identification of two additional v-sea-encoded proteins in avian erythroblastosis virus,...

VIROLOGY 178,232-237 (1990)

Identification of Two Additional v-sea-Encoded Proteins in Avian Erythroblastosis Virus, S134nfected Fibroblasts

JENNIFER KNIGHT,’ DOUGLAS R. SMITH,**’ AND MICHAELJ. HAYMAN

Department of Microbiology, State University of New York at Stony Brook, Stony Brook, New York 11794; and ‘Imperial Cancer Research Fund Laboratories, Dominion House, St. Bartholomew’s Hospital, London EC 1 7BE. United Kingdom

Received March 13, 1990; accepted May 14, 1990

Rabbit antibodies prepared against a v-sea-encoded polypeptide expressed in bacteria were used to characterize the v-sea-encoded proteins in cells transformed by the avian erythroblastosis virus, S13. In addition to the two previously described v-sea-encoded proteins, gp155 and gp70, two additional proteins were identified of molecular weights 38,000 and 36,000 Da. Interestingly, these two proteins were found only in fibroblasts infected with the S13 virus and not in S13-transformed erythroid cells. These two proteins were phosphoproteins, but, unlike the two previously characterized v-sea-encoded proteins, they did not appear to be modified by the addition of N-linked sugars. Possible mechanisms for the biosynthesis of these two new proteins are discussed. o 1990 Academic PWSS, IW.

INTRODUCTION

The avian retrovirus, S13, is a replication-defective virus that has the ability to transform fibroblasts and erythroblasts in in vitro tissue culture systems (Bene- dict et al., 1985; Beug et al., 1985). S13 virally infected chickens develop fibrosarcomas, erythroblastosis, and an associated anemia in vivo (Stubbs and Furth, 1935). Analysis of the S13 viral genome demonstrated that the viral RNA is 8.5 kilobases in length and that in infected cells this RNA encoded normal-sized proteins from the gag and pal genes but the env primary gene product was 155,000 Da rather than the normal size of 95,000 Da (Hayman, 1978; Benedict et al., 1985). Subsequent analysis revealed that this size increase was due to the presence of a cell-derived sequence fused in frame with the normal env gene to generate a glycosylated fusion protein termed gp155 (Hayman et al., 1985; Smith et al., 1989). The cell-derived sequence was termed sea, an acronym derived from the disease potential of the S13 virus, namely sarcoma erythroblastosis, and anemia (Hayman, 1987).

Characterization of the gp155 protein revealed that this protein was subsequently cleaved to generate a 70,000-Da protein, gp70, and an 85,000-Da protein gp85 (Hayman et a/., 1985). gp85 is encoded solely by the env gene, whereas the gp70 protein results from a fusion of the env and sea gene products (Smith et al.,

1 Present address: Institute of Cancer Research, Chester Beatty Laboratories, Fulham Road, London SW3 6JB, UK.

* Present address: Collaborative Research Incorporated, 2 Oak Park, Bedford, MA 01730.

’ To whom requests for reprints should be addressed.

1989). Intracellular localization and topographical analysis demonstrated gp155 to be a glycosylated transmembrane protein oriented such that the env-encoded sequences were extracellular and the sea-encoded sequences were cytoplasmic (Hayman et al., 1985). Fur- thermore, analysis of these protein products revealed that both gp155 and gp70 had an associated protein tyrosine kinase activity (Hayman et a/., 1985). Molecu- lar cloning and nucleic acid sequencing of the S13 virus genome identified sequence motifs within the sea oncogene consistent with its being a member of the protein tyrosine kinase family (Smith et al., 1989). Re- cently a mutant of S13, termed S13 ts-1 , was identified and has been shown to be temperature-sensitive for both transformation and protein tyrosine kinase activity (Knight et al., 1988b). In summation, these data are consistent with the S13 virus transforming cells via an unregulated tyrosine kinase activity that is encoded by the v-sea oncogene.

