Recognition of mos-re la ted Proteins with an Antiserum to a Peptide of the v-mos Gene Product
By G. E. G A L L I C K , 1 J. T. S P A R R O W , 2 B. S I N G H , 3 S. A. M A X W E L L , 3 L. H. S T A N K E R ~ AND R. B. A R L I N G H A U S 3.
UTSCC M.D. Anderson Hospital, Department of Tumor Biology, Houston, Texas 77030, 2Baylor College o f Medicine, Department of Medicine, Houston, Texas 77030, 3Scripps Clinic and Research Foundation, Department o f Molecular Biology, La Jolla, California 92037 and
4Lawrence Livermore National Laboratory, Livermore, California 94550, U.S.A.
(Accepted 21 December 1984)
S U M M A R Y
A mos-specific antiserum was generated by injection of rabbits with a peptide predicted from the sequence of the v-mos gene of Moloney murine sarcoma virus (MuSV) strain 124. The peptide is composed of amino acids 37-55 (cyclized at the cysteine residues) conjugated to keyhole limpet haemocyanin. This serum [anti- mos(37-55c)] specifically recognized p37 m°~ in MuSV-124 acutely infected NIH-3T3 cells, P85gag-mos in 6m2 cells, an NRK clone infected with the temperature-sensitive mutant (tsll0) of Moloney MuSV, and P100 gag . . . . in 54-5A4 cells, an N R K clone infected with a spontaneous revertant of ts110. An additional protein of Mr 55000 from uninfected cells was recognized by this serum. Reactivity of the serum toward the v- inos-containing proteins and the 55K protein was completely inhibited by prior incubation with free peptide. The 55K protein was not recognized by antisera made. from synthetic peptides prepared from the C-terminal eight or 12 amino acids of v-mos.
INTRODUCTION
Among the many oncogenes currently under study, the mouse sarcoma (mos) gene appears anomalous in many respects. Unusual features of the gene include no introns between the coding sequence analogous to v-mos (Jones et aL, 1980; Oskarsson et al., 1980), no apparent transcription of c-mos-specific mRNA in many tissues tested at several stages of development (Muller et aL, 1982) and hypermethylation (Gattoni et al., 1982). The only reported transcription of c-mos occurs after DNA rearrangement (Rechavi et al., 1982). Furthermore, in cell lines infected with wild-type routine sarcoma virus (MuSV), expression of v-mos occurs in minute amounts (Papkoffet al., 1982; Stanker et al., 1983a), possibly because the gene product is toxic (Papkoff et al., 1982). Thus, studying the normal expression of c-mos, the potential for transformation by aberrant expression of this gene, and even transformation induced by MuSV has proven difficult.
Analysis of transformation by the v-mos gene product has been greatly facilitated by a temperature-sensitive mutant, tsll0, which previous studies in this laboratory have demonstrated produces easily detectable levels of an unstable 85 000 mol. wt. gag-mos fusion protein (Horn et al., 1981). A revertant of this mutant, designated 54-5A4, produces a stable 100000 tool. wt. protein in even larger amounts (Stanker et al., 1983b). Studies of cell lines infected with these mutants have revealed several interesting properties of gag mos proteins. P85g~g . . . . has an associated protein kinase activity (Kloetzer et al., 1983, 1984), is structurally labile at the non-permissive temperature (Stanker et al., 1983 c), is associated with lipid (Gallick & Arlinghaus, 1984), and appears to be produced from a 3.5 kb gag-mos mRNA derived from the larger 4-0 kb viral genome by a splicing mechanism (Nash et al., 1984; Junghans et al., 1982).
To analyse transformation in cell lines containing MuSV and variants, and to characterize more accurately which portions of the transforming protein are involved in the above properties,
peptides from the predicted v-mos sequence (Van Beveren et aL, 1981) have been synthesized, and antiserum against them has been generated in rabbits. This communication reports the detection of m o s transforming proteins with an antiserum to a peptide comprising amino acids 37 to 55, cyclized at cysteine residues, as well as the surprising result that a normal cellular protein of about 55000 mol. wt. specifically reacts with this antiserum.
