Phosphorylation of a 36,000 Mr cellular protein in cells infected with partial transformation mutants of rous sarcoma virus.

We have isolated and characterized mutants of Rous sarcoma virus which induce some parameters of transformation but fail to fully induce other parameters. We believe these mutants code for a pp60src which phosphorylates some targets well but phosphorylates others poorly. Using these mutants, we examined the phosphorylation of a 36,000 Mr protein which is phosphorylated on a tyrosine in cells transformed by Rous sarcoma virus, in an attempt to correlate this phosphorylation with the expression of specific transformation parameters. We found that phosphorylation of the 36,000 Mr protein was neither necessary nor sufficient for loss of fibronectin or for loss of density-dependent inhibition of growth. Phosphorylation of the protein was not sufficient for morphological alterations, increased hexose transport, or loss of adhesiveness. For the parameters measured, the best correlation was with increased plasminogen activator. In addition, it is noteworthy that cells infected with the mutant CU2 displayed low levels of phosphorylation of the 36,000 Mr protein and also were deficient in anchorage-independent growth and tumorigenicity, raising the possibility that the phosphorylation of the 35,000 Mr protein may be required for malignant growth properties.     

Cholera toxin treatment stimulates tumorigenicity of Rous sarcoma virus-transformed cells.

Chinese hamster ovary cells transformed by Rous sarcoma virus form tumors poorly in nude mice. Tumorigenicity was markedly stimulated by pretreatment of the cells with cholera toxin, which raises cyclic AMP levels and activates cyclic AMP-dependent protein kinase. Increased tumorigenicity was manifested by a severalfold increase in the rate of tumor formation, as well as earlier appearance and more rapid growth of tumors. In contrast, spontaneously transformed Chinese hamster ovary cells showed decreased tumorigenicity after cholera toxin treatment. The activation of tumorigenic potential in Rous sarcoma virus-transformed Chinese hamster ovary cells by cholera toxin correlated with increased phosphorylation of the viral oncogene product pp60src and stimulation of its tyrosine kinase activity.     

Molecular events leading to enhanced glucose transport in Rous sarcoma virus-transformed cells

Transformation by Rous sarcoma virus results in a dramatic increase in the rate at which the transformed cells transport glucose across the cell membrane. The increased transport rate is a consequence of an increased number of transporters in the transformed cells. Utilizing antibody raised against the purified human erythrocyte glucose transporter, we have identified the glucose transporter as a membrane glycoprotein with a monomer Mr of approximately 41,000. The increased rate of glucose transport is dependent on the activity of pp60src, the transforming protein of Rous sarcoma virus. This protein has been shown to be a protein kinase that phosphorylates on tyrosine residues. We have examined the tyrosine phosphorylation of a major cellular protein of Mr 36,000 in cells infected with a panel of partially transforming mutants of Rous sarcoma virus. One of these mutants (CU2) increases the rate of glucose transport only slightly and does not render the infected cells fully anchorage independent or tumorigenic (although other transformation parameters are fully induced). Cells infected with this mutant display a 36,000-dalton protein that is phosphorylated to a considerably lesser extent than cells infected with wild-type virus. Analyses of this sort may help to identify the cellular targets of pp60src whose phosphorylation is necessary for the increased glucose transport rate.     

Role of the mitogenic property and kinase activity of p60src in tumor formation by Rous sarcoma virus

