Recovery of avian sarcoma virus from tumors induced by transformation-defective mutants.

Transformation-defective (td) mutants of the Schmidt-Ruppin strain of Rous sarcoma virus (RSV), which contains deletions in the gene responsible for transformation (src gene), are unable to transform chicken embryo fibroblasts in vitro. Injection of some of these td mutants into newborn chickens resulted in the formation of sarcomas from which sarcoma virus was unfailingly recovered. The possibility that transforming RSV was present in the td virus preparations was excluded by further purification of the td viruses. Morphology of the foci induced by the newly recovered sarcoma virus was distinct from that of foci induced by the parental Schmidt Ruppin strain of RSV. It is suggested that the new sarcoma virus was generated as a result of the genetic interaction between the genomes of td virus and chicken cells.

p60c-src is complexed with a cellular protein in subcellular compartments involved in exocytosis.

We found high levels of the c-src gene product in neuroendocrine tissues from adult animals. To understand the role of this proto-oncogene product, the subcellular localization of p60c-src was studied in neuroendocrine tissue from adrenal medulla. The results indicate that p60c-src was highly enriched in chromaffin granule membranes, in stable association with a protein of 38 kD. The complex with the 38-kD protein was also detected in brain, a tissue known to carry high levels of p60c-src. The 38-kD protein is not calpactin I, II, or synaptophysin. Comparison of its peptide map showed a high degree of conservation among the different species and tissues examined. The interaction between p60c-src and the 38-kD protein involves disulphide bonds that are stable even when the cell fractionation is performed in the presence of a reducing agent. Since the presence of disulphide bonds among cytoplasmic proteins is very unlikely, the possibility of a noncovalent association between p60c-src and the 38-kD protein in vivo is discussed. The 38-kD protein may be involved in a function of p60c-src related to secretory organelles.

v-src transformation of rat embryo fibroblasts. Inefficient conversion to anchorage-independent growth involves heterogeneity of primary cultures.

To clarify whether a single oncogene can transform primary cells in culture, we compared the transforming effect of a recombinant retrovirus (ZSV) containing the v-src gene in rat embryo fibroblasts (REFs) to that in the rat cell line 3Y1. In the focus assay, REFs exhibited resistance to transformation as only six foci were observed in the primary cultures as opposed to 98 in 3Y1 cells. After G418 selection, efficiency of transformation was again somewhat lower with REFs compared to that with 3Y1 cells, but the number of G418-resistant REF colonies was much greater than the number of foci in REF cultures. Furthermore, while 98% of G418-resistant colonies of ZSV-infected REFs were morphologically transformed, only 25% were converted to anchorage-independent growth, as opposed to 100% conversion seen in ZSV-infected 3Y1 cells. The poor susceptibility of REFs to anchorage-independent transformation did not involve differences in expression and subcellular distribution of p60v-src, or its kinase activity in vitro and in vivo. It rather reflected a property of the primary cultures, as cloning of REFs before ZSV infection demonstrated that only 2 out of 6 REF clones tested were permissive for anchorage-independent growth. The nonpermissive phenotype was dominant over the permissive one in somatic hybrid cells, and associated with organized actin filament bundles and a lower growth rate, both before and after ZSV infection. These results indicate that the poor susceptibility of REFs to anchorage-independent transformation by p60v-src reflects the heterogeneity of the primary cultures. REFs can be morphologically transformed by p60v-src with high efficiency but only a small fraction is convertible to anchorage-independent growth. REF resistance seems to involve the presence of a suppressor factor which may emerge from REF differentiation during embryonic development.

Binding of transforming protein, P47gag-crk, to a broad range of phosphotyrosine-containing proteins.

Although the oncogene product of CT10 virus, P47gag-crk, does not itself phosphorylate proteins at tyrosine residues, it elevates phosphotyrosine in transformed cells. The P47gag-crk oncoprotein contains SH2 and SH3 domains, which are conserved in several proteins involved in signal transduction, including nonreceptor tyrosine kinases. P47gag-crk bound in vitro to phosphotyrosine-containing proteins from crk-transformed cells and from cells transformed by oncogenic tyrosine kinases. The association between P47gag-crk and p60v-src, a phosphotyrosine-containing protein, was abolished by dephosphorylation of p60v-src. This suggests that the SH2 and SH3 regions function to regulate protein interactions in a phosphotyrosine-dependent manner.