METHODS
Cells and virus. The 6m2 cell line, a non-producer clone of normal rat kidney (NRK) cells infected with ts110 MuSV (Mo-MuSV), has been previously described (Horn et aL, 1981 ). 54-5A4, a cell line containing a spontaneous revertant of tsl l 0, has also been described (Stanker et aL. 1983b). MuSV-124 used in this study was isolated by Ball et al. (1973) from a thymus bone marrow cell mixture (Ball et aL, 1964) infected with Mo-MuSV murine leukaemia virus. NIH/3T3 cells were obtained from the American Type Culture Collection. LeE9 rat myoblasts (Nadal- Ginard, 1978), NIH/3T3 cells and HeLa cells were grown in Dulbecco's modified Eagle's medium containing 10~o foetal calf serum. N RK cells and N IH Swiss mouse embryo fibroblasts (JLS-V 16) (Naso et al., 1975) were grown in McCoy's medium containing 15°;; foetal calf serum.
Virus injection. NIH/3T3 cells were infected with virus-containing supernatant of MuSV-124 cells as described by Wong & Gallick (1978), except that virus was diluted in 10 gg/ml Polybrene rather than in DEAE-dextran.
Preparation o f synthetic peptides. A peptide comprising residues 37 to 55 of the predicted v-mos sequence was used. The sequence was as follows: NHz-Ser-Leu-Cys-Arg-Tyr-Leu-Pro-Arg-Glu-Leu-Ser-Pro-Ser-Val-Asp-Ser- Arg-Ser-Cys-COOH. The peptides were synthesized by the solid-phase technique on an improved polystyrene resin (Sparrow, 1976) using a Schwartz Bioresearch Peptide Synthesizer modified for computer control (Edelstein et al., 1981 ). The programme for deprotection and coupling has been reported previously (Bhatnagar et al., 1983 ; Hancock et al., 1981). Resin was removed after the attachment of residue 37. The peptide was deprotected except for Cys which was protected with acetamidomethyl and simultaneously cleaved from the resin by treatment of 1 g of resin for 30 min at 0 "C with 20 ml anhydrous HF containing 2 ml anisole and 0.2 ml ethanedithiol. The HF was removed under vacuum and the peptide and resin washed with ether. The peptide was dissolved in trifluoroacetic acid. The trifluoroacetic acid was evaporated under vacuum and the peptide precipitated with ether, After centrifugation, the peptide was dissolved in 1 M-Tris 6 M-guanidine-HCl and desalted on Bio-Gel P-2 equilibrated in 0.1 M-ammonium bicarbonate. The peptide-containing fractions were lyophilized.
Purification o f the peptides. The 37 to 55 residue peptide was purified by ion-exchange chromatography at 4 °C on a 2.6 x 30 cm column of SP-Sephadex equilibrated at pH 3.74 with 0.02 M-NaHzPO ~ 6 M-urea. After loading the peptide (200 rag) in the starting buffer, a linear gradient between 800 ml of starting buffer and 800 ml of 0,185 M- NaCI, 0.02 M-NaH2PO4 was used to elute peptide 37 55. The peptides were located by measuring the absorbance at 275 nm.
The fractions were pooled, the pH adjusted to 8, and desalted on a column of Bio-Gel P-2 equilibrated with 0.1 M-ammonium bicarbonate. The peptide-containing fractions were lyophilized. Yields of peptides averaged 25 % based on the attachment of the first residue to the resin. The amino acid composition of each purified peptide was as expected (Table 1). The peptides were greater than 95°.~, pure as judged by C t 8 reverse-phase HPLC using a gradient of 0.15 N-triethylammonium phosphate to 500/o propan-2-ol (Bhatnagar et al., 1983).