Expression of the src gene of Rous sarcoma virus in chicken embryo neuroretinal cells results in morphological transformation and sustained proliferation of this normally resting cell population. PA101 and PA104 are two mutants of Rous sarcoma virus which induce neuroretinal cell proliferation in the absence of morphological transformation. Their mitogenic property is temperature sensitive, and they both encode p60src proteins with low kinase activity. To study the role of the mitogenic function and protein kinase activity of p60src in tumorigenesis, we investigated the oncogenicity of PA101 and PA104. Both mutants were less tumorigenic than wild-type virus when injected into chicks. Tumorigenicity was further assayed by inoculating infected chicken embryo fibroblasts and neuroretinal cells onto the chorioallantoid membrane of embryonated duck eggs. This system provides a nonpermissive and immunodeficient environment for xenogenic cell grafting and allows the study of cell tumorigenicity within a temperature range of 37 to 39.5 degrees C. Chicken embryo fibroblasts and neuroretinal cells infected with PA101 were as tumorigenic as wild type-infected cells at 37 degrees C, but tumor development was significantly reduced at 39.5 degrees C. In contrast, both cell types infected with PA104 displayed sharply reduced tumorigenicity. Cell cultures derived from PA101 tumors induced on the chorioallantoid membrane were similar to the corresponding cells maintained in vitro in terms of morphology, production of plasminogen activator, relative amounts of phosphotyrosine in total cellular proteins, and phosphorylation of 34,000-molecular-weight protein. These results indicate that the expression of the mitogenic function of src does not account per se for cell tumorigenicity and that tumor formation is compatible with low levels of p60src protein kinase activity.     

In vitro phosphorylation of the 36K protein in extract from Rous sarcoma virus-transformed chicken fibroblasts

When cell-free extracts of chickens embryo fibroblasts transformed by Rous sarcoma virus (RSV) were incubated with [gamma-32P]ATP, a protein having a Mr of 36,000 was phosphorylated. Two-dimensional electrophoresis of a mixture of phosphorylated proteins formed in vitro and in vivo showed that they are indistinguishable. The in vitro phosphorylation of the Mr = 36,000 protein was completely inhibited by serum isolated from rabbit bearing tumor formed by RSV. In addition, phosphorylation of the 36K protein does not occur if the extract was made from fibroblasts transformed by RSV tsNY68 and cultured at 42 degrees C or from fibroblasts infected with transformation defective RSV. The cell free-phosphorylation of 36K protein was dependent on Mg2+ ions but not dependent on exogenously added cyclic AMP.     

Phosphorylation of cellular proteins in Rous sarcoma virus-infected cells: analysis by use of anti-phosphotyrosine antibodies.

The protein substrates for the tyrosine protein kinases in cells transformed by avian sarcoma viruses were analyzed by gel electrophoresis in combination with immunoblotting or immunoprecipitation by antibodies against phosphotyrosine. We found that greater than 90% of phosphotyrosine-containing cellular proteins can be immunoprecipitated by these antibodies. The level of phosphotyrosine-containing cellular proteins detectable by this method markedly increased upon transformation with Rous sarcoma virus, and more than 20 distinct bands of such proteins were found in lysates of Rous sarcoma virus-transformed cells. Most of these phosphotyrosine-containing proteins had not been identified by other methods, and their presence appeared to correlate with morphological transformation in cells infected with various Rous sarcoma virus mutants and Y73, PRCII, and Fujinami sarcoma viruses. However, considerably different patterns were obtained with cells infected with nontransforming Rous sarcoma virus mutants that encode nonmyristylated src kinases, indicating that most substrates that correlate with transformation can only be recognized by p60v-src associated with the plasma membrane.     

Lack of induction of neuroretinal cell proliferation by Rous sarcoma virus variants that carry the c-src gene.

Expression of p60v-src of Rous sarcoma virus in cultured chicken embryo neuroretinal cells was previously shown to result in the transformation and sustained proliferation of normally quiescent cell populations. We show here that Rous sarcoma virus variants that encode p60c-src, the cellular homolog of p60v-src, lack the ability to induce morphological transformation and cell proliferation of cultured neuroretinal cells. Neuroretinal cells infected with c-src-containing viruses, however, possess no less p60 protein kinase activity assayed in the immune complex than those infected with the transformation-defective Rous sarcoma virus mutants PA101 or PA104, which do stimulate the growth of these cells.     

Identification of a transformation-specific protein induced by a Rous sarcoma virus.

