Evidence that the Rous sarcoma virus transforming gene product phosphorylates phosphatidylinositol and diacylglycerol.

The ability of purified Rous sarcoma virus transforming gene product, pp60v-src, to phosphorylate phosphatidylinositol and diacylglycerol was investigated. Phosphatidylinositol was phosphorylated to form both mono- and diphosphorylated derivatives. 1,2-Diacylglycerol was phosphorylated to form phosphatidic acid. These activities showed the same thermolability and the same sensitivity to inhibitors as shown by the casein kinase activity of pp60v-src. In addition, when serum-starved chicken embryo fibroblasts transformed by a virus mutant temperature-sensitive for transformation were shifted from the nonpermissive to permissive temperature, an increase of 50-100% in the labeling of phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, and phosphatidic acid was observed, as compared to uninfected cells.     

In vitro translation yields a possible Rous sarcoma virus src gene product.

In vitro translation of Rous sarcoma virus (RSV) virion RNA in the messenger-dependent reticulocyte lysate system yielded polypeptides that were not synthesized by translation of RNA from a transformation-defective deletion mutant of RSV. These RSV-specific products migrated on sodium dodecyl sulfate/polyacrylamide gels as two doublets of approximately 25,000 and 17,000 daltons. Synthesis of these proteins was not sensitive to inhibition by m7GTP; however, synthesis of the 76,000-dalton precursor of the internal structural proteins was sensitive to inhibition by m7GTP. Tryptic peptide maps showed the 25,000- and 17,000-dalton proteins to be related to one another but to be distinct from the 76,000-dalton protein. The 25,000-dalton protein was translated only from a polyadenylylated RNA of approximately 2500 nucleotides, whereas the 76,000-dalton protein was translated from 38S RNA, corresponding to the entire viral genome. A 180,000-dalton protein was also synthesized from 38S RSV virion RNA. From the absence of the 25,000- and 17,000-dalton proteins in the translation products of transformation-defective RSV RNA and the size of their RNA templates, we conclude that these proteins may be derived from coding sequences within the RSV src gene.     

Assay for early cytoplasmic effects of the src gene product of Rous sarcoma virus

When microinjected into normal fibroblasts, cytoplasmic extracts of cells transformed by Rous sarcoma virus caused dissolution of microfilament bundles. This activity was not found in extracts of normal cells. The maximum effect was seen within 30 min of injection, and the activity could still be measured after a 10-fold dilution of the cytoplasmic extracts (14 mg/ml original protein concentration). The activity was trypsin sensitive and was destroyed by boiling, but was not RNase sensitive. Protein synthesis was not required for the disruption of actin-containing stress fibers by the injected activity. Microinjected cytoplasts prepared from normal 3T3 cells also showed dissolution of microfilament bundles, indicating that the cell nucleus was not required for expression of activity. Extracts made from fibroblasts transformed by Rous sarcoma virus having a temperature-sensitive mutation in the src gene were also temperature sensitive in the microinjection assay. Thus, the activity of extracts from cells infected with src mutant virus, but not from cells infected with wild-type virus, was destroyed either by in vitro incubation of the extract at the nonpermissive temperature before injection or by incubation of recipient cells at the nonpermissive temperature after injection.We conclude that the microinjection assay can detect a cytoplasmic activity coded for by the src gene of Rous sarcoma virus and that an early direct or indirect target of the src gene product is the cytoskeleton and cell motility system. This result is discussed in relation to the hypothesis that submembranous arrays of microfilaments, microtubules, and their associated proteins interact with cell surface receptors to form a surface modulating assembly that functions as a key regulator of cell growth.     

Association of the src gene product of Rous sarcoma virus with cytoskeletal structures of chicken embryo fibroblasts.

We have prepared cytoskeletons from normal and Rous sarcoma virus-transformed cells by extraction with nonionic detergents in a buffered salt solution designed to preserve the structure as it exists in vivo. Virtually all of the phosphoprotein pp60src in the cell is bound to such cytoskeletons. Furthermore, when these cytoskeletons are incubated in situ with [gamma-32P]ATP, pp60src is phosphorylated. Labeling of other apparently transformation-specific cytoskeletons phosphoproteins is also observed. These results directly demonstrate an association between pp60src and elements of the cytoskeleton and suggest that pp60src may exert at least some of its effects as a consequence of its interaction with this cellular framework.     

Adhesion plaques of Rous sarcoma virus-transformed cells contain the src gene product.