Cyclization and characterization oJ)'-mos peptide 37-55c. Ten mg of peptide in 5 ml 5 % acetic acid was added in one portion to 15 mg iodine in 10 ml glacial acetic acid. After stirring for 1 h, ascorbic acid was added to discharge the excess iodine and the peptide desalted on a Bio-Gel P-2 column in 5%0 acetic acid; the peptide-containing fractions were lyophilized. The lyophilized material was dissolved in 5 ml 6 ~l-guanidine HCI, 0-1 M-Na2HPO4, pH 7.5, and applied to a 2.6 x 100 cm column of Bio-Gel P-10 equilibrated in 0-1 M-NH4HCO3, pH 7.8. Some polymeric material eluted at a volume of 200 ml: uncyclized material eluted at a volume of 243 ml and cyclic peptide at 262 ml. The identity of three peaks was confirmed by amino acid analysis and C~ 8 reverse-phase H PLC. The cyclic peptide is termed 37--55c.
Preparation ofantisera. Monospecific goat antisera prepared against Rauscher leukaemia virus p15 and pl0 were obtained through the Office of Program Resources and Logistics, Viral Oncology, NIH and were absorbed with excess uninfected JLS-VI6 cell extracts as previously described (Jamjoom et al., 1977). The cyclic peptide comprising amino acids 37 to 55 was conjugated to keyhole limpet haemocyanin (KLH ; Biochem, Los Angeles, Ca., U.S.A.) as described by Sanchez et al. ( 1980, 1982). Briefly, 500 gg ofpeptide was activated with 5 mg 1-ethyl- 3-(3-dimethylaminopropyl)carbodiimide-HCl (Pierce Chemical Company, Rockford, II1., U.S.A.) in 0.0125 N- sodium phosphate buffer pH 5, by reacting for 2 rain at 24 °C. The pH was adjusted to 8.0, after which 500 gg KLH in phosphate-buffered saline (PBS) pH 8-0 was added. Conjugation was allowed to occur for 18 h at 24 °C. Free peptide was separated by precipitation with 1 0 ° alum (Sanchez et al., 1980: Dreesman et al., 1981) for 2 h, followed by centrifugation for 10 min at 10000 r.p.m, in a Dupont-Sorvall SS-34 rotor. Conjugated peptide was mixed 1:1 with Freund's complete adjuvant (Difco) and injected into New Zealand white rabbits (Jackson Laboratories, Bar Harbor, Me., U.S.A.) subcutaneously and intraperitoneally. Approximately 500 gg of peptide
v - r n o s protein recognition by an t i -mos (37-55c ) 947
T a b l e l . A m i n o acid composit ion o f synthet ic 37 -55 v - m o s pept ide
* Amino acid analyses were performed on a Beckman 119 amino acid analyser after hydrolysis at 110 °C for 24 h. The values are uncorrected for destruction. Numbers in parentheses are the theoretical values.
~" Present in this analysis but not integrated.
was used per injection. Rabbits were boosted at 2-week intervals until detectable immunoprecipitation ofgag-mos proteins was achieved, the antiserum is identified as anti-mos(37-55c) serum.
Anti-C3 serum was prepared as described by Papkoffet al. (1982). It is made from a v-mos peptide representing the 12 C-terminal amino acids of p37 m°s as predicted by the Van Beveren et al. (1981) sequence. Anti-C2 serum and C2 peptide were kindly provided by T. Hunter of the Salk Institute (La Jolla, Ca., U.S.A.). C2 is the C- terminal eight amino acids (Papkoff et al., 1983) of v-mos predicted by Van Beveren et al. (1981).
Metabolic labelling oJ cells. Cell lines infected with mutant strains of MuSV were allowed to grow to 70 to 80°/0 confluency in T25 tissue culture flasks (Corning), rinsed with Earle~s balanced salt solution, and then incubated for 20 min in Earle's balanced salt solution containing 500 laCi/ml L-[3H]leucine (New England Nuclear; sp. act. 40 to 60 Ci/mmol). For NIH/3T3 cells acutely infected with MuSV-124 supernatant, cells were labelled in MEM containing 5% of the normal amount of methionine, to which 100 laCi/ml L-[3sS]methionine (New England Nuclear; 800 Ci/mmol) was added. Cells were incubated for 12 h in this medium.
