Rous sarcoma virus mutant dlPA105 induces different transformed phenotypes in quail embryonic fibroblasts and neuroretina cells.

dlPA105 is a spontaneous variant of Rous sarcoma virus, subgroup E, which carries a deletion in the N-terminal portion of the v-src gene coding sequence. This virus was isolated on the basis of its ability to induce proliferation of quiescent quail neuroretina cells. The altered v-src gene encodes a phosphoprotein of 45,000 daltons which possesses tyrosine kinase activity. DNA sequencing of the mutant v-src gene has shown that deletion extends from amino acid 33 to 126 of wild-type p60v-src. We investigated the tumorigenic and transforming properties of this mutant virus. dlPA105 induced fibrosarcomas in quails with an incidence identical to that induced by wild-type virus. Quail neuroretina cells infected with the mutant virus were morphologically transformed and formed colonies in soft agar. In contrast, dlPA105 induced only limited morphological alterations in quail fibroblasts and was defective in promoting anchorage-independent growth of these cells. Synthesis and tyrosine kinase activity of the mutant p45v-src were similar in both cell types. These data indicate that the portion of the v-src protein deleted in p45v-src is dispensable for the mitogenic and tumorigenic properties of wild-type p60v-src, whereas it is required for in vitro transformation of fibroblasts. The ability of dlPA105 to induce different transformation phenotypes in quail fibroblasts and quail neuroretina cells is a property unique to this Rous sarcoma virus mutant and provides evidence for the existence of cell-type-specific response to v-src proteins.     

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.     

Distinct protease pathways control cell shape and apoptosis in v-src-transformed quail neuroretina cells

Intracellular proteases play key roles in cell differentiation, proliferation and apoptosis. In nerve cells, little is known about their relative contribution to the pathways which control cell physiology, including cell death. Neoplastic transformation of avian neuroretina cells by p60(v-src) tyrosine kinase results in dramatic morphological changes and deregulation of apoptosis. To identify the proteases involved in the cellular response to p60(v-src), we evaluated the effect of specific inhibitors of caspases, calpains and the proteasome on cell shape changes and apoptosis induced by p60(v-src) inactivation in quail neuroretina cells transformed by tsNY68, a thermosensitive strain of Rous sarcoma virus. We found that the ubiquitin-proteasome pathway is recruited early after p60(v-src) inactivation and is critical for morphological changes, whereas caspases are essential for cell death. This study provides evidence that distinct intracellular proteases are involved in the control of the morphology and fate of v-src-transformed cells.     

Low level of cellular protein phosphorylation by nontransforming overproduced p60c-src.

We have previously found that Rous sarcoma virus variants in which the viral src (v-src) gene is replaced by the cellular src (c-src) gene have no transforming activity. In this study, we analyzed the basis for the inability of the p60c-src overproduced by these variants to transform cells. Phosphorylations of tyrosine residues in total cell protein or in cellular 34K protein are known to be markedly enhanced upon infection with wild-type Rous sarcoma virus. We found that these tyrosine phosphorylations were only slightly increased in the c-src-containing virus-infected cells, whereas both levels were significantly increased by infection with wild-type Rous sarcoma virus, or transforming mutant viruses which are derived from c-src-containing viruses by spontaneous mutation. Phosphorylation at tyrosine 416 of p60 itself was also extremely low in overproduced p60c-src and high in p60s of transforming mutant viruses. In immunoprecipitates with monoclonal antibody, the overproduced p60c-src had much lower casein tyrosine kinase activity than did p60v-src. We previously showed that p60 myristylation and plasma membrane localization may be required for cell transformation. p60c-src was similar to transforming p60s in these properties. These results strongly suggest that the low level of tyrosine phosphorylation by overproduced p60c-src accounts for its inability to transform cells.     

A glycoprotein in the plasma membrane matrix as a major potential substrate of p60v-src.