Another intracellular location of the Rous sarcoma virus (RSU) src gene product (pp60src) has been detected within RSV-transformed cells by indirect immunofluorescence. By using rabbit anti-tumor serum specific for pp60src, a speckled pattern of fluorescence was found on the ventral surface of RSV (Schmidt-Ruppin strain)-transformed normal rat kidney cells. Several tests indicated that this pattern was specific for pp60src. In addition, interference-reflection microscopy was used to visualize cellular adhesion plaques, which are the points at which cells attach to the substratum. Simultaneous immunofluorescence and interference-reflection microscopy indicated that the speckles of pp60src fluorescence corresponded exactly to the adhesion plaque structures. The presence of pp60src within the adhsion plaques was further demonstrated by indirect immunofluorescences on isolated adhesion plaques that remained bound to glass after removal of the cells. pp60src also was observed in adhesion plaques of RSV-tranformed chicken embryo fibroblasts (CEF) and mouse fibroblasts, as well as CEF infected with the temperature-sensitive RSV mutant tsNY68 and grown at permissive temperature. At nonpermissive temperature, pp60src was not detectable in adhesion plaques of the tsNY68-infected CEF. Adhesion plaques serve as focal points of microfilament bundle attachment, and thse results suggest that pp60src interacts directly with cellular cytoskeletal components.     

Multiple forms of sarc gene proteins from Rous sarcoma virus RNA.

In a previous study we were able to identify two proteins of 25,000 and 18,000 daltons that were made from RNA of transforming virions of Rous sarcoma virus (RSV) and that were missing from the translation products of a transformation-defective deletion mutant of RSV. In the present study we have separated RSV virion RNA on sucrose gradients and have determined that the two putative sarc gene products are synthesized as doublets from an mRNA of approximately 18 S. There also appear to be several other sizes of virion mRNA that direct the synthesis of other viral proteins. These data are discussed in terms of the structure of the RSV genome. In addition to the 25,000- and 18,000-dalton doublets, there also is a 60,000-dalton protein whose synthesis is directed by 18S viral RNA from transforming virion of RSV. Peptide mapping has shown that the 60,000- and 25,000-dalton doublet are structurally related. In addition, the use of two-dimensional gel electrophoresis has allowed us to resolve both bands of the 25,000-dalton doublet into several differently charged species.     

Talin is phosphorylated on tyrosine in chicken embryo fibroblasts transformed by Rous sarcoma virus.

We have examined the extent of tyrosine phosphorylation of talin, a component of the cytoskeleton localized in the focal adhesions and, therefore, a potential substrate of p60v-src, the transforming protein of Rous sarcoma virus. p60v-src is a tyrosine kinase that induces high levels of phosphotyrosine and the disorganization of the cytoskeleton in transformed cells. With a polyclonal antibody utilized in a previous study [Maher, P. A., Pasquale, E. B., Wang, J. Y. J. & Singer, S. J. (1985) Proc. Natl. Acad. Sci. USA 82, 6576-6580] for the detection of tyrosine-phosphorylated proteins, we have detected phosphotyrosine residues in talin molecules immunoprecipitated from Rous sarcoma virus-transformed, but not normal, chicken embryo fibroblasts. Phospho amino acid analysis of talin from the infected cells confirmed the presence of phosphotyrosine, in addition to phosphoserine and phosphothreonine. The extent of tyrosine modification in talin was compared to that in vinculin, the other focal adhesion component previously found to contain enhanced levels of phosphotyrosine in various retrovirus-transformed cells. A considerably (3 times) larger fraction of the talin than of the vinculin molecules was found to be phosphorylated on tyrosine. The phosphorylation of talin on tyrosine may be crucial for the expression of the abnormal morphology characteristic of cells transformed by Rous sarcoma virus.     

Cytosolic malic dehydrogenase activity is associated with a putative substrate for the transforming gene product of Rous sarcoma virus.

A cellular protein of apparent Mr 34,000--36,000 was suggested as a possible physiological substrate for the protein kinase (EC 2.7.1.37) activity associated with the transforming gene product of Rous sarcoma virus. We find this protein to migrate with an apparent Mr of 38,000 in NaDodSO4/polyacrylamide gels. It was not separable from cytosolic malic dehydrogenase activity when purified by chromatography on DEAE-Sephacel, hydroxylapatite, poly(A)-Sepharose, and blue Sepharose, by gel filtration, and by isoelectric focusing. The Mr 38,000 protein as well as cytosolic malic dehydrogenase activity focused with a pI of 7.5. In gel filtration experiments, both displayed an apparent native Mr of 68,000. The male dehydrogenase activity contained in homogeneous preparations of the Mr 38,000 protein had a specific activity of up to 130 units/mg of protein. The recovery of the enzyme was 5--10% of the activity in the extract. Antiserum against the Mr 38,000 protein inactivated the malic dehydrogenase activity associated with the Mr 38,000 protein.     

RNA of replication-defective strains of Rous sarcoma virus.

