Fibroblasts transformed by Abelson murine leukemia virus differ from normal fibroblasts in that they contain several cellular proteins, including one of 29 and one of 36 kilodaltons, which are phosphorylated at tyrosine residues. Since it has been shown before that these proteins also become phosphorylated at tyrosine after transformation of fibroblasts by a number of other retroviruses, their phosphorylation may play an important role in the transformation of these cells. In contrast, the 36-kilodalton phosphoprotein was not detectable in three of the four lines of Abelson virus-transformed B lymphoma cell lines studied here. These three cell lines, RAW307.1.1, 18-48, and 18-81, and a B lymphoma induced by mineral oil, WEHI 279, were all found to lack both the phosphorylated and unphosphorylated forms of the 36-kilodalton protein. It thus appears that expression of this major cell protein is not essential for the survival of B lymphoma cells in culture and that the phosphorylation of the 36-kilodalton protein at tyrosine is not essential for transformation of pre-B lymphocytes by Abelson virus.

Both lymphocytes and fibroblasts that have been transformed by ABelson murine leukemia virus contain 6- to 12-fold increased levels of the rare modified amino acid phosphotyrosine in their proteins. This observation, coupled with the fact that the p120 protein encoded by this virus has been shown to undergo an apparent autophosphorylation to yield phosphotyrosine in vitro, suggests that Abelson virus encodes a protein kinase that phosphorylates tyrosine in transformed cells. These results are similar to those obtained previously with Rous sarcoma virus and suggest, by analogy, that the modification of cellular polypeptides through the phosphorylation of tyrosine may be involved in cellular transformation by Abelson virus. p120 isolated from transformed cells contains phosphoserine, phosphothreonine, and phosphotyrosine. The phosphotyrosine is found at two sites in the protein. p120 therefore may be a protein kinase that undergoes autophosphorylation in vivo.

Different variants of Moloney murine sarcoma virus (MSV) were examined by nucleotide sequencing to compare the junctions between the acquired cellular sequence, v-mos, and the adjacent virus-derived sequences. These variants included 124-MSV, m1-MSV, and HT1-MSV and also the purportedly independent isolate Gazdar MSV. These four strains have an identical 5' junction between the murine leukemia virus env gene and the v-mos gene. This junction lies within the sixth codon of the chimeric env-mos coding region that encodes the transforming gene product. In contrast, at the 3' junction between the v-mos gene and the murine leukemia virus env gene, the three variants examined here were all different. A small deletion was found in the COOH-terminal portion of the m1-MSV env-mos coding region, indicating that the COOH terminus of this transforming gene product must be different from that of 124-MSV or HT1-MSV. The data presented here are consistent with the thesis that a virus closely related to HT1-MSV was the primordial Moloney MSV, and that all other related strains evolved from it by deletion or rearrangement. The variability observed in the Moloney MSV family is discussed in terms of possible mechanisms for the initial capture of mos sequences by the parental retrovirus and also in comparison with other transforming retrovirus families, such as Abelson murine leukemia virus and Rous sarcoma virus.

Chronic myelogenous leukemia (CML) is characterized by a translocation involving the c-abl protein-tyrosine kinase gene. A chimeric mRNA is formed containing sequences from a chromosome 22 gene (bcr) at its 5' end and all but the variable exon 1 of c-abl sequence. The product of this mRNA, p210bcr-abl, has constitutively high protein-tyrosine kinase activity. We examined K562 cells and other lines established from CML patients for the presence of phosphotyrosine (P-Tyr)-containing proteins which might be p210bcr-abl substrates. Two-dimensional gel separation of 32P-labeled proteins followed by phosphoamino acid analysis of 25 phosphoproteins, which comprised the major alkali-stable phosphoproteins, indicated that three related proteins of 41 kDa are the most prominent P-Tyr-containing proteins detected by this method. The 41-kDa phosphoproteins are found in two other CML lines that we examined but not in lines of similar lineage isolated from patients with distinct leukemic disease. A protein that comigrates with the major form of pp41 (pp41A) and contains P-Tyr is also found in murine fibroblasts and B-lymphoid cells transformed by Abelson murine leukemia virus, which encodes the v-abl protein, and in platelet-derived growth factor-treated fibroblasts, in which it has been described previously. We analyzed three pairs of Epstein-Barr virus-immortalized B-cell lines from individual CML patients and found that only the lines in which active p210bcr-abl was present contained detectable pp41. We also performed immunoblotting with anti-P-Tyr antibodies on the same CML cell lines and detected at least four other putative substrates of p210bcr-abl, which were undetected with use of the two-dimensional gel technique.

Chicken embryo cells transformed by the related avian sarcoma viruses PRC II and Fujinami sarcoma virus, or by the unrelated virus Y73, contain three phosphoproteins not observed in untransformed cells and increased levels of up to four other phosphoproteins. These same phosphoproteins are present in increased levels in cells transformed by Rous sarcoma virus, a virus which is apparently unrelated to the three aforementioned viruses. In all cases, the phosphoproteins contain phosphotyrosine and thus may be substrates for the tyrosine-specific protein kinases encoded by these viruses. In one case, the site(s) of tyrosine phosphorylation within the protein is the same for all four viruses. A homologous protein is also phosphorylated, at the same major site, in mouse 3T3 cells transformed by Rous sarcoma virus or by the further unrelated virus Abelson murine leukemia virus. A second phosphotyrosine-containing protein has been detected in both Rous sarcoma virus and Abelson murine leukemia virus-transformed 3T3 cells, but was absent from normal 3T3 cells and 3T3 cells transformed by various other viruses. We conclude that representatives of four apparently unrelated classes of transforming retroviruses all induce the phosphorylation of tyrosines present in the same set of cellular proteins.