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Detection and characterization of the protein encoded by the v-rel oncogene
VIROLOGY
149,217-229
(1986)
Detection and Characterization of the Protein Encoded by the v-rel Oncogene’ NANCY
R. RICE,*r2 TERRY D. COPELAND,* STEPHANIE SIMEK,” STEPHEN OROSZLAN,* AND RAYMOND V. GILDENt
*Laboratory of Molecular Virology and Carcinogenesis, Litton Bionetics, Inc., Btic Program, and TProgram Resources, Ivzc., NatiMzal Cancer Institute-Frederick Cancer Research Facility, Frederick, Maryland 21701 Received
August
8, 1985; accepted
November
Research
13, 1985
To identify the protein encoded by v-rel, the oncogene of reticuloendotheliosis virus (REV-T), antisera have been raised to three synthetic peptides derived from the translation of our previously published v-rel DNA sequence [R. M. Stephens, N. R. Rice, R. R. Hiebsch, H. R. Bose, Jr., and R. V. Gilden, Proc. Natl. Acad. Sti. USA 80,6229-6233 (1983)]. Sera to all three peptides precipitate a 59,000 Da protein from REV-T-transformed chicken lymphoid cells. This protein is not detectable in uninfected chick embryo fibroblasts, and its observed size is in good agreement with the 56,000 Da predicted by the DNA sequence. We conclude that this protein is the v-rel product and designate it p5gre’. To search for evidence of post-translational processing of this protein, cells were grown in the presence of glycosylation inhibitors. These resulted in no detectable difference in the size of p5gre’. Nor was its size detectably altered during the course of a pulse-chase experiment. Growth of cells in the presence of [“Plorthophosphate, however, revealed that p59’“’ is a phosphoprotein. It is also closely associated with a protein kinase activity, for precipitation with one of the peptide antisera (but not the other two) resulted in strong kinase activity in p59 re’ itself becomes phosphorylated. the immune complex pellet. During this reaction, Kinase activity was retained in the immune complex following detergent and high salt washes, leaving open the possiblity that p59”’ is itself a kinase.
et al., 1983). The sequence showed that vrel (1415 nucleotides) is inserted into the REV-A erbv gene such that the predicted rel protein (503 amino acids) employs the env initiator and contains the first 12 residues of the envelope polyprotein signal sequence. The rel protein is predicted to terminate within env, with 19 amino acids translated out-of-frame with respect to p20E. This structure explained why antibodies to purified REV-A proteins do not detect the product of v-rel (Hoelzer et al., 1980; Lewis et al, 1981). In order to identify the v-rel protein, we have raised antisera to several synthetic peptides derived from the translated DNA sequence. In this communication we report that these sera detect a protein of about 59,000 Da in transformed chicken spleen
INTRODUCTION
Reticuloendotheliosis virus strain T (REV-T) is an acute leukemia virus of chickens and turkeys. The virus consists of a nontransforming helper virus REV-A and a replication-defective transforming genome (Hoelzer et al., 1979) carrying the oncogene v-reL Our efforts to undertand the mechanism of oncogenesis of REV-T have led us to molecularly clone the viral genomes (Rice et al., 1982) and to determine the nucleotide sequence of v-rel (Stephens 1 The U. S. Government’s right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for governmental purposes, is acknowledged. “To whom requests for reprints should be addressed. 217
0042-6822/86
$3.00
218
RICE
cells. This protein is phosphorylated and has a closely associated kinase activity.
ET
AL.
[35S]methionine at 30 Ci/ml for about 90 min, and immune precipitates with antiserum III were collected (see below). These were washed twice in 0.2 M sodium acetate, MATERIALS AND METHODS pH 5, suspended in 50 ~1 of the same f 10 Cells. Chicken lymphoid cell lines trans~1 endoglycosidase H (Miles, 1 p/ml), and formed in vitro by REV-T were established incubated at 30” for 3 hr. Pellets were coland described by Hoelzer et al. (1980). In lected, washed, and analyzed electrophothis study we used both virus producing retieally. To test the enzyme for activity, and nonproducer lines, grown in suspen- its effect on the envelope precursor protein sion in RPM1 1640 plus 10% fetal calf serum was assayed. For this purpose, dog cells at 37”. Chick embryo fibroblasts were producing REV-A were incubated in grown in DMEM plus 10% serum at 37”. [35S]methionine and treated as above using The REV-A-producing fetal canine thymus antiserum to purified viral p20E. line Cdth has been described previously Immune precipitatiom. Cells were (Gonda et ab, 1980); it was grown in DMEM washed twice in PBS and except for kinase plus 10% serum at 37”. assays were lysed in RIPA buffer [20 mM Metabolic labeling with S5methionine. Tris, pH 7.5,2 mM EDTA, 150 mM sodium Cells were washed twice in phosphatechloride, 1% sodium deoxycholate (DOC), buffered saline (PBS) and incubated in me- 1% Triton X-100, 0.25% sodium dodecyl thionine-free minimum essential medium sulfate (SDS)] containing 1 mM phenyl(MEM) for 10 min at 3’7”. r5S]Methionine methylsulfonyl fluoride (PMSF) and 180 (1000 Ci/mmol, Amersham or New En- Kallikrein units (KU) per milliliter of gland Nuclear) was added to a final con- aprotinin. Lysates were centrifuged in the centration of 30 to 50 Ci/ml, and incubation Eppendorf microfuge for 10 min and the continued for periods ranging from 15 min supernatants were incubated with preimto several hours. mune antiserum overnight at 4’ in the Metabolic labeling with”P0,. Virus-propresence of protein A-Sepharose (Pharducing REV-transformed chicken cells maria). After centrifugation for 1 min, SUwere pelleted, washed twice in phosphatepernatants were incubated at 4” for 3 to 6 free RPM1 1640, resuspended in 15 ml hr with immune serum plus protein A-Sephosphate-free RPM1 1640 supplemented pharose. For lysate from l-2 X lo6 cells, lwith 2% dialyzed fetal calf serum, and in10 ~1 of serum was used. Protein A-Secubated at 37” for 3 hr. They were pelleted pharose was made up as a slurry of 0.1 g again, resuspended in 3 ml phosphate-free plus 0.75 ml RIPA. The ratio of this susRPM1 1640 which was adjusted to 0.1 mM pension to serum was 8:l (vol/vol). AntiNaH2P04 and 200 Ci/ml [32P]orthophosgen-antibody complexes were collected by phate (Amersham, carrier free), and in- centrifugation for 1 min and were washed cubated at 37” for 2 hr. several times at room temperature with Treatment with glycosylation inhibitors. RIPA. The final pellet was resuspended in Virus-producing transformed chicken cells gel loading buffer (20% glycerol, 40% /Iwere pretreated in complete medium with mercaptoethanol, 0.2 M Tris, pH 7, 8% so20 mM 2-deoxyglucose for 2 hr or with 5 dium dodecyl sulfate, and 0.04% bromg/ml tunicamycin (Calbiochem) for 1 hr. phenol blue), boiled for 5 min, and loaded They were then pelleted, washed twice onto 10 to 20% SDS-polyacrylamide gels with PBS, resuspended in methionine-free (Laemmli, 1970). Molecular weight markMEM, and incubated at 37” for 30 min in ers were purchased from Bethesda Rethe presence of [35S]methionine at 50 Ci/ search Laboratories. After electrophoresis, ml. Labeling of the 2-deoxyglucose-treated 35S-containing gels were soaked in dimethsample was done in the presence of 20 mM ylsulfoxide (DMSO) for 1 to 2 hr, then in 2-deoxyglucose. DMSO plus 22% 2,5-diphenyloxazole (PPO) Endoglycosidase H. Nonproducer transfor 3 hr with gentle agitation. The gel was formed chicken cells were incubated in then washed in running water for 1 hr,
v-rel
PROTEIN
dried, and XAR film. dried and tensifying
exposed for 1 to 7 days to Kodak 32P-Containing gels were simply exposed, sometimes with an inscreen (DuPont Lightning Plus). In vitro kinase assays. For kinase assays, nonradioactive cells were washed in PBS and lysed in 20 mM Tris, pH 7.5, 200 mM sodium chloride, 1% Triton X-100 (TNT), containing aprotinin and PMSF as above. Lysates were clarified as above and subjected to immune precipitation immediately. Immune complexes bound to protein A-Sepharose were washed several times in TNT and then incubated at 30” for 10 min in 30 yl TNT containing 10 mM manganese chloride, 180 KU per ml aprotinin, 1 mM PMSF, and 10 &i [T-~~P]ATP (Amersham, 3000 Ci/mmol). The reaction was stopped by addition of 1 ml RIPA and immune pellets were collected by centrifugation for 1 min in the Eppendorf microfuge and washed several times with RIPA. The final pellets were resuspended in gel loading buffer as above, boiled, and loaded onto 10% or 10 to 20% gels. Gels were dried and autoradiographed with an intensifying screen for 2 to 24 hr. Synthesis
of peptides and immunizations.
The protected carboxyl terminal amino acid of each peptide was covalently attached to a solid support, 1% crosslinked divinylbenzene-polystyrene, via the cesium salt (Gisin, 1973). Peptides I and II were then assembled by solid phase synthesis (Merrifield, 1963) in a semiautomated Vega peptide synthesizer, Model 96, as described (Copeland and Oroszlan, 1981). Protected amino acids were purchased from Peninsula Laboratories (San Carlos, Calif.). The amino terminus of each amino acid was protected with tert-butyloxycarbonyl except for arginine which was the amyloxycarbonyl derivative. The side chain protecting groups were tosyl for arginine, benzyl for glutamic acid and threonine, and o-bromobenzyloxycarbonyl for tyrosine. Double couplings with a two fold molar excess were employed. If the ninhydrin assay (Kaiser et al., 1970) indicated that coupling was incomplete, a third coupling was performed. Each peptide was cleaved from the solid support and protecting groups with hydrogen fluoride containing 10% anisole
219
in a Teflon apparatus at 0” for 60 min. Peptides were extracted into 50% acetic acid, diluted with water, and lyophilized. Peptide III was purchased in crude form from Peninsula Laboratories. Each crude peptide was desalted and purified by reversed phase liquid chromatography (RPLC) on a Waters Associates 7.8 mm X 30 cm PBondapak Cl8 column using an aqueous acetonitrile gradient containing 0.05% trifluoroacetic acid. The amino acid composition of each purified peptide was determined and each was found to be correct. Prior to immunization, each RPLC purified peptide was covalently bound to keyhole limpet hemocyanin (KLH) (Calbiochem) utilizing approximately 5 mg of peptide to 5 mg of KLH. Peptide I was coupled with difluorodinitrobenzene (Tager, 1976), peptide II with bis-diazotized benzidine (Bassiri et ah, 1974) and peptide III with m-maleimidobenzoyl-N-hydroxysuccinimide ester (Liu et al, 1979). Following the reactions, each peptide KLH conjugate was dialyzed against PBS at 4”. Rabbits were initially inoculated with 1 mg of the peptide KLH conjugate in PBS diluted to 2 ml with Freund’s complete adjuvant and administered intradermally to several locations on the back. Boosts were made at 2-week intervals with 0.1 mg of the peptide-KLH conjugate in incomplete adjuvant. Test bleeds were begun just prior to the second boost. Antibody titer was determined with peptide bound to diazotized paper strips as described in detail elsewhere (Schultz et al., 1985). Once positive sera to the peptide was obtained, each animal was exsanguinated. Other antisera. Antisera to purified REVA p30 and p20E are described elsewhere (Tsai et ah, 1985; W.-P. Tsai, T. D. Copeland, and S. Oroszlan, in preparation). Affinity purification of antiserum III was performed by Alan Schultz. Serum III was passed over an Affigel 10 column (Bio-Rad) to which peptide III was bound, as described by Schultz et aZ. (1985). RESULTS
Peptides representing three segments of the predicted v-rel protein were synthe-
220
RICE II
I
Peptide
Number
ET
weakly positive and was not used in these studies.