In all of the above studies, the S13-encoded proteins were detected by virtue of the fusion between the sea and env genes, in that antibodies specific to the env gene were used for immunochemical analyses. In this report we describe the production of antibodies specific to the sea gene using bacterially expressed protein. Using these antibodies we have detected novel sea-encoded proteins of molecular weights 36,000 and 38,000 Da in virally transformed fibroblasts. Inter- estingly these two proteins are not expressed at detectable levels in virally transformed erythroblasts.

MATERIALS AND METHODS Cells and viruses

The origin and characterization of the S13 virus has been documented previously (Benedict et al., 1985;

0042-6822190 $3.00 CopyrIght Q 1990 by Academic Press, Inc. All rights of reproduction in any form reserved

232

v-sea-ENCODED POLYPEPTIDES

Beug et al., 1985). The isolation and characterization of the S13 ts-I virus strain was described previously (Knight eta/., 1988b).

Chicken embryo fibroblasts transformed by the S13 virus were produced by infecting freshly prepared chicken embryo cells derived from 8-day-old embryos (Graf, 1973). Both infected and uninfected fibroblasts were grown in standard growth medium (Dulbecco’s modified Eagle’s medium supplemented with 8% fetal bovine serum and 2% chicken serum). Erythroblasts transformed byS13 were isolated bycocultivating S13- transformed CEFs with bone marrow from 1 -week-old chickens in S13 medium (Kowenz et al., 1987) supplemented with 2% anemic chicken serum as a source of growth factors. After 2 days the nonadherent cells were removed and grown in S13 medium. Virally transformed erythroblasts grew out from these cultures within 7-l 0 days; no cells grew out in the absence of infection.

Bacterial expression and preparation of antisera

The Pstl to C/al fragment was isolated from the pS13 plasmid clone (Smith et a/., 1989) and made blunt- ended with T4 polymerase. This fragment contains the entire v-sea coding sequence, except for the first 21 amino acids. This fragment was cloned into the Smal site of the expression vector pJS413tac (Hayman et al., 1986). Plasmrds containing the insert in the correct ori- entation were identified by restriction enzyme analysis and sequencing of the junction confirmed that the correct reading frame for fusion with the first 24 amino acids of the cro gene was maintained. This plasmid was then transformed into the bacterial strain NFl829. Protein expression via the tat promoter was induced by the addition of isopropyl-@-p-thiogalactoside (IPTG) to a final concentration of 1 mM. An IPTG-inducible protein of the predicted molecular weight of 41,000 Da was isolated by SDS-polyactylamide gel electrophore- sis and used to immunize New Zealand White rabbits as described previously (Hayman et al., 1986).

Radioactive cell labeling and immune precipitations

[35S]Methionine and [32P]orthophosphate labeling of cells and subsequent immune precipitations were car- ried out exactly as described previously (Knight et al., 1988a,b). Pulse-chase analysis was performed as described (Hayman, 1978). Antisera against the envgene product and erbB have been described previously (Hayman, 1978; Hayman eta/., 1986).

In vitro kinase assays

The in vitro immune complex kinase assays were performed on immune precipitates formed with the

1 23 4

gp155--

gP70 -

P38-- p36--

FIG. 1. ldentrfrcatron of p36 and ~38 tn S 13-transformed rat cells by Immune precipitahon. S13-transformed rat cells were labeled with [%]methionine and analyzed by Immune precipitation followed by SDS-PAGE as described under Material and Methods. The antisera used were for lanes 1 and 2, two drfferent bleeds from a rabbrt rmmu ntzed wrth the bacterially expressed v-sea polypeptrde; for lane 3, anti-env serum, and for lane 4, normal rabbit serum.

different rabbit antibodies as described (Hayman eta/., 1985, 1986).

One-dimensional peptide mapping

Peptide maps were performed on phosphate-labeled proteins according to the method of Cleveland et al. (1977).