Immunoprecipitation and gel electrophoresis. For cell lines infected with the mutant strains of MuSV, lysates were prepared in a detergent-containing buffer as described by Naso et al. (1975). Acutely infected cells were washed after the labelling period with Tris-buffered saline, and solubilized for 10 min at 4 °C with RIPA buffer (0-15 M- NaCI, 0.01 M-sodium phosphate pH 7-0, 1 °/o NP40, 1% sodium deoxycholate, 0-1 ° o SDS, 1 °/o trasylol). Blocking of the antibody was done with the cyclic peptide at a ratio of 1.6 lag peptide per 40 lal antiserum. Lysates were clarified by centrifuging at 20000 r.p.m, for 30 rain in a Beckman J2-21 centrifuge using type JA-20 rotor, indirect precipitation was performed on all samples using formalin-inactivated Staphylococcus aureus (Cowan I strain) as described by Kessler (1975). The precipitates were washed three times, the proteins dissolved by boiling in sample buffer containing 10°,~, 2-mercaptoethanol and analysed by SDS PAGE on 8!'/o acrylamide gels (Arcement et al., 1976). Gels were dried and radioactive proteins were visualized using preflashed films (Jamjoom et al., 1977).
Western blotting. Western blotting of proteins was done by a modification of the procedure of Anderson et al. (1982). Confluent cells from a T25 tissue culture flask were rinsed in PBS pH 8-0 and lysed for 10 min at 4°C in SDS-PAGE buffer containing 2°.~ SDS, 10?.o 2-mercaptoethanol 0-001 M-EDTA, 0.005% bromophenol blue, 5°//O glycerol, 0.01 M-Tris-HCl pH 8.0. The sample was boiled for 5 min, cooled to 20 °C and centrifuged at 40000 r.p.m, for 30 min in a Beckman Ti-50 rotor. The supernatant was run by standard SDS-PAGE techniques, after which proteins were electroblotted from the gel onto nitrocellulose filter paper (Towbin et at., 1979) for 1 h at 100 V, Blotted protein standards were separated from the rest of the nitrocellulose and stained in a 0-1 °,o~ amido black solution. The rest of the nitrocellulose filter was rinsed in TNE/NP40 buffer (0.15 M-NaC1, 0.002 M-EDTA, 0-1 ~ NP40, 0.05 M-Tris, pH 7.5), then preblocked for 3 h in a plastic bag containing 2 ml per gel lane of TNE/NP40 with 3°/o bovine serum albumin (BSA). After blocking, fresh buffer containing the antiserum to be tested (1:400 dilution) was added and the binding of antibody was allowed to proceed for 12 h at 4 ~C. Filters were washed five times with T N E/N P40 buffer, placed in a plastic bag and incubated with 0.02 laCi/ml 12 s I-labelled staphylococcal Protein A (sp. act. 40 mCi/mg: Amersham) in TNE/NP40 buffer containing 3 ,° o BSA. After incubation, filters were washed four times with TNE/NP40 and then once in TNE/NP40 containing 1.2% glycerol. Filters were dried and autoradiographed at - 70 °C with Kodak Lightning-Plus intensifying screens (Bonner & Laskey, 1974).
948 G. E. G A L L I C K AND OTHERS
1 2 3 4
200
9 2 - 5 - -
Q , a D
. . . .
43 g q l l p p37 m°s-'-'~
2 5 , 7 ~ t f
Fig. 1. Immunoprecipitation of p37 m°~ with anti-mos(37-55c). NIH/3T3 cells were infected with MuSV-124 and 3 days later labelled with [35S]methionine (0.1 mCi/ml) for 12 h. The cell lysate (3 ml), from a T75 flask, was made as described in Methods. Immunoprecipitation was done using 0.75 ml extract and 40 gl an t i -mos (37 55c) (lane 1) or 80 gl anti-C3 (lane 3), For blocking, the excess of corresponding peptides, 1.6 lag v - m o s peptide 37-55c (lane 2) or 8 j_tg C3 v - m o s peptide (residues 363 374; lane 4), was used.