III
I
*
lh
2&l Amino
3kl
Acid Residue
4do
AL.
do
Detection of the v-rel Protein
Number
1. Location of the three peptides in the predicted v-rel protein. Based on the DNA sequence, the v-rel protein has 503 amino acids, the N-terminal 12 and C-terminal 19 of which are coded for by regions derived from the helper viral genome REV-A (Stephens et al, 1983; Wilhelmsen et al., 1984). Sequences of the peptides proceeding from NHa- to COOH-terminal are: I, Tyr-Tyr-Glu-Ala-Glu-Phe-Gly-Pro-GluArg-Gln-Val-Leu; II, Glu-Gln-Pro-Arg-Gln-Arg-GlyThr-Arg-Phe-Arg-Tyr; III, Pro-Ala-Tyr-Asn-Pro-LeuAsn-Trp-Pro-Asp-Glu-Lys-Asn-Cys. The C-terminal cys residue in peptide III is not found in the v-rel protein sequence; it was added to the peptide to facilitate coupling. FIG.
Antisera to each of the peptides were used in immune precipitation experiments with lysates of [35S]methionine-labeled REV-transformed chicken lymphoid cells. As shown in Fig. 2, each serum precipitates a small number of proteins, several of which also appear with preimmune serum or with immune serum in the presence of excess competing peptide; these are judged to be the result of nonspecific binding. A few additional proteins do not appear with preimmune serum and are competable by excess homologous peptide but not by heterologous peptide; these are judged to represent specific antigen-antibody binding. Of these proteins, only two appear with sera to all three peptides. The first has an apparent molecular mass of about 59,000 Da, in good agreement with the 56,000 Da predicted for the v-rel protein by the DNA sequence. It is not precipitated by any of several other antisera raised to unrelated
sized, based on the DNA sequence of v-rel. Their location in the rel protein is shown in Fig. 1. The peptides were coupled to hemocyanin and each was used to immunize two rabbits. All six resulting sera recognize their respective peptides, and five are able to precipitate the v-rel protein. One of the sera raised against peptide I was only Antiserum II I
111 Ir
1
2
3
4
I 5
6
7
6 MW x10-3
-93-66--)r -43-
-26-
-16-
FIG. 2. Detection of the rel protein with antisera to synthetic peptides. Nonproducer transformed chicken cells were grown in the presence of [%]methionine for 2 hr and subjected to immune precipitation as described under Materials and Methods. Each lane received the immune pellet from about lo6 cells. Lysates for lanes 1, 5, and 12 were treated with preimmune serum; for lane 2 with antiserum II alone; for lane 3 with serum II plus competing peptide II; for lane 4 with serum II plus competing peptide III; for lanes 6 and 13 with serum III alone; for lane 7 with serum III plus competing peptide III; for lane 8 with serum III plus competing peptide II; for lane 9 with serum I alone; for lane 10 with serum I plus competing peptide I; for lane 11 with serum I plus competing peptide III. Arrows indicate the putative v-rel encoded protein.
v-rel
221
PROTEIN
proteins or peptides. The second, of about 110,000 Da, does not appear reproducibly and its identity is unknown at present. If p59 is the v-rel protein it should not appear in uninfected chicken cells, and as shown in Fig. 3, it does not. Antiserum I precipitates a protein of about 60,000 Da from the uninfected cells (lane l), but this reaction is not competable by peptide I (lane 2). Antiserum II is negative. Based on its presence in REV-transformed cells, its specific reactivity with three independent peptide antisera, and its size, we conclude that p59 is the v-rel product. Lysates of uninfected chicken cells do contain several other proteins specifically precipitable by each of the sera. With the exception of p59 and ~110 each of the proteins competable by homologous but not by heterologous peptide in the experiment shown in Figs. 2 and 3 is also present in the Fig. 3 study with uninfected cells. As has been reported previously (Nigg et al., 1982), that antisera to synthetic peptides may cross-react with a small number of other proteins, presumably by virtue of their sequence homology to the peptide.
Alternatively one or more of these proteins may be related to the c-rel protein. p5Ye1 Product
Immune precipitation following metabolic labeling with 32P04 demonstrated that the rel protein is phosphorylated. As shown in Fig. 4, all three sera precipitate a protein of approximately 60,000 Da. This protein is not seen with preimmune sera; nor is it present when the immune precipitation is performed in the presence of excess homologous peptide. Since this experiment employed virus-producing cells, it was also possible to detect the gag precursor, which is known to be phosphorylated (Tsai et al., 1985), using antiserum to purified ~30. In several such experiments the intensity of the Pr55g”g band was about the same as that of the p5gre1 band (data not shown). p.!Nez Product
p59”
2
is not Glycosylated
To investigate whether p59’“’ is likely to be a membrane-bound protein, we tested it for glycosylation. If oligosaccharide side chains account for any significant portion
REV-T 1
is a Phosphoprotein
CEF 3
4
1
2
3
4
-t
FIG. 3. Reactivity of peptide antisera with proteins in normal chicken cells. REV-transformed chicken cells and uninfected chick embryo fibroblasts were grown in the presence of methioninefree MEM, [?J]methionine, and 5% dialyzed fetal calf serum for 2 hr and subjected to immune precipitation. Each lane received the immune pellet from about 10” cells. Lysates in lane 1 were treated with antiserum I; in lane 2 with serum I plus competing peptide I; in lane 3 with serum II; and in lane 4 with serum II plus competing peptide II. Notice that except for p5gTe’ and one high molecular weight band, all competable bands are also present in CEFs.
RICE
222
ET
AL.
Antiserum I
I1
I
III
f )
1
2
/ /
3
4
5
6
1
7
MW
a
x 10-3
-
93
-
66
-
43
is phosphorylated. Cells were metabolically labeled with 32P04 as described FIG. 4. The rel protein under Materials and Methods. Aliquots of the lysate were subjected to immune precipitation (about 2 X 10’ cells per lane) with the indicated sera and pellets were analyzed on a 10% polyacrylamide gel. Exposure was for 3 days with an intensifying screen. Lane 1, antiserum I alone; lane 2, antiserum I plus competing peptide I; lane 3, preimmune antiserum II; lane 4, antiserum II alone; lane 5, antiserum II plus competing peptide II; lane 6, antiserum II plus competing peptide I; lane 7, preimmune antiserum III; lane 8, antiserum III. The p59 band in lane 1 is faint but clear on the X-ray film; it reproduces poorly.
of the rel product’s molecular weight, then prevention of their addition to the polypeptide chain should result in the appearante of a rel protein of reduced size. Both tunicamycin and 2-deoxyglucose are known to block glycosylation, and as shown in Fig. Tunicamycin
-
5 each is effective in preventing glycosylation of the viral envelope precursor protein. In untreated cells, antiserum to the purified transmembrane protein p20E precipitates the glycosylated envelope precursor, whose size is about 74,000 Da. Pre-
2-Deoxy-Glucose
+
Envelope
+
Precursor +DG
+T
p59”’
is not detectably glycosylated. Virus-producing transformed cells were FIG. 5. The rel protein treated with tunicamycin or 2-deoxyglucose and labeled in the presence of [35S]methionine as described under Materials and Methods. After immune precipitation (about 2 X lo6 cells per lane) with either antiserum III or antiserum raised against purified viral p20E, samples were analyzed on a lo-20% gradient polyacrylamide gel.
v-rel
PROTEIN
of carbohydrate, p59’“’ cannot be detected in the culture medium following a several hour incubation of transformed cells in [35S]methionine (data not shown). Since 35Slabeled REV-A p30 is easily detected in the medium from these same cells, we conclude that little or no p5gre’ is secreted.
treatment of the cells with either of the inhibitors, however, prevents the synthesis of gPr74”“‘, and only the unglycosylated 57,000-Da polypeptide chain is detectable. If aliquots of these same cellular lysates are incubated not with anti-p20E but with antiserum III, no effect of the inhibitors on the size of the rel protein is detectable. The same result was obtained in experiments with endoglycosidase H, an enzyme which is able to cleave oligosaccharide side chains from a mature protein. When gPr74”“” is precipitated by anti-p20E from lysates of virus-producing cells, treatment of the immune pellet with endoglycosidase H reduces its molecular weight to about 59,000 Da. Under the same conditions in immune precipitates using antiserum III, the enzyme has no effect on the size of the rel product (data not shown). We conclude that there is no detectable glycosylation of p59’? We also looked for evidence of secretion of p59’“‘. Consistent with its apparent lack Antiserum Label 15’
Chase, 60’
2
Pulse-chase
6
Experiment
To determine whether the newly synthesized rel protein is the same size as the presumably mature form observed after a 2-hr incubation with radioactive precursors, a pulse-chase experiment was performed. Cells were grown in the presence of [35S]methionine for 15 or 60 min and then incubated in nonradioactive complete medium for varying times up to 48 hours. As shown in Fig. 6, the size of the newly synthesized rel product observed in the cells labeled for only 15 min is not detectably different from that observed at later times. Even in much longer exposures of the films Antiserum
II Hours
Label
II
I
223
26
46
’
MW x10-3
-’
15’
60’
60’
2
III Chase,
Hours
2
6
I 26
FIG. 6. The rel protein in a pulse-chase experiment. With the exception of an aliquot withdrawn after 15 min, cells were incubated in [35S]methionine (50 &X/ml) for 60 min, washed in PBS, and resuspended in nonradioactive complete medium plus serum. Aliquots were taken at times indicated in the figure and lysates were subjected to immune precipitation with antiserum II or III. Lysates for the third and fifth lanes in the serum III experiment were treated with preimmune serum. Lysates from an approximately constant number of cells (about 3 X 106) were analyzed in each of the lanes shown; the only exception is the sample labeled for only 15 min, from which about 1.5 times the usual number of cells was taken. The doubling time of these tranformed chicken cells is approximately 24 hr.