Endoglycosidase digestions

Immune precipitates were digested with the enzyme endo-@-lv-acetylglucosaminidase H (1 U/ml; Boehrin- ger-Mannheim), endo H, as described (Schmidt et al., 1985).

RESULTS

Identification of two novel sea-encoded proteins

To prepare antisera specific for the v-sea oncogene, a fragment encoding all of the v-sea gene (except the first 2 1 amino acids) was excised from the S13 plasmid clone (Smith et a/., 1989) and ligated in frame with the first 24 amino acids of the ci-o gene in the expression vector pJS413tac (Hayman et a/., 1986). The resultant fusion protein was then expressed and the 41,OOODa protein purified and used to immunize rabbits. These antisera were then tested for their ability to precipitate the gp155 env-sea protein from S13-transformed rat 1 ceils (Fig. 1). As described previously, antisera against the env gene product precipitate the gp155 protein and, less efficiently, the gp70 protein (see Fig. 1, lane 3). In contrast, two different bleeds from rabbits immu- nized with the sea-encoded bacterial protein precipi-

234 KNIGHT, SMITH, AND HAYMAN

FIG. 2. Tyrosine kinase activity and phosphorylation of p36 and ~38. (A) S13 rat cells were labeled with [3zP]orthophosphate and analyzed by immune precipitation followed by SDS-PAGE as described by Knight eta/. (1988a). The immune precipitates were prepared with anti-sea, lane 1, or normal rabbit serum, lane 2. (6) S13 rat cells were lysed and immune precipitates prepared with anti-sea, lane 1, normal rabbit, lane 2, or anti-env serum, lane 3. The immune precipitates were then used for in vitro kinase assays as described previously (Knight et a/., 1988a). The labeled proteins were then visualized by SDS-PAGE and autoradiography.

tate gp155 and gp70 very efficiently and, in addition, precipitate two proteins of apparent molecular weights of 36,000 and 38,000 Da (see Fig. 1, lanes 1 and 2). These proteins will be referred to as p36 and ~38. Pre- immune serum did not precipitate any of these products (see Fig. 1, lane 4).

The finding that the anti-sea serum precipitated two additional proteins can be explained in one of two possible ways. Either these two proteins are encoded by the v-sea gene, or they represent cellular proteins that the antibodies recognize. The latter explanation was considered unlikely since the antibodies did not precipitate these proteins from either normal rat-l or uninfected chicken cells (data not shown) or from cells transformed by other oncogenes, for example, WC- transformed rat cells (see Fig. 6, lane 7 below) or erb- B-transformed chicken cells (data not shown). These cell types were all negative whether the assay used was metabolic labeling or the more sensitive in vitro kinase assay (see below).

If these proteins were encoded by the v-sea gene, it was considered possible that they would be phosphorylated and may have associated tyrosine kinase activity, since the previously characterized v-sea proteins have been shown to have these properties. To test this, S13-transformed rat fibroblasts were labeled with C3’P]- orthophosphate and immune precipitated with either the antiserum against v-sea or a control antiserum. Fig- ure 2A, lane 1, shows that, in addition to gp155 and

gp70, both p38 and p36 are phosphorylated [although the doublet is not well resolved on this gel, these two bands could be resolved and both shown to be phosphorylated on subsequent analysis (data not shown)]. The S13 rat cells were also used to prepare samples for an immune complex kinase assay (Knight et al., 1988a). Figure 26, lane 3, shows the proteins labeled in a kinase assay performed with an immune complex isolated using anti-env serum. The predominant protein phosphorylated is gp155. In contrast, in the immune complex isolated with anti-sea serum, gp70, ~38, and p36 are phosphorylated as well as gp155 (see Fig. 28, lane 1). None of these proteins are labeled in the control precipitate (lane 2). Phosphoamino acid analysis of all four of these proteins confirmed that the amino acid being phosphorylated was tyrosine (data not shown). These data demonstrate that p38 and p36 are phosphorylated proteins found in S13-transformed fibroblasts. From the results of the immune complex assay it is not clear whether they have intrinsic kinase activity or are merely serving as substrates for the known kinases gp155 or gp70.