RESULTS
Immunoprecipitation o f p37 '.°~
Because of the minute amounts of p37 m°s synthesized in MuSV-transformed cells, this protein has been difficult to detect with specific antisera. However, in acutely infected cells, a transient increase of the v-mos product is observed (Papkoff et al., 1982). Therefore, to detect p37m% immunoprecipitations were performed 72 h after de novo infection of NIH/3T3 cells with supernatant of MuSV-124. The results of immunoprecipitation from the cells labelled for 12 h with [3SS]methionine are shown in Fig. 1. A number of bands were detected in the gel when immunoprecipitation with the anti-mos(37-55c) serum was performed (Fig. 1, lane 1). However, when the specific reactivity of the serum was inhibited by prior addition of excess free 37-55c peptide, only one band, of M~ 37000, was specifically blocked (Fig. 1, lane 2). A doublet of about 70000 to 75000 mol. wt. appeared to be blocked in this experiment but it was not reproducible.
v-mos protein recognition by anti-mos(37-55c) 949
Thus, the antiserum to the 37-55c peptide appears to react specifically with the wild-type mos product in acutely infected cells. The anti-mos sera prepared as described by Papkoff et al. (1982), termed anti-C3, was also used to detect p37 m°~. Lane 3 shows a faint band of p37 m°~ at this exposure which was not seen when the antibody was blocked with the C3 peptide. Thus, the antisera to cyclic v-mos peptide 37-55c specifically recognized p37 ~°~. A key element in this experiment is the use of 10~ 2-mercaptoethanol in the sample buffer necessary to release the p37 m°~ from the immune complex.
Immunoprecipitation of gag-mos proteins
The difficulties in detecting and therefore studying the function of p37 ~°~ in prototype MuSV strains have prompted this laboratory to investigate two cell lines, designated 6m2 and 54-5A4, which produce gag-mos fusion proteins in easily detectable amounts. Previous studies from this laboratory have demonstrated that 6m2 cells (an NRK clone containing ts110 virus) grown at 33 °C express two virus-specific proteins with Mr values of 85000 (P85) and 58000 (P58). Immunoprecipitation experiments and tryptic mapping studies have shown that P58 is a product of the gag gene and contains p15, p12 and truncated p30 sequences, but no pl0 sequences and has thus been designated P58 gag. P85 contains additional tryptic peptides which are identical to the v-mos gene product observed in translations in vitro of viral RNA from either MuSV-124 (Arlinghaus et al., 1980) or Mo-MuSV 349 (Horn et al., 1981 ; Murphy & Arlinghaus, 1982). Therefore, the protein has been designated P85 gag . . . . . Thus, antisera to the gag proteins p15, p12 and p30 would be expected to precipitate both P58 gag and P85 gag . . . . , whereas mos- specific antisera should only specifically precipitate P85 gag . . . . . 54-5A4 ceils, infected with a spontaneous revertant of tsl 10, synthesize a major virus protein of MT 100 000 which contains tryptic peptides ofpl5 , p12 and p30 (more p30 peptides than are observed in P85 gag . . . . ) as well as v-mos peptides (Stanker et al., 1983b). This protein has thus been designated P100 gag . . . . . The results of immunoprecipitation experiments conducted on 6m2 and 54-5A4 cells are shown in Fig. 2. Lanes 1 are the result of immunoprecipitation with anti-pl0 serum. As expected, no virus-specific bands were recognized by this serum. Lanes 2 show the results of precipitation with anti-p 15 serum. In 6m2 ceils (Fig. 2 a), both P58 gag and P85 g~g . . . . were recognized, and in 54- 5A4 cells (Fig. 2b), P100ga9 . . . . was recognized, in agreement with previous results (Stanker et al., 1983 b). Lanes 3 demonstrate that antiserum directed against the 37-55c peptide precipitated P85gag . . . . in 6m2 cells (Fig. 2a) and P100 g~g . . . . in 54-5A4 ceils (Fig. 2b). Lanes 4 are the result of immunoprecipitations identical to lanes 3, except that the reactivity of the antisera was blocked by addition of excess free 37-55c peptide. The only bands not seen in lanes 4 which are observed in lanes 3 are P85 gag . . . . in 6m2 cells (Fig. 2a) and P100 gag . . . . in 54-5A4, indicating that these proteins specifically react with this antiserum. Thus, the antiserum to the synthetic mos peptide specifically immunoprecipitates only mos-coded proteins.