224
RICE
there is no indication of an additional rel band, either larger or smaller. Thus, if there is any processing of the nascent rel protein at all, it must either be very closely coupled to its synthesis, for it is not observable even on the time scale of a few minutes, or it must result in a very small change in apparent molecular weight, which is unresolvable in these experiments. A rel protein of the same size is also seen throughout the cold-chase following a 60min incubation in [35S]methionine. The intensity of the band has already diminished after a few hours but it is still detectable after 26 hr and even after 48 hr on films exposed for longer times. There is no indication of the accumulation of stable breakdown products. Since this experiment was performed separately with all three peptide antisera, whose reactivities span the molecule, cleavage products would most likely have been detected. The rel Protein Has an Associated Kinase Activity In vitro kinase assays were performed on immune precipitates of p59”’ using each of the three peptide sera independently. As shown in Fig. 7, the precipitate with serum I was negative, with serum II very weakly positive, but with serum III strongly positive for kinase activity. Serum from both rabbits immunized with peptide III resulted in kinase activity in the immune precipitate (lanes 4 and 5). This activity was also observed after immune precipitation with serum III which had been heated to 56” for 30 min (data not shown) and with affinity-purified serum III (lane 7). To assess the purity of the affinity-purified serum, an aliquot equal to that used in the precipitation experiments was electrophoresed on a 10% polyacrylamide gel. Following silver staining of the gel, the only bands visible were of the size expected for immunoglobulin. We conclude that it is very unlikely that the kinase observed in these experiments is derived from the serum. The level of kinase activity is quite high. Each lane shown in Fig. 7 received the immune precipitate from 1-2 X lo6 cells. Each kinase assay included 10 &i [y-32P]ATP at
ET
AL. Antiserum
III
I
$-IA 1
2
3
4
5
6
7
MW x 10-3
-93
-66 -43
-26
-48 FIG. 7. In vitro kinase assay with p59’“’ immune precipitates. Following immune precipitation of REVtransformed chicken lymphoid cells lysates (1-2 X lo6 cells per lane) with the indicated sera, in vitro kinase assays were performed as described under Materials and Methods. Products were analyzed on a lo-20% gradient SDS-polyacrylamide gel and autoradiographed for about 20 hr with an intensifying screen. Lane 1, preimmune serum; lane 2, antiserum I; lane 3, antiserum II; lanes 4 and 5, antiserum III from two different rabbits; lane 6, antiserum III plus peptide III; lane 7, affinity-purified antiserum III.
3000 Ci/mmol, and the exposure time of the film was about 2 hr with an intensifying screen. This is roughly comparable to the activity reported for p160gagmab’(Konopka et aZ., 1984) and p75gag-‘“f (Moelling et ah, 1984) immune precipitates. The reaction appeared absolutely dependent on the presence of manganese ion. Assays performed in the presence of Mgf+ or Ca++ were negative (data not shown). Among several other strongly labeled bands which appeared following incubation with [T-~‘P]ATP was one of about 59,000 Da. To ascertain whether this protein was the p59’“‘, we tested whether it could be reprecipitated, after the kinase reaction, by each of three sera. This was done by boiling the products of the kinase reaction in 1% SDS, thereby liberating the p59 from antibody and protein A-Sepharose, and then incubating aliquots with serum I, II, or III. As shown in Fig. 8, 32P-labeled p59 is precipitable with all the sera. We conclude therefore that the rel protein is being
v-rel Boiled, 12’3
4
reprecipitated 5
6
7
0
I
-
225
PROTEIN
p59”’
FIG. 8. Phosphorylation of the v-rel protein during an in vitro kinase assay. After immune precipitation with antiserum III of a lysate of transformed chick cells, an in vitro kinase assay was performed as described under Materials and Methods. Following extensive washing to remove unincorporated [32P]ATP, the product (except for aliquots saved for lanes 1 and 2) was boiled for 5 min in 1% SDS. Protein A-Sepharose was pelleted and discarded, and the supernatant was diluted with TNT, aliquoted, and subjected to immune precipitation in the presence of fresh antibody and protein A-Sepharose. Precipitates were collected and analyzed on a 10% SDS-polyacrylamide gel. Exposure to X-ray film was for 24 hr with an intensifying screen. Lane 1, immune precipitation with preimmune serum; lane 2, with antiserum III; lanes 3-8, sample boiled in 1% SDS after kinase reaction. Lane 3, no fresh serum added; lane 4, preimmune serum added; lane 5, fresh serum I added; lane 6, serum II added; lane 7 serum II plus peptide II added; lane 8, serum III added. The precipitate in each lane was derived from about lo6 cells.
phosphorylated during the in vitro kinase assay, but that this occurs only when the immune precipitation is performed with serum III. The identity of the other phosphorylated proteins is unknown. The dark band at about 59,000 Da actually appears to be a doublet, the larger member of which is p59’“‘. The smaller member can be seen very faintly even in assays performed after immune precipitation with preimmune serum (Figs. 7 and 8). It is thus a nonspecific component of the immune pellet and can act as a phosphate acceptor if a kinase is also present. The other dark band, at about 50,000 Da, is the appropriate size for the immunoglobulin heavy chain, but we have not investigated its identity further.
As one approach to identifying the kinase, we attempted to dissociate p59’“’ from the kinase activity. The usual protocol for the assay involves lysis of the cells, immune precipitation, and washing of the precipitates, all in TNT (which contains 0.2 M NaCl and 1% Triton X-100). We therefore tried more stringent washing conditions before the kinase assay to see if a hypothetical associated kinase could be dissociated from the immune pellet. As shown in Fig. 9, kinase activity was retained in the pellet after washing in 0.5% sodium deoxycholate (DOC), in 0.1% SDS, and in 2 M NaCl. Washing in TNT plus 10 mM EDTA or in 20 mMTris plus 1% Triton X100 with no added salt also failed to dislodge kinase activity from the immune complex (data not shown). In addition, we varied conditions during the immune pre-
Before I
Wash Kinase
Assay I
FIG. 9. The effect of various washing conditions before the in vitro kinase assay. Cells were lysed, immune precipitated (l-2 X lo6 cells per lane), and prepared for a kinase assay as described under Materials and Methods. Just before the assay the pellets were incubated for 15 min in 1 ml of TNT supplemented with the indicated reagents. Pellets were then collected, washed once in TNT, and assayed for kinase activity as usual. Lanes 1 and 2 are controls which were treated with TNT throughout the procedure. Lane 1, preimmune serum. Lanes 2-6, antiserum III. The dried gel was autoradiographed for about 6 hr with an intensifying screen.
226
RICE
cipitation, but as shown in Fig. 10, kinase activity was still present in the precipitate when 0.5% DOC and 2 M NaCl were added to the usual buffer. If the kinase is a protein associated with p59’“‘, therefore, the two must be very tightly complexed.
ET
AL.
is about 110 kDa, and it is clearly visible in Figs. 2 and 4. The identity of this protein is unknown at present. It could be an unrelated protein which is specifically associated with p5gre’ in transformed cells, or it could represent undissociated dimers of p5gre1, or it could conceivably be the protein DISCUSSION encoded by c-rel. Peptide mapping studies In this paper we report the detection of will be required to resolve this point. Regarding c-rel, the nucleotide sequence p59’“‘, the protein product of the v-rel onof at least most of the turkey gene is known cogene of reticuloendotheliosis virus. The from the work of Wilhelmsen et al. (1984), protein can be precipitated by antisera and the chicken gene is expected to be raised to three different peptides derived highly related (Simek and Rice, 1980; Wong from the translated DNA sequence of v-rel (Stephens et ab, 1983; Wilhelmsen et al, and Lai, 1981). Each of the three peptides used in this study differs from the corre1984). This precipitation is specific in that it can be competed by homologous, but not sponding region in turkey c-rel. At the poresidue in peptide by heterologous, peptide. Since p59 is not sition of the glutamine I, there is an arginine in c-rel; at the threseen in uninfected chick embryo fibroblasts onine in peptide II, there is a methionine but is seen in several independent REV-Taspargine transformed chicken lymphoid cell lines, in c-rel; and at the C-terminal in peptide III, there is an aspartic acid in we conclude that it is the product of v-rel. c-rel. In addition, peptide III has a threeIts apparent size of 59 kDa is in reasonable residue deletion relative to turkey c-rel. We agreement with the 56 kDa predicted by are thus unable to predict whether any of the DNA sequence. our three sera should recognize the chicken One other protein appears to be precipitable with all three peptide sera. Its size c-rel product. We are hopeful that antisera raised to portions of v-rel expressed in bacteria will afford a better opportunity to identify the c-rel protein. Since we now 2M NaCl 0.2 M NaCl i I I I know that human DNA contains sequences 0.5% DOC 0.5% DOC highly related to at least two of the turkey i 2 3 4 5 6 7 8 ’ MW c-rel exons (Brownell et al., 1985), such antisera should be useful in the search for a human c-rel protein as well. The v-rel protein has a closely associated protein kinase activity observable when immune precipitation is performed with antiserum to peptide III. Antiserum to peptide I results in a background level of activity comparable to that seen with preimmune serum, and antiserum to peptide II results in very low activity. Sera from both rabbits immunized with peptide FIG. 10. The effect on kinase activity of varying conIII, however, result in strong kinase activditions during immune precipitation. Cells were lysed ity in the immune complex. This activity in TNT and clarified as usual. For immune precipiis also observable in affinity-purified antation, lysates (1-2 X lo6 cells per lane) were adjusted tiserum III and is reduced to background to the indicated salt and detergent concentrations. levels when precipitation is carried out in Subsequent washing and assay conditions were as dethe presence of competing peptide III. We scribed under Materials and Methods. Lanes 1, 3, 5, conclude that the kinase activity is due to 7, preimmune serum; lanes 2, 4, 6, 8 antiserum III. a specific component of the cell lysate Exposure was for about 2 hr with an intensifying which is either precipitated by serum III screen. I
I