Using antisera obtained from rats bearing Schmidt-Ruppin strain Rous sarcoma virus-induced tumors, we have idnetified a protein with an apparent molecular weight of 56,000 daltons and an isoelectric point of 6.3 in extracts of chick embryo fibroblasts transformed by a wild-type nondefective Rous sarcoma virus (Schmidt-Ruppin strain). This protein was not found in cells infected by trnasformation-defective mutants with either a partial or complete deletion of the src gene, nor in cells infected by a nontransforming avian leukosis virus. The 56,000 dalton molecular weight protein was found to be synthesized at both the permissive and nonpermissive temperatures in cells infected by either of two conditionallethal mutants that are temperature-sensitive in cell transformation. The amount of this protein, however, accumulated in cells infected by these temperature-sensitive mutants, relative to the structural polypeptides, differed significnatly from that seen with the nondefective virus. Pulsechase experiments indicate that the protein is extremely unstable, with a half-life of about 20 min, and does not serve as a precursor to any of the detectable virion polypeptides. Furthermore, incubation of the rat antiserum with purified, disrupted virus did not affect its immunoreactivity to this particular protein. We conclude that this 56,000 dalton molecular weight protein is a nonstructural protein specific to cells transformed by Rous sarcoma virus.     

Assay of noninfectious fragments of DNA of avian leukosis virus-infected cells by marker rescue.

A marker rescue assay of noninfectious fragments of avian leukosis virus DNAs is describe. DNA fragments were prepared either by sonication of EcoRI-digestion of DNAs of chicken cells infected with wild-type Rous sarcoma virus, with a nontransforming avian leukosis virus, and with a mutant of Rous sarcoma virus temperature sensitive for transformation. Recipient cultures of chicken embryo fibroblasts were treated with noninfectious DNA fragments and infected with temperature-sensitive mutants of Rous sarcoma virus defective in DNA polymerase or in an internal virion structural protein. Wild-type progeny viruses which replicated at the nonpermissive temperature were isolated. Some of the wild-type progeny acquired both the wild-type DNA polymerase and the subgroup specificity of the Rous sarcona virus strain used for preparation of sonicated or EcoRI-digested DNA fragments. Therefore the genetic markers for DNA polymerase and envelope were linked and appeared to be located on the same EcoRi fragment of the DNA of Rous sarcoma virus-infected cells.     

Transformation of rat cells by fusion-infection with Rous sarcoma virus

The transformation of a rat cell line, 3Y1, by nonmammalian tropic strains of avian sarcoma virus was tested using cell-virus fusion mediated by Sendai virus or polyethylene glycol. Furthermore, the establishment of several transformed 3Y1 cell clones induced by the Schmidt-Ruppin strain of Rous sarcoma virus (RSV), its derivative mutants, and the Bryan high-titer strain of RSV is reported. The presence and expression of the viral genomes in these cells were examined, and all transformed cell clones tested were found to contain rescuable RSV genomes when they had been fused with normal chicken embryo fibroblast cells or those preinfected with Rous-associated virus type 1. However, the gag gene product, pr76, was barely detectable in wild-type RSV-transformed cells, whereas it was produced in considerable amounts in cells transformed by env-deleted mutants, the Bryan high-titer strain of RSV and NY8 derived from the Schmidt-Ruppin strain of RSV.     

Construction and biological analysis of deletion mutants of Fujinami sarcoma virus: 5''-fps sequence has a role in the transforming activity.

Fujinami sarcoma virus (FSV) genome codes for the gag-fps fusion protein FSV-P130. The amino acid sequence of the 3' one-third portion in v-fps is partially homologous to the 3' half of pp60src, or the kinase domain, but the sequence of the 5' portion is unique to v-fps. To identify a possible domain structure in the v-fps sequence responsible for cell transformation, we constructed various deletion mutants of FSV with molecularly cloned viral DNA. Their transforming activities were assayed by measuring focus formation on chicken embryo fibroblasts and rat 3Y1 cells and tumor formation in chickens. The mutants carrying a deletion at the 3' portion in v-fps, the kinase domain, lost transforming activity. The mutants carrying an approximately 1-kilobase deletion within the 5' portion of the v-fps sequence retained focus-forming activity and tumorigenicity in the chicken system, but the efficiency of focus formation was about 10 times lower than that of the wild type. The morphology of these transformed cells was distinct from that observed in cells infected with wild-type FSV. Furthermore, these mutants could not transform rat 3Y1 cells, although wild-type FSV DNA transformed rat 3Y1 cells at a high frequency. The mutants carrying a larger deletion in the 5' portion of fps completely lacked the transforming activity. These results suggest that the 3' portion of the v-fps sequence is necessary but not sufficient for cell transformation and that the 5' portion of v-fps has a role in the transforming activity.     

