-C488 (Fig. 4). Finally, the biological role of STP-C488 phosphorylation may be cell specific. Since herpesvirus saimiri is T lymphotropic, phosphorylation of STPC488 may be important for the action of this oncoprotein in T lymphocytes. Further studies are thus needed to clarify the importance of phosphorylation for STP-C488 activity. We thank Beverly Blake for critical reading of the manuscript. This work was supported by Public Health Service grant 31363 from the National Cancer Institute and RR00168 from the Division of Research Resources.
REFERENCES 1. Barbosa, M. S., C. Edmonds, C. Fisher, J. T. Schiller, D. R. Lowy, and K. H. Vousden. 1990. The region of the HPV E7 oncoprotein homologous to adenovirus Ela and SV40 large T antigen contains separate domains for Rb binding and casein kinase II phosphorylation. EMBO J. 9:153-160. 2. Biesinger, B., J. J. Trimble, R. C. Desrosiers, and B. Fleckenstein. 1990. The divergence between two oncogenic Herpesvirus
3.
4. 5.
6. 7.
8. 9.
10. 11.
12. 13.
14. 15. 16.
17.
saimiri strains in a genomic region related to the transforming phenotype. Virology 176:505-514. Chesters, P. M., K. H. Vousden, C. Edmonds, and D. J. McCance. 1990. Analysis of human papillomavirus type 16 open reading frame E7 immortalizing function in rat embryo fibroblast cells. J. Gen. Virol. 71:449-453. Cooper, J. A., B. M. Sefton, and T. Hunter. 1983. Detection and quantification of phosphotyrosine in proteins. Methods Enzymol. 99:387-402. Desrosiers, R. C., A. Bakker, J. Kamine, L. A. Falk, R. D. Hunt, and N. W. King. 1985. A region of the Herpesvirus saimini genome required for oncogenicity. Science 228:184-187. Desrosiers, R. C., R. L. Burghoff, A. Bakker, and J. Kamine. 1984. Construction of replication-competent Herpesvirus saimin deletion mutants. J. Virol. 49:343-348. Desrosiers, R. C., D. P. Silva, L. M. Waldron, and N. L. Letvin. 1986. Nononcogenic deletion mutants of herpesvirus saimiri are defective for in vitro immortalization. J. Virol. 57:701-705. Edelman, A. M., D. K. Blumenthal, and E. G. Krebs. 1987. Protein serine/threonine kinases. Annu. Rev. Biochem. 56:567-613. Fleckenstein, B., and R. C. Desrosiers. 1982. Herpesvirus saimiri and herpesvirus ateles, p. 253-332. In B. Roizman (ed.), The herpesviruses, vol. 1. Plenum Press, New York. Jung, J. U., and R. C. Desrosiers. 1991. Identification and characterization of the herpesvirus saimiri oncoprotein STPC488. J. Virol. 65:6953-6960. Jung, J. U., J. J. Trimble, N. W. King, B. Biesinger, B. W. Fleckenstein, and R. C. Desrosiers. 1991. Identification of transforming genes of subgroup A and C strains of Herpesvirus saimin. Proc. Natl. Acad. Sci. USA 88:7051-7055. Koomey, J. M., C. Mulder, R. L. Burghoff, B. Fleckenstein, and R. C. Desrosiers. 1984. Deletion of DNA sequences in a nononcogenic variant of Herpesvirus saimiri. J. Virol. 50:662-665. Medveczky, P., E. Szomolanyi, R. C. Desrosiers, and C. Mulder. 1984. Classification of herpesvirus saimiri into three groups based on extreme variation in a DNA region required for oncogenicity. J. Virol. 52:938-944. Miller, A. D., and G. J. Rosman. 1989. Improved retroviral vectors for gene transfer and expression. BioTechniques 7:980-990. Murthy, S. C. S., J. J. Trimble, and R. C. Desrosiers. 1989. Deletion mutants of herpesvirus saimiri define an open reading frame necessary for transformation. J. Virol. 63:3307-3314. Schneider, J., and E. Fanning. 1988. Mutations in the phosphorylation sites of simian virus 40 (SV40) T antigen alter its origin DNA-binding specificity for sites I or II and affect SV40 DNA replication activity. J. Virol. 62:1598-1605. Storey, A., N. Almond, K. Osborn, and L. Crawford. 1990. Mutations of the human papillomavirus type 16 E7 gene that affect transformation, transactivation and phosphorylation by the E7 protein. J. Gen. Virol. 71:965-970.