v-rel
PROTEIN
or complexed to a protein which is precipitated by serum III. The identity of the kinase is unknown. A trivial possibility is that the kinase is unrelated to p59’“’ but cross-reacts with antiserum to peptide III. We have attempted to discount this possibility with two additional experiments. First, we assayed chick embryo fibroblasts for kinase activity using antiserum III. The result was a few faint bands visible only after a 4-day exposure with an intensifying screen. Admittedly, this is an imperfect control since a hypothetical cross-reacting kinase could be present in lymphoid cells but not in fibroblasts. Second, we performed the kinase assay with a serum directed against a 262 amino acid segment of the v-rel protein expressed in bacteria (S. Simek, R. Stephens, and N. R. Rice, to be published). When this serum is used to precipitate p59’“’ from lysates of the transformed chicken lymphoid cells, kinase activity in the immune complex is comparable to that seen with antiserum III. This precipitation of kinase activity by the new serum is unaffected by a concentration of peptide III 100 times higher than that required to completely block antiserum III, despite the fact that the new serum binds peptide III in an ELISA assay. These results show that the kinase activity can also be recognized by antisera directed against regions of p59’“’ other than that defined by peptide III. Thus, it is unlikely that the results using peptide III antiserum alone are artifactual. A second possibility is that the kinase is a protein specifically associated with p59’“’ which is dislodged during precipitation with serum I or II. Such a situation would be analogous to that observed with polyoma middle T antigen, where middle T antisera precipitate a kinase activity which appears to be due to closely associated pp60c+rc (Courtneidge and Smith, 1983). In an attempt to dissociate such a hypothetical complex, detergent and salt concentrations have been varied both during the immune precipitation step and during the washing of the immune pellets prior to the kinase assay. None of these conditions (including 0.5% DOC, 0.1% SDS, 10 mM EDTA, and even 2 M NaCl) reduced the
227
kinase activity appreciably. If such a complex exists, therefore, it must be unusually stable. This conclusion is analogous to that reached by Kloetzer et al. (1984) for the pgjPw”o~- associated kinase activity. We are currently using nondenaturing polyacrylamide gels and sucrose gradients to look for evidence of complexes containing p59’“‘. The third possibility is that p59’“’ is a protein kinase, able to phosphorylate itself as well as a number of other proteins found in the immune complex. The failure of sera I and II to result in kinase activity might then be interpreted in terms of their binding to functionally important sites. Sera raised to peptides within the kinase domain of pp60”-“‘” (Gentry et al, 1983), of ~160~“~-“~’ (Ko nopka et al, 1984; Davis et ah, 1985), and of p85gag-fesand p108gag-fes(Sen et aZ., 1983) block kinase activity in the in vitro assay, whereas sera to peptides outside this domain in pp60V+Fe (Gentry et aZ., 1983), in p160gag-“b1(Konopka et al., 1984), and in p37”” (Maxwell and Arlinghaus, 1985) do not inhibit this activity. In p59’“‘, however, kinase domain homologous to that of the src family cannot be identified. While there may be very distant relatedness between several regions of p5gre’ and pp60”” (Stephens et ah, 1983), these do not include sequences in pp60src around the ATP binding site (lysinezg5) (Kamps et ah, 1984). Nor does p5gre’ possess the Ala430Pro431-G1u432 sequence common to most members of the src family (Bishop and Varmus, 1985) and known to be essential in pp60src for kinase activity and for transformation (Bryant and Parsons, 1984). Based on the sequence, therefore, there is no compelling reason to predict that p5gre1 is a kinase of the src family. The identity of the kinase will be satisfactorily resolved only after expression and purification of p59’“’ from bacteria and/or purification from eukaryotic cells with retention of kinase activity. We also wish to know which amino acid(s) is phosphorylated in the in vitro kinase reaction. While our preliminary results indicate the presence of phosphoserine and phosphothreonine and essentially no phosphotyrosine, the analysis has not been carried out on puritied p5gre’. This work is in progress.
228
RICE ET AL. ACKNOWLEDGMENTS
Research was sponsored in part by the National Cancer Institute, DHHS, under Contract NOl-CO23909 with Litton Bionetics, Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U. S. Government. We are grateful to Ronald R. Hiebsch for invaluable assistance in the early stages of this study. We thank Wen-Po Tsai for antiserum to REV-A p30 and p20E; Alan Schultz for affinity purification of antiserum III; and Jeannie Clarke and Margaret Fanning for preparation of the manuscript. REFERENCES BASSIRI, R. M., and UTIGER, R. D. (1974). Thyrotropinreleasing hormone. In “Methods of Hormone Radioimmunoassay” (B. M. Jaffe and H. R. Behrman, eds.), pp. 37-44. Academic press, New York. BISHOP, J. M., and VARMUS, H. (1985). Functions and origins of retroviral transforming genes. In “RNA Tumor Viruses, Molecular Biology of Tumor Viruses,” 2nd Ed., pp. 249-356. Cold Spring Harbor Laboratory. BROWNELL, E., 0-BRIEN, S. J., NASH, W. G., and RICE, N. R. (1985). Genetic characterization of human crel sequences. Mol. Cell. E&l., 5,282.6-2831. BRYANT, D. L., and PARSONS,J. T. (1984). Amino acid alterations within a highly conserved region of the Rous sarcoma virus ST(:gene product ~~60’” inactive tyrosine protein kinase activity. Mol. Cell. Bid. 4, 862-866. COPELAND,T. D., and OROSZLAN,S. (1981). A synthetic dodecapeptide substrate for type C RNA tumor virus associated proteolytic enzyme. In “Peptides: Synthesis-Structure-Function” (D. H. Rich and E. Gross, eds.), pp. 497-500. Pierce Chemical, Rockford, Ill. COURTNEIDGE,S. A., and SMITH, A. E. (1983). Polyoma virus transforming protein associates with the product of the c-src cellular gene. Nature (London) 303,435-439. DAVIS, R. L., KONOPKA, J. B., and WITTE, 0. N. (1985). Activation of the e-&l oncogene by viral transduction or ehromosomal translocation generates altered c-abl proteins with similar in vitro kinase properties. Mol. Cell. Biol. 5,204-213. GENTRY, L. E., ROHRSCHNEIDER,L. R., CASNELLIE, J. E., and KREBS, E. G. (1983). Antibodies to a defined region of pp60”’ neutralize the tyrosine-specific kinase activity. J. Biol. Chem 258,11219-11228. GISIN, B. F. (1973). The preparation of Merrifield-resins through total esterification with cesium salts. Helv. Chim. Acta 56,1476-1481. GONDA, M. A., RICE, N. R., and GILDEN, R. V. (1980). Avian reticuloendotheliosis virus: Characterization
of the high molecular weight viral RNA in transforming and helper virus populations. J. Viral. 34, 743-751. HOELZER, J. D., FRANKLIN, R. B., and BOSE, H. R. JR. (1979). Tranformation by retieuloendotheliosis virus, development of a focus assay and isolation of nontransforming virus. Virology 93,20-30. HOELZER, J. D., LEWIS, R. B., WASMUTH, C. R., and BOSE, H. R., JR. (1980). Hematopoietic cell transformation by reticuloendotheliosis virus: Characterization of the genetic defect. Virology 100, 462474. KAISER, E., COLESCOTT,R. L., BOSSINGER, C. D., and COOK, I’. I. (1970). Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal. Biochem. 34,595-598. KAMPS, M. P., TAYLOR, S. S., and SEFTON, B. M. (1984). Direct evidence that oneogenic tyrosine kinases and cyclic AMP-dependent protein kinase have homologous ATP-binding sites. Nature (London) 310,589592. KLOETZER, W. S., MAXWELL, S. A., and ARLINGHAUS, R. B. (1984). Further characterization of the p858”8”“_ associated protein kinase activity. Virology 138,143-155 KONOPKA, J. B., DAVIS, R. L., WATANABE, S. M., PONTICELLI, A. S., SCHIFF-MAKER, L., ROSENBERG,N., and WITTE, 0. N. (1984). Only site-directed antibodies reactive with the highly conserved src-homologous region of the v-abl protein neutralize kinase activity. J. Irirol. 51, 223-232. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 2?7,680-685. LEWIS, R. B., MCCLURE, J., RUP, B., NIESEL, D. W., GARRY, R. F., HOELZER, J. D., NAZERIAN, K., and BOSE, H. R. JR. (1981). Avian reticuloendotheliosis virus: Identification of the hematopoietic target cell for transformation. Cell 25,421-431. LIU, F.-T., ZINNECKER, M., HAMAOKA, T., and KATZ, D. H. (1979). New procedures for preparation and isolation of conjugates of proteins and a synthetic copolymer of D-amino acids and immunoehemical characterization of such conjugates. Biochemistry l&690-697. MAXWELL, S. A., and ARLINGHAUS, R. B. (1985). Serine kinase activity associated with Moloney murine sarcoma virus-124-encoded ~37~. I%-oZogy143,321333. MERRIFIELD, R. B. (1963). Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Amer. Chem. Sot. S&2149-2154. MOELLING, K., HEIMANN, B., BEIMLING, P., RAPP, U. R., and SANDER,T. (1984). Serine- and threoninespecific protein kinase activities of purified gag-mil and gag-raf proteins. Nature (London) 312,558-561. NIGG, E. A., WALTER, G., and SINGER, S. J. (1982). On the nature of cross-reactions observed with anti-
v-rel PROTEIN bodies directed to defined epitopes. Proc. Natl. Acad Sci. USA 79,5939-5943.
RICE, N. R., HIEBSCH, R. R., GONDA, M. A., BOSE, H. R., JR., and GILDEN, R. V., (1982). Genome of reticuloendotheliosis virus: Characterization by use of cloned proviral DNA. J. Viral. 42,237-252. SCHULTZ,A. M., COPELAND, T. D., MARK, G. E., RAPP, U. R., and OROSZLAN, S. (1985). Detection of the myristylated gag-raftransforming protein with raf specific antipeptide sera. virology 146, ‘78-89. SEN, S., HOUGHTEN, R. A., SHERR, C. J., and SEN, A. (1983). Antibodies of predetermined specificity detect two retroviral oneogene products and inhibit their kinase activities. Proc. Natl. Acacl. Sci. USA 80,1246-1250. SIMEK, S., and RICE, N. R. (1980). Analysis of the nucleic acid components in reticuloendotheliosis virus. J. viol. 33,320-329. STEPHENS, R. M, RICE, N. R., HIEBSCH, R. R., BOSE, H. R., JR., and GILDEN, R. V., (1983). Nucleotide se-
229
quence of v-rel: The oncogene of reticuloendotheliosis virus. Proc. Natl. Acud Sei. USA 80, 62296233. TAGER, H. S. (1976). Coupling of peptides to albumin with difluorodinitrobenzene. Anal. Biochem. 71,367375. TSAI, W.-P., COPELAND,T. D., and OROSZLAN,S. (1985). Purification and chemical and immunological characterization of avian reticuloendotheliosis virus guggene-encoded structural proteins. virology 140,289312. WILHELMSEN, K. C., EGGLETON, K., and TEMIN, H. M. (1984). Nucleic acid sequence of the oncogene v-rel in reticuloendotheliosis virus strain T and its cellular homolog, the protooncogene c-rel. J. ViroZ. 52, 172-182. WONG, T. C., and LAI, M. M. C. (1981). Avian reticuloendotheliosis virus contains a new class of oncogene of turkey origin. Virology 111,289-293.