To determine directly if these two proteins were structurally related to the v-sea proteins, we decided to subject them to one-dimensional peptide mapping (Cleveland et a/., 1977). Phosphate-labeled proteins were isolated from S13-transformed rat 1 cells and digested with increasing amounts of Sraphylococcus aureus V8 protease. Figure 3 shows a comparison of the maps obtained with the p38 and gp155 proteins. The near identity of these maps clearly indicated that

gPl35

P38

FIG. 3. gpl55 and ~38 are structurally related. S13 rat cells were labeled for 4 hr with 4 mCi of [3zP]orthophosphate; the cells were lysed and gp155 and ~38 isolated by immune precipitation followed by SDS-PAGE. The two proteins were then mapped by the method of Cleveland et al. using V8 protease at 0 pg/ml (lane I), 0.5 pg/ml (lane 2) 1 .O pglml (lane 3). and 2.0 Mg/ml (lane 4). Panel A shows the patterns seen with gp155 and panel 6. those with ~38.

v-sea-ENCODED POLYPEPTIDES 235

1 2 3

gp155- - p100

= P38 P3'3

- - +

FIG. 4. p36 and p38 are resistant to Endo H digestion. S13 rat cells were labeled wrth 250 &i of [35S]methionine for 90 min and then immune precipitated with either normal rabbit serum, lane 1, or anti- sea serum, lanes 2 and 3. The proteins precipitated with the anti-sea serum were divided into two alrquots and one was digested wrth Endo H, 0.001 units, at 37” for 16 hr as described previously (Schmidt et al., 1985). The labeled proteins were visualized by SDS- PAGE followed by fluorography.

these two proteins are structurally related. The one-dimensional map of the p36 protein was indistinguish- able from that of ~38 (data not shown).

p36 and p38 are not glycosylated

The two previously identified v-sea-encoded proteins, gp155 and gp70, have both been shown to be glycosylated by the addition of N-linked carbohydrate (Hayman et a/., 1985). Therefore, it was of interest to determine if p38 and p36 were also modified in this manner. To test this, we used the enzyme endo-H which specifically removes N-linked core sugars from glycoproteins. S13-transformed rat 1 cells were pulse- labeled with [35S]methionine and immune complexes formed with either normal rabbit serum (lane 1) or anti- sea serum (lanes 2 and 3). The anti-sea immune precipitate was then divided into two equal parts, and one treated with the endo-H (see Fig. 4, lane 3). The gpl55 protein was specifically converted into a lOO,OOO-Da protein, ~100, by the enzyme treatment but the molecular weights of p38 and p36 were unchanged. These two proteins were also found to be resistant to digestion with the endoglycosidase endo-F, which digests essentially all N-linked carbohydrate side chains (data not shown). Thus, by these criteria ~38 and p36 do not appear to be glycosylated.

Pulse-chase analysis

gp70 has been shown to be derived from gpl55 by proteolytic processing (Hayman et a/., 1985). To determine if there was any possible precursor-product relationship between gp155 and the formation of either

p36 or ~38, a pulse-chase analysis was performed. Parallel dishes of S13-transformed rat 1 cells were labeled for 30 min and then either lysed immediately or chased for various times and then lysed. The lysates were then subjected to immune precipitation analysis, the results of which are shown in Fig. 5. Following the pulse-label, ~38 and p36 were already apparent as is gp155 (Fig. 5, lane 2). During the chase periods the amount of radioactivity associated with gp155 declined and there was an increase in the amount in gp70. In contrast, the amount of radioactivity in p38 and p36 declined steadily during the chase periods and there was no precursor-product relationship evident, either between gp155 and these two proteins or even between the two proteins themselves. Similar experiments using shorter pulse-labeling time periods yielded identical conclusions (data not shown).