Transformation by Rous sarcoma virus: a cellular substrate for transformation-specific protein phosphorylation contains phosphotyrosine

Transformation of chicken embryo fibroblasts by Rous sarcoma virus (RSV) is caused by a single viral gene, src, which encodes a phosphoprotein, pp60src, with the enzymatic activity of a protein kinase. The relative abundance of a 36,000 molecular weight (36K) phosphorylated polypeptide which can be detected by two-dimensional electrophoresis of 32P-labeled phosphoproteins is greatly increased in RSV-transformed fibroblasts. We have reported previously that phosphorylation of the 36K polypeptide is an early event in the process of transformation and that protein synthesis is not required for its appearance. Here we identify a nonphosphorylated 36K polypeptide, present in both uninfected and transformed cells, which is homologous to the 36K phosphoprotein as judged by limited proteolysis and by tryptic peptide mapping. We conclude that the 36K phosphoprotein is generated by phosphorylation of this 36K polypeptide. It has recently been shown that pp60src phosphorylates tyrosine residues in vitro: phosphotyrosine and also phosphoserine are present in the 36K phosphoprotein isolated from RSV-transformed cells. On the basis of these results we propose that the 36K polypeptide present in chicken fibroblasts is a substrate for the protein kinase activity of pp60src. Phosphorylation of this polypeptide may be important in cellular transformation by Rous sarcoma virus.     

N-terminal deletion in the src gene of Rous sarcoma virus results in synthesis of a 45,000-Mr protein with mitogenic activity.

Expression of the v-src gene of Rous sarcoma virus in avian embryo neuroretina cells results in transformation and sustained proliferation of these normally resting cells. Transformed neuroretina cells are also tumorigenic upon inoculation into immunodeficient hosts. We have previously described conditional mutants of Rous sarcoma virus encoding p60v-src proteins which induce proliferation of neuroretina cells in the absence of transformation and tumorigenicity. These results suggest that p60v-src is composed of functionally distinct domains which may interact with multiple cellular targets. In this study, we describe a spontaneous variant of Rous sarcoma virus, subgroup E, which carries a deletion of 278 base pairs in the 5' portion of the v-src gene but which has retained the ability to induce proliferation of quail neuroretina cells. The deleted v-src gene encodes a 45,000-molecular-weight phosphoprotein which contains both phosphoserine and phosphotyrosine, is myristylated, and possesses tyrosine kinase activity indistinguishable from that of wild-type p60v-src. Molecular cloning and sequence analysis of the mutant v-src gene have shown that this deletion extends from amino acid 33 to 126 of the wild-type p60v-src. Therefore, this portion of the v-src protein is dispensable for the mitogenic activity of Rous sarcoma virus in neuroretina cells.     

Phosphorylation of talin at tyrosine in Rous sarcoma virus-transformed cells.

The cytoskeletal protein talin was found to undergo enhanced phosphorylation at tyrosine residues in chicken embryo fibroblasts following transformation by Rous sarcoma virus. An increase in the tyrosine phosphorylation of talin was also observed within 6 h in cells infected by the temperature-sensitive mutant tsNY68 after a shift from the nonpermissive to the permissive temperature. The overall extent of phosphorylation was 0.07 mol of phosphate per mol of talin and was not appreciably altered by transformation. In uninfected cells talin was shown to be phosphorylated at multiple sites by tryptic peptide mapping. Following transformation most of these sites remained phosphorylated, to the same or to a lesser extent, while novel, phosphotyrosine-containing phosphopeptides appeared. Talin was phosphorylated at tyrosine in cells infected by Rous sarcoma virus mutants which induce altered or partial transformation morphologies; thus the increased phosphorylation of talin at tyrosine occurred irrespective of the morphology induced. Transformation by Y73 also induced elevated levels of phosphotyrosine in talin, whereas transformation by the avian erythroblastosis and Fujinami sarcoma viruses did not.     