149,217-229
(1986)
Detection and Characterization of the Protein Encoded by the v-rel Oncogene’ NANCY
R. RICE,*r2 TERRY D. COPELAND,* STEPHANIE SIMEK,” STEPHEN OROSZLAN,* AND RAYMOND V. GILDENt
*Laboratory of Molecular Virology and Carcinogenesis, Litton Bionetics, Inc., Btic Program, and TProgram Resources, Ivzc., NatiMzal Cancer Institute-Frederick Cancer Research Facility, Frederick, Maryland 21701 Received
August
8, 1985; accepted
November
Research
13, 1985
To identify the protein encoded by v-rel, the oncogene of reticuloendotheliosis virus (REV-T), antisera have been raised to three synthetic peptides derived from the translation of our previously published v-rel DNA sequence [R. M. Stephens, N. R. Rice, R. R. Hiebsch, H. R. Bose, Jr., and R. V. Gilden, Proc. Natl. Acad. Sti. USA 80,6229-6233 (1983)]. Sera to all three peptides precipitate a 59,000 Da protein from REV-T-transformed chicken lymphoid cells. This protein is not detectable in uninfected chick embryo fibroblasts, and its observed size is in good agreement with the 56,000 Da predicted by the DNA sequence. We conclude that this protein is the v-rel product and designate it p5gre’. To search for evidence of post-translational processing of this protein, cells were grown in the presence of glycosylation inhibitors. These resulted in no detectable difference in the size of p5gre’. Nor was its size detectably altered during the course of a pulse-chase experiment. Growth of cells in the presence of [“Plorthophosphate, however, revealed that p59’“’ is a phosphoprotein. It is also closely associated with a protein kinase activity, for precipitation with one of the peptide antisera (but not the other two) resulted in strong kinase activity in p59 re’ itself becomes phosphorylated. the immune complex pellet. During this reaction, Kinase activity was retained in the immune complex following detergent and high salt washes, leaving open the possiblity that p59”’ is itself a kinase.
et al., 1983). The sequence showed that vrel (1415 nucleotides) is inserted into the REV-A erbv gene such that the predicted rel protein (503 amino acids) employs the env initiator and contains the first 12 residues of the envelope polyprotein signal sequence. The rel protein is predicted to terminate within env, with 19 amino acids translated out-of-frame with respect to p20E. This structure explained why antibodies to purified REV-A proteins do not detect the product of v-rel (Hoelzer et al., 1980; Lewis et al, 1981). In order to identify the v-rel protein, we have raised antisera to several synthetic peptides derived from the translated DNA sequence. In this communication we report that these sera detect a protein of about 59,000 Da in transformed chicken spleen
INTRODUCTION
Reticuloendotheliosis virus strain T (REV-T) is an acute leukemia virus of chickens and turkeys. The virus consists of a nontransforming helper virus REV-A and a replication-defective transforming genome (Hoelzer et al., 1979) carrying the oncogene v-reL Our efforts to undertand the mechanism of oncogenesis of REV-T have led us to molecularly clone the viral genomes (Rice et al., 1982) and to determine the nucleotide sequence of v-rel (Stephens 1 The U. S. Government’s right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for governmental purposes, is acknowledged. “To whom requests for reprints should be addressed. 217
0042-6822/86
$3.00
218
RICE
cells. This protein is phosphorylated and has a closely associated kinase activity.
ET
AL.
[35S]methionine at 30 Ci/ml for about 90 min, and immune precipitates with antiserum III were collected (see below). These were washed twice in 0.2 M sodium acetate, MATERIALS AND METHODS pH 5, suspended in 50 ~1 of the same f 10 Cells. Chicken lymphoid cell lines trans~1 endoglycosidase H (Miles, 1 p/ml), and formed in vitro by REV-T were established incubated at 30” for 3 hr. Pellets were coland described by Hoelzer et al. (1980). In lected, washed, and analyzed electrophothis study we used both virus producing retieally. To test the enzyme for activity, and nonproducer lines, grown in suspen- its effect on the envelope precursor protein sion in RPM1 1640 plus 10% fetal calf serum was assayed. For this purpose, dog cells at 37”. Chick embryo fibroblasts were producing REV-A were incubated in grown in DMEM plus 10% serum at 37”. [35S]methionine and treated as above using The REV-A-producing fetal canine thymus antiserum to purified viral p20E. line Cdth has been described previously Immune precipitatiom. Cells were (Gonda et ab, 1980); it was grown in DMEM washed twice in PBS and except for kinase plus 10% serum at 37”. assays were lysed in RIPA buffer [20 mM Metabolic labeling with S5methionine. Tris, pH 7.5,2 mM EDTA, 150 mM sodium Cells were washed twice in phosphatechloride, 1% sodium deoxycholate (DOC), buffered saline (PBS) and incubated in me- 1% Triton X-100, 0.25% sodium dodecyl thionine-free minimum essential medium sulfate (SDS)] containing 1 mM phenyl(MEM) for 10 min at 3’7”. r5S]Methionine methylsulfonyl fluoride (PMSF) and 180 (1000 Ci/mmol, Amersham or New En- Kallikrein units (KU) per milliliter of gland Nuclear) was added to a final con- aprotinin. Lysates were centrifuged in the centration of 30 to 50 Ci/ml, and incubation Eppendorf microfuge for 10 min and the continued for periods ranging from 15 min supernatants were incubated with preimto several hours. mune antiserum overnight at 4’ in the Metabolic labeling with”P0,. Virus-propresence of protein A-Sepharose (Pharducing REV-transformed chicken cells maria). After centrifugation for 1 min, SUwere pelleted, washed twice in phosphatepernatants were incubated at 4” for 3 to 6 free RPM1 1640, resuspended in 15 ml hr with immune serum plus protein A-Sephosphate-free RPM1 1640 supplemented pharose. For lysate from l-2 X lo6 cells, lwith 2% dialyzed fetal calf serum, and in10 ~1 of serum was used. Protein A-Secubated at 37” for 3 hr. They were pelleted pharose was made up as a slurry of 0.1 g again, resuspended in 3 ml phosphate-free plus 0.75 ml RIPA. The ratio of this susRPM1 1640 which was adjusted to 0.1 mM pension to serum was 8:l (vol/vol). AntiNaH2P04 and 200 Ci/ml [32P]orthophosgen-antibody complexes were collected by phate (Amersham, carrier free), and in- centrifugation for 1 min and were washed cubated at 37” for 2 hr. several times at room temperature with Treatment with glycosylation inhibitors. RIPA. The final pellet was resuspended in Virus-producing transformed chicken cells gel loading buffer (20% glycerol, 40% /Iwere pretreated in complete medium with mercaptoethanol, 0.2 M Tris, pH 7, 8% so20 mM 2-deoxyglucose for 2 hr or with 5 dium dodecyl sulfate, and 0.04% bromg/ml tunicamycin (Calbiochem) for 1 hr. phenol blue), boiled for 5 min, and loaded They were then pelleted, washed twice onto 10 to 20% SDS-polyacrylamide gels with PBS, resuspended in methionine-free (Laemmli, 1970). Molecular weight markMEM, and incubated at 37” for 30 min in ers were purchased from Bethesda Rethe presence of [35S]methionine at 50 Ci/ search Laboratories. After electrophoresis, ml. Labeling of the 2-deoxyglucose-treated 35S-containing gels were soaked in dimethsample was done in the presence of 20 mM ylsulfoxide (DMSO) for 1 to 2 hr, then in 2-deoxyglucose. DMSO plus 22% 2,5-diphenyloxazole (PPO) Endoglycosidase H. Nonproducer transfor 3 hr with gentle agitation. The gel was formed chicken cells were incubated in then washed in running water for 1 hr,
v-rel
PROTEIN
dried, and XAR film. dried and tensifying
exposed for 1 to 7 days to Kodak 32P-Containing gels were simply exposed, sometimes with an inscreen (DuPont Lightning Plus). In vitro kinase assays. For kinase assays, nonradioactive cells were washed in PBS and lysed in 20 mM Tris, pH 7.5, 200 mM sodium chloride, 1% Triton X-100 (TNT), containing aprotinin and PMSF as above. Lysates were clarified as above and subjected to immune precipitation immediately. Immune complexes bound to protein A-Sepharose were washed several times in TNT and then incubated at 30” for 10 min in 30 yl TNT containing 10 mM manganese chloride, 180 KU per ml aprotinin, 1 mM PMSF, and 10 &i [T-~~P]ATP (Amersham, 3000 Ci/mmol). The reaction was stopped by addition of 1 ml RIPA and immune pellets were collected by centrifugation for 1 min in the Eppendorf microfuge and washed several times with RIPA. The final pellets were resuspended in gel loading buffer as above, boiled, and loaded onto 10% or 10 to 20% gels. Gels were dried and autoradiographed with an intensifying screen for 2 to 24 hr. Synthesis
of peptides and immunizations.
The protected carboxyl terminal amino acid of each peptide was covalently attached to a solid support, 1% crosslinked divinylbenzene-polystyrene, via the cesium salt (Gisin, 1973). Peptides I and II were then assembled by solid phase synthesis (Merrifield, 1963) in a semiautomated Vega peptide synthesizer, Model 96, as described (Copeland and Oroszlan, 1981). Protected amino acids were purchased from Peninsula Laboratories (San Carlos, Calif.). The amino terminus of each amino acid was protected with tert-butyloxycarbonyl except for arginine which was the amyloxycarbonyl derivative. The side chain protecting groups were tosyl for arginine, benzyl for glutamic acid and threonine, and o-bromobenzyloxycarbonyl for tyrosine. Double couplings with a two fold molar excess were employed. If the ninhydrin assay (Kaiser et al., 1970) indicated that coupling was incomplete, a third coupling was performed. Each peptide was cleaved from the solid support and protecting groups with hydrogen fluoride containing 10% anisole
219
in a Teflon apparatus at 0” for 60 min. Peptides were extracted into 50% acetic acid, diluted with water, and lyophilized. Peptide III was purchased in crude form from Peninsula Laboratories. Each crude peptide was desalted and purified by reversed phase liquid chromatography (RPLC) on a Waters Associates 7.8 mm X 30 cm PBondapak Cl8 column using an aqueous acetonitrile gradient containing 0.05% trifluoroacetic acid. The amino acid composition of each purified peptide was determined and each was found to be correct. Prior to immunization, each RPLC purified peptide was covalently bound to keyhole limpet hemocyanin (KLH) (Calbiochem) utilizing approximately 5 mg of peptide to 5 mg of KLH. Peptide I was coupled with difluorodinitrobenzene (Tager, 1976), peptide II with bis-diazotized benzidine (Bassiri et ah, 1974) and peptide III with m-maleimidobenzoyl-N-hydroxysuccinimide ester (Liu et al, 1979). Following the reactions, each peptide KLH conjugate was dialyzed against PBS at 4”. Rabbits were initially inoculated with 1 mg of the peptide KLH conjugate in PBS diluted to 2 ml with Freund’s complete adjuvant and administered intradermally to several locations on the back. Boosts were made at 2-week intervals with 0.1 mg of the peptide-KLH conjugate in incomplete adjuvant. Test bleeds were begun just prior to the second boost. Antibody titer was determined with peptide bound to diazotized paper strips as described in detail elsewhere (Schultz et al., 1985). Once positive sera to the peptide was obtained, each animal was exsanguinated. Other antisera. Antisera to purified REVA p30 and p20E are described elsewhere (Tsai et ah, 1985; W.-P. Tsai, T. D. Copeland, and S. Oroszlan, in preparation). Affinity purification of antiserum III was performed by Alan Schultz. Serum III was passed over an Affigel 10 column (Bio-Rad) to which peptide III was bound, as described by Schultz et aZ. (1985). RESULTS
Peptides representing three segments of the predicted v-rel protein were synthe-
220
RICE II
I
Peptide
Number
ET
weakly positive and was not used in these studies.