p38 and p36 are not found in S13-transformed erythroblasts

The S13 virus is capable of transforming er-ythroid cells as well as fibroblasts. Therefore, we analyzed S13-transformed avian erythroid cells for the presence of ~38 and p36 by immune precipitation of [35S]methionine-labeled cell extracts. Much to our surprise, we were unable to detect either of these proteins in the S13 erythroblasts (data not shown). Since the in vitro immune complex kinase assay is a more sensitive indi- cator of the presence of these two proteins, we reex- amined S 13-transformed etythroblasts using this as-

12345678 9 10 11 12 13 14

P lsmin 30min 6omin 12Omin l6Omin 300min

FIG. 5. Pulse-chase analysis. S13 rat cells were labeled with 150 &I of [%]methionrne for 30 min. The cells were then erther lysed immedrately, P, or the labeling medrum was replaced wrth normal growth medrum and the cells were Incubated for the times shown under the lanes prior to lysis. The lysates were then analyzed by rm mune preciprtatron usrng either normal serum, lanes 1, 3. 5, 7, 9. 1 1, and 13. or antr-sea serum, lanes 2, 4, 6, 8, 10, 12, and 14. The labeled proterns were displayed by SDS-PAGE followed by fluorography. The 55,000.Da protein precrpitated by the anti-sea serum was also precipitated from normal rat 1 cells (data not shown).

236 KNIGHT, SMITH, AND HAYMAN

gp155 -

gp70 -

P38 = p35

FIG. 6. p36 and ~38 are not found in S134ransformed erythroblasts. S13-transformed chicken erythroblasts. lanes 1 and 2, 513 rat cells, lanes 3-5, or RSV-transformed rat cells, lanes 6-8, were lysed and immune precipitated with normal rabbit serum (lanes 1, 3, and 6) anti-sea serum, (lanes 2, 4, and 7) or anti-erbB serum (lanes 5 and 8). The immune precipitates were then incubated with [3’P]- ATP (5 &i/reaction) in an in vitro kinase assay (Knight et al., 1988a). The labeled proteins were visualized by SDS-PAGE followed by autoradiography.

say. Figure 6 shows a comparison of S13 erythroid cells with S13-transformed rat 1 cells together with Rous sarcoma virus-transformed rat 1 cells as a negative control. Figure 6, .lane 2, demonstrates that, whereas the S13 erythroid cells contain readily detectable amounts of gpl55 and gp70, we could not detect either ~38 or ~36. In contrast, the S13 rat 1 cells contain easily detectable amounts of these two proteins (Fig. 6, lane 4) as do S13-transformed chickfibroblasts (data not shown). The Rous sarcoma virus-transformed rat cells were negative for the expression of these two proteins demonstrating that they are found only in S 13- infected fibroblasts (see Fig. 6, lane 7). Immune precipitation with an antiserum against bacterially expressed erbB protein prepared in an identical manner to the anti-sea serum was also negative on all cell types tested (see Fig. 6, lanes 5 and 8) indicating the speci- ficity of the anti-sea antiserum.

DISCUSSION

In this report, we describe the production of poly- clonal rabbit antiserum specific for the v-sea gene products. Using this serum, two previously unidentified proteins were found in fibroblasts transformed by the S13 virus. These proteins were shown to be phosphoproteins with apparent molecular weights of 38,000 and 36,000 Da. Unlike the previously identified v-sea- encoded proteins, gp155 and gp70, neither of these two proteins could be shown to be modified by the addition of N-linked sugars. One-dimensional peptide

mapping of the proteins demonstrated that they are structurally related to the v-sea-encoded proteins and thus are also likely to be virally encoded. Pulse-chase analysis revealed that unlike the gp155 and gp70 which have relatively long half lives (in the range of 2- 4 hr), the half lives of ~38 and ~36 were relatively short (in the range of 30-60 min). Furthermore, there was no obvious precursor-product relationship between gp155 and these proteins or between ~38 and ~36 themselves. Surprisingly, ~38 and p36 were detected only in fibroblasts transformed by Sl3 and were not detected in S13-transformed erythroid cells.