Proteins p37 m°~, P85 gag . . . . and P 100 gag . . . . are also recognized by anti-mos(37-55c) in Western blots. However, while gag-mos proteins can be detected in chronically transformed cells, p37 ~°~ can only be detected by Western blotting in acutely infected cells (data not shown) apparently because of its low abundance (Papkoff et al., 1982). Recently, we have been able to detect p37 m°~ in acutely infected as well as chronically infected cells using anti-mos(37-55c) in immune complex kinase assays (data not shown).
Recognition o f a cellular protein by anti-mos(37-55c)
Because the denatured protein from an SDS-polyacrylamide gel may expose antigenic sites buried in the native protein, detection of proteins with antisera directed against synthetic peptides is often facilitated by Western blotting. Thus, specific reactivity of antiserum directed against the 37-55c peptide with gag-mos proteins was first demonstrated by this procedure before immunoprecipitating antibodies were detected during the immunization schedule (data not shown). Further, this antiserum was found to recognize strongly a protein of Mr 55000 (termed P55) in a variety of normal cell lines tested by Western blotting. Fig. 3 (a) demonstrates reactivity of the Mr 55000 band with rat cells (lanes 1, 2), mouse cells (lanes 3, 4) and human cells (lane 5). When normal rabbit serum (Fig. 3d) or immune serum whose specific reactivity with
950
(a) M
gPr90 gPr80 ~ l
P r 6 5 ~ b
w
1
G. E. GALLICK AND OTHERS
2 3 4 (b) M 1
~1~- ~1~ • P85
~ D " ~ : p 5 8
, I b ~ m ~ ' ~ a ~
gPr90 gPr80
Pr65
P100
Fig. 2. Virus-specific proteins recognized by anti-mos(37-55c) serum from ts110-infected and wild-type revertant-infected cells. Mutant ts110 MuSV-infected 6m2 cells were grown at 28 °C (a) and wild-type revertant 54-5A4 cells were grown at 37 °C (b). Cultures at 80% confluency were pulse-labelled with 500 ~tCi/ml L-[3H]leucine. Cells were immediately lysed, and equal aliquots of cytoplasmic extract were reacted with anti-Rauscher pl0 serum (lane 1), anti-Rauscher p15 serum (lane 2), anti-37-55c serum (lane 3) and anti-37-55c serum whose specific reactivity was blocked by addition of excess 37-55c peptide (lane 4). Lane M represents anti-Rauscher murine leukaemia virus (MuLV)-precipitated extract of Rauscher MuLV-infected cells pulse-labelled with L-[3H]leucine mixed with extracts of similarly labelled chase-incubated cells to serve as markers. The immunoprecipitates were analysed by SDS PAGE on 8% acrylamide gels.
mos had been blocked by prior addi t ion of excess free 37-55c pept ide (Fig. 3b) was employed in identical immunoblot t ing experiments, reactivity with the Mr 55 000 protein was not observed. However, when reactivity to the carrier K L H was blocked by pr ior addi t ion of an excess of this protein (Fig. 3c) reactivity of the serum with the Mr 55000 protein was unaffected, indicat ing that the cross-reactivity of this protein is specific to the 37-55c peptide.
Anti-mos(37-55c) serum has been shown to immunoprecipi ta te P55. Immunoprec ip i ta t ion of [35S]methionine-pulsed cell extracts indicated that P55 contained antigenic determinants reactive with anti-mos(37-55c) serum (Fig. 4, lanes 1,2 and 4) but not with an ant iserum directed against the C-terminal eight amino acids (anti-C2 serum) of v-mos (Fig. 4, lane 5), When anti- mos(37-55c) serum was blocked with excess 37-55c peptide, P55 disappeared (lane 3). Blocking with an unrelated pept ide had no effect on the reactivity of the serum towards P55 (lane 4). Maximal detection of P55 was obtained when cells were pulse-labelled with 1 mCi/ml [35S]methionine for 15 min and were lysed in NP40 lysis buffer. Detect ion of p37 m°s was very poor under these conditions due to the presence of non-specific proteins in the 37000 to 40000 mol. wt. region.