III
I
*
lh
2&l Amino
3kl
Acid Residue
4do
AL.
do
Detection of the v-rel Protein
Number
1. Location of the three peptides in the predicted v-rel protein. Based on the DNA sequence, the v-rel protein has 503 amino acids, the N-terminal 12 and C-terminal 19 of which are coded for by regions derived from the helper viral genome REV-A (Stephens et al, 1983; Wilhelmsen et al., 1984). Sequences of the peptides proceeding from NHa- to COOH-terminal are: I, Tyr-Tyr-Glu-Ala-Glu-Phe-Gly-Pro-GluArg-Gln-Val-Leu; II, Glu-Gln-Pro-Arg-Gln-Arg-GlyThr-Arg-Phe-Arg-Tyr; III, Pro-Ala-Tyr-Asn-Pro-LeuAsn-Trp-Pro-Asp-Glu-Lys-Asn-Cys. The C-terminal cys residue in peptide III is not found in the v-rel protein sequence; it was added to the peptide to facilitate coupling. FIG.
Antisera to each of the peptides were used in immune precipitation experiments with lysates of [35S]methionine-labeled REV-transformed chicken lymphoid cells. As shown in Fig. 2, each serum precipitates a small number of proteins, several of which also appear with preimmune serum or with immune serum in the presence of excess competing peptide; these are judged to be the result of nonspecific binding. A few additional proteins do not appear with preimmune serum and are competable by excess homologous peptide but not by heterologous peptide; these are judged to represent specific antigen-antibody binding. Of these proteins, only two appear with sera to all three peptides. The first has an apparent molecular mass of about 59,000 Da, in good agreement with the 56,000 Da predicted for the v-rel protein by the DNA sequence. It is not precipitated by any of several other antisera raised to unrelated
sized, based on the DNA sequence of v-rel. Their location in the rel protein is shown in Fig. 1. The peptides were coupled to hemocyanin and each was used to immunize two rabbits. All six resulting sera recognize their respective peptides, and five are able to precipitate the v-rel protein. One of the sera raised against peptide I was only Antiserum II I
111 Ir
1
2
3
4
I 5
6
7
6 MW x10-3
-93-66--)r -43-
-26-
-16-
FIG. 2. Detection of the rel protein with antisera to synthetic peptides. Nonproducer transformed chicken cells were grown in the presence of [%]methionine for 2 hr and subjected to immune precipitation as described under Materials and Methods. Each lane received the immune pellet from about lo6 cells. Lysates for lanes 1, 5, and 12 were treated with preimmune serum; for lane 2 with antiserum II alone; for lane 3 with serum II plus competing peptide II; for lane 4 with serum II plus competing peptide III; for lanes 6 and 13 with serum III alone; for lane 7 with serum III plus competing peptide III; for lane 8 with serum III plus competing peptide II; for lane 9 with serum I alone; for lane 10 with serum I plus competing peptide I; for lane 11 with serum I plus competing peptide III. Arrows indicate the putative v-rel encoded protein.
v-rel
221
PROTEIN
proteins or peptides. The second, of about 110,000 Da, does not appear reproducibly and its identity is unknown at present. If p59 is the v-rel protein it should not appear in uninfected chicken cells, and as shown in Fig. 3, it does not. Antiserum I precipitates a protein of about 60,000 Da from the uninfected cells (lane l), but this reaction is not competable by peptide I (lane 2). Antiserum II is negative. Based on its presence in REV-transformed cells, its specific reactivity with three independent peptide antisera, and its size, we conclude that p59 is the v-rel product. Lysates of uninfected chicken cells do contain several other proteins specifically precipitable by each of the sera. With the exception of p59 and ~110 each of the proteins competable by homologous but not by heterologous peptide in the experiment shown in Figs. 2 and 3 is also present in the Fig. 3 study with uninfected cells. As has been reported previously (Nigg et al., 1982), that antisera to synthetic peptides may cross-react with a small number of other proteins, presumably by virtue of their sequence homology to the peptide.
Alternatively one or more of these proteins may be related to the c-rel protein. p5Ye1 Product
Immune precipitation following metabolic labeling with 32P04 demonstrated that the rel protein is phosphorylated. As shown in Fig. 4, all three sera precipitate a protein of approximately 60,000 Da. This protein is not seen with preimmune sera; nor is it present when the immune precipitation is performed in the presence of excess homologous peptide. Since this experiment employed virus-producing cells, it was also possible to detect the gag precursor, which is known to be phosphorylated (Tsai et al., 1985), using antiserum to purified ~30. In several such experiments the intensity of the Pr55g”g band was about the same as that of the p5gre1 band (data not shown). p.!Nez Product
p59”
2
is not Glycosylated
To investigate whether p59’“’ is likely to be a membrane-bound protein, we tested it for glycosylation. If oligosaccharide side chains account for any significant portion
REV-T 1
is a Phosphoprotein
CEF 3
4
1
2
3
4
-t
FIG. 3. Reactivity of peptide antisera with proteins in normal chicken cells. REV-transformed chicken cells and uninfected chick embryo fibroblasts were grown in the presence of methioninefree MEM, [?J]methionine, and 5% dialyzed fetal calf serum for 2 hr and subjected to immune precipitation. Each lane received the immune pellet from about 10” cells. Lysates in lane 1 were treated with antiserum I; in lane 2 with serum I plus competing peptide I; in lane 3 with serum II; and in lane 4 with serum II plus competing peptide II. Notice that except for p5gTe’ and one high molecular weight band, all competable bands are also present in CEFs.
RICE
222
ET
AL.
Antiserum I
I1
I
III
f )
1
2
/ /
3
4
5
6
1
7
MW
a
x 10-3
-
93
-
66
-
43
is phosphorylated. Cells were metabolically labeled with 32P04 as described FIG. 4. The rel protein under Materials and Methods. Aliquots of the lysate were subjected to immune precipitation (about 2 X 10’ cells per lane) with the indicated sera and pellets were analyzed on a 10% polyacrylamide gel. Exposure was for 3 days with an intensifying screen. Lane 1, antiserum I alone; lane 2, antiserum I plus competing peptide I; lane 3, preimmune antiserum II; lane 4, antiserum II alone; lane 5, antiserum II plus competing peptide II; lane 6, antiserum II plus competing peptide I; lane 7, preimmune antiserum III; lane 8, antiserum III. The p59 band in lane 1 is faint but clear on the X-ray film; it reproduces poorly.
of the rel product’s molecular weight, then prevention of their addition to the polypeptide chain should result in the appearante of a rel protein of reduced size. Both tunicamycin and 2-deoxyglucose are known to block glycosylation, and as shown in Fig. Tunicamycin
-
5 each is effective in preventing glycosylation of the viral envelope precursor protein. In untreated cells, antiserum to the purified transmembrane protein p20E precipitates the glycosylated envelope precursor, whose size is about 74,000 Da. Pre-
2-Deoxy-Glucose
+
Envelope
+
Precursor +DG
+T
p59”’
is not detectably glycosylated. Virus-producing transformed cells were FIG. 5. The rel protein treated with tunicamycin or 2-deoxyglucose and labeled in the presence of [35S]methionine as described under Materials and Methods. After immune precipitation (about 2 X lo6 cells per lane) with either antiserum III or antiserum raised against purified viral p20E, samples were analyzed on a lo-20% gradient polyacrylamide gel.
v-rel
PROTEIN
of carbohydrate, p59’“’ cannot be detected in the culture medium following a several hour incubation of transformed cells in [35S]methionine (data not shown). Since 35Slabeled REV-A p30 is easily detected in the medium from these same cells, we conclude that little or no p5gre’ is secreted.
treatment of the cells with either of the inhibitors, however, prevents the synthesis of gPr74”“‘, and only the unglycosylated 57,000-Da polypeptide chain is detectable. If aliquots of these same cellular lysates are incubated not with anti-p20E but with antiserum III, no effect of the inhibitors on the size of the rel protein is detectable. The same result was obtained in experiments with endoglycosidase H, an enzyme which is able to cleave oligosaccharide side chains from a mature protein. When gPr74”“” is precipitated by anti-p20E from lysates of virus-producing cells, treatment of the immune pellet with endoglycosidase H reduces its molecular weight to about 59,000 Da. Under the same conditions in immune precipitates using antiserum III, the enzyme has no effect on the size of the rel product (data not shown). We conclude that there is no detectable glycosylation of p59’? We also looked for evidence of secretion of p59’“‘. Consistent with its apparent lack Antiserum Label 15’
Chase, 60’
2
Pulse-chase
6
Experiment
To determine whether the newly synthesized rel protein is the same size as the presumably mature form observed after a 2-hr incubation with radioactive precursors, a pulse-chase experiment was performed. Cells were grown in the presence of [35S]methionine for 15 or 60 min and then incubated in nonradioactive complete medium for varying times up to 48 hours. As shown in Fig. 6, the size of the newly synthesized rel product observed in the cells labeled for only 15 min is not detectably different from that observed at later times. Even in much longer exposures of the films Antiserum
II Hours
Label
II
I
223
26
46
’
MW x10-3
-’
15’
60’
60’
2
III Chase,
Hours
2
6
I 26
FIG. 6. The rel protein in a pulse-chase experiment. With the exception of an aliquot withdrawn after 15 min, cells were incubated in [35S]methionine (50 &X/ml) for 60 min, washed in PBS, and resuspended in nonradioactive complete medium plus serum. Aliquots were taken at times indicated in the figure and lysates were subjected to immune precipitation with antiserum II or III. Lysates for the third and fifth lanes in the serum III experiment were treated with preimmune serum. Lysates from an approximately constant number of cells (about 3 X 106) were analyzed in each of the lanes shown; the only exception is the sample labeled for only 15 min, from which about 1.5 times the usual number of cells was taken. The doubling time of these tranformed chicken cells is approximately 24 hr.