Immune complex protein kinase assays using the sea-specific serum demonstrated that the ~38 and ~36 proteins became phosphorylated, and phosphoamino acid analysis identified this as being on tyrosine resi- dues. Whether these two proteins have intrinsic tyrosine kinase activity remains an open question. It could be demonstrated that the kinase activity associated with the immune complexes was v-sea-encoded by using a temperature-sensitive mutant of S13, ts-I (data not shown). However, since gpl55 and gp70 are present in the immune complex, this experiment does not resolve the question as to whether ~38 or p36 have intrinsic kinase activity, since these two proteins could be serving as substrates for the other v-sea-encoded proteins. To clarify this issue, conditions will have to be determined under which the ~38 and ~36 proteins can be purified away from gpl55 and gp70 and then as- sayed for their intrinsic kinase activity.

The pulse-chase analysis raises questions concern- ing the biosynthesis of ~38 and ~36. There are at least three possible mechanisms for their synthesis: (1) They could be post-translational products derived by prote- olysis of the primary translational product gp 155. How- ever, the pulse-chase analysis is not easily reconciled with this option since their turnover appears to be inde- pendent of that of gpl55. If synthesized by this mecha- nism, the lack of these two proteins in erythroid cells could be explained by the cell type-specific expression of the cellular protease. (2) Alternatively, the two proteins could be translated from subgenomic messenger RNAs that are generated only by alternative splicing in fibroblasts. Previous analysis of S13-specific RNAs found in fibroblasts by Northern blotting would argue against this alternative since no candidate mRNAs for these two proteins were detected (Smith et a/., 1989; Benedict et al., 1985). (3) The third possibility would be cell type-specific internal initiation of translation from one of the v-sea mRNAs. If this latter possibility were the case, then the size of the two proteins would dic- tate where translation would have to initiate within the S13 genome. Examination of this region within the S13 sequence reveals that there are no methionine codons

v-sea-ENCODED POLYPEPTIDES 137

in positions that are compatible with the sizes of these two proteins. However, there are several in-frame CUG codons in this region and recently CUG codons have been demonstrated to serve as initiation codons in a variety of systems (Hann et al., 1988; Acland et al., 1990). Hence, it is possible that these two proteins are the products of internal initiation from v-sea-specific CUG codons. Obviously, further experimentation using site-directed mutagenesis of the S13 genome will be necessary to distinguish between these and other al- ternatives.

Transformation by S13 can be attributed directly to the v-sea oncogene. Thus, the identification of novel v- sea-encoded polypeptides raises the question of their possible role in transformation. The lack of their expression in erythroblasts would indicate that they are not needed for erythroid cell transformation. However, analysis of various mutants in the e&B oncogene has demonstrated that erythroid cells and fibroblasts have differing genetic requirements for transformation (re- viewed in Hayman, 1986). Thus, it is possible that p38 and p36 may play a role in fibroblast transformation while having no effect on erythroid transformation. It has recently been demonstrated that the glucosidase inhibitor, castanospermine, can inhibit transformation by S13 (Knight et al., 1988a). p38 and p36 do not contain any N-linked carbohydrate side chains and therefore should not be directly affected by this inhibitor. This data indicates that p38 and p36 are not sufficient to transform fibroblasts, but further experimentation is needed to determine if these two proteins are necessary for fibroblast transformation.

ACKNOWLEDGMENTS

The authors thank Allson Crowe, Kathy Donnelly, Paula Enrietto, Neil Kabrun, and Alyssa Monmoto for their helpful Comments on the manuscript. These experiments were supported in Part by grants from the National Cancer Institute (ROlCA42573 and 5POlCA28146)to M.J.H.

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identification of two additional v-sea-encoded proteins in avian erythroblastosis virus,...

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