A potential substrate of p60v-src in Rous sarcoma virus-transformed cells was found to be a 130-kilodalton (kDa) glycoprotein which binds to lectin-Sepharose and can be immunoprecipitated by an anti-phosphotyrosine antibody. This glycoprotein was shown to be distinct from the fibronectin receptor and a cellular protein phosphorylated in p60v-src immune complexes. The protein was a transmembrane protein localized in the plasma membrane and resistant to extraction with Triton X-100. The 130-kDa protein was also highly phosphorylated in cells transformed by Fujinami sarcoma virus or Y73 but not in cells infected with Rous sarcoma virus mutants that encode p60v-src lacking myristoylated N termini. Phosphorylation of this glycoprotein was temperature dependent in cells infected with temperature-sensitive mutants. The good correlation between its phosphorylation and morphological transformation, together with its relative abundance among phosphorylated proteins and its subcellular localization, suggests that phosphorylation of the 130-kDa glycoprotein is one of the primary events important for cell transformation by p60v-src and related oncogene products.     

Genetic analysis of p60v-src domains involved in the induction of different cell transformation parameters.

The expression of p60v-src in chicken cells infected with Rous sarcoma virus causes stimulation of cell proliferation, morphological alteration, and anchorage independence. PA101 and PA104 are temperature-sensitive variants encoding mutant p60v-src proteins that are partially defective in the induction of these transformation parameters. To define the structural basis for the transformation defectiveness of the p60v-src mutants, the v-src genes of PA101 and PA104 were molecularly cloned and analyzed. Amino- and carboxy-terminal coding regions of the cloned mutant genes were exchanged with the corresponding regions of cloned wild-type v-src and chicken c-src genes, reconstructed into viral DNA, and expressed in infected cells maintained at various temperatures. This analysis revealed that lesions within the tyrosine kinase domains of the two mutant proteins confer temperature sensitivity on all three transformation functions of p60v-src. An amino-terminal region of the PA101 mutant protein, which coincides with the proposed modulatory domain and appears to interact with the kinase domain, affects morphological alteration in a temperature-independent manner. Our results suggest that the function of the kinase domain is essential to all three parameters examined, whereas the amino-terminal domain is important in determining cell morphology.     

Intracellular localization and processing of pp60v-src proteins expressed by two distinct temperature-sensitive mutants of Rous sarcoma virus.

The transforming protein of Rous sarcoma virus, pp60v-src, is known to be a tyrosine protein kinase, but the mechanism of cell transformation remains unclear. In further investigating pp60v-src structure and function, we have analyzed two temperature-sensitive (ts) Rous sarcoma virus src gene mutants, tsLA29 and tsLA32. The mutations in tsLA29 and tsLA32 map in the carboxy-terminal region and the amino-terminal half of pp60v-src, respectively, and encode mutant proteins with either temperature-labile (tsLA29) or -stable (tsLA32) kinase activities. Here we examined the intracellular processing and localization of these pp60v-src mutants and extended our characterization of transformation parameters expressed by cells infected by the Rous sarcoma virus variants. No obvious defects in functional integrity of the tsLA32 pp60v-src could yet be demonstrated, whereas the tsLA29 pp60v-src was perturbed not only in kinase activity, but also in aspects of protein processing and localization. Analysis of transformation parameters expressed by infected cells demonstrated the complete temperature lability of both mutants.     

Phosphorylation of the transforming protein of Rous sarcoma virus: direct demonstration of phosphorylation of serine 17 and identification of an additional site of tyrosine phosphorylation in p60v-src of Prague Rous sarcoma virus.