Amino terminal myristylation of the protein kinase p60src, a retroviral transforming protein.

The transforming protein of Rous sarcoma virus, p60src, was shown to be acylated at its amino terminus with the long-chain fatty acid myristic acid by isolation of a tryptic peptide with the following structure: myristylglycylserylseryllysine. The occurrence of this unusual posttranslational modification in the cyclic adenosine monophosphate-dependent protein kinase and in several transforming protein kinases of mammalian retroviruses suggests that myristylation of the amino terminal glycyl residue may be critical for the function of certain proteins related to cell transformation and growth control.

Absence of polymerase protein in virions of alpha-type rous sarcoma virus.

Noninfectious particles of a mutant of Rous sarcoma virus failed to exhibit DNA polymerase activity even with the use of the most sensitive synthetic template-primer complexes. A neutralization blocking test against antibody to DNA polymerase revealed that these mutants did not contain protein immunologically related to the DNA polymerase.

SH2 and SH3 domains as molecular adhesives: the interactions of Crk and Abl.

The Src homology domains SH2 and SH3 are modular components present in many signal transduction proteins. They allow rapid formation of stable protein complexes and may also regulate protein function through intramolecular binding events. SH2 domains recognize phosphotyrosyl residues in a specific sequence context, while SH3 domains recognize a PxxP motif and additional residues that mediate binding specificity.

The Ras-MAPK pathway downregulates Caveolin-1 in rodent fibroblast but not in human fibroblasts: implications in the resistance to oncogene-mediated transformation.

Normal human diploid fibroblasts (HDFs) are refractory to oncogene-mediated transformations in vitro, compared with rodent fibroblasts. As successful oncogene-mediated transformations of normal HDFs have been reported using the human telomerase catalytic subunit, it has been considered that telomerase activity contributes to the species-specific transformability. However, these transformed HDFs are much less malignant compared with those of rodent cells, suggesting the existence of undefined mechanisms that render HDFs resistant to malignant transformation. Here, cDNA microarray analysis identified caveolin-1 as one of the possible cellular factors involved in such mechanisms. The mitogen-activated protein kinases (MAPK) pathway downregulates Caveolin-1 in rodent fibroblasts, transformed by coexpression of the SV40 early region and activated H-Ras. In contrast, the coexpression of these two oncogenes in HDFs failed to reduce the expression level of Caveolin-1. These results strongly suggest the presence of critical differences in events following the phosphorylation of ERK during the activation process of the MAPK signaling pathway between human and rodent cells, as the ERK protein was similarly phosphorylated in both systems. Furthermore, the small interfering RNA-mediated suppression of Caveolin-1 facilitated the oncogene-mediated transformation of normal HDFs, clearly indicating that the differences in the transformability between human and rodent cells are due, at least in part, to the mechanism responsible for the resistance to Ras-induced Caveolin-1 downregulation in HDFs.

Phosphatidylinositol kinase activity associates with viral p60src protein.

Immunoprecipitates of p60v-src proteins from chicken embryo fibroblasts infected with Rous sarcoma virus were assayed for phosphatidylinositol (PI) kinase activity in the absence of detergents. The product of the PI kinase reaction, phosphatidylinositol monophosphate (PIP), migrated slightly slower than did the authentic phosphatidylinositol-4-monophosphate marker in thin-layer chromatography and was indistinguishable from phosphatidylinositol-3-monophosphate produced by PI kinase type I. Furthermore, the deacylated product comigrated with glycerophosphoinositol-3-phosphate in high-performance liquid chromatography. Both sucrose gradient fractionation and the heat stability of PI kinase activity from cells infected with temperature-sensitive mutants suggest that the PI kinase activity is not intrinsic to p60v-src but is a property of another molecule complexed with p60v-src. All transforming variants of p60src were associated with PI kinase activity, whereas this enzyme activity was hardly detectable in immunoprecipitates from cells infected with nontransforming viruses encoding p60c-src or an enzymatically inactive variant. However, PI kinase activity was found in p60src immunoprecipitates from cells infected with nonmyristylated, nontransforming mutants as well as temperature-sensitive mutants at the nonpermissive temperature, which indicated that simple association of PI kinase activity with p60src is not sufficient for cell transformation.