Cellular tumorigenicity in nude mice. Role of susceptibility to natural killer cells

The athymic nude mouse is a useful animal model for assaying the neoplastic growth potential in vivo of animal cells transformed in vitro. Despite the demonstrated absence of thymus-dependent immunological functions, however, the nude mouse has now been shown to reject transplants of certain highly malignant heterologous tumors. In addition, a few transformed mammalian cell lines that exhibit all or most of the cellular phenotypes usually associated with malignancy fail to grow as tumors when injected into nude mice. In a continuing study to identify the in vitro phenotypes associated with tumor-forming ability in vivo, we investigated the role of cellular susceptibility to the naturally occurring, thymus-independent lymphocytes (natural killer or NK cells) in determining tumor induction by animal cells in nude mice. A representative collection of animal cells (ranging from normal human diploid cell strains to highly tumorigenic clonal cell lines, either transformed in vitro or derived from experimental tumors) was tested to see if the ability of cells to form tumors is consistently correlated with their susceptibility to NK cell-mediated lysis measured in vitro with splenic leukocytes from nude mice. If the physiological role of the NK cells in vivo were to recognize, and possibly to destroy, incipient tumor cells in situ, a direct association between cellular tumorigenicity and susceptibility to NK activity, might be expected. If, on the other hand, the formation of growing tumors by animal cells in nude mice depended on their ability to escape the cytolytic activity of NK cells, cellular tumorigenicity would be associated with cellular resistance to NK cells. Results obtained in this study failed to confirm either of these associations. Thus, cellular suscepbibility to NK cells, at least as determined by direct cytotoxicity assay in vitro, is not a useful predictive indicator of cellular tumorigenicity in nude mice.     

Fibrin overlay methods for the detection of single transformed cells and colonies of transformed cells.

Fibrin overlay methods are described which can detect the plasminogen activator produced by single transformed cells or small colonies of transformed cells. These methods were applied to malignant cells derived from humans, mice, hamsters, rats, and chicks. The lysis observed was plasminogen dependent. Transformation of chicken cells by Rous sarcoma virus was detected 4 days after infection. The number of lysis zones produced was proportional to the virus inoculum and was identical to the number of morphologically determined foci. These methods may also have application in model systems for scoring transformation by chemicals. Transformed mouse and chicken cells were detected at the single cell level and the number of lysis zones produced was dependent on the number of cells present, the time of incubation, and the concentration of plasminogen.     

Temperature-sensitive transformation by Rous sarcoma virus and temperature-sensitive protein kinase activity

The transforming protein of Rous sarcoma virus, p60src, has associated with it a protein kinase activity. We examined whether a correlation exists between the cellular concentration of enzymatically active p60src and the degree to which chick cells are transformed by mutants of Rous sarcoma virus which are temperature-sensitive for transformation. Such a correlation does exist, but cells infected with some mutants could be shown to contain, at the nonpermissive temperature, an amount of protein kinase activity equal to 30 to 40% of that in a wild-type transformed cell. We quantified the amount of virus-induced protein kinase activity by precipitation of p60src with an excess of antitumor antiserum. Our initial measurements of activity were serious underestimates, due to the lability of the protein kinase activity associated with p60src of at least four temperature-sensitive mutants. In fact, no activity at all was associated with p60src of tsLA90 when immunoprecipitation was performed by standard means. However, when immunoprecipitation was performed with procedures which minimize inactivation, it became apparent both that cells transformed by tsLA90 contained protein kinase activity and that cells infected with either NY68 or BK5 contained at the nonpermissive temperature, one-third to one-half as much activity as wild-type transformed cells. This level of activity was much more than that arising from p60sarc in uninfected cells. In uninfected cells we found an amount of protein kinase activity which varied from 3 to 5% as much as that in a virally transformed cell. The lability of the protein kinase activity of each of these mutants is a further demonstration that this activity is essential for the transformation of cells by Rous sarcoma virus. So as to explain the high protein kinase levels in cells infected with NY68 and BK5 at the nonpermissive temperature, the idea that transformation may be a response to a small quantitative change in the total activity of p60src and the possibility that there may be more than one viral function which is essential for transformation are discussed.     