To examine further the extent of homology between v-mos protein sequences and P55, we examined cell extracts containing P55 with anti-C3 serum by Western blotting. As expected,
v-mos protein recognition by an t i -mos (37-55c )
(a) (b) (c) (d)
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4
!
200-- i '~
r i
6 8 - - " '
} 4 3 - - -~ ~" , " ~ ,,
z d
25.7-- - ~: " ,
Fig. 3. Detection of P55 with anti-mos(37-55c) !n various cell lines. Cell extracts were made from the subconfluent cultures of L6E9 rat myoblasts (lane 1), NRK (lane 2), NIH/3T3 (lane 3), JLS-V16 (lane 4) and HeLa (lane 5) cells. The proteins were separated on 8 ~ polyacrylamide gels and blotted. The blots were reacted with 40 pl anti-mos(37-55c) (a), 40 pl anti-mos(37-55c) + 1.6 pg v-mos 37-55c peptide (b), 40 pl anti-mos(37 55c) + 5 pg KLH (c) or 40 pl normal rabbit serum (d).
92.5--
951
5
anti-C3 serum antibodies recognized P85 gag-m°s in Western blots and the reactivity was blocked with an excess of peptide (Fig, 5, lanes 1, 2). However, anti-C3 serum failed to react with P55 (Fig. 5, lane 3). This result suggests than homology between p37 and P55 is only partial and may be limited to the 37 to 55 amino acid sequence of v-mos.
DISCUSSION
Antisera directed against synthetic peptides have proven useful reagents in the specific recognition and subsequent analyses of transforming proteins of retroviruses as well as their cellular counterparts. Antisera against a peptide (termed C3) representing the carboxy-terminus of v-mos has been demonstrated to recognize specifically p37 m°s in MuSV-124-infected cells (Papkoffet al., 1982) as well as P850ag . . . . in ts l 10-infected cells (Stanker et al., 1983a; Papkoff& Hunter, 1983) and P 100 gag . . . . in a revertant cell line (Stanker et al., 1983 b). However, because of the variability in the various C-termini of viral (Papkoff et al., 1982; Brown et al., 1984) and cellular mos genes (Van Beveren et al., 1981), antisera directed against conserved regions of the gene may be more broadly reactive. Furthermore, to study the effect of post-translational modifications of mos and g a g - m o s proteins, such as addition of lipid (Gallick & Arlinghaus, 1984), and activities of the protein, such as kinase activity (Kloetzer et al., 1983, 1984), antisera directed against several portions of the molecule may provide useful tools. The data presented in this report indicate that antiserum to a cyclized peptide comprising amino acids 37 to 55 of the predicted v-mos sequence (Van Beveren et al., 1981) specifically recognized the e n v - m o s protein p37 m°s and g a g - m o s proteins in cells infected with variants of MuSV- 124. A n t i - m o s ( 3 7 - 5 5 c ) was somewhat less efficient in recognizing P85 and P100 than the anti-pl5 serum (Fig. 2), possibly due to lower avidity of the antibody or lower titre.
952 G. E. G A L L I C K AND OTHERS
1 2 3 4
Fig. 4. Immunoprecipitation of P55 from [3sS]methionine-labelled NIH/3T3 cell extracts. Lane 1, immune complexes from uninfected 3T3 cell extracts; lanes 2 to 5, immunoprecipitates from MuSV- 124 acutely infected 3T3 cell extracts. Lanes 1, 2, anti-mos(37-55c); lane 3, immune complexes derived using 37-55c v-mos peptide-blocked anti-mos(37 55c) serum; lane 4, myc peptide-blocked anti-mos(37 55c) immunoprecipitate; lane 5, anti-C2 immunoprecipitate. Cells were incubated for 15 min at 37 ~C in methionine-deficient MEM containing l mCi/ml ~3sS]methionine and 10,°~ dialysed foetal calf serum. Radioactive media were thoroughly decanted and 0-5 ml NP40 lysis buffer (1% NP40, 150 mM- NaCI, 1 mM-EDTA, 100 KIU aprotinin/ml, 10 mM-sodium phosphate, pH 7.2) added per 25 cm-' of cells. After incubating cells in lysis buffer on ice, cells were scraped and the lysate clarified by centrifuging at 50000 g for 30 min. Immunoprecipitation was performed for 1 h and the immune complexes washed once with NP40/PBS (0.1% NP40, 150 mM-NaCl, 10 mM-sodium phosphate, pH 7-2), followed by two washings in RIPA buffer. Samples were then prepared for electrophoresis as described in Methods. The gel was prepared for autoradiography and exposed to Kodak XAR~5 film for 36 h.