The RNA of a replication-defective (rd) mutant, isolated from stocks of nondefective (nd) Schmidt-Ruppin Rous sarcoma virus of subgroup A (SR-A) and termed SR-N8, was compared to the RNAs of SR-A, of a transformation-defective derivative of SR-A (td SR-A) and of rd Bryan Rous sarcoma virus, RSV (minus). The molecular mass of the 30-40S species of SR-N8 RNA was estimated to be 21% (congruent to 7.5 to 8 times 10-5 daltons) smaller than that of SR-A by (i) electrophoresis in polyacrylamide gels and (ii) analyses of RNA complexity based on RNase T1-resistant oligonucleotides. ST-N8 shares probably all (=14) of its large RNase T1-resistant oligonucleotides with the RNA of SR-A as judged from the chromatographic distribution and the RNase A-resistant fragments obtained from RNase T1-resistant oligonucleotides. However, SR-N8 RNA lacked six large oligonucleotides which were present in the RNAs of SR-A and td SR-A. Conversely, the RNAs of SR-A, and of SR-N8 contained two oligonucleotides not found in td SR-A. The RNA of SR-N8 was found to differ from that of RSV (minus) in its electrophoretic mobility and its fingerprint pattern. It is concluded that the RNA of SR-N8 was generated by a deletion of SR-A. The extent of this deletion is compatible with the notion that the genetic information for the large viral envelope glycoprotein (molecular mass = 70,000-85,000 daltons) has been lost from the RNA of SR-A to yield SR-N8 RNA. From a comparison of td and rd deletion mutants, it appears that loss of different functions corresponds to the absence of different oligonucleotides in their RNA.     

Association of the src-gene product of Rous sarcoma virus with a pyruvate-kinase inactivation factor

A casein-kmase preparation was isolated from extracts of mouse tumors induced by Rous sarcoma virus with the aid of affinity chromatography. The preparation was several hundredfold enriched with respect to casein-inase as well as pp60^src-kinase activity and contained a pyruvate-kinase inactivating factor. The pp60^src- kinase is expressed by a phosphoprotein (pp60^src) which is the product of the transforming src gene of the Rous sarcoma virus (RSV). Thepp60^src- kinase phosphorylates IgG from sera of rabbits bearing the RSV tumor (TBR sera) in free solution exclusively in tyrosine residues. The activity ofpp60^src- kinase, as well as casein phosphorylation, was inhibited by μmolar concentrations of quercetin.The protein-kinase preparation caused MgATP-dependent inactivation of co-eluting pyruvate kinase. The activation of pyruvate kinase was blocked by phosphoribosyl pyrophosphate, fructose 1,6-diphosphate and P¹,p⁵-di (adenosine-5')-pentaphosphate. These compounds also inhibited tyrosine phosphorylation of TBR-IgG and the casein-kinase activity. Immunoadsorption of the protein-kinase preparation by TBR-IgG was accompanied by a loss of the protein-kinase preparation's ability to cause MgATP-dependent inactivation of pyruvate kinase. Essentially the same results were obtained when tumor extracts were first passed over Blue Sepharose and subsequently over the casein-affinity column. Inactivation of purified chicken-liver pyruvate kinase, type M₂, was observed with an 800-fold purifiedpp60^src-kinase preparation obtained from RSV-transformed chicken-embryo fibroblasts. Incubation of the pyruvate kinase with pp60^srcin the presence of MgATP resulted in increased tyrosine phosphorylation of proteins with sub-unit molecular weights of 52 000 and 56 000 dalton. The latter corresponded to the sub-unit molecular weight of chicken-liver pyruvate kinase.The results show that the pyruvate-kinase inactivating factor is closely associated with pp60^src-kinase. The findings support the hypothesis that substrates of pp60^src, essential for cell proliferation, are among the enzymes that control glycolysis.     

Phosphorylation of tyrosine residues of calmodulin in Rous sarcoma virus-transformed cells.

Calmodulin, a wide-spread eukaryotic Ca2+-binding protein, was phosphorylated at its tyrosine residues in Rous sarcoma virus (RSV)-transformed chicken and rat cells but not in normal chicken embryo fibroblasts. In contrast, serine and threonine phosphorylation of calmodulin was found to occur in both normal and virus-transformed cells. In an in vitro system containing purified src kinase from RSV-transformed cells, tyrosine phosphorylation of calmodulin by the src kinase was inhibited by Ca2+. Furthermore, the tyrosine-phosphorylated calmodulin showed slower mobility than that of nonphosphorylated calmodulin in NaDodSO4/polyacrylamide gel electrophoresis when Ca2+ was present. These results suggest that the structure of calmodulin Ca2+ complex may be altered by tyrosine phosphorylation. It is thus inferred that Ca2+ may regulate the level of tyrosine phosphorylation of calmodulin in RSV-transformed cells, and phosphorylation in turn may attenuate the function of this protein in vivo.     

pp60v-src tyrosine kinase is expressed and active in sarcoma-free avian embryos microinjected with Rous sarcoma virus.

