Avian Sarcoma Virus Src Research Articles

Altered stem cell (CFU-S) function following infection of hematopoietic cells with a virus carrying V-src

Long-term murine bone marrow cultures were used to support the growth and development of hematopoietic cells. After hematopoiesis was established, the cultures were infected with a recombinant murine amphotropic virus carrying the avian sarcoma virus src gene and the CFU- S kinetics were examined. The CFU-S from the src-infected cultures displayed a reduced seeding efficiency in the standard spleen colony assay. The self-renewal capacity of these CFU-S was tested by their ability to reestablish hematopoiesis when serially transplanted on irradiated bone marrow cultures and by serial passage in spleens of irradiated mice. In both tests, cells from the src-infected cultures exhibited an enhanced ability to sustain a high level of self-renewal. The other property of stem cells which may be measured is the probability of self-renewal at each cell division which dictates the distribution between stem cells and differentiated type progeny. CFU-S from the src-infected cultures had higher average probabilities of self- renewal and therefore reduced differentiation. These differences suggest that expression of src had indirectly or directly altered the normal differentiation program of the stem cells.

Altered Stem Cell (CFU-S) Function Following Infection of Hematopoietic Cells With a Virus Carrying V-src

Long-term murine bone marrow cultures were used to support the growth and development of hematopoietic cells. After hematopoiesis was established, the cultures were infected with a recombinant murine amphotropic virus carrying the avian sarcoma virus src gene and the CFU-S kinetics were examined. The CFU-S from the src-infected cultures displayed a reduced seeding efficiency in the standard spleen colony assay. The self-renewal capacity of these CFU-S was tested by their ability to reestablish hematopoiesis when serially transplanted on irradiated bone marrow cultures and by serial passage in spleens of irradiated mice. In both tests, cells from the src-infected cultures exhibited an enhanced ability to sustain a high level of self-renewal. The other property of stem cells which may be measured is the probability of self-renewal at each cell division which dictates the distribution between stem cells and differentiated type progeny. CFU-S from the src-infected cultures had higher average probabilities of self-renewal and therefore reduced differentiation. These differences suggest that expression of src had indirectly or directly altered the normal differentiation program of the stem cells.

A mouse homolog to the avian sarcoma virus src protein is a member of a protein kinase cascade

Recent work has identified a cascade of membrane-bound protein kinases in Ehrlich ascites tumor cells. These enzymes, designated PK L, PK S and PK M, are present in both Ehrlich tumor and mouse brain, but the cascade is active only in the tumor tissue. We have now purified a fourth protein kinase, PK F, that is also associated with this cascade. Protein kinase F phosphorylates PK L and is phosphorylated by PK S. The position of this kinase in the cascade is as follows, where the arrows denote phosphorylation: PK F → PK L → PK M → Na + K + − ATPase The phosphorylation by PK F, like phosphorylation by the other kinases, is at a tyrosine residue and causes the substrate kinase (PK L) to become active. The role of the tyrosine phosphorylation in activating these kinases is described in detail elsewhere. One result of activation of the cascade is the phosphorylation of the β subunit of the Na +K +-ATPase, which causes inefficient Na + pumping and is at least in part responsible for the high aerobic glycolysis of Ehrlich ascites tumor cells. By several criteria protein kinase F from Ehrlich cells is homologous to the src gene product (pp60 src) from avian sarcoma viruses. Antiserum raised against PK F and sera from rabbits bearing Rous sarcoma virus (RSV)-induced tumors quantitatively precipitate the same 60 kd phosphoprotein from cell lysates of three different RSV-transformed cell lines. Both proteins phosphorylate PK L and a 130 kd cytoskeletal protein (vinculin). The tryptic maps of these proteins are closely similar. Both proteins bind specifically to PK L covalently coupled to Sepharose. We used this latter observation to facilitate the purification of pp60 src from RSV-transformed cells.

Cell-free translation of purified avian sarcoma virus src mRNA

Avian sarcoma virus 21 S RNA, purified by hybridization from virus-infected cells, was translated in a cell-free system. The major product of translation was a protein of 60,000 daltons. This protein was the same as authentic pp60 src , the product of the ASV src gene, when compared by electrophoretic mobility in polyacrylamide gels, immunological reactivity and partial protease digestion. These findings confirm that the 21 S ASV RNA serves as mRNA for pp60 src . Furthermore, pp60 src is the only major product of translation of the src gene and is apparently synthesized without a cleavable signal sequence.

Sites of synthesis of viral proteins in avian sarcoma virus-infected chicken cells.