Phosphatidylinositol kinase activity in virus-transformed and nontransformed cells.

We assayed phosphatidylinositol (PI) kinase (EC 2.7.1.67) activity in detergent extracts of nontransformed or virus-transformed cells. Nontransformed chicken embryo fibroblasts (CEF) contain PI kinase activity with an apparent specific activity of 20 pmol/min per mg of protein. This activity sedimented as a single peak with a molecular weight of approximately 60,000 in a glycerol gradient, although immunoprecipitation with anti-p60src sera showed that the PI kinase activity is distinct from p60c-src. Extracts from CEF transformed by Rous sarcoma virus, Fujinami sarcoma virus, or avian sarcoma virus UR2 showed no elevation of PI kinase activity over nontransformed CEF. Removal of the oncogene products from extracts by immunoprecipitation did not change the level of PI kinase activity in extracts, suggesting that putative virus-coded PI kinases do not make a significant contribution to overall levels of PI kinase activity in transformed cells. Additionally, P140gag-fps was separated from cellular PI kinase by phosphocellulose chromatography. This partially purified fraction contained low PI kinase activity distinct from P140gag-fps, indicating that P140gag-fps has no detectable PI kinase activity.

Expression of 9E3 mRNA is associated with mitogenicity, phosphorylation, and morphological alteration in chicken embryo fibroblasts.

Transformation of chicken embryo fibroblasts (CEF) with viruses encoding src, ros, yes, and fps as well as ras, mos, middle T, erbA and erbB, myc, and crk stimulated 9E3 mRNA expression. Treatment of CEF with agents that modulate cell shape or attachment to the substratum caused an increase in 9E3 mRNA without an increase in tyrosine phosphorylation. 9E3 mRNA was also increased in CEF in response to several agents which modulate phosphorylation, including phorbol myristic acetate, vanadate, and okadaic acid, which suggests that the rapid induction of 9E3 mRNA expression in CEF by the src protein occurs downstream of morphological or phosphorylation events.

Requirement of phosphatidylinositol-3 kinase modification for its association with p60src.

When purified p60v-src was mixed with lysates of chicken embryo fibroblasts and immunoprecipitated with anti-Src antibody, phosphatidylinositol (PI)-3 kinase activity was found to be present in the Src protein immunoprecipitates. The level of bound PI-3 kinase activity was 5 to 10 times higher in lysates obtained from cells transformed by the src, fps, or yes oncogene than in lysates of uninfected cells. This increase in associated PI-3 kinase activity appears to be due to increased binding of this enzyme to p60v-src. This change most likely resulted from tyrosine phosphorylation of PI-3 kinase or an associated protein, since the PI-3 kinase activity that can bind to p60v-src was depleted by antiphosphotyrosine antibody. Binding of PI-3 kinase did not require either p60src protein kinase activity or autophosphorylation of p60v-src tyrosine residues. Furthermore, binding was markedly decreased by deletions in the N-terminal SH2 region but unchanged by deletion of the C-terminal half of p60v-src containing the catalytic domain. Taking these data together, it appears that PI-3 kinase or its associated protein is phosphorylated on tyrosine and that the phosphorylated form can bind to the N-terminal half of p60v-src, which contains the SH2 domain.

Cellular proteins homologous to the viral yes gene product.

We raised antibodies in rabbits against the amino-terminal portion of the viral yes protein produced in bacteria with the use of an expression vector based on the lac operon. The anti-yes serum thus obtained precipitated P90gag-yes from Yamaguchi 73 virus-transformed chicken embryo fibroblasts, and this immunoprecipitation was blocked by the purified antigen. The anti-yes serum did not recognize viral src, fps, or fgr proteins. Affinity-purified anti-yes immunoglobulin G (IgG) precipitated two proteins of 59 and 62 kilodaltons from lysates of normal chicken embryo fibroblasts. Two-dimensional tryptic peptide mapping showed that these proteins are closely related to P90gag-yes and that they are different from pp60c-src. Similar to P90gag-yes, the 59- and 62-kilodalton proteins were phosphorylated exclusively on tyrosine in an in vitro kinase reaction, whereas in vivo they were phosphorylated on serine and, to a lesser extent, on tyrosine as well. Expression of the 59- and 62-kilodalton proteins, determined by the immune complex kinase assay, was relatively high in brain, retina, kidney, and liver. The presence in normal chicken embryo fibroblasts and in chicken kidney of two transcripts, 3.7 and 3.9 kilobases in length, that hybridize with a yes-specific DNA probe, as well as the two proteins recognized by anti-yes IgG, suggests either differential splicing of cellular yes gene transcripts or the existence of another yes-related gene.

