Cell, Vol. 29. 417-426. June 1982. Copyright 0 1962 by MIT Detection of a Transforming Gene Product in Cells Transformed by Moloney Murine Sarcoma Virus Jackie Papkoff Department of Biology University of California, San Diego La Jolla, California 92093 lnder M. Verma and Tony Hunter Tumor Virology Laboratory The Salk Institute P. 0. Box 85800 San Diego, California 92138 Summary We identified, in cells transformed by Moloney mu- rine sarcoma virus (M-MuSV clone 124), a protein encoded by the M-MuSV transforming gene, V-mos. An antiserum against a synthetic peptide corre- sponding to the C terminus of a protein predicted from the v-mos nucleotide sequence specifically recognizes a protein doublet of approximately 37,000 daltons from 35S-methioninalabeled M- MuSV 124-transformed producer cells. By peptide mapping, this protein is almost identical to the 37 kd in vitro translation product from the M-MuSV V-mos gene. lmmunoprecipitates from 32P-labeled cells contain a single v-mos-specific phosphopro- tein, which has at least six sites of phosphorylation containing phosphoserine. Pulse-chase experi- ments show that the lower band in the 35S-methio- nine-labeled doublet is the primary translation prod- uct, which is modified, probably by phosphoryla- tion, to yield the upper band. A similar mos protein is immunoprecipitated from HTl -MuSV-transformed cells, but not from uninfected NIH/3T3 cells. These mos proteins are present at very low levels in trans- formed cell lines. Cells acutely infected with M- MuSV 124, however, transiently contain much higher levels of the mos protein. These high levels coincide with extensive cell mortality. Introduction Moloney murine sarcoma virus (M-MuSV), a replica- tion-defective retrovirus, is capable of producing fibro- sarcomas in animals and transforming fibroblasts in vitro (Moloney, 1966; Aaronson and Rowe, 1970; Aaronson et al., 1970). The M-MuSV genome is ap- parently the product of a naturally occurring recom- bination event between a nonsarcomagenic retrovirus (Moloney murine leukemia virus, M-MuLV) and normal mouse cellular sequences (Frankel and Fischinger, 1976, 1977; Tronick et al., 1979; Jones et al., 1980; Oskarsson et al., 1980). The structure of the genome of M-MuSV is exemplified by that of M-MuSV clone 124 (Ball et al., 1973) an isolate that has been studied in great detail. During the genesis of M-MuSV 124, a block of 1157 nucleotides of cellular sequence (re- ferred to as v-mos) was incorporated into the viral genome near the 3’ end (Reddy et al., 1980, 1981; Van Beveren et al., 1981 a. 1981 b; Donoghue, 1982). In addition, portions of the viral structural genes were deleted from the MuSV genome (Hu et al., 1977; Donoghue et al., 1979). Transfection experiments with MuSV-derived DNA fragments demonstrate that the v-mos portion of the M-MuSV genome, when ap- propriately combined with a retroviral control element, contains sufficient information to induce malignant transformation (Andersson et al., 1979; Canaani et al., 1979; Vande Woude et al., 1979; Blair et al., 1980; Verma et al., 1980). The cellular gene (c-mos), from which the v-mos gene was derived, is also ca- pable of transforming cells when transfected together with a viral control element (Blair et al., 1981). Initial attempts to identify the protein or proteins coded for by the v-mos gene were made by in vitro translation of virion RNA from M-MuSV 124 (Dina and Nadal-Ginard, 1979; Lyons et al., 1980; Papkoff et al., 1980, 1981; Cremer et al., 1981). In this manner, an overlapping set of four v-mos-coded proteins of approximately 37, 33, 24 and 18 kd was identified. The complete nucleotide sequence of the v-mos gene of M-MuSV has been determined (Reddy et al., 1980, 1981; Van Beveren et al., 1981 a, 1981 b; Donoghue, 1982). The structure of the 37 kd in vitro translation product is consistent with that of the 41 kd protein predicted from the single long open reading frame in the v-mos nucleotide sequence (Van Beveren et al., 1981a; Donoghue, 1982). The AUG codon used to initiate synthesis of the 37 kd protein is not actually located in the cellular sequences acquired by M-MuSV 124, but instead is in the viral sequences immediately upstream of the cellular insert (Reddy et al., 1980; Van Beveren et al., 1981a; Donoghue, 1982). This viral sequence originated from the N terminus of the env gene, and as a result the 37 kd protein shares its N-terminal five amino acids with the parental MuLV env gene product. The three smaller in vitro products of 33, 24 and 18 kd are initiated from AUG codons within the v-mos gene (Papkoff et al., 1981). All four in vitro proteins share a common C terminus defined by a terminator codon within the cell- derived sequences of M-MuSV 124 (Van Beveren et al., 1981a; Donoghue, 1982; Donoghue and Hunter, 1982). In a previous report, we described the produc- tion of an antiserum to a synthetic peptide correspond- ing to the 12 amino acids upstream of this terminator codon. This serum specifically precipitates all four of the v-mos in vitro translation products (Papkoff et al., 1981). In this study we used the antipeptide serum to identify and characterize proteins coded for by the v- mos gene in MuSV-transformed cells. A phosphopro- tein very similar in size and sequence to the 37 kd in vitro translation product was detected in several dif- ferent MuSV-transformed cell lines.