224
RICE
there is no indication of an additional rel band, either larger or smaller. Thus, if there is any processing of the nascent rel protein at all, it must either be very closely coupled to its synthesis, for it is not observable even on the time scale of a few minutes, or it must result in a very small change in apparent molecular weight, which is unresolvable in these experiments. A rel protein of the same size is also seen throughout the cold-chase following a 60min incubation in [35S]methionine. The intensity of the band has already diminished after a few hours but it is still detectable after 26 hr and even after 48 hr on films exposed for longer times. There is no indication of the accumulation of stable breakdown products. Since this experiment was performed separately with all three peptide antisera, whose reactivities span the molecule, cleavage products would most likely have been detected. The rel Protein Has an Associated Kinase Activity In vitro kinase assays were performed on immune precipitates of p59”’ using each of the three peptide sera independently. As shown in Fig. 7, the precipitate with serum I was negative, with serum II very weakly positive, but with serum III strongly positive for kinase activity. Serum from both rabbits immunized with peptide III resulted in kinase activity in the immune precipitate (lanes 4 and 5). This activity was also observed after immune precipitation with serum III which had been heated to 56” for 30 min (data not shown) and with affinity-purified serum III (lane 7). To assess the purity of the affinity-purified serum, an aliquot equal to that used in the precipitation experiments was electrophoresed on a 10% polyacrylamide gel. Following silver staining of the gel, the only bands visible were of the size expected for immunoglobulin. We conclude that it is very unlikely that the kinase observed in these experiments is derived from the serum. The level of kinase activity is quite high. Each lane shown in Fig. 7 received the immune precipitate from 1-2 X lo6 cells. Each kinase assay included 10 &i [y-32P]ATP at
ET
AL. Antiserum
III
I
$-IA 1
2
3
4
5
6
7
MW x 10-3
-93
-66 -43
-26
-48 FIG. 7. In vitro kinase assay with p59’“’ immune precipitates. Following immune precipitation of REVtransformed chicken lymphoid cells lysates (1-2 X lo6 cells per lane) with the indicated sera, in vitro kinase assays were performed as described under Materials and Methods. Products were analyzed on a lo-20% gradient SDS-polyacrylamide gel and autoradiographed for about 20 hr with an intensifying screen. Lane 1, preimmune serum; lane 2, antiserum I; lane 3, antiserum II; lanes 4 and 5, antiserum III from two different rabbits; lane 6, antiserum III plus peptide III; lane 7, affinity-purified antiserum III.
3000 Ci/mmol, and the exposure time of the film was about 2 hr with an intensifying screen. This is roughly comparable to the activity reported for p160gagmab’(Konopka et aZ., 1984) and p75gag-‘“f (Moelling et ah, 1984) immune precipitates. The reaction appeared absolutely dependent on the presence of manganese ion. Assays performed in the presence of Mgf+ or Ca++ were negative (data not shown). Among several other strongly labeled bands which appeared following incubation with [T-~‘P]ATP was one of about 59,000 Da. To ascertain whether this protein was the p59’“‘, we tested whether it could be reprecipitated, after the kinase reaction, by each of three sera. This was done by boiling the products of the kinase reaction in 1% SDS, thereby liberating the p59 from antibody and protein A-Sepharose, and then incubating aliquots with serum I, II, or III. As shown in Fig. 8, 32P-labeled p59 is precipitable with all the sera. We conclude therefore that the rel protein is being
v-rel Boiled, 12’3
4
reprecipitated 5
6
7
0
I
-
225
PROTEIN
p59”’
FIG. 8. Phosphorylation of the v-rel protein during an in vitro kinase assay. After immune precipitation with antiserum III of a lysate of transformed chick cells, an in vitro kinase assay was performed as described under Materials and Methods. Following extensive washing to remove unincorporated [32P]ATP, the product (except for aliquots saved for lanes 1 and 2) was boiled for 5 min in 1% SDS. Protein A-Sepharose was pelleted and discarded, and the supernatant was diluted with TNT, aliquoted, and subjected to immune precipitation in the presence of fresh antibody and protein A-Sepharose. Precipitates were collected and analyzed on a 10% SDS-polyacrylamide gel. Exposure to X-ray film was for 24 hr with an intensifying screen. Lane 1, immune precipitation with preimmune serum; lane 2, with antiserum III; lanes 3-8, sample boiled in 1% SDS after kinase reaction. Lane 3, no fresh serum added; lane 4, preimmune serum added; lane 5, fresh serum I added; lane 6, serum II added; lane 7 serum II plus peptide II added; lane 8, serum III added. The precipitate in each lane was derived from about lo6 cells.
phosphorylated during the in vitro kinase assay, but that this occurs only when the immune precipitation is performed with serum III. The identity of the other phosphorylated proteins is unknown. The dark band at about 59,000 Da actually appears to be a doublet, the larger member of which is p59’“‘. The smaller member can be seen very faintly even in assays performed after immune precipitation with preimmune serum (Figs. 7 and 8). It is thus a nonspecific component of the immune pellet and can act as a phosphate acceptor if a kinase is also present. The other dark band, at about 50,000 Da, is the appropriate size for the immunoglobulin heavy chain, but we have not investigated its identity further.
As one approach to identifying the kinase, we attempted to dissociate p59’“’ from the kinase activity. The usual protocol for the assay involves lysis of the cells, immune precipitation, and washing of the precipitates, all in TNT (which contains 0.2 M NaCl and 1% Triton X-100). We therefore tried more stringent washing conditions before the kinase assay to see if a hypothetical associated kinase could be dissociated from the immune pellet. As shown in Fig. 9, kinase activity was retained in the pellet after washing in 0.5% sodium deoxycholate (DOC), in 0.1% SDS, and in 2 M NaCl. Washing in TNT plus 10 mM EDTA or in 20 mMTris plus 1% Triton X100 with no added salt also failed to dislodge kinase activity from the immune complex (data not shown). In addition, we varied conditions during the immune pre-
Before I
Wash Kinase
Assay I
FIG. 9. The effect of various washing conditions before the in vitro kinase assay. Cells were lysed, immune precipitated (l-2 X lo6 cells per lane), and prepared for a kinase assay as described under Materials and Methods. Just before the assay the pellets were incubated for 15 min in 1 ml of TNT supplemented with the indicated reagents. Pellets were then collected, washed once in TNT, and assayed for kinase activity as usual. Lanes 1 and 2 are controls which were treated with TNT throughout the procedure. Lane 1, preimmune serum. Lanes 2-6, antiserum III. The dried gel was autoradiographed for about 6 hr with an intensifying screen.
226
RICE
cipitation, but as shown in Fig. 10, kinase activity was still present in the precipitate when 0.5% DOC and 2 M NaCl were added to the usual buffer. If the kinase is a protein associated with p59’“‘, therefore, the two must be very tightly complexed.
ET
AL.
is about 110 kDa, and it is clearly visible in Figs. 2 and 4. The identity of this protein is unknown at present. It could be an unrelated protein which is specifically associated with p5gre’ in transformed cells, or it could represent undissociated dimers of p5gre1, or it could conceivably be the protein DISCUSSION encoded by c-rel. Peptide mapping studies In this paper we report the detection of will be required to resolve this point. Regarding c-rel, the nucleotide sequence p59’“‘, the protein product of the v-rel onof at least most of the turkey gene is known cogene of reticuloendotheliosis virus. The from the work of Wilhelmsen et al. (1984), protein can be precipitated by antisera and the chicken gene is expected to be raised to three different peptides derived highly related (Simek and Rice, 1980; Wong from the translated DNA sequence of v-rel (Stephens et ab, 1983; Wilhelmsen et al, and Lai, 1981). Each of the three peptides used in this study differs from the corre1984). This precipitation is specific in that it can be competed by homologous, but not sponding region in turkey c-rel. At the poresidue in peptide by heterologous, peptide. Since p59 is not sition of the glutamine I, there is an arginine in c-rel; at the threseen in uninfected chick embryo fibroblasts onine in peptide II, there is a methionine but is seen in several independent REV-Taspargine transformed chicken lymphoid cell lines, in c-rel; and at the C-terminal in peptide III, there is an aspartic acid in we conclude that it is the product of v-rel. c-rel. In addition, peptide III has a threeIts apparent size of 59 kDa is in reasonable residue deletion relative to turkey c-rel. We agreement with the 56 kDa predicted by are thus unable to predict whether any of the DNA sequence. our three sera should recognize the chicken One other protein appears to be precipitable with all three peptide sera. Its size c-rel product. We are hopeful that antisera raised to portions of v-rel expressed in bacteria will afford a better opportunity to identify the c-rel protein. Since we now 2M NaCl 0.2 M NaCl i I I I know that human DNA contains sequences 0.5% DOC 0.5% DOC highly related to at least two of the turkey i 2 3 4 5 6 7 8 ’ MW c-rel exons (Brownell et al., 1985), such antisera should be useful in the search for a human c-rel protein as well. The v-rel protein has a closely associated protein kinase activity observable when immune precipitation is performed with antiserum to peptide III. Antiserum to peptide I results in a background level of activity comparable to that seen with preimmune serum, and antiserum to peptide II results in very low activity. Sera from both rabbits immunized with peptide FIG. 10. The effect on kinase activity of varying conIII, however, result in strong kinase activditions during immune precipitation. Cells were lysed ity in the immune complex. This activity in TNT and clarified as usual. For immune precipiis also observable in affinity-purified antation, lysates (1-2 X lo6 cells per lane) were adjusted tiserum III and is reduced to background to the indicated salt and detergent concentrations. levels when precipitation is carried out in Subsequent washing and assay conditions were as dethe presence of competing peptide III. We scribed under Materials and Methods. Lanes 1, 3, 5, conclude that the kinase activity is due to 7, preimmune serum; lanes 2, 4, 6, 8 antiserum III. a specific component of the cell lysate Exposure was for about 2 hr with an intensifying which is either precipitated by serum III screen. I