Early embryonic avian tissue is resistant to transformation by Rous sarcoma virus. To determine the nature of this resistance, we examined the expression and properties of the Rous sarcoma virus transforming protein pp60v-src, in infected embryonic chicken limbs in ovo. Lysates from Rous sarcoma virus-infected limbs contained the viral structural protein p19gag, as detected by immunoblot analysis, and showed pp60v-src kinase activity in vitro. Immunoblot analysis of lysates with anti-phosphotyrosine antibodies revealed a number of phosphotyrosine-containing proteins present in lysates of Rous sarcoma virus-infected embryos but not in lysates of control, uninfected embryos. Anti-phosphotyrosine immunoreactivity was observed in frozen sections in the same cell types that expressed pp60v-src and p19gag. These studies demonstrate that pp60v-src is co-expressed with viral structural determinants in infected embryonic avian tissue. Furthermore, pp60v-src is active in ovo as a tyrosine-specific phosphotransferase, despite the apparent lack of sarcoma induction. The localization pattern of the major src gene substrate p36 (calpactin I) was compared with that of p19gag by double-label immunofluorescence and found to be generally nonoverlapping. These observations are consistent with the concept that the induction of tumors in ovo requires complementation between viral determinants and host factors. These host factors, which may be critical substrates of pp60v-src, are subject to developmental regulation in the avian embryo.     

Decrease of tyrosine-O-sulfate-containing proteins found in rat fibroblasts infected with Rous sarcoma virus or Fujinami sarcoma virus.

Our interest was aroused by the recent report by Huttner [ Huttner , W. B. (1982) Nature (London) 299, 273-276] on general sulfation of tyrosine residues of proteins in normal and malignantly transformed tissues. Here we report on the reduction of sulfation in embryonic rat fibroblasts, line 3Y1, infected with Rous sarcoma virus or Fujinami sarcoma virus. In view of the instability of tyrosine O-sulfate in strong acid, the protein sulfation was tested for after incubation with [35S]sulfate and exhaustive Pronase hydrolysis. We found in general a reduction of sulfation in transformed tissue. It was greatest in the fibroblasts permanently transformed with Rous sarcoma virus. When fibroblasts transformed by the temperature-sensitive Fujinami sarcoma virus, line ts225 -3Y1, were used for comparison of sulfation at nonpermissive and permissive temperatures, the latter showed a strong reduction. Furthermore, we tested these cells for the uptake of inorganic [35S]sulfate. Uptake appeared highly reduced in the permanently infected fibroblasts, but ts225 -3Y1 grown at permissive and nonpermissive temperatures exhibited no difference. Uptake at both temperatures was comparable to uptake by normal 3Y1 cells. A recently much investigated cell surface protein, fibronectin, was reported to be lost on malignant transformation and to contain sulfate in an undetermined location. We found that ts225 -3Y1 cells grown at permissive temperature released fibronectin that contained tyrosine O-sulfate.     

Rous sarcoma virus activates embryonic globin genes in chicken fibroblasts.

Complementary DNA (cDNA) specific for chick globin mRNA sequences fails to hybridize to total RNA extracted from chicken fibroblasts. After infection by Rous sarcoma virus, RNA complementary to globin cDNA is detectable in 100-500 copies per cell. Infection of fibroblasts with the transformation defective (td) deletion mutant of Rous sarcoma virus leads to normal virus production, but not to host cell transformation or accumulation of RNA sequences complementary to globin cDNA. Our evidence shows that the globin genes activated by Rous sarcoma virus are those specified by embryonic chick red cells; adult-specific globin sequences were not detected.     

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.     

Hematopoietic cell transformation by a murine recombinant retrovirus containing the src gene of Rous sarcoma virus.

A recombinant murine retrovirus (MRSV) containing the src gene of avian Rous sarcoma virus (RSV) was shown to induce hematopoietic colonies in infected mouse bone marrow. MRSV-induced colony formation followed single-hit kinetics and required mercaptoethanol in the agar medium. Cells from the colonies induced by MRSV could be established as continuous cell lines that demonstrated unrestricted self-renewal in vitro and tumorigenicity in vivo. The transformants, all of which expressed high levels of the Rous sarcoma virus transforming protein, pp60src, appeared to be at an early stage in lymphoid cell differentiation. They lacked Fc receptors and detectable immunoglobulin mu heavy chain synthesis, markers normally associated with committed B cells. The majority of the MRSV-transformed cell lines contained high levels of terminal deoxynucleotidyl transferase, an enzyme present in lymphoid progenitor cells committed to the T-cell lineage. One cell line expressed Thy-1 antigen, but none expressed Lyt-1 and Lyt-2, markers of more differentiated T cells. These findings demonstrate that the src gene is capable of transforming cells of hematopoietic origin.