Processing of 9E3 mRNA and regulation of its stability in normal and Rous sarcoma virus-transformed cells.

We studied the expression of 9E3 mRNA, which is known to be induced in chicken embryo fibroblasts by p60v-src activity and by serum. In addition to full-length 9E3 mRNA, we identified several smaller RNAs that hybridized with 9E3 cDNA. One of these RNAs hybridized with a 5' 9E3 cDNA probe but not with a 3' cDNA probe. The other hybridized with a 3' cDNA probe but lacked 5' sequences, including the entire 9E3 coding region. Only the latter RNA was polyadenylylated, as determined by RNase H digestion in the presence of oligo(dT). The level of the small RNAs increased after treatment with cycloheximide and actinomycin D, indicating that the small RNAs were produced by processing of preexisting transcripts. The derivation of the small RNAs from 9E3 mRNA rather than from a related gene was confirmed by S1 nuclease analysis. The 3' terminus of the 5' RNA and the 5' terminus of the 3' RNA mapped to the same position, which suggested that the small RNAs were formed by endonucleolytic cleavage of 9E3 mRNA at a specific site in the 3' noncoding region. We also found that the stability of 9E3 mRNA was increased after serum stimulation and was greater in Rous sarcoma virus-transformed than in uninfected cells. The relative amount of the small RNAs as compared with the full-length transcript was greatest under conditions in which the full-length transcript was least stable. These data suggest that site-specific endonucleolytic cleavage regulates the stability of 9E3 mRNA.

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.

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.

Novel tyrosine kinase identified by phosphotyrosine antibody screening of cDNA libraries.

In an attempt to clone protein tyrosine kinases, antiphosphotyrosine antibodies were used to screen lambda gt11 cDNA expression libraries. By this method, a 2.5-kilobase cDNA encoding a novel tyrosine kinase was isolated from a mouse liver cDNA library. This new gene is most closely related to the receptor tyrosine kinases ret, fms, and kit.

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.

Preferential expression of the c-fps protein in chicken macrophages and granulocytic cells.

We have studied the expression of the protein kinase activity of NCP98, the c-fps gene product, in several hemopoietic tissues of chickens as a function of the developmental stage of these organs. We found that in bone marrow, spleen, and bursa, maximum NCP98 kinase activity on a per-cell basis correlates with the peak of granulopoiesis in these organs. Furthermore, in a bovine serum albumin density gradient fractionation of bone marrow cells, granulocytic cells appeared to account for most of the NCP98 kinase activity. No correlation was found between the distribution of erythrocytic, lymphocytic, or thrombocytic cells and the distribution of the expression of NCP98 kinase activity. However, NCP98 protein and kinase activity were 10-fold higher in macrophages than in bone marrow. In addition, depletion by complement-mediated lysis of erythrocytic cells in bone marrow did not significantly reduce the total recovery of NCP98 kinase activity. These results argue for the specific expression of the c-fps gene product in granulocytic cells and macrophages.

Identification of domains of the v-crk oncogene product sufficient for association with phosphotyrosine-containing proteins.

The oncogene product of the avian sarcoma virus CT10, P47gag-crk, contains the SH2, SH2', and SH3 domains and binds proteins in a phosphotyrosine (ptyr)-dependent manner. In this study, we have determined the region of P47gag-crk essential for binding to ptyr-containing proteins. Mutant P47gag-crk proteins expressed in Escherichia coli that have the intact SH2 and SH2' regions retained the capacity to bind ptyr-containing proteins obtained from cells transformed by crk and src. The deletion of SH2 resulted in the loss of binding activity. Other mutants that have altered SH2 or SH2' bound few, if any, of the ptyr-containing proteins. Those mutants that bound ptyr-containing proteins associated with tyrosine kinase activity. We also found that polypeptides containing SH2, SH2', and SH3 of p60v-src and p60c-src associated with ptyr-containing proteins from crk-transformed cells. Thus, the SH2 and SH2' domains of P47gag-crk are responsible for their binding to ptyr-containing proteins.