We provide direct evidence that serine 17 is the major site of serine phosphorylation in p60v-src, the transforming protein of Rous sarcoma virus, and in its cellular homolog, p60c-src. The amino acid composition of the tryptic peptide containing the major site of serine phosphorylation in p60v-src was deduced by peptide map analysis of the protein labeled biosynthetically with a variety of radioactive amino acids. Manual Edman degradation revealed that the phosphorylated serine in this peptide was the amino terminal residue. These data are consistent only with the phosphorylation of serine 17. The major site of serine phosphorylation in chicken p60c-src, the cellular homolog of p60v-src, is contained in a tryptic peptide identical to that containing serine 17 in p60v-src of Schmidt Ruppin Rous sarcoma virus of subgroup A. Serine 17 is therefore also phosphorylated in p60c-src. The p60v-src protein encoded by Prague Rous sarcoma virus was found to contain two sites of tyrosine phosphorylation. The previously unrecognized site of tyrosine phosphorylation may be tyrosine 205 or possibly tyrosine 208. Treatment of Prague Rous sarcoma virus-infected cells with vanadyl ions stimulated the protein kinase activity of p60v-src and increased the phosphorylation of tyrosine 416 but not the phosphorylation of the additional site of tyrosine phosphorylation.     

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.     

Differentiation of retrovirus-infected avian neuroretina cells

The effects of oncogenic retroviruses on the expression of differentiation markers were studied in monolayer cultures of chick and quail embryo neuroretinas. Transformation by Rous sarcoma virus (RSV) did not affect the appearance of synapses, and the expression of glutamic acid decarboxylase was stimulated by pp60v-src, the product of the src gene. Quail embryo neuroretina cells transformed by Mill Hill 2 (which contains the two oncogenes v-mil and v-myc) were induced to proliferate into permanent cultures that synthesized crystallins and produced lentoid bodies. In contrast, transformation with a temperature-sensitive mutant of RSV reversibly blocked the production of crystallins and lentoid bodies. These data show that given cellular genes can respond differently to distinct oncogenes.     

The membrane-binding domain and myristylation of p60v-src are not essential for stimulation of cell proliferation.

Previous studies showed that the amino-terminal domain of Rous sarcoma virus p60v-src involved in myristylation and membrane association of the protein is required for morphological transformation and anchorage independence. Analysis of src delection mutants revealed that the amino-terminal one-third of p60v-src, including the membrane-binding domain, is not essential for induction of cell proliferation. These results demonstrated that, in contrast to the cellular target(s) involved in morphological transformation and anchorage independence, the target(s) involved in mitogenic activity is accessible to nonmyristylated src proteins.     

Rous sarcoma virus does not induce sarcoma in early chick embryos by lack of the v-src gene expression.

The Schmidt-Ruppin or the B77 strain of Rous sarcoma virus (RSV) was inoculated into limb buds of 4.5-days-old avian embryos. No sarcoma but blister formation was observed in those RSV-inoculated embryos. Protein kinase activity of pp60v-src in RSV-inoculated embryos, even in the site of virus inoculation, was the same as that in mock-infected embryos. This indicated that the expression of the v-src gene did not attain superiority over that of the c-src gene in RSV-inoculated embryos. The v-src gene was detected in every DNA from tissues of RSV-inoculated embryos but not in DNAs from tissues of RSV-inoculated chicken except for the DNA from Rous sarcoma. Those results confirmed that the lack of sarcoma induction in early avian embryos by RSV was due to the lack of the expression of the v-src gene which was present in the target cells.     

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.     

A mutation in v-src that removes a single conserved residue in the SH-2 domain of pp60v-src restricts transformation in a host-dependent manner.

The v-src oncogene of Rous sarcoma virus (RSV) is able to transform both avian and mammalian cells, but the mutant allele v-src-L displays a host range dependence for transformation, transforming chicken but not rat cells with wild-type efficiency. This host range restriction can be detected by measuring growth in low serum, saturation density, and anchorage independent growth. In addition, rat cells expressing v-src-L do not form tumors in syngeneic rats or nude mice, but RSV carrying the mutant allele causes tumors in chicks, although at a reduced efficiency and with increased latency. To determine the lesion responsible for this phenotype, we sequenced the entire v-src gene from the parental B77 strain of RSV, as well as the mutant allele. v-src-L is missing 3 nucleotides present in the wild-type parent, RSV B31, eliminating Phe-172, an invariant residue in a conserved region of src-related proteins known as SH-2. The kinase activity of pp60v-src-L was indistinguishable from that of the wild type in chicken cells but was significantly reduced in rat cells as assayed by an in vitro immune complex assay; in vivo phosphorylation of one specific substrate, p36 (calpactin I heavy chain); and total phosphotyrosine-containing proteins. In addition, the pattern of phosphotyrosine-containing proteins in rat cells was qualitatively different when cells containing pp60v-src-L were compared with cells with wild-type pp60v-src, even though both pp60v-src proteins were membrane associated. The data are consistent with a role for the SH-2 region in substrate specificity.     