I
v-rel
PROTEIN
or complexed to a protein which is precipitated by serum III. The identity of the kinase is unknown. A trivial possibility is that the kinase is unrelated to p59’“’ but cross-reacts with antiserum to peptide III. We have attempted to discount this possibility with two additional experiments. First, we assayed chick embryo fibroblasts for kinase activity using antiserum III. The result was a few faint bands visible only after a 4-day exposure with an intensifying screen. Admittedly, this is an imperfect control since a hypothetical cross-reacting kinase could be present in lymphoid cells but not in fibroblasts. Second, we performed the kinase assay with a serum directed against a 262 amino acid segment of the v-rel protein expressed in bacteria (S. Simek, R. Stephens, and N. R. Rice, to be published). When this serum is used to precipitate p59’“’ from lysates of the transformed chicken lymphoid cells, kinase activity in the immune complex is comparable to that seen with antiserum III. This precipitation of kinase activity by the new serum is unaffected by a concentration of peptide III 100 times higher than that required to completely block antiserum III, despite the fact that the new serum binds peptide III in an ELISA assay. These results show that the kinase activity can also be recognized by antisera directed against regions of p59’“’ other than that defined by peptide III. Thus, it is unlikely that the results using peptide III antiserum alone are artifactual. A second possibility is that the kinase is a protein specifically associated with p59’“’ which is dislodged during precipitation with serum I or II. Such a situation would be analogous to that observed with polyoma middle T antigen, where middle T antisera precipitate a kinase activity which appears to be due to closely associated pp60c+rc (Courtneidge and Smith, 1983). In an attempt to dissociate such a hypothetical complex, detergent and salt concentrations have been varied both during the immune precipitation step and during the washing of the immune pellets prior to the kinase assay. None of these conditions (including 0.5% DOC, 0.1% SDS, 10 mM EDTA, and even 2 M NaCl) reduced the
227
kinase activity appreciably. If such a complex exists, therefore, it must be unusually stable. This conclusion is analogous to that reached by Kloetzer et al. (1984) for the pgjPw”o~- associated kinase activity. We are currently using nondenaturing polyacrylamide gels and sucrose gradients to look for evidence of complexes containing p59’“‘. The third possibility is that p59’“’ is a protein kinase, able to phosphorylate itself as well as a number of other proteins found in the immune complex. The failure of sera I and II to result in kinase activity might then be interpreted in terms of their binding to functionally important sites. Sera raised to peptides within the kinase domain of pp60”-“‘” (Gentry et al, 1983), of ~160~“~-“~’ (Ko nopka et al, 1984; Davis et ah, 1985), and of p85gag-fesand p108gag-fes(Sen et aZ., 1983) block kinase activity in the in vitro assay, whereas sera to peptides outside this domain in pp60V+Fe (Gentry et aZ., 1983), in p160gag-“b1(Konopka et al., 1984), and in p37”” (Maxwell and Arlinghaus, 1985) do not inhibit this activity. In p59’“‘, however, kinase domain homologous to that of the src family cannot be identified. While there may be very distant relatedness between several regions of p5gre’ and pp60”” (Stephens et ah, 1983), these do not include sequences in pp60src around the ATP binding site (lysinezg5) (Kamps et ah, 1984). Nor does p5gre’ possess the Ala430Pro431-G1u432 sequence common to most members of the src family (Bishop and Varmus, 1985) and known to be essential in pp60src for kinase activity and for transformation (Bryant and Parsons, 1984). Based on the sequence, therefore, there is no compelling reason to predict that p5gre1 is a kinase of the src family. The identity of the kinase will be satisfactorily resolved only after expression and purification of p59’“’ from bacteria and/or purification from eukaryotic cells with retention of kinase activity. We also wish to know which amino acid(s) is phosphorylated in the in vitro kinase reaction. While our preliminary results indicate the presence of phosphoserine and phosphothreonine and essentially no phosphotyrosine, the analysis has not been carried out on puritied p5gre’. This work is in progress.
228
RICE ET AL. ACKNOWLEDGMENTS
Research was sponsored in part by the National Cancer Institute, DHHS, under Contract NOl-CO23909 with Litton Bionetics, Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U. S. Government. We are grateful to Ronald R. Hiebsch for invaluable assistance in the early stages of this study. We thank Wen-Po Tsai for antiserum to REV-A p30 and p20E; Alan Schultz for affinity purification of antiserum III; and Jeannie Clarke and Margaret Fanning for preparation of the manuscript. REFERENCES BASSIRI, R. M., and UTIGER, R. D. (1974). Thyrotropinreleasing hormone. In “Methods of Hormone Radioimmunoassay” (B. M. Jaffe and H. R. Behrman, eds.), pp. 37-44. Academic press, New York. BISHOP, J. M., and VARMUS, H. (1985). Functions and origins of retroviral transforming genes. In “RNA Tumor Viruses, Molecular Biology of Tumor Viruses,” 2nd Ed., pp. 249-356. Cold Spring Harbor Laboratory. BROWNELL, E., 0-BRIEN, S. J., NASH, W. G., and RICE, N. R. (1985). Genetic characterization of human crel sequences. Mol. Cell. E&l., 5,282.6-2831. BRYANT, D. L., and PARSONS,J. T. (1984). Amino acid alterations within a highly conserved region of the Rous sarcoma virus ST(:gene product ~~60’” inactive tyrosine protein kinase activity. Mol. Cell. Bid. 4, 862-866. COPELAND,T. D., and OROSZLAN,S. (1981). A synthetic dodecapeptide substrate for type C RNA tumor virus associated proteolytic enzyme. In “Peptides: Synthesis-Structure-Function” (D. H. Rich and E. Gross, eds.), pp. 497-500. Pierce Chemical, Rockford, Ill. COURTNEIDGE,S. A., and SMITH, A. E. (1983). Polyoma virus transforming protein associates with the product of the c-src cellular gene. Nature (London) 303,435-439. DAVIS, R. L., KONOPKA, J. B., and WITTE, 0. N. (1985). Activation of the e-&l oncogene by viral transduction or ehromosomal translocation generates altered c-abl proteins with similar in vitro kinase properties. Mol. Cell. Biol. 5,204-213. GENTRY, L. E., ROHRSCHNEIDER,L. R., CASNELLIE, J. E., and KREBS, E. G. (1983). Antibodies to a defined region of pp60”’ neutralize the tyrosine-specific kinase activity. J. Biol. Chem 258,11219-11228. GISIN, B. F. (1973). The preparation of Merrifield-resins through total esterification with cesium salts. Helv. Chim. Acta 56,1476-1481. GONDA, M. A., RICE, N. R., and GILDEN, R. V. (1980). Avian reticuloendotheliosis virus: Characterization
of the high molecular weight viral RNA in transforming and helper virus populations. J. Viral. 34, 743-751. HOELZER, J. D., FRANKLIN, R. B., and BOSE, H. R. JR. (1979). Tranformation by retieuloendotheliosis virus, development of a focus assay and isolation of nontransforming virus. Virology 93,20-30. HOELZER, J. D., LEWIS, R. B., WASMUTH, C. R., and BOSE, H. R., JR. (1980). Hematopoietic cell transformation by reticuloendotheliosis virus: Characterization of the genetic defect. Virology 100, 462474. KAISER, E., COLESCOTT,R. L., BOSSINGER, C. D., and COOK, I’. I. (1970). Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal. Biochem. 34,595-598. KAMPS, M. P., TAYLOR, S. S., and SEFTON, B. M. (1984). Direct evidence that oneogenic tyrosine kinases and cyclic AMP-dependent protein kinase have homologous ATP-binding sites. Nature (London) 310,589592. KLOETZER, W. S., MAXWELL, S. A., and ARLINGHAUS, R. B. (1984). Further characterization of the p858”8”“_ associated protein kinase activity. Virology 138,143-155 KONOPKA, J. B., DAVIS, R. L., WATANABE, S. M., PONTICELLI, A. S., SCHIFF-MAKER, L., ROSENBERG,N., and WITTE, 0. N. (1984). Only site-directed antibodies reactive with the highly conserved src-homologous region of the v-abl protein neutralize kinase activity. J. Irirol. 51, 223-232. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 2?7,680-685. LEWIS, R. B., MCCLURE, J., RUP, B., NIESEL, D. W., GARRY, R. F., HOELZER, J. D., NAZERIAN, K., and BOSE, H. R. JR. (1981). Avian reticuloendotheliosis virus: Identification of the hematopoietic target cell for transformation. Cell 25,421-431. LIU, F.-T., ZINNECKER, M., HAMAOKA, T., and KATZ, D. H. (1979). New procedures for preparation and isolation of conjugates of proteins and a synthetic copolymer of D-amino acids and immunoehemical characterization of such conjugates. Biochemistry l&690-697. MAXWELL, S. A., and ARLINGHAUS, R. B. (1985). Serine kinase activity associated with Moloney murine sarcoma virus-124-encoded ~37~. I%-oZogy143,321333. MERRIFIELD, R. B. (1963). Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Amer. Chem. Sot. S&2149-2154. MOELLING, K., HEIMANN, B., BEIMLING, P., RAPP, U. R., and SANDER,T. (1984). Serine- and threoninespecific protein kinase activities of purified gag-mil and gag-raf proteins. Nature (London) 312,558-561. NIGG, E. A., WALTER, G., and SINGER, S. J. (1982). On the nature of cross-reactions observed with anti-
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RICE, N. R., HIEBSCH, R. R., GONDA, M. A., BOSE, H. R., JR., and GILDEN, R. V., (1982). Genome of reticuloendotheliosis virus: Characterization by use of cloned proviral DNA. J. Viral. 42,237-252. SCHULTZ,A. M., COPELAND, T. D., MARK, G. E., RAPP, U. R., and OROSZLAN, S. (1985). Detection of the myristylated gag-raftransforming protein with raf specific antipeptide sera. virology 146, ‘78-89. SEN, S., HOUGHTEN, R. A., SHERR, C. J., and SEN, A. (1983). Antibodies of predetermined specificity detect two retroviral oneogene products and inhibit their kinase activities. Proc. Natl. Acacl. Sci. USA 80,1246-1250. SIMEK, S., and RICE, N. R. (1980). Analysis of the nucleic acid components in reticuloendotheliosis virus. J. viol. 33,320-329. STEPHENS, R. M, RICE, N. R., HIEBSCH, R. R., BOSE, H. R., JR., and GILDEN, R. V., (1983). Nucleotide se-
229
quence of v-rel: The oncogene of reticuloendotheliosis virus. Proc. Natl. Acud Sei. USA 80, 62296233. TAGER, H. S. (1976). Coupling of peptides to albumin with difluorodinitrobenzene. Anal. Biochem. 71,367375. TSAI, W.-P., COPELAND,T. D., and OROSZLAN,S. (1985). Purification and chemical and immunological characterization of avian reticuloendotheliosis virus guggene-encoded structural proteins. virology 140,289312. WILHELMSEN, K. C., EGGLETON, K., and TEMIN, H. M. (1984). Nucleic acid sequence of the oncogene v-rel in reticuloendotheliosis virus strain T and its cellular homolog, the protooncogene c-rel. J. ViroZ. 52, 172-182. WONG, T. C., and LAI, M. M. C. (1981). Avian reticuloendotheliosis virus contains a new class of oncogene of turkey origin. Virology 111,289-293.