We determined the sites of synthesis of avian sarcoma virus-specific proteins in infected chicken cells by immunoprecipitation of the products synthesized in vitro by free and membrane-bound polyribosomes; 85% of Pr76, the precursor of the viral internal structural proteins (group-specific antigens), was synthesized on free polyribosomes, and 15% was synthesized on membrane-bound polyribosomes. Pr92, the lycosylated precursor of the viral glycoproteins (gp85 and gp35), was synthesized exclusively on membrane-bound polyribomes, which is consistent with its role as a membrane protein. When we investigated the site of synthesis of pp60src, the product of the avian sarcoma virus src gene, we found that 90% was synthesized on free polyribosomes, whereas 10% was detected on membrane-bound polyribosomes. The implications of these results with respect to the subcellular location of pp60src are discussed.

Characterization of the protein kinase activity of avian sarcoma virus src gene product.

The avian sarcoma virus src gene product, p60src, has been purified 650-fold from cytoplasmic extracts of the rat tumor cell line RR1022 by using ammonium sulfate fractionation, hydrophobic chromatography on omega-aminohexyl agarose, and ion exchange chromatography on phosphocellulose. Partially purified p60src is a monomer, with a native molecular weight of about 60,000 and an apparent pI of 6.0. In immunoprecipitates, p60src catalyzed phosphorylation of anti-p60src IgG heavy chains within the variable (VH) domain, which contains the heavy chain portion of the antigen combining site. Crude preparations of p60src contained phosphatase activity able to cleave phosphate from IgG heavy chains; this activity was removed by the purification procedure, and partially purified p60src could phosphorylate the heavy chain of specific antibody in solution. Furthermore, purified p60src catalyzed phosphorylation in solution of the general protein kinase substrate, alpha-casein, strengthening the hypothesis that it may in fact function as a protein kinase in vivo.

Polymorphism of avian sarcoma virus src proteins

The src gene products of seven different avian sarcoma viruses were compared. In vitro translation of virion RNA yielded products identified unambiguously as p60src in the case of two stocks of the Schmidt-Ruppin strain, three stocks of the Prague strain, the Bryan strain, and the Bratislava 77 strain of avian sarcoma virus. Differences in the electrophoretic mobility of these seven p60src proteins in sodium dodecyl sulfate-polyacrylamide gels, corresponding to variation in the apparent molecular weights ranging from 56,000 to 60,500, were observed. Antigenic variability was also found; only three of the seven viruses tested encoded a p60src, which was precipitated by antisera derived from rabbits bearing tumors induced by the Schmidt-Ruppin strain of Rous sarcoma virus. Examination of the methionine-containing tryptic peptides of the seven ;60src proteins by two-dimensional mapping revealed four common peptides but marked variability in the five to eight other peptides in each protein. Clear differences in the peptide maps of p60src were observed, both between different strains of virus and within strains. In the three cases examined, p60src synthesized in transformed cells was found to be essentially identical to that synthesized in vitro. We conclude that there is significant polymorphism in the p60src proteins of the avian sarcoma viruses.

Protein kinase activity associated with the avian sarcoma virus src gene product.

Incorporation of phosphorus from [gamma-32P]ATP into protein was catalyzed by specific immunoprecipitates from avian sarcoma virus (ASV)-transformed avian and mammalian cells. This incorporation was observed only when antiserum from tumor-bearing rabbits able to specifically precipitate the ASV sarcoma gene product, p60src, was used to immunoprecipitate antigens from transformed cell lysates. Immunoprecipitates of extracts from normal cells or cells infected with a transformation-defective ASV mutant showed no activity in this assay, nor did any immune complexes formed with normal rabbit serum and any of the cell extracts tested. The expression of the protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) was growth temperature-dependent in cells infected with an ASV mutant temperature-sensitive for the transformation. These results on an enzymatic activity associated with the ASV transforming protein are discussed in terms of protein phosphorylation as a mechanism for viral transformation.

Identification of a polypeptide encoded by the avian sarcoma virus src gene.

Two techniques were used to search for the polypeptide encoded by the avian sarcoma virus (ASV) src gene. First, antiserum from rabbits bearing ASV-induced fibrosarcomas was used to immunoprecipitate a transformation-specific antigen from ASV-transformed chick embryo fibroblasts. This antigen has an apparent molecular weight (Mr) of 60,000. Second, the 3' one-third of the ASV genome, selected by oligo(dT)-cellulose chromatography and sucrose gradient sedimentation, was translated in a mRNA-dependent reticulocyte cell-free lysate. This RNA species programmed the synthesis of a polypeptide that comigrated with the transformation-specific antigen of Mr 60,000 immunoprecipitated from transformed cells. The methionine-containing tryptic peptides from the polypeptides of Mr 60,000 obtained from translation in vitro and from immunoprecipitation were found to be identical upon two-dimensional fractionation.