Detection and Assay of Avian Tumor Virus Group-Specific Antigen and Antibody by the Paired Radioiodine-Labeled Antibody Technique

The paired radioiodine-labeled antibody technique (PRILAT) was applied to the detection and quantitation of avian tumor virus group-specific (gs) antigens and antibody. The technique proved to be specific, repeatable, and appreciably more sensitive than the microcomplement-fixation test for avian leukosis (COFAL). The PRILAT facilitated direct measurement of comparative antigen content of several types of transformed, neoplastic, or virus-infected cells and the magnitude of nonspecific antibody binding by appropriate control cells. The versatility of the technique was illustrated by application to the detection and quantitation of gs antibody content of chicken, turkey, pigeon, and hamster sera. Antibodies were detected in COFAL-negative sera from hamsters bearing tumors induced by the Schmidt-Ruppin strain of Rous sarcoma virus. Sera from chickens bearing similar tumors were not positive for gs antibodies, although sera from turkeys and chickens immunized with avian leukosis virus did contain gs antibodies.     

Morphological transformation, autonomous proliferation and colony formation by chicken heart mesenchymal cells infected with avian sarcoma, erythroblastosis and myelocytomatosis viruses

Normal chicken heart mesenchymal cells at low density in monolayer culture in plasma-containing medium have a polygonal shape and are proliferatively quiescent. The combination of epidermal growth factor and insulin at hyperphysiological concentration, an insulin-like growth factor surrogate, causes these cells to assume a fusiform shape and to increase 40-fold in number during four days of incubation. These mitogenic hormones do not, however, induce normal chicken heart mesenchymal cells to form colonies in agarose suspension culture. Chicken heart mesenchymal cells infected with the Schmidt-Ruppin or Prague-A strains of Rous sarcoma virus or with the Fujinami or Y73 avian sarcoma viruses assume spindle and round shapes, increase 50-100 fold in number during four days of monolayer culture in the absence of mitogenic hormones and form macroscopic colonies during 3-4 days of agarose suspension culture. The autonomous (mitogenic hormone-independent) proliferation, in monolayer culture, of cells infected with temperature-sensitive transformation mutants of Rous sarcoma virus (tsNY68, tsNY72, tsLA24, tsLA29) is temperature-sensitive. Chicken heart mesenchymal cells infected with avian erythroblastosis virus assume spindle shapes and proliferate in monolayer culture at a rate comparable to that of sarcoma virus-infected cells but do not, however, form colonies in agarose suspension culture. Cells infected with the myelocytomatosis virus MC29 assume stellate shapes and increase 18-fold in number during four days of monolayer culture. Cells infected with the myelocytomatosis virus MH2 assume fusiform shapes and increase fourfold in number during four days of monolayer culture. Neither MC29 nor MH2 renders chicken heart mesenchymal cells capable of colony formation in agarose suspension culture. Infection with avian leukosis viruses (RAV-1, RAV-2, RPL-42) or with transformation-defective mutants of Rous sarcoma virus (tdNY105, 107, 109) does not affect the morphology or proliferative behavior of chicken heart mesenchymal cells. Monolayer culture of chicken heart mesenchymal cells in plasma-containing medium appears, therefore, to define the ability of onc genes of acute transforming avian retroviruses to induce autonomous (mitogenic hormone-independent) cell proliferation, the essential characteristic of neoplasia. The differences in transformed morphology and rates of autonomous proliferation between cells infected with different acute transforming retroviruses probably reflects differences in the modes of action of the transforming proteins encoded by the onc genes of the respective viruses.(ABSTRACT TRUNCATED AT 400 WORDS)