v-mos protein recognition by anti-mos(37-55c)
1 2 3 4 4~
953
200
I
9 2 . 5 - -
P85gag . . . . .~
6 8 ~
P55 v
4 3 ~
Fig. 5. P55 is not recognized by anti-C3 serum. The extracts from 6m2 ceils maintained at 28 °C (lanes 1, 2) and L~E,~ rat myoblasts (lanes 3, 4) were subjected to SDS PAGE followed by Western blotting. The filters were reacted with anti-C3 serum (lanes 1, 3) or anti-C3 preblocked with C3 peptide (4 gg per 50 gl serum; lanes 2, 4). The arrows mark the location of either P55 or P85 g~g .. . . based upon relative position of markers.
The anti-mos(37-55c) sera also recognized a cellular prote in of M r 55000 in un infec ted cells. Because of its apparen t ubiqui tous expression among several cell lines, this prote in is probably not the t ranslat ion product o f the c-mos gene, which is apparen t ly t ranscr ip t ional ly silent in most cell l ines and mouse tissues tested (Gat ton i et al., 1982). However , since reac t iv i ty to this pro te in was p reven ted by blocking the ant isera wi th excess pept ide while react iv i ty was unaffected by blocking with carr ier K L H , the Mr 55000 prote in appears to be structural ly re la ted to the v-mos product . P55 was, however , not recognized by two C- te rmina l mos ant isera m a d e f rom ei ther a C- te rmina l 12 amino acid or eight amino acid peptide. Thus, the re la t ionship of P55 to v-mos
954 G. E, GALLIC K AND OTHERS
p r o t e i n s m a y on ly l ie in t he 37 to 55 a m i n o ac id d o m a i n o f v-mos p r o t e i n s . T h e s e resu l t s s u g g e s t
t h a t P55 is n o t a c-mos g e n e p r o d u c t . F u r t h e r m o r e , P55 d e c r e a s e s in L 6 E 9 r a t m y o b l a s t s a c u t e l y i n f e c t e d w i t h M u S V - 1 2 4 as t h e v-mos p r o d u c t i n c r e a s e s , i n d i c a t i n g t h a t t h e p r o t e i n m a y p l a y a role in t r a n s f o r m a t i o n by M u S V (B. S i n g h & R. B. A r l i n g h a u s , u n p u b l i s h e d resul ts ) . T h u s , P55 m a y be i n v o l v e d in n o r m a l f u n c t i o n s o f cel ls a n d ye t be r e l a t e d to a p r o t e i n r e s p o n s i b l e fo r t r a n s f o r m a t i o n . S u c h a p o s s i b i l i t y w o u l d be o f i m p o r t a n c e in t h e u n d e r s t a n d i n g o f t h e m e c h a n i s m o f a c t i o n o f t r a n s f o r m i n g p r o t e i n s . S t u d i e s a r e in p r o g r e s s to u n d e r s t a n d b e t t e r t h i s
resu l t a n d to i d e n t i f y t h e P55 p r o t e i n .
We acknowledge the excellent technical assistance of Nila Parikh, Alan R. Calwell, Cynthia Furlong, and J. Syrewicz as well as Michael Nash for assistance with the rabbits, and Rebecca Bertrand for manuscript preparation. This research was supported in part by a grant from the Robert A. Welch Foundation (G-429), Public Health Service grant CA-36714 and core grant CA-16672 from the National Institutes of Health. J.T.S. is an established investigator of the American Heart Association. G.E.G. and L.H.S. were supported by Public Health Service Training Grant (CA-09299) from the National Institutes of Health.
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