Generation of a recombinant Moloney murine leukemia virus carrying the v-src gene of avian sarcoma virus: transformation in vitro and pathogenesis in vivo.

A Moloney murine leukemia virus (M-MuLV) recombinant carrying the v-src gene of avian sarcoma virus was generated by the introduction of a cloned portion of v-src from Schmidt-Ruppin A avian sarcoma virus into a molecular clone of M-MuLV provirus at the recombinant DNA level. The v-src sequences (lacking a portion of the 5' end of v-src) were inserted into the p30 region of the M-MulV gag gene so that M-MuLV gag and v-src were in the same reading frame. Transfection of this chimeric clone, pMLV(src), into NIH 3T3 cells which were constitutively producing M-MuLV gag and pol protein resulted in the formation of foci of transformed cells. Infectious and transforming virus could be recovered from the transformed cells. This virus was designated M-MuLV(src). M-MuLV(src)-transformed cells contained two novel proteins of 78 and 90 kilodaltons. The 78-kilodalton protein, p78gag-src, contained both gag and src determinants, exhibited kinase activity in an immune kinase assay, and is probably a fusion of Pr65gag and src. The 90-kilodalton protein, which is of the appropriate size to be the gPr80gag fused to src, contained gag determinants as well as a V8 protease cleavage fragment typical of the carboxy terminus of avian sarcoma virus pp60src. However, it could not be immunoprecipitated with an anti-v-src serum. M-MuLV(src)-transformed cells showed elevated levels of intracellular phosphotyrosine in proteins, although the elevation was intermediate compared with cells transformed with wild-type v-src. M-MuLV and amphotropic murine leukemia virus pseudotypes of M-MuLV(src) were inoculated into newborn NIH Swiss mice. Inoculated mice developed solid tumors at the site of inoculation after 3 to 6 weeks, with most animals dying by 14 weeks. Histopathological analysis indicated that the solid tumors were mesenchymally derived fibrosarcomas that were both invasive and metastatic.     

Irreversible inhibition of v-src tyrosine kinase activity by herbimycin A and its abrogation by sulfhydryl compounds

Herbimycin A, an antibiotic which reverses Rous sarcoma virus transformation, inhibited irreversibly the auto- and trans-phosphorylation activities of p60v-src in in vitro immune complex kinase assays. The addition of a sulfhydryl compound such as dithiothreitol, 2-mercaptoethanol, glutathione (reduced form) or cysteine abolished the ability of herbimycin A to inactivate p60v-src kinase as well as the ability to reverse transformed cell morphology, whereas the addition of oxidized glutathione, cystine or methionine showed no effect. The sulfhydryl alkylating reagent N-ethylmaleimide also, although less effectively, inactivated p60v-src kinase activity in vitro. These results suggest the likelihood that sulfhydryl groups of p60v-src are involved in the inactivation of v-src tyrosine kinase activity by herbimycin A.     

Specific proteolytic fragmentation of p60v-src in transformed cell lysates.

Work involving the transforming protein, p60v-src, of Rous sarcoma virus has resulted in the extensive characterization of its protein structure and associated phosphotransferase activity. However, in many investigations proteolytic fragments (principally p52v-src) of the src protein are actually studied. Here, we emphasize potential problems in the interpretation of experimental results in which the proteolytic fragmentation of p60v-src may be involved and offer several means for the complete prevention of this p60v-src degradation.